PERSISTENCE OF TWO BACTERIAL BIOPESTICIDES ON Bombus terrestris L. AND TOMATO BLOSSOMS

Year 2025, Volume: 25 Issue: 2, 251 - 262, 17.11.2025

Asiye Uzun Yiğit , İsmail Yaşhan Buluş , Ozan Demirözer , Ayhan Gösterit

Abstract

In the present study, the persistence of the biological control agent bacteria Bacillus thuringiensis var. kurstaki (Rebound, Hektaş) and Bacillus velezensis QST 713 (Serenade SC, Bayer) on tomato flowers and B. terrestris worker bees was determined after application. This study consists of two parts. In the first part, these bacterial agents were applied to tomato flowers under greenhouse conditions. Suspensions prepared at the recommended field dose of each bacterial isolate were applied by spraying the ten flowering tomato plants under greenhouse conditions. Ten flowers from each treated tomato plant were collected 24, 48, and 72 hours after application. Surface sterilization was performed using 70% alcohol on tomato flowers collected from the greenhouse at various observation times. These flowers were then transferred separately to petri dishes containing Nutrient Agar (NA) and Sabouraud Dextrose Agar (SDA). In the second part, bacterial agents were applied to Bombus terrestris worker bees under laboratory conditions and then transferred to hives under field conditions. The field öşe of each bacterial isolate was applied to 50 B. terrestris workers for 20 seconds (0.5 ml) using a system capable of spraying at 4 atm pressure. These workers were then transferred to a separate hive for each bacterial isolate. Re-isolation was carried out by mechanically killing ten worker bees taken from these hives at the 24th, 48th, and 72nd hours. During the reisolation phase, worker bees were individually dissected in sterile distilled water after surface sterilization for each observation time. A drop of haemolymph obtained from the dissected insect was diluted 100 times with sterile water and then spread on Nutrient Agar. According to the results obtained from the study, it was determined that Bacillus thuringiensis var. kurstaki and Bacillus velezensis QST 713 were found in both tomato flowers and the hemolymph of B. terrestris worker bees 24, 48, and 72 hours after application.

Keywords

Entomopathogen , Spore viability , Biocontrol agent , Bumble bee

Ethical Statement

This study does not require ethical committee approval.

Supporting Institution

This study was funded by the Scientific and Technological Research Council of Türkiye (TUBITAK, Project number: 221O580).

Project Number

221O580

Thanks

We thank the Scientific and Technological Research Council of Türkiye (TÜBİTAK, Project No: 221O580) for financial support.

İki Bakteriyal Biyopestisitlerin Bombus terrestris L. ve Domates Çiçeklerinde Kalıcılığı

Abstract

Çalışmada biyolojik mücadele etmeni olan bakterilerden Bacillus thuringiensis var. kurstaki (Rebound, Hektaş) ve Bacillus velezensis QST 713 (Serenade SC, Bayer)’in uygulandıktan sonra domates çiçekleri ve B. terrestris işçi arılarında kalıcılığı belirlenmiştir. Çalışma iki kısımdan oluşmaktadır. Birinci kısımda bu bakteriyal etmenler domates çiçeklerine sera koşullarında uygulanmıştır. Sera koşullarında 10 adet çiçekli domates bitkisine her bir bakteri izolatına ait arazi tavsiye dozunda hazırlanmış süspansiyonlar püskürtme yöntemi ile uygulanmıştır. Uygulamadan sonra 24., 48. ve 72. saatlerde uygulama yapılmış domates bitkilerinden her bir izolat için onar adet çiçek toplanmıştır. Seradan farklı gözlem zamanlarında alınan domates çiçeklerine %70’lik alkol ile yüzey sterilizasyonu uygulanmıştır. Daha sonra bu çiçekler Besin Agarı (NA) ve Sabouraud Dekstroz Agarı (SDA) içeren petri kaplarına ayrı ayrı aktarılmıştır. İkinci kısımda ise, Bombus terrestris işçi arılarına laboratuvar koşullarında uygulama yapıldıktan sonra işçi arılar arazi koşullarında kovan içerisine aktarılmıştır. Her bir bakteri izolatının arazi dozu 50 adet B. terrestris işçi bireyine 4 atm basınçta püskürtme yapabilen sistem yardımıyla 20 sn süreyle (0,5 ml) uygulanmıştır. Bu işçi arılar her bir bakteri izolatı için ayrı birer kovan içerisine aktarılmıştır. Bu kovanlardan 24., 48. ve 72. saatlerde alınan onar adet işçi arı mekanik yöntemle öldürülerek re-izolasyon gerçekleştirilmiştir. Re-izolasyon aşamasında, her bir gözlem zamanı için işçi arılar ayrı ayrı yüzey sterilizasyonu yapıldıktan sonra steril damıtılmış suda disekte edilmiştir. Disekte edilen böcekten elde edilen bir damla haemolymf 100 kez steril su ile seyreltildikten sonra Nutrient Agar'a yayılmıştır. Çalışmadan elde edilen sonuçlara göre; B. thuringiensis var. kurstaki ve B. amyloliquefaciens QST 713’in uygulandıktan 24, 48 ve 72 saat sonra hem domates çiçeklerinde hem de B. terrestris işçi arılarının hemolenfinde bulunduğu saptanmıştır.

Keywords

Entomopatojen , Spor canlılığı , Biyokontrol etmeni , Bombus arısı

Project Number

221O580

References

• Abdalla SA, Algam SAA, Ibrahim EA, Naim AME. In vitro screening of Bacillus isolates for biological control of early blight disease of tomato in shambat soil. World J. Agric. Res. 2014; 2 (2): 47-50. https://doi:10.12691/wjar-2-2-3
• Alkassab AT, Beims H, Janke M, Pistorius J. Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product. Environ. Sci. Pollut. Res. 2022; 29: 25995–26001, doi.org/10.1007/s11356-022-19414-5
• Alsaedi G, Ashouri A, Talaei-Hassanloui R. Evaluation of Bacillus thuringiensis to Control Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) under Laboratory Conditions. Agricultural Sciences. 2017; 8: 591-599, DOI: 10.4236/as.2017.87045
• Babendreier D, Reichhart B, Romeis J, Bigler F. Impact of insecticidal proteins expressed in transgenic plants on bumblebee microcolonies. Entomol. Exp. Appl. 2008; 126: 148–157. doi: 10.1111/j.1570-7458.2007.00652.x
• Basit A, Ahmad I, Rahim K, Shafiq M. Isolation and identification of phytopathogens from diseased vegetables at district Kohat, Khyber Pakhtunkhwa, Pakistan. J. Curr. Res. Sci., 2014; 2(4): 521-525.
• Benzina F, Oulebsir-Mohandkacı H, Belaid M, Irnatene H, Mammeri S. Isolation of entomopathogenic bacteria from larvae of a Lepidopteran species; Galleria mellonella and study of their insecticidal effect. J. Agric. For. Sci.. 2017; 63(4): 59-68, doi.org/10.17707/AgricultForest.63.4.06
• Bizzarri M, Bishop A. The ecology of Bacillus thuringiensis on the phylloplane: colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae. Microb. Ecol. 2008; 56: 133–139, doi: 10.1007/s00248-007-9331-1
• Brar S, Verma M, Tyagi RD, Valéro JR. Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides. Process. Biochem. 2006; 41 (2): 323–342, doi.org/10.1016/j.procbio. 2005.07.015
• Bravo A, Likitvivatanavong S, Gill SS, Soberón M. Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem. Mol. Biol. 2011; 41(7): 423–431, doi: 10.1016/j.ibmb.2011.02.006
• Brittain CA, Vighi M, Bommarco R, Settele J, Potts S. Impacts of a pesticide on pollinator species richness at different spatial scales. Basic Appl. Ecol. 2010; 11(2): 106–115, DOI: 10.1016/j.baae.2009.11.007
• Cappa F, Baracchi D, Cervo R. Biopesticides and insect pollinators: Detrimental effects, outdated guidelines, and future directions. Sci. Total Environ. 2022; 837 155714 1-14, doi: 10.1016/j.scitotenv.2022.155714
• Dafni A, Kevan P, Gross CL, Goka K. Bombus terrestris, pollinator, invasive and pest: An assessment of problems associated with its widespread introductions for commercial purposes. Appl. Entomol. Zool. 2010; 45 (1): 101–113, DOI: 10.1303/aez.2010.101
• Demirözer O, Uzun YA, Yanık G, Buluş İY, Gösterit A. Investigation of the efficacy of some biopesticides by food exposure on Bombus terrestris L. (Hymenoptera: Apidae). J. Apic. Res. 2023; 62: 1153-1157, doi.org/10.1080/00218839.2022.2054538
• Dunkle RL, Shasha BS. Response of starch-encapsulated Bacillus thuringiensis containing ultraviolet screens to sunlight. Envir. Entomol. 1989; 18(6): 1035–41.
• D’urso V, Mazzeo G, Vaccalluzzo V, Sabella G, Bucchier F, Viscuso R, Vitale DGM. Observations on midgut of Apis mellifera workers (Hymenoptera: Apoidea) under controlled acute exposures to a Bacillus thuringiensis-based biopesticide. Apidologie. 2017; 48: 51–62.
• EFSA BIOHAZ Panel. (EFSA Panel on Biological Hazards). Scientific opinion on the risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs. EFSA Journal. 2016; 14(7): 4524, 93 pp.
• Egan PA, Dicks LV, Hokkanen HM, Stenberg JA. Delivering integrated pest and pollinator management (IPPM). Trends Plant Sci. 2020; 25: 577–589.
• Gonzalez-Cabrera J, Molla O, Monton H, Urbaneja A. Efficacy of Bacillus thuringiensis (Berliner) for Controlling the Tomato Borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). BioControl. 2011; 56: 71-80, DOI: 10.1007/s10526-010-9310-1
• Goulson D, Nicholls E, Botías C, Rotheray EL. Bee decline is driven by combined stress from parasites, pesticides, and lack of flowers. Science. 2015; 347: 1435–1444.
• Haddad MDL, Polanczyk RA, Alves SB, Garcia MDO. Field persistence of Bacillus thuringiensis on maize leaves (Zea mays L.). Braz. J. Microbiol. 2005; 36(4): 309–314, DOI: 10.1590/S1517-83822005000400001
• Ignoffo CM. Environmental factors affecting persistence of entomopathogens. The Fla. Entomol. 1992; 75(4): 516-525, doi.org/10.2307/3496133
• Ignoffo CM, Garcia C. UV-photoinactivation of cells and spores of Bacillus thuringiensis and effects of peroxidase on inactivation. Environ. Entomol. 1978; 7(2): 270–272, doi.org/10.1093/ee/7.2.270
• Ignoffo CM, Hostetter DL, Pinnell RE. Stability of Bacillus thuringiensis and Baculovirus heliothis on soybean foliage. Environ. Entomol. 1974; 3(1): 117–119, doi.org/10.1093 /ee/3.1.117
• Jangir M, Pathaka R, Sharma S, Sharma S. Biocontrol mechanisms of Bacillus sp., isolated from tomato rhizosphere, against Fusarium oxysporum f. sp. lycopersici. Biological Control. 2018; 123: 60–70, https://doi.org/10.1016/j.biocontrol.2018.04.018
• Jangir M, Sharma S, Sharma S. Target and non-target effects of dual inoculation of biocontrol agents against Fusarium wilt in Solanum lycopersicum. Biological Control. 2019; 138: 104069. https://doi.org/10.1016/j.biocontrol.2019.104069
• Khorramvatan S, Marzban R, Ardjmand M, Safekordi A, Askary H. The effect of polymers on the stability of microencapsulated formulations of Bacillus thuringiensis subsp. kurstaki (Bt-KD2) after exposure to ultra violet radiation. Biocontrol Sci Technol. 2014; 24(4): 462-472. doi: 10.1080/09583157.2013.871503
• Kraus FB, Szentgyörgyi H, Rozej E, Rhode M, Moron D, Woyciechowski M, Moritz RFA. Greenhouse bumblebees (Bombus terrestris) spread their genes into the wild. Conserv Genet. 2011; 12: 187–192, DOI 10.1007/s10592-010-0131-7
• Krebs B, Höding B, Kübart S, Workie MA, Junge H, Schmiedeknecht G, Grosch R, Bochow H, Hevesi M. Use of Bacillus subtilis as biocontrol agent. I. activities and characterization of Bacillus subtilis strains. J. Plant Dis. Prot. 1998; 105(2): 181-197.
• Kuzina LV, Peloquin JJ, Vacek DC, Miller TA. Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae). Curr. Microbiol. 2001; 42: 290-294, doi: 10.1007/s0 02840110219.
• Larpent JP. 1997. Microbiologie alimentaire, Technique de laboratoire. Ed. Lavoisier, Paris.
• Malone LA, Scott-Dupree CD, Todd JH, Ramankutty P. No sub-lethal toxicity to bumblebees, Bombus terrestris, exposed to Bt-corn pollen, captan, and novaluron. N. Z. J. Crop Hortic. Sci. 2007; 35: 435–439, doi.org/10. 1080 /01140670709510211.
• Mommaerts V, Jans K, Smagghe G. Impact of Bacillus thuringiensis strains on survival, reproduction and foraging behaviour in bumblebees (Bombus terrestris). Pest Manag. Sci. 2010; 66: 520–525, doi: 10.1002/ps.1778
• Mommaerts V, Sterk G, Smagghe G. Side effects of commercial Bacillus thuringiensis insecticides on micro-colonies of Bombus terrestris. Hazards of pesticides to bees. 10th International Symposium of the ICP-Bee Protection Group; Bucharest, Romania, January 2009a, p. 68.
• Mommaerts V, Sterk G, Hoffmann L, Smagghe GA. Laboratory evaluation to determine the compatibility of microbiological control agents with the pollinator Bombus terrestris. Pest Manag. Sci. 2009b; 65(9): 949-955, doi: 10.1002/ps.1778.
• Morandin LA, Winston ML. Effects of novel pesticides on bumblebee (Hymenoptera: Apidae) colony and foraging ability. Environ. Entomol. 2003; 32(3): 555-563. doi: 10.1603/0046-225X-32.3.555.
• Morsy EM, Abdel-Kawi KA, Khalil MNA. Efficiency of Trichoderma viride and Bacillus subtilis as biocontrol agents against Fusarium solani on tomato plants. Egypt. J. Phytopathol. 2009; 37 (1): 47-57, DOI: 10.21608/ejar.2019.111021.
• Ndakidemi B, Mtei K, Ndakidemi PA. Impacts of synthetic and botanical pesticides on beneficial insects. Agric. Sci. 2016; 7(6): 364-372, doi: 10.4236/as.2016.76038.
• Pedersen JC, Hansen BM, Damgaard PH, Eilenberg J. Dispersal of Bacillus thuringiensis var. kurstaki in an experimental cabbage field. Can. J. Microbiol. 1995; 41(2): 118–125, DOI: 10.1139/m95-016.
• Pinnock DE, Brand RJ, Milstead JE. The field persistence of Bacillus thuringiensis spores. J. Invertebr. Pathol. 1971; 18(3): 405–411, doi: 10.1016/0022-2011(74)90099-8.
• Ramanaidu K, Cutler GC. Different toxic and hormetic responses of Bombus impatiens to Beauveria bassiana, Bacillus subtilis and spirotetramat. Pest Manag. Sci. 2013; 69: 949–954, https://doi:10.1002/ps.3456.
• Ramyabharathi SA, Meena B. Raguchander T. Induction of chitinase and ß-1,3-glucanase PR proteins in tomato through liquid formulated Bacillus subtilis EPCO 16 against Fusarium wilt. J. Today's Biol. Sci. Res. Rev. 2012; 1: 50–60.
• Raymond B, Wyres KL, Sheppard SK, Ellis RJ, Bonsall MB. Environmental factors determining the epidemiology and population genetic structure of the Bacillus cereus group in the field. PLoS. 2010; 6(5): 1-13, doi: 10.1371/journal.ppat.1000905.
• Sabo R, Kopčáková A, Hamarová Ľ, Cingeľová Maruščáková I, Mudroňová D, Sabová L, Javorský P, Legáth J. Sublethal effects of commercial plant protection product containing spores Bacillus amyloliquefaciens QST 713 (formerly subtilis) on winter adult honeybees. Apidologie, 2020; 51 (2): 226-239, DOI: 10.1007/s13592-019-00705-9.
• Sansinenea E, Salazar F, Ramirez M, Ortiz A. An ultraviolet-tolerant wild-type strain of melanin-producing Bacillus thuringiensis. Jundishapur J. Microbiol. 2015; 8(7): e20910, doi: 10.5812/jjm.20910v2.
• Shanmugam V, Kanoujia N. Biological management of vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycospersici by plant growth-promoting rhizobacterial mixture. Biological Control. 2011; 57, 85–93, https://doi.org/10.1016/j.biocontrol.2011.02.001.
• Singleton P. Bactériologie pour la médecine, biologie et biotechnologie. Dunod, 2005, p. 541.
• Soumia PS, Krishna R, Jaiswal DK, Verma JP, Yadav J, Singh M. Entomopathogenic Microbes for Sustainable Crop Protection: Future Perspectives. Eds. Yadav AN, Singh J, Singh C, Yadav N. "Current Trends in Microbial Biotechnology for Sustainable Agriculture", Springer Nature Singapore Pte Ltd., 2021, p. 469-497.
• Sponsler DB, Grozinger CM, Hitaj C, Rundlöf M, Botías C, Code A, Douglas MR. Pesticides and pollinators: a socioecological synthesis. Sci. Total Environ. 2019; 662: 1012–1027, DOI: 10.1016/j.scitotenv.2019.01.016.
• Steinigeweg C, Alkassab AT, Beims H, Eckert JH, Richter D, Pistorius J. Assessment of the impacts of microbial plant protection products containing Bacillus thuringiensis on the survival of adults and larvae of the honeybee (Apis mellifera). Environ. Sci. Pollut. Res., 2021; 28 (11):1-8, DOI: 10.1007/s11356-021-12446-3.
• Surgeoner GA, Farkas MJ. Review of Bacillus thuringiensis var. kurstaki (Btk) for use in forest pest management programs in Ontario, with special emphasis on the aquatic environment. Ontario Ministry of the Environment, 1990, p. 1-79.
• Thiery I, Frachon E. Identification, isolation, culture and preservation of entomopathogenic bacteria. Ed.LA Lacey, "Manual of Techniques in Insect Pathology", Academic Press, London 1997, p. 55-77.
• Topakcı N, Keçeci M. Türkiye’de örtüaltında zararlılara karşı biyolojik mücadele uygulamalarının gelişimi: araştırmadan pratiğe Antalya örneği. Türkiye Biyolojik Mücadele Dergisi. 2017; 8 (2): 161-174.
• Yaman M, Nalçacıoğlu R, Demirbağ Z. Studies on bacterial flora in the population of fall webworm, Hyphantria cunea Drury. (Lepidoptera: Arctiidae). J. Appl. Entomol., 2002; 126: 470-474, DOI: 10.1046/j.1439-0418.2002.00681.x
• Young JO. Effects of Bacillus thuringiensis subsp. kurstaki application on non-target nocturnal macromoth biodiversity in the eastern boreal forest, Canada. 2023; Preprint, DOI: 10.1101/2024.07.19.604320.
• Zhou Z, Zhang H, Mashilingi SK, Jie C, Guo B, Guo Y, Hu X, Iqbal S, Wei B, Liu Y. Jiandong An. Bombus terrestris prefer mixed-pollen diets for a better colony performance: A laboratory study. Insects. 2024; 15 (4): 285-297, DOI: 10.3390/insects15040285.