Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

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.

Owlet moths (Lepidoptera: Noctuoidea) associated with Bt and non- Bt soybean in the brazilian savanna / Noctuóides (Lepidoptera: Noctuoidea) associados a soja Bt e não-Bt no Cerrado brasileiro

Abstract The use of GMO expressing Bt toxin in soybean production has increased significantly in the last years in Brazil in order to manage the damage caused by lepidopteran pests. In this study, we compared the richness and abundance of owlet moths (Noctuoidea) associated with Bt and non-Bt soybean. We determined the temporal variations as a function of phenology, and correlated the population variations of the most common species with meteorological variables. The research was conducted at the experimental area of Embrapa Cerrados. The collection method used was differentiated being suppressive and absolute. A total of 13 species were collected, of which eight occurred on Bt soybeans. The most representative taxa were Chrysodeixis includens (72.87%), Anticarsia gemmatalis (18.17%) and Spodoptera spp (5.22%). The number of larvae belonging to species targeted by the Bt technology was 10 times lower on Bt than on non-Bt soybeans. Utetheisa ornatrix and Elaphria deltoides were recorded on soybean for the first time, observing larvae of both species in non-Bt soybean and those of U. ornatrix also in Bt soybean. Only A. gemmatalis larvae correlated (p <0.05) negatively with precipitation. This study provided field information on the abundance and species richness of owlet moths on non-Bt soybeans, associated with the effects of Bt soybean. When considering the different levels of infestation between cultivars as a criterion, larvae monitoring is of substantial importance in order to develop the lost control program.

Resumo O uso de OGM que expressam toxina Bt na produção de soja tem aumentado significativamente nos últimos anos no Brasil e são utilizados para conter os danos causados ​​pelos lepidópteros pragas. Neste estudo comparamos a riqueza e a abundância de Noctuoides (Noctuoidea) associados à soja Bt e não-Bt. Determinamos as variações temporais em função da fenologia e correlacionamos às variações populacionais das espécies mais comuns com variáveis ​​meteorológicas. A pesquisa foi conduzida na área experimental da Embrapa Cerrados. O método de coleta utilizado foi diferenciado sendo supressivo e absoluto. Um total de 13 espécies foram coletadas, das quais oito ocorreram em soja Bt. Os taxa mais representativos foram Chrysodeixis includens, Anticarsia gemmatalis e Spodoptera spp. O número de larvas pertencentes às espécies alvo da tecnologia Bt foram 10 vezes menores na soja Bt do que em soja não-Bt . Utetheisa ornatrix e Elaphria deltoides foram registradas na soja pela primeira vez, observando-se larvas de ambas espécies na soja não-Bt e as de U. ornatrix também na soja Bt. Somente as larvas de A. gemmatalis se correlacionaram (p <0,05) negativamente com a precipitação. Este estudo forneceu informações em campo sobre a abundância e riqueza de espécies na soja não- Bt, associada aos efeitos da soja Bt. A importância do monitoramento das lagartas é substancial, a fim de tomar a melhor decisão de controle, considerando-se os diferentes níveis de infestação entre cultivares como critério.

Expression of cry1Ab gene from a novel Bacillus thuringiensis strain SY49-1 active on pest insects

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.

Chloroplast localization of Cry1Ac and Cry2A protein- an alternative way of insect control in cotton

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

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.

Transformation of coffee (Coffea Arabica L. cv. Catimor) with the cry1ac gene by biolistic, without the use of markers / Transformação do café (Coffea arabica L. cv. Catimor) com o gene cry1ac por bombardeamento, sem usar marcadores

The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.

A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.

Bacillus thuringiensis (Bt) transgenic crop: an environment friendly insect-pest management strategy.

Introduction of DDT (dichloro-diphenyl-trichloroethane) and following move towards indiscriminate use of synthetic chemical insecticides led to the contamination of water and food sources, poisoning of non-target beneficial insects and development of insect-pests resistant to the chemical insecticides. Increased public concems about the adverse environmental effects of indiscriminate use of chemical insecticides prompted search of altemative methods for insect-pest control. One of the promising alternatives has been the use of biological control agents. There is well-documented history of safe application of Bt (B. thuringiensis, a gram positive soil bacterium) as effective biopesticides and a number of reports of expression of delta-endotoxin gene(s) in crop plants are available. Only a few insecticidal sprays are required on Bt transgenic crops, which not only save cost and time, but also reduce health risks. Insects exhibit remarkable ability to develop resistance to different insecticidal compounds, which raises concern about the unsystematic use of Bt transgenic technology also. Though resistance to Bt products among insect species under field conditions has been rare, laboratory studies show that insects are capable of developing high levels of resistance to one ormore Cry proteins. Now it is generally agreed that 'high-dose/refuge strategy' is the most promising and practical approach to prolong the effectiveness of Bt toxins. Although manybiosafety concerns, ethical and moral issues exist, area under Bt transgenic crops is rapidly increasing and they are cultivated on more than 32 million hectares world over Even after reservation of European Union (EU) for acceptance of geneticaly modified (GM) crops, 6 out of 25 countries have already adopted Bt crops and many otherindustrial countries will adopt Bt transgenic crops in near future. While the modem biotechnology has been recognized to have a great potential for the promotion of human well-being, adoption of biosafety protocol is necessary to protect human health and environment from the possible adverse effects of the products of genetic engineering. The debate between proponents and opponents of GM technology has created major obstacles in hamessing benefits of the technology It has now become clear that transgenics willbe accepted by the public only when doubts related with general risks and environmental safety are adequately dispelled. Thus, there is need to organize public awareness and present the benefits of Bt transgenic crops to improve social attitude for their rational deployment. In this review, an attempt has been made to discuss social and environmental safety issues of Bt transgenic crops.

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...

Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee berry borer Hypothenemus hampei

The coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) was first reported infecting Costa Rican coffee plantations in the year 2000. Due to the impact that this plague has in the economy of the country, we were interested in seeking new alternatives for the biological control of H. hampei, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 202 B. thuringiensis isolates obtained from Costa Rican coffee plantations infested with H. hampei were analyzed through crystal morphology of the crystal inclusions and SDS-PAGE of 6-endotoxins, while 105 strains were further evaluated by PCR for the presence cry, cyt and vip genes. Most of the Bt strains showed diverse crystal morphologies: pleomorphic (35%), oval (37%), bipyramidal (3%), bipyramidal and oval (12%), bipyramidal, oval and pleomorphic (10%) and bipyramidal, oval and cubic (3%). The SDS-PAGE analyses of the crystal preparations showed five strains with delta-endotoxin from 20 to 40 kDa, six from 40 to 50 kDa, seven from 50 to 60 kDa, 19 from 60 to 70 kDa, 29 from 70 to 100 kDa and 39 from 100-145 kDa. PCR analyses demonstrated that the collection showed diverse cry genes profiles having several genes per strain: 78 strains contained the vip3 gene, 82 the cry2 gene, 45 the cry1 and 29 strains harbored cry3-cry7 genes. A total of 13 strains did not amplified with any of the cry primers used: cry1, cry2, cry3-7, cry5, cry11, cry12 and cry14. Forty-three different genetic profiles were found, mainly due to the combination of cry1A genes with other cry and vip genes. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against H. hampei and other insect pests of agricultural importance.

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae.

A sporulating culture of Bacillus thuringiensis subsp. kenyae strain HD549 is toxic to larvae of lepidopteran insect species such as Spodoptera litura, Helicoverpa armigera and Phthorimaea operculella, and a dipteran insect, Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against Helicoverpa armigera, and lower mortality against third-instar larvae of dipteran insects Culex fatigans, Anopheles stephensi and Aedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus thuringiensis var. kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.