Akademik Ziraat Dergisi 7(1):21-28 (2018) Araştırma ISSN: 2147-6403 DOI: http://dx.doi.org/10.29278/azd.440586 (Research) Isolation and characterization of Bacillus megaterium isolates from dead pentatomids and their insecticidal activity to Palomena prasina nymphs Hasan Murat AKSOY1, Celal TUNCER1, Islam SARUHAN1, Ismail ERPER1, Murat OZTURK1, Izzet AKCA1 1Ondokuz Mayis University, Faculty of Agriculture, Department of Plant Protection, SAMSUN , Kabul tarihi 06 Nisan 2018 Sorumlu yazar: , e-posta:[email protected] Alınış tarihi: 19 Aralık 2017 İslam SARUHAN Abstract Ölü pentatomidlerden Bacillus megaterium Bacillus megaterium isolates, were demonstrated to izolatlarının izolasyonu, karekterizasyonu ve be efficient biocontrol agents against the green Palomena prasina nimflerine insektisital etkisi shield bug (Palomena prasina Pentatomidae). Firstly hazelnut orchards were Öz surveyed and four B. megateriumL., isolatesHeteroptera: were Bacillus megaterium obtained from P. prasina. The morphological, Palomena prasina physiological and biochemical characteristics of B. Bu çalışma, izolatlarının fındık megaterium isolates were determined according to kokarcasına ( L., Heteroptera: the standardized methodology. Additionally 16S Pentatomidae) karşıP. etkinprasina biyokontrol Bacillusajanları to megateriumolduğu gösterilmiştir. Öncelikle fındıkB. bahçelerindemegaterium survey yapılarak, 'dan dört rRNA gene sequence analyse was performed gene confirmed that isolates Sa-1, Sa-5, SAk-2 and izolatı elde edilmiştir. determine the isolates. Analysis of the 16S rRNA SAkc-19 are B. megaterium izolatlarının morfolojik, fizyolojik ve biyokimyasal homology to the type strains of B. megaterium. özellikleri standart tanı yöntemlerine göre Effectiveness of these isolates, withwas tested100% againstsequence P. sonucu;belirlenmiştir. Sa-1, Sa Ayrıca-5, SAk izolatların-2 ve SAkc tanısı- için 16S rRNAB. prasina nymphs in laboratory, at 25±1°C and 70±5 megateriumgen dizi analizi yapılmıştır. 16S rRNA geninin analizi 19 izolatlarının dead individuals were counted daily, following a 12 olduğu, tip ırkları ile % 100 dizi RH. Isolates were bioassayed against nymphs and izolatl 5 days lasting treatment. Lethal time (LT50 and LT90) benzerliğiP. prasinagöstermesi ile doğrulanmıştır. Bu values of B. megaterium isolates were calculated. arın etkinliği, laboratuarda 25±1 °C ve 70± LT50 values of the two most active isolates were 1.91 veRH'de son uygulamadannimflerine 12 gün sonkarşı denenmiştir. and 11.18 days. The highest mortality rate was 98% İzolatlar, nimflere karşı biyolojikB. megaterium olarak test edilmiş obtained with the treatment of SAkc-2 isolates at ra ölen bireyler 50 ve LT90) concentrations of 108 cfu ml-1 on the 12th day of post günlük olarak sayılmıştır. izolatlarının 50 treatment. Lethal zaman değerleri (LT Key words: Bacillus megaterium, Palomena prasina, hesaplanmıştır. Enk caktif- iki izolatın LT8 cfudeğerleri ml-1 biological control, entomopathogen 1.91 ve 11.18 gün olarak belirlenmiştir. En yüksek ölüm oranı, SA .2 izolatının 10 konsantrasyon uygulanması ile 12. günde % 98 Anahtar kelimeler: Bacillus megaterium, Palomena olarak elde edilmiştir prasina , biyolojik mücadele, entomopatojen 22 Aksoy, H.M., Tuncer, C., Saruhan, İ., Erper, İ., Öztürk, M., Akca, I. Introduction is the first study investigating the effects of B. megaterium against P. prasina. Turkey is the largest producer of hazelnut in the world. The country, account for around 75 percent of Materials and Methods total global hazelnut production. The total area of Insect culture hazelnut orhards is currently around 618 000 tons a Fourth stage nymphs of P. prasina were used in year and 2.3 billion US dollars yearly from hazelnut bioassays. The nymphs were collected from different exports in 2014 in Turkey (Tuncer et al., 2002; hazelnuts orchards by beating-sheet method during Anonymous, 2016a, b). July in Samsun province. The insects were conserved Several insect pests infest hazelnut orchards. Among in climate chamber with 25±1 ºC, 70±5 relative these insect pests are some pentatomids which effect humidity and photoperiodic lighting (16 hours of light: 8 hours of dark). After the bugs were Tuncer et al., 2004; Tuncer et al., 2009). Feeding of transferred to the laboratory conditions, the insect pentatomidson hazelnut causeskernel emptyquality nuts (Tavella during etearly al., se2001;ason culture was fed with fresh bean pods (Phaseolus and kernel damage during later period. Kernels vulgaris) and food was changed daily basis. damaged by pentatomids lost their shape and taste Bacterial isolation bitter. Among pentatomids, the predominant species Bacillus isolates were isolated from dead male and is the green shield bug, Palomena prasina L., female P. prasina individuals found in hazelnut orchards in Giresun, Düzce, Ordu and Samsun cities, in hazelnut orchards of Black Sea region of Turkey located in Black Sea region of Turkey. These (Tuncer(Heteroptera: et al., Pentatomidae)2005). The public with concern85% prevalence over the orchards had not been previously treated with any dangerous effects of insecticides on the living Bacillus biopesticide. The collected samples were organisms and atmosphere has enhanced the search kept at about 4°C in the refrigerator until they are of safer and ecologically friendly biological control used for bacterial isolation. To eliminate external alternatives. One of alternative control may contamination, dead P. prasina male and female individuals were disinfected in 1% NaOCl within 3 management of P. prasina. Few antagonistic min. Thereafter, the samples were kept several times microosuggestionrganisms an areadditional positively technique applied tofor control the in sterile distilled water and moved aseptically into a pests in contaminated soil. These microorganisms sterile mortar and macerated with a sterile pestle. consist of Bacillus species, B. thuringiensis (Bt), The macerate was placed in 1.5 ml of sterile distilled Lysinibacillus sphaericus (Ls) and B. megaterium water. The suspension was then heated to 75°C for (Bm). The usage of Bm, which can produce 10-15 min, and diluted in the ratios of 1×10-2 and insecticidal metabolites, also deliberated to be a 1×10-4. The dilutions were streaked on nutrient agar promising tool for controlling the chemical pests and the plates were incubated at 30°C for 48 h (Aksoy and Ozman-Sullivan, 2008). It is also a (Cavados et al., 2001). The colony characteristic are potential biological control agent of nematodes observed for selection of candidate isolates. is a Gram-positive, mostly aerobic spore-forming Biochemical characteristics bacterium(Padgham andfound Sikora, broadly 2007; in Huangdiverse et habitatsal., 2010). from Bm The biochemical characteristics, morphological and soil to seawater, river, sea food and salt lake (Gu et physiological of Bm isolates were identified based on al., 2007; Patricia et al., 2007). Even though there the standardized methods recommended in Bergey’s have been various researches on microorganism as a Manual of Systematic Bacteriology (Logan and Vos, possible bacterial control agent (Sezen et al., 2004; 2009). Buresova et al., 2006; Gokce et al., 2010; Ozsahin et 16S rRNA gene sequencing al., 2014), none have used on against B. megaterium The bacterial total genomic DNA was extracted from . P. prasina bacterial suspensions (after 12 h incubation in LB) The aim of this study was to obtain bacterial isolates using Qiagen DNA extraction kit and DNA from dead pentatomids in hazelnut orchards in Düzce, Samsun, Giresun and Ordu provinces in Black Four bacterial Sea region of Turkey, to characterize these isolates concentration was standardized at about 50 ng/μl by molecular methods and to determine their prior to performing PCR assay. insecticidal activity against P. prasina nymphs. This isolates were sequenced for the 16S rRNA region. 16S rRNA gene was amplified in 50 µl volume as described for PCR tests above, using the universal Isolation and characterization of Bacillus megaterium isolates from dead pentatomids and their insecticidal… ___________ 23 primers 27F (AGAGTTTGATC(AC)TGGCTCAG; days in a Binder incubator (Model KBWF 240, Germany). Polyethylene sheets were used together TGTTACGACTT; positions 1508 to 1492, (Weisburg with rubber in order to cover the open sides of cups. positions 8 to 27 and 1492R (ACGGTTACCT The causal agents were again re-isolated from dead nymphs according to Cavados et al. (2001) and et al., 1991). Amplifications of the 16S rRNA shown to be identical to the organisms characterized fragments were achieved in a final volume of 50 µl bioassays were with 5 µl of 10× PCR buffer (Qiagen), 1.5 mM MgCl2, conducted twice, with three replications. 1 µl PCR Nucleotide Mix Plus (Roche), 0.2 µM of each by sequencing of 16S rRNA gene. The Statistical analysis primer, 1.25 U Taq polymerase- (250 U HotStarTaq DNA Polymerase, Qiagen) and 3 µl of template DNA. The mortality data of isolates on nymphs of P. prasina were corrected using Abbott’s formula 94°C,Reactions 35 cycles were of used 30 s atin 94°C,a Bio 1Rad min T100at 52°C Thermal and 90 (Abbott, 1925) and percentages of mycosed insect sCycler. at 72°C, The followed PCR conditions by a final extusedension were at 1572°C min for at7 cadavers were calculated. Due to the limited availability of insect material for experiment, strains was performed in both directions and the min. Sequencing for PCR results of the selected multiple observations were made for each of the dose groups at a series of times after