Phenotypic plasticity in adults of Anticarsia gemmatalis exposed to sub-doses of Bt-based bioinsecticide / Plasticidade fenotípica morfométrica em adultos de Anticarsia gemmatalis Hübner, 1818 expostos a subdoses de Bacillus thuringiensis Berliner, 1911

Anticarsia gemmatalis Hünber, 1818 is one of the main defoliating species in the soybean crop. Bacillus thuringiensis Berliner, 1915, is a bacterium used in the biological control of this pest species. Resistant populations and their sublethal effects caused by the use of the bacteria have already been reported; however, there are no studies on phenotypic plasticity in adulthood exposed to Bt-based bioinsecticide sub-doses. This study aimed to evaluate the morphometry of A. gemmatalis adults under laboratory conditions submitted to the Bt-based bioinsecticide Dipel® over the three generations. The body segments mensuread were width, length, and area of the anterior and posterior wings, the weight of the integument, chest, abdomen, wings, and the whole adult of males and females. Among the treatments, LC5 in the first generation and LC10 in the second generation were those with lower thresholds in relation to the weight of the chest and abdomen, considering the proportions of the body smaller than the females. The female's weight adulthood was reduced by 10% about males, and, only in the first generation. Males have larger body size and more pronounced phenotypic plasticity than females. Here, we demonstrate the first study assessing the phenotypic plasticity of A. gemmatalis adults.

Anticarsia gemmatalis Hünber, 1818 é uma das principais espécies desfolhadoras da cultura da soja. Bacillus thuringiensis Berliner, 1915, é uma bactéria utilizada no controle biológico dessa espécie de praga. Populações resistentes e seus efeitos subletais causados pelo uso da bactéria já foram relatados, no entanto, não há estudos sobre a plasticidade fenotípica na idade adulta exposta a subdoses de bioinseticida à base de Bt. Este trabalho teve como objetivo avaliar a morfometria de adultos de A. gemmatalis em condições de laboratório submetidos ao bioinseticida Dipel® ao longo de três gerações. Os segmentos corporais mensuráveis eram largura, comprimento e área das asas anterior e posterior, o peso do tegumento, tórax, abdômen, asas e todo o adulto de machos e fêmeas. Dentre os tratamentos, CL5 na primeira geração e CL10 na segunda geração foram aqueles com limiares mais baixos em relação ao peso do tórax e abdômen, considerando as proporções do corpo menores que as do sexo feminino. O peso da fêmea na idade adulta foi reduzido em 10% em relação aos machos e, apenas na primeira geração. Os machos têm tamanho corporal maior e plasticidade fenotípica mais pronunciada do que as fêmeas. Este estudo demonstra o primeiro estudo avaliando a plasticidade fenotípica de adultos de A. gemmatalis.

Phenotypic plasticity in adults of Anticarsia gemmatalis exposed to sub-doses of Bt-based bioinsecticide

Abstract Anticarsia gemmatalis Hünber, 1818 is one of the main defoliating species in the soybean crop. Bacillus thuringiensis Berliner, 1915, is a bacterium used in the biological control of this pest species. Resistant populations and their sublethal effects caused by the use of the bacteria have already been reported; however, there are no studies on phenotypic plasticity in adulthood exposed to Bt-based bioinsecticide sub-doses. This study aimed to evaluate the morphometry of A. gemmatalis adults under laboratory conditions submitted to the Bt-based bioinsecticide Dipel® over the three generations. The body segments mensuread were width, length, and area of the anterior and posterior wings, the weight of the integument, chest, abdomen, wings, and the whole adult of males and females. Among the treatments, LC5 in the first generation and LC10 in the second generation were those with lower thresholds in relation to the weight of the chest and abdomen, considering the proportions of the body smaller than the females. The females weight adulthood was reduced by 10% about males, and, only in the first generation. Males have larger body size and more pronounced phenotypic plasticity than females. Here, we demonstrate the first study assessing the phenotypic plasticity of A. gemmatalis adults.

Resumo Anticarsia gemmatalis Hünber, 1818 é uma das principais espécies desfolhadoras da cultura da soja. Bacillus thuringiensis Berliner, 1915, é uma bactéria utilizada no controle biológico dessa espécie de praga. Populações resistentes e seus efeitos subletais causados pelo uso da bactéria já foram relatados, no entanto, não há estudos sobre a plasticidade fenotípica na idade adulta exposta a subdoses de bioinseticida à base de Bt. Este trabalho teve como objetivo avaliar a morfometria de adultos de A. gemmatalis em condições de laboratório submetidos ao bioinseticida Dipel® ao longo de três gerações. Os segmentos corporais mensuráveis eram largura, comprimento e área das asas anterior e posterior, o peso do tegumento, tórax, abdômen, asas e todo o adulto de machos e fêmeas. Dentre os tratamentos, CL5 na primeira geração e CL10 na segunda geração foram aqueles com limiares mais baixos em relação ao peso do tórax e abdômen, considerando as proporções do corpo menores que as do sexo feminino. O peso da fêmea na idade adulta foi reduzido em 10% em relação aos machos e, apenas na primeira geração. Os machos têm tamanho corporal maior e plasticidade fenotípica mais pronunciada do que as fêmeas. Este estudo demonstra o primeiro estudo avaliando a plasticidade fenotípica de adultos de A. gemmatalis.

Research progress on the structure of crystal protein of Bacillus thuringiensis and its mechanism / 中国热带医学

@#Abstract: Mosquitoes are involved in the transmission of serious diseases such as malaria, dengue, chikungunya, yellow fever, Zika virus disease, and filariasis, and their prevention and control have always been a research hotspot. Currently, mosquito control methods mainly include physical control, chemical control and biological control. Physical control methods are environmentally friendly, but they are slow to take effect and have unsatisfactory control effects; although chemical control can quickly eliminate mosquitoes, it has been eliminated due to its high pollution, high residual, and easy drug resistance; biological control uses natural enemies or pathogens to kill mosquitoes and reduce their ability to transmit disease. Therefore, environmentally friendly biological control has become the main measure for controlling and preventing mosquitoes. In recent years, significant progress has been made in bacterial mosquito control agents, among which Bacillus thuringiensis has been the most extensively studied. Bacillus thuringiensis is a Gram-positive soil microorganism, which is the pathogenic bacterium of a variety of agricultural pests such as Lepidoptera, Coleoptera and Diptera. During the sporulation process, its strains produce a variety of insecticidal crystal proteins (ICPs) or δ-endotoxins with insecticidal activity against mosquito larvae. This review firstly introduces the crystal proteins of Bacillus thuringiensis, describes in detail the types and structures of crystal proteins in detail, and also reveals the mechanism of action of crystal proteins related to receptors.

Bacillus thuringiensis A21, un aislado no tóxico de elevada actividad larvicida residual contra Aedes aegypti / Bacillus thuringiensis A21, a non-toxic isolate of high residual larvicidal activity against Aedes aegypti

Introducción: Los productos derivados de Bacillus thuringiensis se han empleado con éxito en el control de insectos. Sin embargo, la poca residualidad promueve la búsqueda de alternativas. En Cuba diferentes estudios informan la evaluación y selección de aislamientos de B. thuringiensis con actividad larvicida contra Aedes aegypti: el aislado A21 se destaca por su elevada actividad larvicida y ausencia de betaexotoxina. Objetivo: Evaluar la toxicidad/patogenicidad aguda oral y actividad larvicida residual del aislado A21. Métodos: La evaluación de la toxicidad/patogenicidad aguda oral del aislado A21 se estableció en el modelo biológico Rattus norvegicus. Se realizaron observaciones clínicas diarias de los animales y se evaluó el peso corporal. Se estimó la eliminación y la infectividad de B. thuringiensis mediante análisis de muestras de heces, y de fluidos y órganos, respectivamente. Para determinar la actividad larvicida residual del aislado A21 frente a larvas de Ae. aegypti se realizaron bioensayos con diferentes volúmenes de recambios de agua semanal (total, parcial, no recambio) en los recipientes. La mortalidad obtenida se calculó a las 24-72 h. Resultados: Con el aislado A21 no se detectó toxicidad/patogenicidad aguda oral en Rattus norvegicus. En el estudio de residualidad, la mortalidad larvaria a las 72 h se mantuvo elevada (80-100 por ciento) hasta la semana 24. A partir de la semana 25 la mortalidad larvaria disminuyó (p < 0,05). Conclusiones: Se evidencia la baja toxicidad y la elevada actividad larvicida residual del aislado A21 contra Ae. aegypti y lo convierten en un candidato promisorio para el desarrollo de biolarvicidas. Estos productos biológicos podrían contribuir a mejorar las estrategias de control del vector existentes en Cuba(AU)

ABSTRACT Introduction: Bacillus thuringiensis-based products have been successfully used for insect control. However, their low residuality promotes the search for alternatives. In Cuba, different studies have informed about the evaluation and selection of B. thuringiensis isolates with larvicidal activity against Aedes aegypti: isolate A21 highlights for its high larvicidal activity and absence of beta-exotoxins. Objective: To evaluate the acute oral toxicity/pathogenicity and the residual larvicidal activity of isolate A21. Methods: The evaluation of the acute oral toxicity/pathogenicity of isolate A21 was established in the animal model Rattus norvegicus. Daily clinical observations of the animals were carried out, and their body weight was evaluated. The elimination and infectivity of B. thuringiensis were estimated by analyzing feces, and fluids and organs samples, respectively. To determine the residual larvicidal activity of isolate A21 to Ae. aegypti larvae, bioassays were conducted with different volumes of weekly water changes (total, partial, no change) in the containers. Mortality was calculated at 24h-72h. Results: No acute oral toxicity/pathogenicity was detected with isolate A21 in Rattus norvegicus. In the study of residuality, at 72h larval mortality remained high (80 percent-100%) until week 24, decreasing from week 25 (p < 0.05). Conclusions: It is evidenced the low toxicity and high residual larvicidal activity of isolate A21 against Ae. aegypti, which makes it a promising candidate for the development of biolarvicides. These biological products could contribute to the improvements of the existing vector control strategies in Cuba(AU)

Evaluation of Cuban Bacillus thuringiensis (Berliner, 1911) (Bacillales: Bacillacea) isolates with larvicidal activity against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae)

Objective: To evaluate 11 Cuban native Bacillus (B.) thuringiensis isolates in order to select one with the best larvicidal activity against Aedes (Ae.) aegypti and low cytotoxicity. Methods: The cry and cyt genes of the isolates (A21, A51, L95, L910, M29, R84, R85, R87, R89, U81 and X48) were amplified by PCR. The influence of organic matter and NaCl on the larvicidal activity was tested by bioassays. Cytotoxicity was assayed on peritoneal macrophages of BALB/c mice. Results: The cyt1 (Aa, Ab, Ba), cyt2, cry4aA, cry4Ba, cry11 (Aa, Ba, Bb) and cry10 genes were identified in all native Cuban isolates. The larvicidal activity (LC90) of seven isolates was affected by the presence of organic matter in the water, while A21, A51, L910, R84, U81 and X48 had better LC50, LC90, LC95 than the 266/2 9-VI-98 control strain. The LC50 of two isolates was affected by the presence of NaCl and A21, A51, R85 isolate had better larvicidal activity than the 266/2 9-VI-98 control strain. In terms of toxicity against macrophages, the extracts of nine isolates were less cytotoxic than the control strains. Conclusions: Native isolate A21 had the main virulence factors against Ae. aegypti larvae, displayed a good larvicidal activity in presence of different factors related with Ae. aegypti breeding sites, and had low citotoxicity against macrophages. These results can contribute to the improvement of existing biological control strategies and the development of new biolarvicides.

Controladores biológicos para la reducción de índices aédicos / Biological controllers for the reduction of aedic indices

Ante la ausencia de vacunas eficaces para las arbovirosis transmitidas por el vector A. aegypti y la resistencia a insecticidas se hace necesario buscar alternativas, donde resalta el control biológico con el uso del larvicida microbiológico Bacillus thuringiensis var. israelensis (Bti). Como objetivo, se propuso determinar los índices aédicos y el uso de Bacillus thuringiensis israelensis como controlador biológico de Aedes aegypti en la localidad de Huánuco, Perú durante el segundo semestre de 2019. La investigación fue experimental de corte transversal con una muestra de 263 viviendas distribuidas en cinco bloques. Como instrumento se usó la observación y reporte de fichas a través de la inspección de cada vivienda en dos momentos, inicial y final, separadas por un lapso de 90 días. Para el análisis de los datos se usó estadística descripctiva a través de Epidat 3.0, obteniendo IA, IR e IB. Como resultado, de las 236 casas inspeccionadas, 51% (120/236) fueron no intervenidas y 49% (116/236) fueron intervenidas con Bacillus thuringiensis, de las casas no intervenidas (120/236) en la fase inicial 59% fueron positivas (con la presencia de algún estadío de A. aegypti) y en la fase final fue de 63%, por otra parte, en las casas intervenidas con Bacillus thuringiensis (CCB) (116/236) en la fase inicial 61% (71/116) estaban positivas y la fase final hubo un descenso a 32% (37/116). En conclusión, se sugiere que la aplicación de Bti constituye un componente adecuado para el manejo de poblaciones larvales de A. aegypti, en la ciudad de Perú(AU)

In the absence of effective vaccines for arboviruses transmitted by the vector A. aegypti and resistance to insecticides, it is necessary to look for alternatives, where biological control stands out with the use of the microbiological larvicide Bacillus thuringiensis var. israelensis (Bti). As an objective, it was proposed to determine the aedic indices and the use of Bacillus thuringiensis israelensis as a biological controller of Aedes aegypti in the town of Huánuco, Peru during the second half of 2019. The research was experimental cross-sectional with a sample of 263 homes distributed in five blocks. As an instrument, the observation and reporting of records was used through the inspection of each dwelling in two moments, initial and final, separated by a period of 90 days. For data analysis, descriptive statistics were used through Epidat 3.0, obtaining IA, IR and IB. As a result, of the 236 houses inspected, 51% (120/236) were not intervened and 49% (116/236) were intervened with Bacillus thuringiensis, of the houses not intervened (120/236) in the initial phase, 59% were positive (with the presence of some stage of A. aegypti) and in the final phase it was 63%, on the other hand, in the houses intervened with Bacillus thuringiensis (CCB) (116/236) in the initial phase 61% (71 /116) were positive and the final phase there was a decrease to 32% (37/116). In conclusion, it is suggested that the application of Bti constitutes an adequate component for the management of larval populations of A. aegypti, in the city of Peru(AU)

A 6-year field monitoring of fall armyworm, Spodoptera frugiperda, in transgenic Bt maize in Brazil

ABSTRACT A 6-year field monitoring study was designed to compare the presence of fall armyworm Spodoptera frugiperda (J. E. Smith), leaf injury, its parasitoids, 100 seed weight, and seed yield of Bt maize hybrids approved for commercialization in the Brazilian market. Field trials were planted in two municipalities, divided into two periods, from 2011 to 2014 (period I), and due to the approval of different Bt hybrids, from 2015 to 2016 (period II). Treatments were Bt, non-Bt maize and all non-Bt maize sprayed with insecticide methomyl. Six Bt-maize hybrids were planted, expressing the following proteins: Cry1Ab, Cry1F, Cry1A.105 (Cry1Ab +Cry1Ac +Cry1F), Cry2Ab2a and Vip3. In 2015 and 2016 Bt Powercore was planted due to its commercial approval. The number of S. frugiperda larvae for most Bt hybrids was lower than on non-Bt hybrids, except for Fórmula TL and Herculex in period II, in both municipalities. In period I, Bt hybrids VT, VT 2 and Viptera showed almost no injury on their leaves, despite the high number of fall armyworm larvae found in plants. Different parasitoids emerged from larvae from Bt and non Bt hybrids: Archytas sp., Campoletis sp., Chelonus sp., Eiphosoma sp. and Ophion luteus. The average 100 seed weight was higher in municipality from Sete Lagoas for all Bt hybrids, non-Bt hybrids and non-Bt hybrids sprayed with methomyl, when compared to Nova Porteirinha during period I. Fórmula TL, non-Bt Fórmula TL sprayed with methomyl, and non-Bt Fórmula TL showed the lowest seed yield in both municipalities and periods.

Impact of Bacillus thuringiensis on inhibiting certain Alternaria alternata’s mycotoxins isolated from infected potatoes

Aims@#Potatoes are considered one of the most strategic vegetable crops all over the world. Alternaria alternata has recently contaminated certain potatoes farms in different regions in Egypt. Among thirteen samples from fifteen regions were studied in a precedent study. Our study was aimed to investigate the effect of Bacillus thuringiensis subsp. Kurosaki suspension on inhibiting the growth of the three tested isolates of A. alternata and minimizing their mycotoxins production in vitro using three isolates with three levels of highly, moderate and low pathogenicity with unequal amounts of dual mycotoxins production.@*Methodology and results@#Three isolates of A. alternata from three regions, Kom Hamada (KH3), Alamin (Alam1) and Nobaria (NO3), which were determined as a producer of tenuazonic acid (TeA) and alternariol monomethyl ether (AME) toxins. Bacillus thuringiensis (Bt) use as commercial fungicide was applied with three suspension concentrations (75, 150 and 300 μg/mL) as inhibitor for the two mycotoxins. Our results illustrated that the three tested isolates recorded high TeA and AME inhibition efficacies by increasing the Bt suspension concentration. The highest inhibitory concentration of Bt was at concentration 75 μg/mL for isolated from Nobaria third region (NO3) and Alam1 it was (99.83 and 99.74%) for mycotoxin (AME) while, TeA mycotoxin had the most inhibition percentage (99.58%) at concentration 150 μg/mL for the isolate (NO3).@*Conclusion, significance and impact of study@#The preliminary results of the study suggest that B. thuringiensis spores’ suspension with different concentrations can be used as anti-mycotoxigenic agents to inhibit the (TeA) and (AME) mycotoxins produced by Alternaria alternata.

Advances in receptor-mediated resistance mechanisms of Lepidopteran insects to Bacillus thuringiensis toxin / 生物工程学报

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.

Study of Expression of Indigenous Bt cry2AX1 Gene in T3 Progeny of Cotton and its Efficacy Against Helicoverpa armigera (Hubner)

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.

Evaluación de la eficacia larvicida de Rapidall NP3 (Bacillus thuringiensis) contra Aedes aegypti (Linnaeus) (Diptera: Culicidae) en condiciones de laboratorio / Evaluation of the larvicidal efficacy of Rapidall NP3 (Bacillus thuringiensis) against Aedes aegypti (Linnaeus) (Diptera: Culicidae) in laboratory conditions

Los mosquitos son vectores trasmisores de enfermedades como: dengue, zika y chikunguya. El control biológico es una alternativa viable a tener en cuenta por los programas de control. Bacillus thuringiensis es el microorganismo más usado en salud pública. Su efectividad como larvicida contra culícidos depende, en gran medida, de la eficiencia del proceso productivo. El objetivo es evaluar y comparar la eficacia larvicida contra Aedes aegypti de la formulación en polvo Rapidall NP3 con la del producto Bactivec®, formulación líquida utilizada en Cuba. Se realizaron bioensayos de laboratorio según los procedimientos descritos y sugeridos por la OMS para determinar la eficacia, exponiendo las larvas a las concentraciones recomendadas por los fabricantes de los productos Rapidall NP3 y Bactivec®. A los recipientes utilizados se les realizó recambio de agua medio, total y sin recambio, con una frecuencia semanal. A las 24 h se calculó la mortalidad obtenida y se analizó estadísticamente mediante un ANOVA de una vía; cuando existieron diferencias significativas se aplicó un análisis Post-hoc mediante la prueba de Tukey. El producto evaluado Rapidall NP3, provocó 100 por ciento de mortalidad larvaria durante 11 semanas, independientemente de la proporción de recambio de agua, mientras que el Bactivec® solo provocó 100 por ciento de mortalidad hasta las 6 semanas de iniciado el experimento. El biolarvicida Rapidall NP3 mostró buena eficacia y actividad residual prolongada al compararlo con Bactivec®, por lo que recomendamos su evaluación en el terreno en el control larvario de depósitos que constituyen sitios de cría habituales y que no pueden ser eliminados físicamente(AU)

Mosquitoes are vectors of human diseases such as dengue, zika and chikungunya. Biological control is a viable alternative to be taken into account in control programs. Bacillus thuringiensis is the microorganism most commonly used in public health. Its effectiveness as a larvicide against culicides depends to a great extent to the production process. The objectives of the study were to evaluate and compare the larvicidal efficacy against Aedes aegypti of the Rapidall NP3 powder formulation with Bactivec®, a liquid formulation used in Cuba. Laboratory bioassays were conducted to determine efficacy following WHO protocols. Larvae were exposed to the concentrations recommended by Rapidall NP3 and Bactivec® manufacturers. The water in the containers used in the study was replaced once a week: half, whole and no replacement. At 24 hours mortality was estimated and statistically analysis were done by one-way ANOVA. When significant differences were found, post-hoc analysis was performed with Tukey's test. Rapidall NP3 caused 100 percent larval mortality for 11 weeks, regardless of the water replacement proportion, whereas Bactivec® caused 100 percent mortality for only 6 weeks after the start of the experiment. When compared with Bactivec®, biolarvicide Rapidall NP3 displayed better efficacy and more prolonged residual activity. We therefore recommend its field evaluation for larval control of containers constituting habitual breeding sites which cannot be physically eliminated(AU)

Salmonella spp. em pontos críticos da cadeia de produção de hortaliças orgânicas no Estado de São Paulo: contribuição para avaliação de risco / Salmonella spp. at critical points in organic vegetables production chain in the state of São Paulo: contribution for risk assessment

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

Larvicidal and adulticidal effects and ultrastructural changes of larvae midgut epithelium of musca domestica (diptera: muscidae) fed with bacillus thuringiensis var. kyushuensis

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.

Preparation and purification of Cry1Ah protein candidate reference material / 生物工程学报

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.

Selectivity of different biological products to the egg parasitoid Telenomus remus (Hymenoptera: Platygastridae)

ABSTRACT The selectivity of five entomopathogens and a chemical insecticide (positive control) to pupae and adults of the egg parasitoid Telenomus remus (Nixon) was evaluated in the laboratory under controlled environmental conditions according to protocols established by the International Organization for Biological Control. Baculovirus anticarsia (Baculovirus AEE®), Bacillus thuringiensis var. kurstaki (Thuricide®), Bacillus thuringiensis var. aizawai (Agree®), Beauveria bassiana (Boveril®), Metarhizium anisopliae (Metarril®) and Trichoderma harzianum (Trichodermil®) are harmless to T. remus pupae, and adults. Thus, our results suggest that the insect control strategies applied here are compatible since entomopathogens were classified as harmless to T. remus in most examined cases and therefore facilitate a joint application to control different pests. Bacillus thuringiensis var. kurstaki (Dipel®), despite being classified as slightly harmful in some of the evaluations, can still be considered compatible for use together with T. remus, especially when compared with chemical insecticides such as chlorpyrifos that might be considered harmful to the parasitoid survival.

Novel larvicide tablets of Bacillus thuringiensis var. israelensis: Assessment of larvicidal effect on Aedes aegypti (Diptera: Culicidae) in Colombia / Nuevas tabletas larvicidas de Bacillus thuringiensis var. israelensis: evaluación del efecto larvicida sobre Aedes aegypti (Diptera: Culicidae) en Colombia

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.

Susceptibilidad de la larva de aedes aegypti a bacillus thuringiensis var israelensis en Tegucigalpa, Honduras / Susceptibility of the larva de Aedes aegypti to bacillus thuringiensis var israelensis in Tegucigalpa, Honduras

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)

Field testing & evaluation of the efficacy & duration of effectiveness of a biolarvicide, Bactivec® SC (Bacillus thuringiensis var. israelensis SH-14) in Bengaluru, India

Background & objectives: Different formulations of Bacillus thuringiensis var. israelensis (Bti) have been tested against different mosquito vectors and other insects for their residual activity. In the present study, the efficacy and residual activity of a new formulation of Bti (Bactivec Suspension Concentrate) were evaluated against immature stages of Anopheles stephensi Liston (Diptera: Culicidae), Aedes aegypti Linnaeus (Diptera: Culicidae) and Culex quinquefasciatus Say (Diptera: Culicidae), in natural habitats in Phase II and Phase III in Bengaluru, India. Methods: Preferential breeding habitats of the mosquito species were selected and four dosages (0.25, 0.5, 1 and 2 ml/50 l) were tested in Phase II trial. Two most effective dosages, 0.5 and 1 ml/50 l were selected for Phase III trial. The evaluation was carried out essentially following the guidelines of the World Health Organization Pesticide Evaluation Scheme. Pre-treatment and post-treatment densities were recorded at regular intervals, and >80 per cent reduction in pupae was taken as the duration of effectiveness. Results: Bactivec SC treated at the dosage of 1 ml/50 l could produce 10-17 days efficacy (>80% reduction in pupae) in clean water habitats tested, whereas 0.5 ml/50 l dosage showed residual activity from 7 to 14 days against Ae. aegypti and An. stephensi in Phase III studies. In polluted water habitats, 4-7 days efficacy could be recorded against Cx. quinquefasciatus in Phase III. Interpretation & conclusions: The Bactivec SC formulation was operationally feasible and easy to handle. For the control of Anopheles and Aedes mosquitoes in freshwater habitats, 1 ml/50 l dosage was found effective, whereas in polluted water habitats against Cx. quinquefasciatus 5 ml/m2 was found effective.

Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae / Selección de cepas de Bacillus thuringiensis tóxicas para larvas del picudo del algodonero (Anthonomus grandis, Coleoptera: Curculionidae)

Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were β-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of β-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of β-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strainsβ-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., crylla) to produce transgenic cotton plants resistant to this pest.

Se realizaron ensayos preliminares con cultivos completos de 124 cepas de Bacillus thuringiensis utilizando larvas neonatas de Anthonomus grandis, una plaga principal del algodón en Argentina y otras regiones de América. Se seleccionaron 3 cepas exóticas y 4 nativas por producir mortalidad superior al 50%, todas ellas productoras de β-exotoxina. Las cepas nativas presentan la misma morfología de cristales, un perfil de proteínas similar y los mismos genes insecticidas. Estas características hacen que se parezcan a cepas tóxicas para lepidópteros. Además, mostraron un perfil de Rep-PCR idéntico al de la cepa lepidoptericida HD-1, lo que indica que podrían pertenecer al serovar kurstaki. Sin embargo, se observaron diferencias en el perfil plasmídico y en la producción de β-exotoxina. Para determinar en qué fracción del cultivo se encontraban los metabolitos responsables de la toxicidad, se compararon los resultados de bioensayos en los que se utilizó biomasa, sobrenadante filtrado (SF) o cultivos completos. Ambas fracciones mostraron cierto grado de toxicidad. Los resultados indican que los principales factores tóxicos se encuentran en el sobrenadante y estarían directamente relacionados con la presencia de β-exotoxina. De acuerdo con los bioensayos de SF y SF autoclavado, no se descarta también la participación en la toxicidad de factores de virulencia termolábiles, como CrylIa. En las cepas seleccionadas, el principal factor de virulencia es la β-exotoxina, por lo que su uso debería restringirse para el control biológico de A. grandis. No obstante, estas podrían ser fuente de genes (p. ej., crylIa) para la producción de plantas de algodón transgénicas resistentes a dicha plaga.

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina / Diversidad y distribución de genes de toxinas insecticidas Lepidoptera-específicos en cepas de Bacillus thuringiensis de Argentina

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cryl, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

Se analizaron 268 cepas de Bacillus thuringiensis obtenidas de diferentes fuentes de Argentina con el objeto de determinar la diversidad y distribución de genes cryl, cry2, cry8, cry9 y vip3A, que codifican proteínas insecticidas lepidóptero-específicas. Se excluyeron las cepas gemelas. Se detectaron solo diez perfiles diferentes entre los 80 B. thuringiensis seleccionados. Dos de estos perfiles, el cry1Aa, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (35/80) y el cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (25/80), comprendieron el 75% de las cepas seleccionadas. La existencia de esta baja diversidad es una rareza, ya que en la mayor parte de las colecciones estudiadas se ha descrito una gran diversidad de perfiles de genes de toxinas insecticidas. El perfil detectado con mayor frecuencia se obtuvo principalmente de cepas procedentes de suelo (el 70% de los de esa fuente lo tenían), también fue mayoritario entre los procedentes de polvo de producto almacenado (59%) y en los que procedían de telas de arana (50%). En cambio, el perfil cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa se detectó principalmente en las cepas aisladas de hojas (40%) y de larvas de insectos muertos (50%). Seis de los perfiles identificados fueron encontrados en cepas aisladas de polvo de producto almacenado y de hojas, lo que indica una mayor diversidad de perfiles en estas fuentes que en otras. Se identificaron algunas cepas con alta actividad insecticida contra larvas de Epinotia aporema (Lepidoptera), hallazgo importante para explorar en el futuro estrategias microbianas para el control de esta plaga en la región.