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Abstract:The alder leaf beetle (Agelastica alni L., Coleoptera: Chrysomelidae) causes approximately 10% of total economic damage to hazelnut product per year in Turkey. A. alni larvae are susceptible to several pathogens indigenous to the area in which these insects occur in Turkey. In the present study, in order to find a more effective and safer biological control agent against this common pest, we evaluated the various biological agents' insecticidal activity during the four hazelnut seasons from 2002 to 2005 on the larvae of the alder leaf beetle collected from the vicinity of Trabzon, Turkey. The tested agents are 25 insect-originating bacteria, 2 bacterial toxins and 1 viral preparation. The results showed that the highest insecticidal activity was obtained by bacterial isolates at 1.8 × 10 9 bacteria/mL dose, within ten days on the larvae of A. alni. These are 90% for Bacillus thuringiensis biovar tenebrionis (4AA1), Bacillus sphaericus (Ar4, isolated from Anoplus roboris L., Col.: Curculionidae), and Bacillus thuringiensis (Mm2, isolated from Melolontha melolontha L., Col.: Scarabaeidae). Our results indicate that these isolates may be valuable as biological control agent.

Melolontha melolontha larvae are susceptible to several pathogens indigenous to the area in which these insects occur in Turkey. We isolated and identified seven bacterial strains from M. melolontha and evaluated their pathogenic activity during three hazelnut seasons from 2002 to 2004 on larvae of M. melolontha. Using various morphological, physiological, and biochemical characteristics in detail, bacterial isolates were identified as Pseudomonas sp., Bacillus thuringiensis, Pseudomonas sp., Enterobacter sp., B. sphaericus, Acinetobacter sp., and B. weihenstephanensis. The insecticidal activity of isolates at 1.8 × 10 9 bacteria/ml dose, within 10 days on the larvae of M. melolontha are 40% for Pseudomonas sp., 80% for Bacillus thuringiensis, 50% for Pseudomonas sp., 20% for Enterobacter sp., 60% for B. sphaericus, and 80% for B. weihenstephanensis. We also purified crystals from B. thuringiensis and B. sphaericus and tested the insecticidal activity on the larvae of M. melolontha. In crystal protein bioassays, the highest insecticidal effect detected was 70% with crystals of B. thuringiensis. Our results indicate that indigenous H05146;

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.

Colorado potato beetle, Leptinotarsa decemlineata (Say), is a devastating pest of potatoes in North America and Europe. L. decemlineata has developed resistance to insecticides used for its control. In this study, in order to find a more effective potential biological control agent against L. decemlineata, we investigated its microbiota and tested their insecticidal effects. According to morphological, physiological and biochemical tests as well as 16S rDNA sequences, microbiota was identified as Leclercia adecarboxylata (Ld1), Acinetobacter sp. (Ld2), Acinetobacter sp. (Ld3), Pseudomonas putida (Ld4), Acinetobacter sp. (Ld5) and Acinetobacter haemolyticus (Ld6). The insecticidal activities of isolates at 1.8×109 bacteria/mL dose within five days were 100%, 100%, 35%, 100%, 47% and 100%, respectively, against the L. decemlineata larvae. The results indicate that Leclercia adecarboxylata (Ld1) and Pseudomonas putida (Ld4) isolates may be valuable potential biological control agents for biological control of L. decemlineata.

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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.

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.