Identification and characterization of the Sudanese Bacillus thuringiensis and related bacterial strains for their efficacy against Helicoverpa armigera and Tribolium castaneum.

Forty-four isolates of Bacillus thuringiensis like bacteria from various sources in different locations from Sudan were tested for their insecticidal activity. The toxicity of these isolates ranged from 6.6 to 70% to the neonates of cotton bollworm, Helicoverpa armigera at 10 ppm concentration. The most effective ones are Kb-29, St-6 and Wh-1 comparable with HD-1.  Toxicity of isolates to larvae of the red flour beetle, Tribolium castaneum ranged from 20 to 100%. Isolates St-2 and St-23 gave 100% larval mortality within 15 days of exposure and were at par with Ab-8, Ab-12, Kb-26, Kb-30, Om-4, Po-2, Po-5, Po-7, Sa-8 and Wh-5 and were also comparable with E. coli clone expressing Cry3 toxin. The most effective five isolates viz., Kb-29, St-2, St-6, St-23 and Wh-1 belonged to B. thuringiensis. The St-6 isolate, which also showed high toxicity to T. castaneum larvae, had cry1 genes along with coleopteran active cry28 genes, but not cry3 genes. Of the 25 isolates characterized with 16s DNA sequencing, seven belonged to Paenibacillus spp., one Lysinibacillus sphaericus, one Bacillus pumilus, four Bacillus spp., and rest 12 belonged to B. thuringiensis. Biochemical characterization in each species showed variation. The present study shows potential of some isolates like Kb-29, St-2, St-6, St-23 and Wh-1 as promising bioinsecticides.

Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests

The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations.

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples / Isolamento de linhagens de Bacillus thuringiensis contendo genes cry díptero-específicos a partir de amostras de solo oriundas de Ilha Bela-São Paulo, Brasil

The entomophatogenic bacterium Bacillus thuringiensis produces crystal proteins, named Cry proteins which are encoded by the cry genes. This bacterium is used on biological control of important economical pests, as well as in the control of disease´s vectors, such as Aedes aegypti, a mosquito that transmits the dengue viruses. Isolates of this bacterium can be characterized by the content of cry genes and this prediction helps target different insect orders. In this research, we isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela (SP, Brazil), a place where simulids are already biologically controlled by B. thuringiensis, to find bacterial isolates that were capable of controlling A. aegypti. The 16S ribosomal subunit genes of the selected isolates were sequenced, and the isolates were molecularly characterized based on their Dipteran-specific cry gene contents. Eight of the 76 isolates (10.52%) contained the cry4Aa, cry4Ba or cry10Aa genes, these isolates were carried out against A. aegypti larvae on bioassay. The presence or absence of specific cry genes was associated with the observed average larval mortalities. From the 76 isolates, seven (9.2%) were potentially able to control A. aegypti larvae. Therefore these are promising isolates for the biological control of A. aegypti larvae.

Bacillus thuringiensis é entomopatogênica, por produzir proteínas cristais, denominadas proteínas Cry, as quais são codificadas pelos genes cry. Essa bactéria atua no controle biológico de insetos-praga de culturas economicamente importantes, bem como no controle de insetos vetores causadores de doenças, como o Aedes aegypti, mosquito transmissor do vírus da dengue. Os isolados dessa bactéria podem ser caracterizados pelo conteúdo de genes cry que possuem e, assim, predizer o alvo de controle dos mesmos às diferentes ordens de insetos. Com o objetivo de encontrar isolados eficientes no controle do vetor A. aegypti, o presente trabalho isolou 76 colônias de B. thuringiensis a partir de 30 amostras de solo oriundas de Ilhabela-SP, município que se caracteriza por realizar controle biológico de simulídeos com essa bactéria. Os 76 isolados foram sequenciados na região da subunidade ribossomal 16S e caracterizados molecularmente quanto ao conteúdo de genes cry díptero-específicos. No total, oito isolados (10,52% do total) apresentaram bandas para os genes cry4Aa, cry4Ba e cry10Aa, sendo os mesmos testados contra larvas de A. aegypti por meio de bioensaios. A presença e/ou ausência dos genes cry foi associada à mortalidade média de larvas. Dentre os isolados estudados, sete (9,2% do total) apresentaram elevado potencial de controle às larvas de A. aegypti, sendo assim considerados como promissores para o manejo do controle biológico de larvas de A. aegypti com a bactéria B. thuringiensis.

Two new Brazilian isolates of Bacillus thuringiensis toxic to Anticarsia gemmatalis (Lepidoptera: Noctuidae) / Dois novos isolados brasileiros de Bacillus thuringiensis tóxicos para Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Bacillus thuringiensis is a bacterium used for biopesticides production and pest-resistant plants due to the synthesis of protein crystals by cry genes, which are effective in controlling several insect orders such as Lepidoptera. This work aimed at the evaluation and characterisation of two new B. thuringiensis isolates active against A. gemmatalis (Hübner 1818) larvae, which is the soybean major pest. The results showed that Bt117-4 isolate amplified fragments corresponding to cry2 and cry9 genes, and synthesised protein fragments equivalent to 130, 90 and 45 kDa. The Bt3146-4 isolate amplified DNA fragments corresponding to cry9 gene and synthesised protein fragments of 70, 58 and 38 kDa. Transmission electron microscopy revealed the presence of protein crystals in both isolates. CL50 with Cry purified proteins from Bt117-4 and Bt3146-4, corresponded to 0.195 and 0.191 µg larvae-1, respectively. The two B. thuringiensis isolates selected in this study were effective to control velvetbean caterpillar at laboratory conditions. Field tests should be carried on to develop new biopesticides formulation as well for cry genes resource for Anticarsia gemmatalis resistant transgenic plants.

Bacillus thuringiensis é uma bactéria utilizada na produção de biopesticidas e de plantas resistentes às pragas por causa da síntese de cristais proteicos pelos genes cry, os quais são eficazes no controle de diversas ordens de insetos, como os lepidópteros. O presente trabalho objetivou a avaliação e a caracterização de dois novos isolados de B. thuringiensis ativos contra lagartas de A. gemmatalis (Hübner 1818), que é a principal praga da cultura da soja. Os resultados obtidos revelaram que o isolado Bt117-4 amplificou fragmentos correspondentes aos genes cry2 e cry9, sendo que os fragmentos proteicos sintetizados foram equivalentes a 130, 90 e 45 kDa. O isolado Bt3146-4 amplificou fragmentos de DNA que correspondem ao gene cry9 e sintetizou fragmentos proteicos de 70, 58, e 38 kDa. Os dados de microscopia eletrônica de transmissão revelam a presença de cristais proteicos em ambos os isolados. A CL50, com proteínas Cry purificadas de Bt117-4 e Bt3146-4, correspondeu a 0,195 e 0,191 µg lagarta-1, respectivamente. Os dois isolados de B. thuringiensis selecionados neste trabalho mostraram-se eficientes no controle da lagarta-da-soja em laboratório, sendo recomendada sua avaliação a campo para posterior aplicação na formulação de biopesticidas ou como fonte de genes cry para a obtenção de plantas geneticamente modificadas resistentes à Anticarsia gemmatalis.

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec’s isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba. Rev. Biol. Trop. 59 (3): 1007-1016. Epub 2011 September 01.

El uso prolongado de insecticidas ha conducido al desarrollo de resistencia en diferentes especies de mosquitos y al incremento de la degradación del ambiente. El control biológico de insectos ha devenido como una alternativa útil y de bajo impacto ambiental. En nuestro estudio fueron identificados, caracterizados tres aislamientos de suelos procedentes de diferentes regiones del archipiélago cubano y comparados con cepas de referencia: aisladas de los biolarvicidas Bactivec y Bactoculicida, además de IPS82. La diferenciación de los mismos se llevó a cabo mediante métodos morfológicos, bioquímicos y moleculares (SDSPAGE, perfil plasmídico, RAPD). Los aislamientos fueron identificados como Bacillus thuringiensis; A21, A51 y C17 mostraron una mayor actividad contra larvas de Aedes aegypti and Culex quinquefasciatus que la cepa aislada del biolarvicida Bactivec, utilizada como referencia en este estudio. Dos de los aislamientos poseían perfiles proteicos y plasmídicos similares al de la cepa control IPS82, pero el restante difería de ellos. Los tres mostraron patrones de RAPD diferentes lo que nos permitió su diferenciación. Estos patrones de RAPD también diferían del observado para las cepas utilizadas comúnmente en el control biológico en nuestro país.

Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin, China

Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4 percent), followed by cry2Aa (6.8 percent) and cry2Ab alone (2.5 percent), and only one novel type of cry2 gene was cloned from one isolate (JF19-2). The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR), which was designated as cry2Ag1 (GenBank No. ACH91610) by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM) observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera) and Pieris rapae Linnaeus, 1758 (Lepidoptera). Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.

Atividade tóxica de isolados de Bacillus Thuringiensis a larvas de Aedes Aegypti (l.) (diptera: culicidae) / Toxic activity of Bacillus Thuringiensis isolates to Aedes Aegypti (l.) (diptera: culicidae) larvae

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2 percent) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3 percent) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100 percent mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.

Characterization of an Argentine isolate of Bacillus thuringiensis similar to the HD-1 strain

We report the characterization of an Argentine isolate of Bacillus thuringiensis (INTA TA24-6) similar to the HD-1 strain, which harbors a cryptic cry2Ab gene that apparently is transcribed but not translated into a protein. INTA TA24-6 showed a Rep-PCR pattern identical to the HD-1 strain, a plasmid pattern that resembled that of this strain and cry1 and cry2 genes as HD-1. Screening of cry1 and cry2 genes showed that INTA TA24-6 harbors only cry1Ac and cry2Ab genes. Furthermore, crystalline inclusions of INTA TA24-6 exhibit a bipyramidal shape, typical of Lepidoptera-active B. thuringiensis strains, containing a major protein of ca. 130 kDa toxic to Epinotia aporema Wals. (Lepidoptera: Tortricidae) larvae. Neither the flat-square to cuboidal crystal nor a ca. 65 kDa protein typical of strains expressing Cry2 proteins were detected in INTA TA24-6. In agreement with this information, parasporal crystals of INTA TA24-6 did not show toxicity to Aedes aegypti L. (Diptera:Culicidae) larvae. Gene transcription analyses suggested that the cry2A gene might be cryptic in INTA TA24-6 despite its transcription at different sporulation stages.

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.

Associação de bioensaios e caracterização molecular para seleção de novos isolados de Bacillus thuringiensis efetivos contra Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) / Association of bioassays and molecular characterization to select new Bacillus thuringiensis isolates effective against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

A lagarta de Spodoptera frugiperda (J. E. Smith) é uma das principais pragas do milho e para seu controle o Bacillus thuringiensis se destaca por sua atividade entomopatogênica. Este trabalho objetivou a caracterização molecular de isolados de B. thuringiensis quanto à presença do gene cry1 e a avaliação da sua eficiência no controle de lagartas de S. frugiperda. Nas análises da PCR, foi utilizado o Gral-cry1 para confirmação da presença do gene cry1 nos 115 isolados. Uma suspensão de 3 x 10(8) esporos/ml banhou a dieta utilizada para alimentação de 30 lagartas por isolado, com três repetições. A identificação do tipo de genes cry1 dos diferentes isolados foi realizada para cinco sub-classes de genes e análises de regressão linear foram realizadas para verificar possíveis associações entre a presença de um gene cry individual e altos níveis de toxicidade. Ttodos os DNAs amplificados com os iniciadores Gral-cry1 apresentaram produto de amplificação com tamanho esperado. Quanto aos níveis de eficiência inseticida contra a lagarta-do-cartucho, 41 isolados apresentaram 100 por cento de mortalidade e 16 apresentaram índice entre 75 por cento e 90 por cento. O gene cry1Ab esteve presente em 80 isolados, cryB em 69 isolados, cry1Ac em todos os isolados, cryV e cry1E em 93 e 27 isolados, respectivamente. Os valores referentes ao efeito individual de cada gene na mortalidade de larvas foram significativos a 1 por cento de probabilidade, para os genes cry1Ac e cry1E.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main corn pests and Bacillus thuringiensis is important in its control because of its entomopathogenic property. The objective of this study was the molecular characterization of B. thuringiensis isolates for cry1 locus presence and the assessment of the efficiency of these isolates in controlling S. frugiperda caterpillars. Gral-cry1 was used in the PCR analyses to confirm the presence of the cry1 locus in 15 isolates. A 3 x 10(8) spore/ml suspension bathed the diet used to feed 30 caterpillars per isolate, with three replications. The cry1 locus type genes of the different isolates were identified for five gene subclasses; linear regression analyses were carried out to ascertain possible associations between the presence of an individual cry1 locus gene and high levels of toxicity. All the DNAs amplified with Gral-cry1 presented an amplification product with the expected size. Regarding the levels of insecticide efficiency against the cob worm, 41 isolates presented 100 percent mortality and 16 presented an index between 70 percent and 90 percent. The cry1Ab gene was present in 80 isolates, cryb in 69 isolates, cry1Ac in all the isolates and cryv and cry1E in 93 and 27 isolates, respectively. The values regarding the individual effect of each gene on caterpillar mortality were significant at 1 percent probability for the cry1Ac and cry1E genes.

Bacillus thuringiensis in caterpillars and associated materials collected from protected tropical forests in northwestern Costa Rica

Bacillus thuringiensis (Bt) synthesizes crystalline inclusions that are toxic to caterpillars (Lepidoptera) and other orders of invertebrates. Materials associated with 37 caterpillars from 16 species, collected while feeding on 15 different species of host plants in dry, cloud and rain forests located in the Area de Conservación Guanacaste in northwestern Costa Rica, were examined for the presence of Bt. From a total of 101 derived samples, 25 Bt isolates were cultured: 56% from host plant leaves, 8% from caterpillar guts and 36% from caterpillar fecal pellets. Bt was isolated from at least one sample in 38% of the systems constituted by the food plant, gut and fecal pellets corresponding to a single caterpillar. Four different morphologies of crystalline inclusions were observed, with bipyramidal and irregular crystal morphologies being the most prevalent.

Bacillus thuringiensis (Bt) sintetiza inclusiones cristalinas que resultan tóxicas para algunas larvas de lepidópteros y otros órdenes de invertebrados. Su presencia fue examinada en materiales asociados a 37 orugas de mariposas de 16 especies, las cuales fueron colectadas mientras se alimentaban en 15 especies diferentes de plantas hospederas en bosques secos, nubosos y húmedos localizados dentro del Área de Conservación Guanacaste (ACG) en el noroeste de Costa Rica. A partir de un total de 101 muestras se obtuvo 25 aislamientos de Bt: 56% a partir de material foliar de las plantas hospederas, 8% a partir del contenido intestinal de las larvas y 36% a partir de sus excrementos. Esta bacteria fue cultivada a partir de al menos uno de los 3 diferentes tipos de muestra asociados a una oruga particular (planta hospedera, intestino, excremento) en 38% de los casos posibles. En la colección de aislamientos obtenida se observaron cuatro morfologías de inclusiones cristalinas, siendo aquellas bipiramidales e irregulares las más prevalentes.

Characterization of a Bacillus thuringiensis strain collection isolated from diverse Costa Rican natural ecosystems

Costa Rican natural ecosystems are among the most diverse in the world. For this reason, we isolated strains of the entomopathogenic bacteria Bacillus thuringiensis (Bt ) to determine their diversity, distribution and abundance. A total of 146 Bt strains were obtained from environmental samples collected from diverse natural ecosystems and life zones of Costa Rica. We recovered Bt strains from 71%, 63%, 61% and 54% of soil samples, fresh leaves, other substrates and leaf litter respectively. Bt was isolated in 65% of the samples collected in the humid tropical forest in national parks (Braulio Carrillo, Gandoca Manzanillo, Sierpe, Hitoy Cerere, and Cahuita), and in 59% of the samples collected in the dry tropical forest (Parque Nacional Marino las Baulas, Palo Verde and Santa Rosa). In the very humid tropical forest (Tortuguero) Bt was isolated in 75% of the samples and in the very humid tropical forest transition perhumid (Carara) it was found in 69% of the samples. The strains exhibit a diverse number, size and morphology of parasporal inclusion bodies: irregular (47%), oval (20%), bipyramidal (3%), bipyramidal and cubic (1%), bipyramidal, oval and irregular (5%) and bipyramidal, oval and cubic crystals (2%). Strains isolated from Braulio Carrillo, Tortuguero and Cahuita, presented predominantly irregular crystals. On the other hand, more than 60% of the isolates from Térraba-Sierpe and Hitoy-Cerere had medium oval crystals. Strains from Gandoca-Manzanillo, Palo Verde and Carara presented mainly combinations of oval and irregular crystals. Nevertheless, the greatest diversity in crystal morphology was observed in those from Santa Rosa, Llanos del Río Medio Queso and Parque Marino las Baulas. Protein analyses of the crystal-spore preparations showed -endotoxin with diverse electrophoretic patterns, with molecular weights in the range of 20 to 160 kDa. Fifty six percent of the strains amplified with the cry2 primer, 54% with vip3, 20% with cry1...

Como los ecosistemas naturales de Costa Rica figuran entre los más diversos del mundo, se propuso aislar la bacteria entomopatógena Bacillus thuringiensis (Bt ) con el fin de conformar una colección de cepas y caracterizarlas molecularmente. Se obtuvieron 146 cepas a partir de muestras ambientales de diversas áreas protegidas, que incluían 9 de las 12 zonas de vida de Costa Rica. Se recobraron cepas del 71%, 63%, 61%y 54% de las muestras de suelo, hojas frescas, otros sustratos y hojarasca respectivamente. Se aisló Bt del 65% de las muestras del bosque tropical húmedo, un 59% de las muestras del bosque tropical seco. Del bosque tropical muy húmedo se aisló Bt del 75% de las muestras y finalmente del bosque tropical muy húmedo transición perhúmedo se encontró en el 69% de las muestras. Las cepas se caracterizaron según la morfología de los cuerpos paraesporales de inclusión, el peso molecular de las -endotoxinas y de genes cry,cyt y vip que contenían. Las cepas exhibieron cristales de diferente morfología, tamaño y número: irregulares, ovales, bipiramidales, cúbicos o mezclas de uno u otro. No se encontró correlación al comparar la forma de los cristales con el sitio de origen de la cepa. El análisis proteico de las mezclas de esporas y cristales mostró que las cepas contenían -endotoxinas de 20 a 160 kDa.El 66 por ciento de las cepas amplificaron con los imprimadores específicos para el gen cry2,54% con vip3, 20% con el cry1, 9% con el cry3-cry7 y 8% con el gen cry8. Los genes cry11 y cyt se encontraron en el 8%y 7% de las cepas respectivamente. Veinticuato cepas no amplificaron con los imprimadores utilizados por lo que podrían contener genes novedosos. Las cepas que contenían el gen cry1 se amplificaron posteriormente con imprimadores específicos para la subfamilia de dicho gen, obteniéndose 13 perfiles diferentes. En síntesis, la diversidad genética de las cepas sugiere que la colección tiene un gran potencial para el control de diferentes...

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinanting B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.

Identification of entomopathogenic Bacillus isolated from Simulium (Diptera, Simuliidae) larvae and adults

Entomopathogenic bacteria isolated from Simulium larvae and adults from breeding sites in the states of Säo Paulo and Rio de Janeiro, Brazil, were identified as 18 strains of Bacillus thuringiensis and one of B. sphaericus. Most of these strains were serotyped according to their flagellar antigens. However, nine of the B. thuringiensis samples, could not be serotyped and were designated as "autoagglutinating"; they were also shown to be toxic in preliminary tests against Aedes aegypti larvae. Additionally, B. sphaericus was also shown to be toxic towards Culex quinquefasciatus larvae

Isolation of mosquito-pathogenic Bacillus sphaericus & B. thuringiensis from the root surface of hydrophytes.

Attempts were made to isolate B. sphaericus and B. thuringiensis active against mosquito larvae from the root surface of hydrophytes. Out of 139 samples processed, 86 B. sphaericus and 23 B. thuringiensis isolates were obtained. Sixty two of the B. sphaericus isolates belonged to the serotype H5a5b, 2 to H6 and 22 isolates did not agglutinate with any of the 6 antisera tested. Twenty of the B. thuringiensis isolates belonged to the H14 serotype, 1 each to the H10 and H17 serotype(s) and 1 to an unknown serotype. Fifty nine of the B. sphaericus and 20 of the B. thuringiensis isolates fall under highly toxic category with the LC50 dose of 1-50 ng/ml for Culex quinquefasciatus third instar larvae.

Isolation of indigenous larvicidal microbial control agents of mosquitos: the Malaysian experience.

A nationwide screening program searching for microbial control agents of mosquitos was initiated in Malaysia in 1986. A total of 725 samples were collected and 2,394 bacterial colonies were isolated and screened for larvicidal activity. From such screening, 20 Bacillus thuringiensis, 6 B. sphaericus, 1 Clostridium bifermentans and 2 Pseudomonas pseudomallei larvicidal isolates were obtained. Of these, a new B. thuringiensis named as subspecies malaysianensis was found, while the C. bifermentans was also a new anaerobe individualized as serovar malaysia. It was concluded that this screening program was highly successful.