Kazim Sezen

The first study on the bacterial flora of the european spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera, Scolytidae), is one of the most serious pests of oriental spruce (Picea orientalis L.) in Turkey. In this study, we investigated bacterial flora of D. micans collected from different populations of the forests of Eastern Black Sea Region of Turkey from 2002 to 2004. Seven different bacteria were isolated from healthy, diseased and dead specimens based on the color of colony and morphology. According to morphological, physiological and biochemical properties, metobolic enyzme profile by BIOLOG microtiter plate system, and total cellular fatty acid profile by Microbial Identification System (MIS), isolates were identified as Micrococcus luteus, Bacillus thuringiensis subsp. morrisoni, Serratia grimesii, Enterobacter cloaceae, Enterobacter intermedius, Streptococcus sp. and Pseudomonas putida. This is the first study on the bacterial flora of D. micans.

Characterization of a Highly Pathogenic Bacillus thuringiensis Strain Isolated from Common Cockchafer, Melolontha melolontha

A bacterial isolate (Mm2) ofMelolontha melolontha was identified and characterized. Based on various morphological, physiological, biochemical and molecular characteristics, it was identified asBacillus 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 ≈65 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique proteinase-resistant peptide of ≈50 kDa. Plasmid pattern showed similar bands to those of the reference strain, PCR analysis showed that the isolate hascry3 gene. Toxicity tests (against 5 coleopteran species) showed 80 % insecticidal activity against the larvae ofM. melolontha. The isolate Mm2 may be valuable as biological control agent forM. melolontha and other coleopteran insects.

Identification and pathogenicity of bacteria from European shot-hole borer,Xyleborus dispar Fabricius (Coleoptera: Scolytidae)

In this study, we have isolated and identified 5 bacterial isolates from European shot-hole borer,Xyleborus dispar (Coleoptera: Scolytidae), an important pest of hazelnut. After various morphological, physiological, biochemical and molecular characteristics were determined in detail, bacterial isolates were identified asPseudomonas fluorescens (Xd1),Bacillus megaterium (Xd2),Bacillus thuringiensis (Xd3),Pseudomonas rhizosphaerae (Xd4) andPantoea cedenensis (Xd5). Especially, theB. thuringiensis strain was identified in more detail asBacillus thuringiensis subsp.tenebrionis (H8ab). The insecticidal activities were determined against the larvae ofAgelastica alni L. (Col.: Chrysomelidae),Amphimallon solstitiale L. (Col.: Scarabaeidae),Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae), andMelolontha melolontha L. (Col.: Scarabaeidae) larvae at 1.8×109 CFU/ml dose, within five days. The highest insecticidal activities are 100, 100, 80 and 100% forBacillus thuringiensis subsp.tenebrionis (Xd3) onA. alni, A. solstitiale, L. decemlineata, andM. melolontha larvae, respectively. Our results indicate that especiallyB. thuringiensis subsp.tenebrionis (Xd3) may be valuable as a microbial control agent for coleopteran pests, respectively.

The first investigation of the diversity of bacteria associated with Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

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.

The relationship between insecticidal effects and chitinase activities of Coleopteran-originated entomopathogens and their chitinolytic profile

Peritrophic membrane (PM) is present in most insects’ midgut and acts as a mechanical barrier to protect the epithelium from various harmful factors such as pathogens or toxins. Chitinase is a virulence factor due to its ability to degrade the chitin content of PM. Therefore, chitinase is a mediator for easier binding of toxins to gut epithelium and intercepting nutritional absorption in the midgut. One hundred and eight bacterial isolates derived from microbial flora of coleopteran pests were screened to determine chitin-producing entomopathogenic bacteria. The M9 chitin–-agar method and polymerase chain reaction with specific primers for a conserved domain of chitinase genes indicated that 23 of the 108 isolates have chitinase activity. The chitinase activities of the chitinase-positive bacteria were measured. We compared these results with the insecticidal activities results to determine, statistically, the potential relationship between the chitinase activities and the insecticidal activity. Consequently, 21.3% of bacterial isolates showed chitinolytic ability and among these the chitinase-positive bacteria, Serratia marcescens, was found the most active one in the M9-CAD method. More importantly, our study indicated a very strong positive correlation between the insecticidal activities of isolates and the chitinase activities with the M9-CAD method (r2 = 0.96, p ≤ 0.01), but not with the DNS method (r2 = −0.279, p ≤ 0.01). This strong relationship of entomopathogens has a high potential for biocontrol of Coleopteran pests.

Highly pathogenic Bacillus thuringiensis subsp. tenebrionis from European shot-hole borer, Xyleborus dispar (Coleoptera: Scolytidae)

Abstract 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.

High insecticidal activity of Leclercia adecarboxylata isolated from Leptinotarsa decemlineata (Col.: Chrysomelidae)

Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is an important pest on solanaceous crops worldwide. CPB has developed resistance to insecticides used for its control. In this study, in order to find a more effective and safer biological control agent against L. decemlineata, we studied the bacterial flora of CPB, and tested them for insecticidal effects on it. The highest insecticidal effect determined on L. decemlineata within 5 days was 100% and this effect was exhibited by Ld1 isolate. According to the morphological, physiological and biochemical tests, and 16S rRNA sequence homologies, Ld1 was identified as Leclercia adecarboxylata. This is the first time that this bacterium has been isolated from any insect pests. Our results indicate that Lecl. adecarboxylata may be valuable as a biological control agent for L. decemlineata.

Insecticidal effects of some biological agents on Agelastica alni (Coleoptera: Chrysomelidae)

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 × 109 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.

Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae)

Abstract The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDS–PAGE analysis, cry gene contents, H-serotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDS–PAGE in Tp6 and Tp14 isolates, respectively. According to H-serotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests.

Characterisation and toxicity of Bacillus thuringiensis strains from hazelnut pests and fields

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.