PCR-Based Screening of Pathogens in Bombus terrestris Populations of Turkey.

PURPOSE: Bumblebees are an important group of insects in the pollination of various vegetables, fruits, oilseeds, legumes, and the fodder crops. Compared to honeybees, they have a wider choice of hosts and a longer flight period. These bees are used especially for the pollination of plants in greenhouses and are commercially produced for this purpose. Recently, serious decreases have been occurring in bumblebee populations due to various reasons such as pathogens, and some of species are even threatened with extinction. Due to the worldwide decline in pollinator insects, determining the distribution and prevalence of bumblebee pathogens is of great importance. Therefore, this study was conducted to determine the incidence and prevalence of pathogens in Turkish bumblebee populations and how much of each pathogen was in bumblebee samples. METHODS: A total of 172 Bombus terrestris (Linnaeus,1758) samples (21 samples from commercial enterprises, 79 samples from greenhouses and 72 samples from nature) were randomly collected from 3 provinces (Antalya, Mersin and Izmir) where greenhouse cultivation is intensively carried out in Turkey. Eighty-nine of these samples were collected in the spring and eighty-three in the autumn. The presence of four pathogens (Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri) was investigated by PCR using universal primers. RESULTS: The overall prevalence of Nosema bombi, Crithidia bombi, Apicystis bombi, and Locustacarus buchneri was determined as 7.55%, 9.3%, 11.62%, and 4.65%, respectively. Co-infections (5.81%) were only detected in wild-caught (nature) samples. C. bombi and A. bombi infections were detected at higher rates in the spring samples than in the autumn samples (p < 0.05). There was no significant difference between the spring and autumn samples with respect to the presence of N. bombi and L. buchneri (p > 0.05). CONCLUSION: The results obtained could be important in determining the prevalence and spread rates of the bumblebee diseases in Turkey and to determine appropriate protection measures. The information gathered should increase our knowledge about the presence of these pathogens in Turkey and could contribute to improve apiarist's practice. More studies are needed to determine the transmission pathways of these pathogens between the populations. Also, complex pathogen interactions in bumblebee populations should be considered in the future to improve bumblebee health.

PCR-based detection of the honeybee tracheal mite (Acarapis woodi) in Türkiye.

Acarapis woodi (Rennie 1921) (Acari: Tarsonemidae) is one of the mites that settles in the respiratory system of honeybees (Apis mellifera L. (Hymenoptera, Apidae)) and distributed throughout the world. It causes significant economic losses on honey production. In Türkiye, studies on the existence of A. woodi are very limited and so far, no studies on the molecular diagnosis and phylogenetic of it have been reported in Türkiye. This study was conducted to investigate the prevalence of A. woodi in Türkiye, especially in areas where beekeeping is intense. Diagnosis of A. woodi was performed using both microscopic and molecular methods using specific PCR primers. Adult honeybee samples were collected from 1.193 hives in 40 provinces of Türkiye between 2018 and 2019. Based on identification studies, the presence of A. woodi was detected in a total of 3 hives (0.5%) in 2018 and 4 hives (0.7%) in 2019. This is the first report for determination of A. woodi in Türkiye.

Culturable bacterial microbiota of Plagiodera versicolora (L.) (Coleoptera: Chrysomelidae) and virulence of the isolated strains.

Plagiodera versicolora (Laicharting, 1781) (Coleoptera: Chrysomelidae) is an important forest pest which damages many trees such as willow, poplar, and hazelnut. In order to find new microbes that can be utilized as a possible microbial control agent against this pest, we investigated the culturable bacterial flora of it and tested the isolated bacteria against P. versicolora larvae and adults. We were able to isolate nine bacteria from larvae and adults. The isolates were characterized using a combination of morphological, biochemical, and physiological methods. Additionally, we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results. Based on characterization studies, the isolates were identified as Staphylococcus sp. Pv1, Rahnella sp. Pv2, Rahnella sp. Pv3, Rahnella sp. Pv4, Rahnella sp. Pv5, Pantoea agglomerans Pv6, Staphylococcus sp. Pv7, Micrococcus luteus Pv8, and Rahnella sp. Pv9. The highest insecticidal activity against larvae and adults was obtained from M. luteus Pv8 with 50 and 40 % mortalities within 10 days after treatment, respectively. Extracellular enzyme activity of the bacterial isolates such as amylase, proteinase, lipase, cellulose, and chitinase was also determined. Consequently, our results show that M. luteus Pv8 might be a good candidate as a possible microbial control agent against P. versicolora and were discussed with respect to biocontrol potential of the bacterial isolates.

Bacteria from Ips sexdentatus (Coleoptera: Curculionidae) and their biocontrol potential.

Ips sexdentatus (Coleoptera: Curculionidae) is one of the most destructive pests of the spruce trees in Europe. In this study, we have isolated and characterized culturable bacteria from I. sexdentatus and tested their insecticidal activity against the last instar larvae of the pest as a possible biocontrol agent. A total of eight bacterial isolates was determined and four of them were identified at species level, and the others were identified at genus level. Isolates were identified as Stenotrophomonas maltophilia (Is1), Rahnella sp. (Is2), Pseudomonas sp. (Is3), Bacillus sp. (Is4), Alcaligenes faecalis (Is5), Panteoea agglomerans (Is6), Pseudomonas fluorescens (Is7) and Serratia sp. (Is8) based on their morphological, biochemical and molecular characteristics. Insecticidal effects of bacterial isolates were performed on the last instar larvae of the pest. The highest insecticidal activity was obtained from P. fluorescens (Is7) with 73% mortality within 10 days after inoculation (p < 0.05). Mortality values of the other isolates ranged from 20 to 53%. This study suggests that Pseudomonas fluorescens (Is7) seems to be a good candidate as a possible biocontrol agent against I. sexdentatus, and provides suitable strains that can be modified to express insecticidal toxins and/or other detrimental substances to develop new control methods for I. sexdentatus.

Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence.

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

A novel cry2Ab gene from the indigenous isolate Bacillus thuringiensis subsp. kurstaki.

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.

Molecular characterization and virulence of Beauveria spp. from the pine processionary moth, Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae).

The pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) is one of the most harmful pests to pine species in Mediterranean countries including Turkey. Caterpillars of T. pityocampa are not only significantly harmful to forest trees but also responsible for various allergic reactions in humans and animals. In this study, in order to find a more effective and safe biological control agent against T. pityocampa, we investigated fungal pathogens of T. pityocampa in the Black Sea Region of Turkey and tested their pathogenicity on it. Five different fungi were isolated and identified based on their morphological and molecular characteristics including ITS and partial sequence of EF1-[alpha]. Based on these characteristics, four isolates were identified as Beauveria bassiana cf. Clade C (Rehner and Buckley in Mycologia 97:84-98, 2005) and one isolate was identified as Beauveria bassiana. Among these isolates, B. bassiana KTU-24, B. bassiana cf. Clade C KTU-66 and KTU-67 showed the highest virulence with 100% mortality within 10 days after application. B. bassiana isolate KTU-24 produced the highest mycosis value with %100. Consequently, B. bassiana KTU-24 seems to be good candidate for further investigation as a possible biological control agent against this pest.

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey.

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla(oxA-30), and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.

Molecular characterization of antibiotic resistant Escherichia coli strains isolated from tap and spring waters in a coastal region in Turkey.

A hundred and seventeen antibiotic-resistant Escherichia coli strains were isolated from public tap and spring waters which were polluted by fecal coliforms. There were no significant differences between two water sources as to the coliform pollution level (p> 0.05). All E. coli isolates were detected to be resistant to one or more antibiotics tested. Nearly 42% of the isolates showed multiresistant phenotype. Three (2.5%) of these isolates contained class 1 integron. Sequencing analysis of variable regions of the class 1 integrons showed two gene cassette arrays, dfr1-aadA1 and dhfrA17-aadA5. Resistance to ampicillin, tetracycline or trimethoprim-sulfamethoxazole was transferable according to the results of conjugation experiments. The rate of tetracycline resistance was 15%. tet(A)-mediated tetracycline resistance was widespread among tetracycline-resistant E. coli isolates. Genotyping by BOX-polymerase chain reaction (BOX-PCR) showed that some of the strains were epidemiologically related. This is the first report on the prevalence and characterization of class 1 integron-containing E. coli isolates of environmental origin in Turkey.