Zihni Demirbag

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.

Isolation of pathogenic bacteria from Oberea linearis (Coleptera: Cerambycidae)

The bacterial flora of the Oberea linearis (Coleoptera: Cerambycidae) was investigated and 13 different bacteria were isolated from O. linearis larvae. Seven of these bacteria were performed and characterized at species level and the rest of them were characterized at genus level. In this study, we determined morphological and physiological characteristics of the bacterial isolates by conventional and routine techniques, biochemical properties and metabolic enzyme profiles by API20E and Phoenix 1000A panel test systems. Additionally, 16S rRNA gene sequence analysis was also performed to identify the isolates at the molecular level. The isolates were identified as Acinetobacter calcoaceticus (Ol1), Enterobacter aerogenes (Ol2), Pseudomonas sp. (Ol3), Flavobacterium sp. (Ol4), Microbacterium sp. (Ol5), Enterobacter agglomerans (Ol6), Xanthomonas sp. (Ol7), Pseudomonas syringae (Ol8), Pseudomonas sp. (Ol9), Xanthomonas sp. (Ol10), Enterobacter cancerogenus (Ol11), Xanthomonas maltophilia (Ol12), and Serratia marcescens (Ol13). This is the first record of bacterial isolates (Ol5, Ol8, Ol11, Ol12) from any insect. All these bacteria were tested against O. linearis larvae, and Serratia marcescens was found to cause the highest mortality (65%). On the other hand, we determined 90% mortality against this pest within four days by utilizing spore and crystal mixture of Bacillus thuringiensis isolated from Melolontha melolontha.

Screening of entomopathogenic fungi against the European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

Abstract The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera: Scolytidae), is one of the most serious destructive pests of oriental spruce (Picea orientalis L.). In order to find an effective biocontrol agent against this pest, we determined the effectiveness of entomopathogenic fungi against D. micans. Virulence of nine highly pathogenic strains including Beauveria bassiana (2), Beauveria cf. bassiana (2), Metarhizium anisopliae (2), Metarhizium sp. (1), Isaria fumosorosea (1) and Evlachovaea sp. (1) was evaluated on D. micans larvae and adults under laboratory conditions. Mortality values for both larvae and adults ranged from 83 to 100% and from 23 to 100%, respectively. Larvae were more susceptible to fungi than adults (P<0.05). Based on screening tests, B. cf. bassiana isolate KTU-53 was found the most effective isolate. LC50 values were calculated as 1.77×104 and 2.65×104 conidia mL−1 for isolate KTU-53 against larvae and adults, respectively. Consequently, B. cf. bassiana isolate KTU-53 appears to be the most promising microbial control agent for biocontrol of D. micans.

Isolation, characterization and pathogenicity of bacteria from Rhynchites bacchus (Coleoptera: Rhynchitidae)

Abstract The leafroller weevil beetle (Rhynchites bacchus L., Coleoptera: Rhynchitidae) is one of the most serious pests of apple, plum, apricot, cherry and peach (nectarine) fruit worldwide. The aim of this study was to isolate and characterize pathogenic bacteria from this pest for possible use in biocontrol, and to determine their pathogenicity. R. bacchus were collected from the Eastern Black Sea Region of Turkey in 2007. Based on their morphological, physiological and biochemical features, five different bacterial isolates were obtained from adults and larvae. In addition, 16S rRNA gene was amplified and sequenced to confirm isolate identification. According to the numerical and molecular data, the culturable bacterial flora of R. bacchus was determined as Bacillus licheniformis (Rb1), Serratia marcescens (Rb2), Enterobacter hormaechei (Rb3), Paenibacillus sp. (Rb4), and Enterobacter sp. (Rb5). Isolate Rb2 produced the highest mortality (73%) against larvae within 10 days after inoculation (P<0.05). The others, Rb1, Rb3, Rb4 and Rb5, caused 20, 13, 26 and 13% mortality in the larvae within the same period. These results indicate that S. marcescens Rb2 seems to be the most promising biocontrol agent againts this pest.

Isolation and virulence of entomopathogenic fungi against the great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae).

Abstract Twelve fungal strains including Lecanicillium muscarium (Petch.) Zare and Gams, Isaria farinosa (Holmsk.) Fr., Fusarium sp., Beauveria bassiana Sensu Lato and Beauveria sp. were isolated from larvae and adults of D. micans. In addition, virulence of these isolates against this pest was determined. Conidia suspensions of 1×106 conidia mL–1 were applied to larvae and adults. The highest mortality and mycosis for larvae were obtained from isolate ARSEF 9271 (Beauveria bassiana) with 90% mortality and mycosis within 10 days. ARSEF 9271 also produced 93% mortality and mycosis in adults. On the other hand, the highest mortality and mycosis for adults were obtained with isolate ARSEF 9272 (Beauveria sp.), with 100% mortality and 80% mycosis within 10 days. These results indicate that isolates ARSEF 9271 and ARSEF 9272 seem to be the most promising potential fungal biocontrol agents against D. micans.

Open reading frame 193R of Chilo iridescent virus encodes a functional inhibitor of apoptosis (IAP)

Programmed cell death or apoptosis is a major defense mechanism in insects in response to viral infections. The genome of Chilo iridescent virus (CIV) has three ORFs with homology to baculovirus inhibitor of apoptosis (iap) genes. The proteins encoded by the 157L, 193R, and 332L ORFs contain 152, 208 and 234 amino acids, respectively. While all three proteins contain C-terminal RING domains, only the protein encoded by ORF 193R contains a baculoviral iap repeat (BIR) domain, indicative of a putative IAP protein. The 193R protein has 28 and 27% similarity in amino acid sequence to the Orgyia pseudotsugata MNPV and Cydia pomonella granulovirus IAP-3 proteins, respectively. ORF 193R from CIV is the only gene known to exist among the Iridoviridae that encodes a BIR domain. 193R is transcribed early during CIV infection, and its transcription is not dependent on the synthesis of early viral proteins. When this putative CIV IAP was transiently expressed in SPC-BM-36 and Sf21 cells under the control of an immediate early baculovirus promoter it significantly reduced apoptosis induced by actinomycin-D. Silencing of the CIV iap gene (193R) in CIV infected SPC-BM-36 cells with 193R-specific dsRNA resulted in apoptosis. Thus, CIV ORF 193R is the first iap gene identified in an iridovirus, which encodes a functional IAP protein.

Isolation, characterization and virulence of bacteria from Ostrinia nubilalis (Lepidoptera: Pyralidae)

Isolation, characterization and virulence of the culturable bacteria from entire tissues of larval Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) were studied to obtain new microbes for biological control. A total of 16 bacteria were isolated from living and dead larvae collected from different maize fields in the Eastern Black Sea Region of Turkey. The bacterial microbiota of O. nubilalis were identified as Pseudomonas aeruginosa (On1), Brevundimonas aurantiaca (On2), Chryseobacterium formosense (On3), Acinetobacter sp. (On4), Microbacterium thalassium (On5), Bacillus megaterium (On6), Serratia sp. (On7), Ochrobactrum sp. (On8), Variovorax paradoxus (On9), Corynebacterium glutamicum (On10), Paenibacillus sp. (On11), Alcaligenes faecalis (On12), Microbacterium testaceum (On13), Leucobacter sp. (On14), Leucobacter sp. (On15) and Serratia marcescens (On16) based on their morphological and biochemical characteristics. A partial sequence of the 16S rRNA gene was also determined to confirm strain identification. The highest insecticidal activities were obtained from P. aeruginosa On1 (80%), Serratia sp. On7 (60%), V. paradoxus On9 (50%) and S. marcescens On16 (50%) against larvae 14 days after treatment (p < 0.05). Also, the highest activity from previously isolated Bacillus species was observed from Bacillus thuringiensis subsp. tenebrionis Xd3 with 80% mortality within the same period (p < 0.05). Our results indicate that P. aeruginosa On1, Serratia sp. On7, V. paradoxus On9, S. marcescens On16 and B. thuringiensis subsp. tenebrionis Xd3 show potential for biocontrol of O. nubilalis.

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.

Brevibacterium pityocampae sp. nov., isolated from caterpillars of Thaumetopoea pityocampa (Lepidoptera, Thaumetopoeidae).

This work deals with the taxonomic study of a bacterium, strain Tp12(T), isolated from caterpillars of the pine processionary moth (Thaumetopoea pityocampa Denis & Schiffermüller, 1775; Lepidoptera, Thaumetopoeidae). The isolate was assigned to the genus Brevibacterium on the basis of a polyphasic taxonomic study, including morphological and biochemical characteristics, 16S rRNA gene sequence analysis, fatty acid analysis and DNA G+C content. The highest 16S rRNA gene sequence similarity to this isolate was approximately 96 %, with the type strains of Brevibacterium album and Brevibacterium samyangense. Cellular fatty acids of the isolate are of the branched type, with the major components being anteiso-C(15 : 0) and anteiso-C(17 : 0). The DNA G+C content was 69.8 mol%. Although the strain was related to B. album and B. samyangense according to 16S rRNA gene sequence analysis, it differed from any known species of Brevibacterium. Based on this evidence, the novel species Brevibacterium pityocampae sp. nov. is proposed, with strain Tp12(T) (=DSM 21720(T) =NCCB 100255(T)) as the type strain.

Amsacta moorei entomopoxvirus encodes a functional DNA photolyase (AMV025).

The major damage induced in DNA by ultraviolet light is the induction of cyclobutane pyrimidine dimers (CPDs). Amsacta moorei entomopoxvirus (AMEV) encodes a CPD photolyase (AMV025) with a putative role in converting these dimers back into monomers. In infected Lymantria dispar cells transcription of the AMV025 gene started 8h post inoculation (p.i.) and continued through 38hp.i. Transcription was inhibited by a DNA synthesis blocker. Transient expression in an Escherichia coli strain that lacks its endogenous photolyase, rescued growth of the UV-irradiated bacteria in a light-dependent manner, showing that AMV025 encodes a functional DNA photolyase.