ABSTRACT
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.
Subject(s)
Bacillus thuringiensis/metabolism , Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , Endotoxins/pharmacology , Genetic Variation , Hemolysin Proteins/pharmacology , Pest Control, Biological , Spodoptera/drug effects , Spodoptera/genetics , Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis/isolation & purification , Bacillus thuringiensis Toxins , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Endotoxins/genetics , Endotoxins/metabolism , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Latin America , Microvilli/metabolism , Polymerase Chain Reaction , Random Amplified Polymorphic DNA Technique , Soil Microbiology , Spodoptera/classification , Spodoptera/growth & developmentABSTRACT
A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detection and identification of cry1I genes from Bacillus thuringiensis (Bt) was established. Based on the analysis of conserved regions of the cry1I genes, 2 oligonucleotide primers were designed to amplify a 665-bp fragment of the genes. The amplification products were digested with restriction endonuclease HinfI or with RsaI in addition for specific detection of different variants from the known subclasses of cry1I genes. The PCR-RFLP pattern obtained revealed the detection of cry1I genes in 151 of 202 native Bt isolates. Furthermore, cry1I genes were detectable in 10 of 19 standard strains tested. The cry1Ia gene was the most abundant cry1I gene subclass present in 54 of 56 native Bt isolates and in 8 of 10 standard strains. Based on the results obtained, the PCR-RFLP method may be a valuable and reliable tool for specific detection and identification of cry1I genes.
Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Toxins/genetics , DNA, Bacterial/genetics , Endotoxins/genetics , Bacillus thuringiensis Toxins , DNA Primers , DNA, Bacterial/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Hemolysin Proteins , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identification of cry2 genes from Bacillus thuringiensis (Bt) was established. Strains from different sources of Argentina were analyzed to study the distribution of cry2 genes. The results showed that cry2Aa/cry2Ab profile was the most abundant irrespective of source and represented 56 of 59 Bt isolates (94.9%). Three different cry2 profiles were found in this collection, one of which was novel.
Subject(s)
Bacillus thuringiensis/classification , Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Endotoxins/genetics , Animals , Argentina , Bacillus thuringiensis/isolation & purification , Bacillus thuringiensis/metabolism , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , DNA, Bacterial/analysis , Endotoxins/metabolism , Environmental Microbiology , Hemolysin Proteins , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
This work describes a novel rolling-circle replicating (RCR) plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) encoding an insecticidal crystal protein-like gene. pBMBt1 (6700 bp) contains three ORFs and their putative transcription initiation sites and Shine-Dalgarno sequences were localized. ORF1 encodes a 34.6 kDa protein which showed identity with the protein CryC53 from B. thuringiensis subsp. cameroun (24.6%), the Cry15Aa insecticidal crystal protein from B. thuringiensis subsp. thompsoni (21.9%) and the Mtx3 protein from Bacillus sphaericus (27.8%). The ORF2 (52.3 kDa) showed a 74% identity with the Mob protein coded by pUIBI-1 from B. thuringiensis subsp. entomocidus and 64% identity with the Mob protein of pBMY1 from Bacillus mycoides; both Mob proteins belong to the pMV158 superfamily. To evaluate the Mob protein, the plasmid pHTMob14-4 was constructed. This plasmid shows transfer frequencies of 9.1x10(-6) in B. thuringiensis subsp. israelensis (4Q7Gm(R)). The ORF3 (23.6 kDa) gene product is homologous to the Rep protein from the plasmid pBMYdx of B. mycoides (37.6%). A putative double-strand origin with significant homology to that of B. thuringiensis plasmids, and an ssoA-type single-strand origin were also identified. Detection of single-stranded pBMBt1 DNA replicating intermediaries suggests that replication occurs via the rolling-circle mechanism.
Subject(s)
Bacillus thuringiensis/genetics , DNA Replication , DNA, Bacterial , DNA, Circular , Plasmids/genetics , Amino Acid Sequence , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Base Sequence , Conserved Sequence , Endotoxins/chemistry , Endotoxins/genetics , Hemolysin Proteins , Molecular Sequence Data , Molecular Weight , Open Reading Frames , Sequence Homology, Amino AcidABSTRACT
Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.
