Helicosporidium infection of the great European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

An infection of the great European spruce bark beetle Dendroctonus micans (from Turkey) by the parasitic green alga Helicosporidium is described. This is the first time that Helicosporidium has been found to infect a bark beetle (Scolytidae) and the first time that a naturally infected beetle has been reported for the Eurasian continent. The typical cysts of Helicosporidium contain three ovoid cells and one helical, filamentous cell. The morphological characteristics are revealed by light and electron microscopy. Distinct electron-dense inclusions with a peculiar ultrastructure may represent pyrenoids. Since D. micans is an important pest, the discovery of a natural pathogen may offer a chance for biological control.     

Isolation of some pathogenic bacteria from the great spruce bark beetle, <Emphasis Type="Italic">Dendroctonus micans</Emphasis> and its specific predator, <Emphasis Type="Italic">Rhizophagus grandis</Emphasis>

Some bacteria were isolated from Dendroctonus micans and its specific predator, Rhizophagus grandis. Six bacteria from D. micans were identified as Bacillus pumilus, Enterobacter intermedius, Citrobacter freundii, Cellulomonas flavigena, Microbacterium liquefaciens and Enterobacter amnigenus, three bacteria from R. grandis as Klebsiella pneumoniae, Pantoea agglomerans and Serratia grimesii, on the basis of fatty acid methyl ester analysis and carbon utilization profile by using Microbial Identification and Biolog Microplate Systems. Their insecticidal effects were tested on larvae and adults of D. micans.     

Identification,distribution and occurrence of the ascomycete <Emphasis Type="Italic">Metschnikowia typographi</Emphasis> in the great spruce bark beetle, <Emphasis Type="Italic">Dendroctonus micans</Emphasis>

This is the first report on the ascomycete Metschnikowia typographi from the adults and larvae of the great spruce bark beetle Dendroctonus micans in Turkey. In total, 910 of 1928 adults and 44 of 149 larvae investigated during the two years were infected by the pathogen. In a fresh smear the asci of the pathogen measure 18.5 ± 2.05 μm (14.7–22.3) in length and 2.1 ± 0.4 μm in width (n = 35). The ascospores are about 2 μm shorter than asci, having an average length of 16.4 ± 1.5 μm (14.2–18.0). The total infection rate of D. micans was 47.2 %. The prevalence of M. typographi infections differed between localities and years. Different infection rates of male and female beetles of D. micans were not recognized.     

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.     

Screening of entomopathogenic fungi against 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 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 and identification of bacteria from <Emphasis Type="Italic">Thaumetopoea pityocampa</Emphasis> Den. and Schiff. (Lep., Thaumetopoeidae) and determination of their biocontrol potential

The pine processionary moth Thaumetopoea pityocampa (Den. and Schiff.) is one of the most harmful insect pest for pine species in Mediterranean countries including Turkey. The objective of the present study is to find a more effective and safe biological control agent against T. pityocampa. Thus, we investigated the bacterial flora of the pest insect, collected from the Middle Black Sea Region of Turkey from 2003 to 2004. Based on morphological, physiological, biochemical and molecular methods, 14 different bacterial isolates were determined. The identified bacterial flora of T. pityocampa consisted of bacteria belonging to the Enterobacteriaceae (Tp1), Arthrobacter sp. (Tp2), Staphylococcus spp. (Tp3 and 10), Bacillus subtilis (Tp4), Serratia liquefaciens (Tp5), Bacillus thuringiensis subsp. morrisoni (Tp6 and 14), an acrystalliferous form Bacillus thuringiensis (Tp7), Staphylococcus cohnii (Tp8), Bacillus licheniformis (Tp9), Bacillus pumilus (Tp11), Brevibacterium sp. (Tp12) and Bacillus simplex (Tp13). After analysing the conclusions of conventional and molecular tests Tp1 (Enterobacteriaceae), Tp2 (Arthrobacter sp.) and Tp12 (Brevibacterium sp.) were assigned as novel bacterial species. Isolate Tp5 had a promising insecticidal effect on third instar larvae of T. pityocampa (up to 70% mortality within 10 days).     

Isolation of pathogenic bacteria from <Emphasis Type="Italic">Oberea linearis</Emphasis> (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.     

北部湾海洋原甲藻的形态特征及其系统进化分析

为探讨广西北部湾海洋原甲藻(Prorocentrum micans)的形态特征及其系统进化意义,利用光学显微镜、分子生物学方法,对海洋原甲藻BBW-01 藻株的形态特征进行了描述,并分析了其分子系统进化关系.结果表明,各地理株系的海洋原甲藻的形态特征相似,仅在细胞大小上存在差异.海洋原甲藻BBW-01 与采自于广东大亚湾的海洋原甲藻形态特征最为接近,其壳板后端的7 个呈“V”字形对称排列的大孔可作为海洋原甲藻鉴定的重要指标.18S rDNA 序列同源检索和系统进化分析表明,海洋原甲藻BBW-01 与源自中国南海的海洋原甲藻的亲缘关系最近,并与其他2 株海洋原甲藻聚成一支,属于浮游、兼性浮游类原甲藻.因此,对赤潮原因种的准确识别有助于预防和减轻赤潮对海洋环境和人类带来的危害.     

Cost-effectiveness analysis of options within an Integrated Crop Management regime against great spruce bark beetle, Dendroctonus micans, Kug. (Coleoptera: Scolytidae)

1 Great spruce bark beetle Dendroctonus micans (Kug.) (Coleoptera: Scolytidae) has been subject to an Integrated Crop Management (ICM) regime in Great Britain since its first discovery in 1982. The elements of the ICM approach are sanitation felling of the initial infestations, restriction on movement of potentially infested conifers to prevent spread to uninfested parts of the country and biological control through rearing and release of the specific predatory beetle Rhizophagus grandis Gyll. (Coleoptera: Rhizophagidae). Such a programme carries costs that have to be evaluated against the benefits to the ICM strategy. 2 This paper presents a cost-effectiveness analysis of three options – (i) continue with the current policy of restriction on movement of infested timber and use of the imported predator R. grandis, (ii) do nothing (i.e. drop all attempts to reduce the spread of D. micans), or, (iii) introduce a new east–west Dendroctonus Micans Control Area (DMCA) to define the management area. Assumptions on rates of mortality and natural spread were based on research into D. micans in Britain’s spruce forests. 3 Appraisal indicated that the current policy was the most cost-effective, even when subject to sensitivity analysis to test the limits of the assumptions included in the models. It is concluded that the current policy should remain in force.     

野生和人工饲喂条件下甘肃鼢鼠肠道细菌多样性比较

【目的】肠道微生物在宿主代谢和健康方面发挥着重要的作用,已有报道表明甘肃鼢鼠肠道菌群和地域、性别以及季节等因素有关,但对于野生和人工饲喂条件下,取食不同食物的甘肃鼢鼠肠道菌群是否存在差异尚不明确。【方法】本研究选取苜蓿、樟子松根、油松根和云杉根对甘肃鼢鼠进行分组饲喂,同时以野生个体作为对照,采用16S rRNA V3-V4高通量测序技术对野生和人工饲喂甘肃鼢鼠肠道细菌进行比较。【结果】野生和人工饲喂条件下,优势菌门均为Firmicutes和Bacteroidetes,但含量却存在显著性差异。在人工饲喂条件下,樟子松根、油松根和云杉根饲喂的三组甘肃鼢鼠肠道细菌群落结构相似度较高,而与苜蓿组和野生组之间均存在较大差异,樟子松组、油松组和云杉组甘肃鼢鼠肠道细菌多样性高于苜蓿组和野生组。【结论】野生和人工饲喂条件下甘肃鼢鼠肠道菌群组成和结构存在显著性差异。     

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

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.     

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.     

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.     

RAPD variations among pure and hybrid populations ofPicea mariana, P. rubens, andP. glauca (Pinaceae), and cytogenetic stability ofPicea hybrids: Identification of species-specific RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to determine genetic relationships amongP. mariana (black spruce),P. rubens (red spruce), andP. glauca (white spruce) and to assess the degree of polymorphism within populations from different provenances and among spruce hybrids. Eleven arbitrary decamer primers were used to amplify genomic DNAs extracted from embryogenic cultures and seedlings. Species-specific RAPD markers were identified.Picea mariana andP. rubens showed similar RAPD profiles confirming their close genetic relationship. Species-specific RAPD markers were identified and were useful in distinguishing white spruce from black and red spruces. RAPD differentiation between populations within each species was small. The level of polymorphism was much higher in spruce hybrid populations than in the pure species. Cytological analysis ofP. mariana ×P. rubens hybrids showed normal mitotic behaviour at prophase, metaphase, anaphase, and telophase. All the hybrids analyzed from different cross combinations were euploids.     

Bacteria associated with four species of Dacus (Diptera: Tephritidae) and their role in the nutrition of the larvae

Summary The bacteria associated with Dacus tryoni (Froggatt), Dacus jarvisi (Tryon), Dacus neohumeralis (Hardy) and Dacus cacuminatus (Hering) were examined. Bacteria were isolated from the surface of freshly-laid eggs, from within surface sterilised pupae, from heads and abdomens of wild and laboratory-maintained flies, and from decomposed fruits in which the wild larvae were feeding. A more diverse flora was associated with D. tryoni and D. jarvisi (15 and 14 species, respectively) than with D. neohumeralis and D. cacuminatus (9 and 6 species, respectively). Most of the bacteria belonged to the family Enterobacteriaceae and while there were similarities of bacterial associations between fly species there was no evidence of a strict symbiotic association of a particular bacterium or bacteria with each species of fly. The larvae of D. jarvisi were unable to develop normally in an artificial medium containing unhydrolysed protein and free of bacteria and on a medium containing casein and Serratia liquefaciens (isolated from the flies and shown to secrete protease) the larvae died. On the same casein medium containing Enterobacter cloacae (isolated from the flies and shown to be protease negative) the larvae developed normally. Larvae of D. tryoni and D. jarvisi were devoid of protease and cellulase activity, but contained some amylase activity. The significance of these results in terms of current hypotheses concerning symbioses between tephritids and bacteria is discussed.     

Past attacks influence host selection by the solitary bark beetle Dendroctonus micans

1. A spatio‐temporal study of host selection and local spread of a solitary bark beetle attacking live spruce Dendroctonus micans (Kugelann) was carried out using a combination of standard statistical methods, geostatistical analyses, and modelling. The study was based on data from three plots (150–300 trees, 0.3–1 ha) from 1978 to 1993. All trees were mapped and successful and abortive bark‐beetle attacks on each tree were counted annually. Because the attacked trees usually survived, temporal attack patterns as well as spatial patterns could be analysed. 2. The distribution of successful insect attacks on the trees was slightly aggregative, indicating some degree of choice rather than totally random establishment. 3. The level of yearly individual attacks per tree was very stable, suggesting that D. micans usually leave the host in which they develop. 4. The attacked trees were distributed randomly in the plots; at the study's spatial scale, the insects dispersed freely throughout the plot (no spatial dependence). 5. On the other hand, time dependence was strong; some trees were attacked repeatedly while others were left untouched. 6. Among a choice of scenarios (random attack, fixed variability in individual host susceptibility, induced host susceptibility following random attack), the best fit was obtained with the model involving induced individual host susceptibility. This type of relation to the host tree contrasts strongly with patterns generally described in host–plant relationships (including gregarious, tree‐killing bark beetles), where local herbivore damage results in induced resistance. 7. These results suggest that the first attacks in a new stand are made at random, that all or most of the beetles emerging from a tree disperse and resample the stand, and that they settle preferentially on trees that were colonised successfully by previous generations.     

Frankia in the rhizosphere of nonhost plants: A comparison with root-associated N2-fixing Enterobacter,Klebsiella and Pseudomonas

Bacterial growth in the rhizosphere and resulting changes in plant growth parameters were studied in small aseptic seedlings of birch (Betula pendula and B. pubescens) and grasses (Poa pratensis and Festuca rubra). The seedlings were inoculated with three Frankia strains (Ai1a and Ag5b isolated from native Alnus root nodules and Ai17 from a root nodule induced by soil originating from a Betula pendula stand), and three associative N₂-fixing bacteria (Enterobacter agglomerans, Klebsiella pneumoniae and Pseudomonas sp., isolated from grass roots). Microscopic observations showed that all the Frankia strains were able to colonize and grow on the root surface of the plants tested without addition of an exogenous carbon source. No net growth of the associative N₂-fixers was observed in the rhizosphere, although inoculum viable counts were maintained over the experimental period. Changes in both the biomass and morphology of plant seedlings in response to bacterial inoculation were recorded, which were more dependent on the plant species than on the bacterial strain.     

Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.     

Culturable heterotrophic bacteria from the marine sponge <Emphasis Type="Italic">Dendrilla</Emphasis> <Emphasis Type="Italic">nigra</Emphasis>: isolation and phylogenetic diversity of actinobacteria

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “Micromonospora–Saccharomonospora–Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.     

Changes in the phytoplankton community of a coastal,hyposaline lake in western Anatolia,Turkey

The phytoplankton composition of coastal, hyposaline (≅12 g l⁻¹) Lake Bafa in western Turkey, was investigated after the alteration of salinity levels in past decades. Lake Bafa, formerly a bay of the Aegean Sea, was separated from the sea as a result of alluvium deposits of the Meander River. After the construction of a sett for flood control in 1985, freshwater inflow was reduced and salinity increased, which caused some ecological changes. Phytoplankton samples were taken in August and November 2000, and February and May 2001, from the surface and at 5-m intervals in the water column at three stations. The phytoplankton community contained a mixture of freshwater and marine origin species. A total of 45 species from Bacillariophyceae, Chlorophyceae, Cyanophyceae, Chrysophyceae, and Dinophyceae were identified. Marineoriginated species such as Chaetoceros spp. (November and May), Thalassionema nitzschioides and Prorocentrum micans (August and November), and P. minimum (May) were dominant on the basis of numerical abundance. The occurrence of salinity-tolerant species such as T. nitzschioides, P. micans, and P. minimum has not been recorded in previous studies.