Isolation and characterization of Bacillus thuringiensis from olive tree-related habitats

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2005Text in English; Abstract: Turkish and Englishxii,115 leavesAbstract:Bacillus thuringiensis (commonlyreferred to as Bt) is a Gram-positive, sporeforming soil bacterium that produces insecticidal crystal proteins during sporulation.These crystals are referred as Bt toxins or -endotoxins. The most important characteristics of the toxins are their insecticidalactivity against many insects. Since their insecticidal potential has been discovered,it has been produced commercially and used as microbial pesticides all over the world.Therefore, the aim of this study was to isolate Btfrom olive tree-related habitats, and to determine the phenotypic andisolate Btfrom olive tree-related habitats, and to determine the phenotypic and genotypic characteristics of the isolates. To accomplish this purpose, 240 samples were collected in the Aegean Region. The phase-contrast microscopy results showed the presence of crystals in 54 environmental samples, corresponding to 100 Bt isolates. The crystal morphologies were spherical, bipyramidal, cuboidal, irregular pointed, and irregular shaped. The greatest proportion of samples yielding this organism was from the soil. The remaining were from olive leaf residue, green olive leaves, animal faeces,dust samples, and olive pomace. The isolates were characterized on the basis of biochemical characters, cry andcyt gene content, plasmid profiling, 16S-ITS rDNA RFLP.Biochemical tests included protease (caseinase and gelatinase), lecithinase, amylase, nuclease, urease, esculinase, arginine dihydrolyse activity; fermentation of sucrose, salicin, mannose, cellobiose, and maltose, production of acetyl-methyl carbinol, methyl red reaction. Polymerase chain reaction (PCR) has been applied for the identification of cry1, cry2, cry4, cry9, cry11, cry13, cyt1, and cyt2 genes. 68% of the isolates amplified cry1 gene; 57% amplified cry4; 20% amplified cry11; 26% amplified cry9; 20% amplified cry2 genes, andnone of the isolates harbored cry13 gene. Cyt1gene was found in 40% of the isolates while cyt2 gene was present in 80% of the isolates. The most abundant genotype of cry genes was cry1 and cry4. Most of the isolates(58%) possessed more than one cry gene. In addition, different combinations of cry andcytgenes were obtained. Plasmid profiling showed the presence of plasmids in all isolatesand the number of plasmids was usually morethan one. Also, the discrimation effect of 16S-ITS rDNA RFLP was tested to differentiate certain isolates and reference strains, which showed similar biochemical characteristics

Isolation and characterization of Bacillus thuringiensis from olive tree-related habitats

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2005Text in English; Abstract: Turkish and Englishxii,115 leavesAbstract:Bacillus thuringiensis (commonlyreferred to as Bt) is a Gram-positive, sporeforming soil bacterium that produces insecticidal crystal proteins during sporulation.These crystals are referred as Bt toxins or -endotoxins. The most important characteristics of the toxins are their insecticidalactivity against many insects. Since their insecticidal potential has been discovered,it has been produced commercially and used as microbial pesticides all over the world.Therefore, the aim of this study was to isolate Btfrom olive tree-related habitats, and to determine the phenotypic andisolate Btfrom olive tree-related habitats, and to determine the phenotypic and genotypic characteristics of the isolates. To accomplish this purpose, 240 samples were collected in the Aegean Region. The phase-contrast microscopy results showed the presence of crystals in 54 environmental samples, corresponding to 100 Bt isolates. The crystal morphologies were spherical, bipyramidal, cuboidal, irregular pointed, and irregular shaped. The greatest proportion of samples yielding this organism was from the soil. The remaining were from olive leaf residue, green olive leaves, animal faeces,dust samples, and olive pomace. The isolates were characterized on the basis of biochemical characters, cry andcyt gene content, plasmid profiling, 16S-ITS rDNA RFLP.Biochemical tests included protease (caseinase and gelatinase), lecithinase, amylase, nuclease, urease, esculinase, arginine dihydrolyse activity; fermentation of sucrose, salicin, mannose, cellobiose, and maltose, production of acetyl-methyl carbinol, methyl red reaction. Polymerase chain reaction (PCR) has been applied for the identification of cry1, cry2, cry4, cry9, cry11, cry13, cyt1, and cyt2 genes. 68% of the isolates amplified cry1 gene; 57% amplified cry4; 20% amplified cry11; 26% amplified cry9; 20% amplified cry2 genes, andnone of the isolates harbored cry13 gene. Cyt1gene was found in 40% of the isolates while cyt2 gene was present in 80% of the isolates. The most abundant genotype of cry genes was cry1 and cry4. Most of the isolates(58%) possessed more than one cry gene. In addition, different combinations of cry andcytgenes were obtained. Plasmid profiling showed the presence of plasmids in all isolatesand the number of plasmids was usually morethan one. Also, the discrimation effect of 16S-ITS rDNA RFLP was tested to differentiate certain isolates and reference strains, which showed similar biochemical characteristics

Identification and bioactivity of native strains of Bacillus thuringiensis from grain-related habitats in Turkey

A native collection of Bacillus thuringiensis (Bt) strains originated from grain-related habitats in Turkey was characterized according to serotype, cry1 gene content, and bioactivity against Ephestia kuehniella (Lepidoptera: Phycitidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). Twenty-three different serotypes as well as 24 unknown serotypes were obtained from 56 positively agglutinated strains with previously characterized antisera. Most common serovars were sotto, kim, and tochigiensis with the percentages of 14, 14, and 13, respectively. Among the cry1 gene-positive 36 strains, cry1E (100%), cry1Aa (94%), cry1Ac (92%), and cry1D (83%) genes were the most abundant. Bioactivity tests with 56 Bt strains carrying cry1, cry2, and/or cry9 genes indicated that all of them resulted in growth retardation or inhibition of larvae of both E. kuehniella and S. littoralis; however, only one strain, 85PPb (serovar morrisoni), caused high mortality in both insects (84% and 100%, respectively). Different crystal morphology was observed for the strain 85PPb and the standard strain B. thuringiensis subsp. morrisoni. Finally, no correlation was found among serotype, cry gene content and biotoxicity of Bt strains in the collection.State Planning Organization, DPT (2002K-120/390

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

Aims: To isolate Bacillus thuringiensis strains from different olive-related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. Methods and Results: A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S-internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S-ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical-irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S-ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Conclusions: Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. Significance and Impact of the Study: This is the first study on the isolation and characterization of B. thuringiensis from olive-related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.State Planning Organization, DPT (2002K-120390