İsmail Demir

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.

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.

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.

Isolation and identification of entomopathogenic nematodes (Nematoda: Rhabditida) from the Eastern Black Sea region and their biocontrol potential against Melolontha melolontha (Coleoptera: Scarabaeidae) larvae

A survey was conducted for entomopathogenic nematodes in various agricultural fields in the Eastern Black Sea region of Turkey. A total of 77 soil samples were collected from 15 distinct geographic areas during 2010-2011. Seven entomopathogenic nematode isolates (ZET02, ZET04, ZET09, ZET28, ZET31, ZET35, and ZET76) were detected from the soil samples (9.1% positive) using the Galleria baiting technique. Morphological and molecular characterizations of the isolates were performed for species identification. Five isolates were identified as Heterorhabditis bacteriophora (ZET02, ZET04, ZET09, ZET28, and ZET35) and 2 isolates were identified as Steinernema feltiae (ZET31 and ZET76). The efficacy of all isolates was tested on Melolontha melolontha larvae in plastic boxes and pot experiments. Different concentrations of nematodes at 0, 500, 1000, or 2000 infective juveniles (IJs)/mL and 2 different temperature regimes (15 and 25 °C) were used. One hundred percent mortality was obtained from the ZET09 and ZET35 isolates at a concentration of 2000 IJs/mL at 25 °C. The same isolates also provided 100% protection with 100 IJs/cm2 in strawberry planted pot experiments.

Molecular characterization, virulence and horizontal transmission of Beauveria pseudobassiana from Dendroctonus micans (Kug.) (Coleoptera: Curculionidae)

The great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae), has been a potential threat for Turkey and the entire Eurasian spruce forests for many years. Control strategies which have been applied so far are still insufficient to prevent its damage. A previous study has shown that a Beauveria isolate (ARSEF 9271) proved to be an efficient microbial control agent against the great spruce bark beetle. In this study, this isolate was identified as B. pseudobassiana based on the partial sequence of EF1‐α and ITS sequence. A conidial suspension (1 × 108/ml) of this fungus caused 100% mortality on both larvae and adults of D. micans within 5 and 6 days, respectively. Also, it caused 100% mycosis value on both larvae and adults. Mortality values of horizontal transmission experiments between larvae and adults which were contaminated with 1 × 106/ml spore suspension at 25%, 50%, 75% and 100% rates were determined as 100% after 15 days at 20°C under the laboratory conditions. We also determined the decrease of the damage in spruce wood block (15 × 25 cm) when the contamination rate of the larvae was increased. Our results indicate that B. pseudobassiana ARSEF 9271 seems to be a very promising biocontrol agent against D. micans.

Characterization of a nucleopolyhedrovirus variant of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae) in Turkey

ABSTRACT The gypsy moth (Lymantria dispar L., Lepidoptera: Lymantriidae) is one of the most serious pest of various forestal, food and industrial crops worldwide. We have characterized a new Lymantria dispar nucleopolyhedrovirus (LdMNPV-T4) variant, which was isolated from dead L. dispar larvae in Turkey. Scanning electron microscope observations showed that the polyhedral occlusion bodies (OBs) of the LdMNPV-T4 were irregularly shaped. Transmission electron microscopy revealed that OBs of LdMNPV-T4 were occupied with several virions in which multiple nucleocapsids packaged by viral envelope. Restriction analysis of the LdMNPV-T4 DNA purified from the viral inclusion bodies yielded BamHI, BglII, EcoRI and HindIII fragments. The mean size estimated for the complete LdMNPV-T4 genome was calculated to be 163.3 kb. Phylogenetic analysis of amplified polh, lef-8 and lef-9 sequences showed its relation to the other NPVs from Lymantria species. Mortality values of the LdMNPV-T4 at four different concentrations against third instar larvae of L. dispar ranged from 45% to 88%. These results suggest that LdMNPV-T4 isolated from Turkey is a promising microbial control agent to be utilized for the biological control of L. dispar.

Characterisation of three Alphabaculovirus isolates from the gypsy moth, Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

ABSTRACT In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170 kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 103 and 106 OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC50 and LC95 values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC50 value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.

Bacterial isolates from Palomena prasina (Hemiptera: Pentatomidae) include potential microbial control agents

The green shield bug, Palomena prasina (Hemiptera: Pentatomidae), is a pest of deciduous trees and shrubs throughout Turkey and is found in a large variety of habitats, including gardens. We investigated the facultative cultivable bacterial flora of 10 healthy and 5 dead green shield bugs, collected from the vicinity of Trabzon, Turkey, and tested them for insecticidal activity. Based on the conventional and molecular tests, 12 different bacteria were isolated and identified as Curtobacterium sp., Rhodococcus sp., Arthrobacter nicotinovorans, Arthrobacter oxydans, Agrococcus jejuensis, Pseudomonas poae, Raoultella terrigena, Serratia sp., Lysinibacillus sphaericus, Stenotrophomonas rhizophila, Bacillus thuringiensis and Microbacterium oxydans. Mortalities due to the application of 0.5 mL of L. sphaericus, B. thuringiensis and R. terrigena at a density of OD600 1.89 were 60%, 70% and 60%, respectively, on adult P. prasina. This indicates that facultative cultivable bacterial flora isolated from P. prasina have potential for microbial control of this pest.