Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.Fall armyworm, Spodoptera frugiperda (J. E. Smith), is a worldwide pest of economic importance for different crops. This species has a highly polyphagous feeding behavior, which includes the consumption of different cultivated plants, such as maize (Zea mays L.), cotton (Gossypium hirsutum L.), and rice (Oryza sativa L.). To date, the most common method for controlling this pest relies on the use of synthetic insecticides such as methomyl, carbaryl, and cypermethrin (10), in spite of the damage they cause to the environment and to nontarget organisms. Two distinct strains of S. frugiperda, one associated with maize and the other with rice, have been already identified in the United States (17, 19). The detection and characterization of genetic diversity among insect populations is a critical issue for the improvement of pest management strategies, since the evolution of resistance to insecticides among insect populations is dependent on the frequency of resistant alleles, the inheritance of resistance, the relative fitness cost, and the gene flow.Bioinsecticides are viable alternatives for insect control in agriculture, and among them, Bacillus thuringiensis is the most widely used. B. thuringiensis is compatible with sustainable and environmentally friendly agricultural practices. This bacterium produces insecticidal proteins (Cry protoxins) during sporulation as parasporal crystals, which are highly specific to their target insects; safe for humans, other vertebrates, and plants; and biodegradable (12). Moreover, recombinant DNA technology using cry genes has developed insect-resistant transgenic plants that are used extensively for cotton, corn, and rice production, among others (23).Information regarding the suscept...

ABSTRACI?Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18°C for 48 hours, one half of the seedlings was transferred to 0°C for another 48 hour period, the other half being kept at 18°C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with I35Simethionine. The different polypeptides were separated by twodimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0°C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments.During the last few years, it has been demonstrated that higher plants are able to respond to various environmental changes by inducing the synthesis of specific proteins. The best known examples are the induction of heat shock proteins (10,11,17) and anaerobic proteins (4,19,22

A simplified DNA-directed in vitro system has been developed to study the regulation of the synthesis of ribosomal protein L1O by measuring the formation of the first dipeptide, fMet-Ala. The results show that the inhibition of L1O syn. thesis by L1O (autoregulation) occurs at or prior to the formation of the first peptide bond.It is now known that the synthesis ofEscherichia coli ribosomal proteins is under both transcriptional and translational control. At the level of transcription, ribosomal protein synthesis is under stringent control mediated by the unique nucleotide guanosine-3'-diphosphate-5'-diphosphate (ppGpp) (1). More recently, it has been shown that the synthesis ofcertain ribosomal proteins can be autoregulated at the level of translation. In this process, specific ribosomal proteins inhibit their own synthesis and, in some cases, the synthesis of other ribosomal proteins whose genes are on the same operon. Thus, ribosomal proteins S4 (2, 3), S7 (4), S8 (2), Li (2, 3, 5), LA (2, 6), and L10 (7-10) have been shown to be autoregulators, functioning at the level of translation.Our laboratory has recently studied the effect of L10 on its own synthesis in DNA-and mRNA-directed in vitro systems (7). Although considerable progress has been made in using defined components in these in vitro systems (11,12), their complexity to some extent limits their usefulness in studying the mechanism of autoregulation.The present study describes a simplified in vitro system that can be used to study the regulation of gene expression at transcription or translation initiation by measuring the formation of NH2-terminal small peptides characteristic ofthe gene product. Evidence is presented that the inhibition by L10 ofits own synthesis occurs at, or prior to, the formation of the first peptide bond of L10. MATERIALS AND METHODSE. coli containing Arif18 phage was obtained from J. B. Kirschbaum (Harvard University, Cambridge, MA). E. coli JF943 containing either plasmid pNF1337 or 1341 were kindly supplied by J. Friesen (York University, Ontario, Canada). Ribosomal protein L12 was purified as described (13) of Ala-tRNA and Ser-tRNA synthetase (14). N10-Formyl-H4-folate-Met-tRNA transformylase was purified by a described procedure (15). The acylation and transformylation reactions were carried out as described (15)(16)(17)(18). At the end of the incubations, the reaction mixtures were extracted with phenol and the acylated tRNA species were precipitated with alcohol and dialyzed overnight against 2 mM K acetate (pH 5.5).For Ala-and Ser-tRNA synthesis, unfractionated E. coli tRNA was used; purified tRNAP et was used to prepare fMet-and L-methionine-L-serine were purchased from Sigma. N-Formylmethionylserine was synthesized as follows (19). To 71 mg of L-methionyl-L-serine dissolved in 1.26 ml of 98% formic acid was added dropwise 0.42 ml of acetic anhydride at 10°C. The mixture was stirred for 1 hr at room temperature, and 0.5 ml of ice-water was added. The mixture was brought to dryness under vacuum and the white p...

Aims: To introduce a cry gene into microorganisms that naturally colonize the phylloplane of tomato plants to improve the persistence of the Cry proteins for controlling a South American tomato moth (Tuta absoluta, Meyrick, 1917). Methods and Results: A cry1Ab gene isolated from a native Bacillus thuringiensis strain (LM-466), showing a relevant activity against T. absoluta larvae, was cloned into the shuttle vector pHT315 (Arantes and Lereclus 1991). The construct was introduced by electroporation into native Bacillus subtilis and Bacillus licheniformis strains, both natural inhabitants of the tomato phylloplane. Western analysis and toxicity assays against the target larvae proved that the successful expression of the gene was accomplished in host bacteria. Recombinant toxin displayed a similar LC 50 value in comparison to native donor strain LM-466. Both transformed Bacillus survived for at least 45 days on the tomato leaf surface. Conclusions: Plant-associated microorganisms that naturally colonize the phylloplane could be useful as recombinant microbial delivery systems of toxin genes of B. thuringiensis. Significance and Impact of the Study: Modified microorganisms capable of surviving on leaf surfaces for several weeks with insecticidal activity should allow for a reduction in pesticide application.

No abstract