Search

Use of chemical pesticides in agriculture harms humans, non-target organisms and environments, and causes increase resistance against chemicals. In order to develop an effective bio-pesticide against coleopterans, particularly against Agelastica alni (Coleoptera: Chrysomelidae) which is one of the serious pests of alder leaf and hazelnut, we tested the insecticidal effect of 21 Bacillus isolates against the larvae and adults of the pest. Bacillus thuringiensis var. tenebrionis-Xd3 (Btt-Xd3) showed the highest insecticidal effect based on screening tests. For toxin protein production and high sporulation of Xd3, the most suitable medium, pH and temperature conditions were determined as nutrient broth medium enriched with salts, pH 7 and 30 °C, respectively. Sporulated Btt-Xd3 in nutrient broth medium enriched with salts transferred to fermentation medium containing soybean flour, glucose and salts. After fermentation, the mixture was dried in a spray dryer, and spore count of the powder product was determined as 1.6 × 10 10  c.f.u. g -1 . Moisture content, suspensibility and wettability of the formulation were determined as 8.3, 86% and 21 s, respectively. Lethal concentrations (LC 50 ) of formulated Btt-Xd3 were determined as 0.15 × 10 5  c.f.u. ml -1 for larvae at laboratory conditions. LC 50 values were also determined as 0.45 × 10 6  c.f.u. ml -1 at the field condition on larval stage. Our results showed that a new bio-pesticide developed from B. thuringiensis tenebrionis (Xd3) (Btt-Xd3) may be valuable as a biological control agent for coleopteran pests.

The chitinase B (chiB) and C (chiC) genes and flanking regions from a local isolate of Serratia marcescens were cloned individually and sequenced. Results showed that these chiB and chiC genes have a 96 % maximum similarity with chiB and chiC from different S. marcescens species (GenBank numbers Z36295.1 and AJ630582.1, respectively). The amplified chiB fragment, including some upstream and downstream regions, is 1,689-bp long with an open reading frame of 1,500 bp. The amplified fragment of chiC is 1,844 bp with an open reading frame of 1,443 bp. These sequences were submitted to the GenBank with accession numbers JX847796 (chiB) and JX847797 (chiC). Putative promoter regions and Shine-Dalgarno sequences were identified in both genes. The genes were cloned into a shuttle vector and the constructs were designated as pHYSB and pHYSC, respectively. Both plasmids were introduced separately into kurstaki and israelensis strains of Bacillus thuringiensis and the insecticidal activities of the engineered B. thuringiensis strains were assayed in larvae of Galleria mellonella and adult of Drosophila melanogaster. Engineered B. thuringiensis strains showed higher insecticidal activity than parental strain and the parental S. marcescens. In addition, pHYSB and pHYSC were stable over 16 daily passages under non-selective conditions in transformed B. t. israelensis 5724 strain.

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

Background: In order to find and identify more toxic insecticidal Bacillus thuringiensis Berliner (Bt) strains, a survey was carried out of B. thuringiensis isolate pests belonging to Coleoptera, Lepidoptera and Diptera and from soils in hazelnut fields. Of 16 isolates having Bacillus cereus-B. thuringiensis morphology, eight were classified as B. thuringiensis because of the production of parasporal delta-endotoxin crystals., Results: In this study, eight isolates of B. thuringiensis from hazelnut pests (isolates Bn1, Mm2, Mnd and Xd3) and from hazelnut soils (isolates 6, 27, 40 and 46) have been characterised in detail. These isolates were compared with reference strains by electron microscopy, SDS-PAGE analysis, cry gene content, serological test and insecticidal activity., Conclusion: Results indicate that Bn1 and MnD are B. thuringiensis subsp. kurstaki, and Mm2 and Xd3 are B. thuringiensis subsp. tenebrionis. In addition, isolate 6 is B. thuringiensis subsp. israelensis, isolates 27 and 46 are B. thuringiensis subsp. kumamotoensis and isolate 40 is B. thuringiensis subsp. indiana. The four B. thuringiensis isolates from hazelnut pests may be valuable as biological control agents against coleopteran and lepidopteran insects.

In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.

A bacterial isolate (Mm2) of Melolontha melolontha was identified and characterized. Based on various morphological, physiological, biochemical and molecular characteristics, it was identified as Bacillus thuringiensis subsp. tenebrionis. This isolate was compared to the reference strains by electron microscopy, SDS-PAGE analysis, plasmid pattern, cry gene content and insecticidal activity. Cells of the isolate harbored flat square inclusions containing a protein component of approximately equal to65 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique proteinase-resistant peptide of approximately equal to 50 kDa. Plasmid pattern showed similar bands to those of the reference strain, PCR analysis showed that the isolate has cry3 gene. Toxicity tests (against 5 coleopteran species) showed 80 % insecticidal activity against the larvae of M. melolontha. The isolate Mm2 may be valuable as biological control agent for M. melolontha and other coleopteran insects.

A local isolate of Bacillus thuringiensis Mm2 had insecticidal activity against the larvae of Melolontha melolontha, Agelastica alni, Leptinotarsa decemlineata and Amphimallon solstitiale and produced a 65 kDa protein. SDS-PAGE profile of B. thuringiensis Mm2 was compared with those of 29 different Cry3Aa producers which verified Cry3Aa biosynthesis by the isolate. The cry3Aa gene of Mm2 was cloned, sequenced and the deduced amino acid sequence was compared with the cry3Aa sequences of ten different quaternary ranks. Its identity to these sequences ranged between 97.4% and 99.2%. The gene was next cloned into E. coli-Bacillus shuttle vector pNW33N and expressed at a low level in B. subtilis 168.

The entomopathogenic bacterium Bacillus thuringiensis is the most widely used biopesticide. In this study, to find and identify the more toxic B. thuringiensis strains against coleopteran pests, we isolated a B. thuringiensis strain (Xd3) from European shot-hole borer, Xyleborus dispar (Coleoptera: Scolytidae), a higly damaging pest of hazelnut. Based on various morphological, physiological, biochemical, and molecular characteristics, the bacterial isolate was identified as B. thuringiensis subsp. tenebrionis (morrisoni) serovar H8a8b. This isolate was compared with the reference strains by scanning electron microscopy, SDS-PAGE analysis, cry gene content, and insecticidal activity. Isolate Xd3 forms a flat-square inclusion containing a protein component of c. 70 kDa. PCR analysis showed that the Xd3 has a cry gene, cry3. Toxicity tests were performed against coleopteran species. One hundred percent mortality was observed against larvae of Agelastica alni (Coleoptera, Chrysomelidae). The others were 90% for Amphimallon solstitiale (Coleoptera, Scarabaeidae), and Melolontha melolontha (Coleoptera: Scarabaeidae). Our results indicate that B. thuringiensis subsp. tenebrionis (Xd3) may be valuable as biological control agent for coleopteran insects. [ABSTRACT FROM AUTHOR]