Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, Vol. 53, ¹ 1, p. 201-208.
(how to cite this article)

doi: 10.15389/agrobiology.2018.1.201eng

UDC 579.64:632.937.15

Acknowledgements:
This work was supported by the project of applied research and experimental development (PNER) batch 2017-14-579-0030 on the topic: «Creation of microbiological preparations for expanding the adaptive capacity of agricultural crops for nutrition, resistance to stress and pathogens» (code of the application «2017-14-579-0030-013»), Agreement No. 14.607.21.0178, a unique identifier (project) RFMEFI60717X0178

 

ACTIVITY OF INSECTICIDAL Bacillus thuringiensis var. israelensis
STRAINS STORED BY VARIOUS METHODS

V.P. Ermolova, S.D. Grishechkina, A.A. Nizhnikov

All-Russian Research Institute for Agricultural Microbiology, Federal Agency for Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail: Ermolovavalya1940@mail.ru (✉ corresponding author), svetagrishechkina@mail.ru, ant.nizhnicov@gmail.com

ORCID:
Ermolova V.P. orcid.org/0000-0002-9473-8334
Nizhnikov A.A. orcid.org/0000-0002-8338-3494
Grishechkina S.D. orcid.org/0000-0002-4877-705X

Received December 20, 2017

 

Microbiological method of insecticidal pests control is an alternative to chemical pesticides. Insect control agents are based on different microorganisms, which should be stably effective against target pest organism. There are different origins of industrial strains including isolation from natural objects, screening of collections, selection of existing strains, genetic engineering etc. but in all cases beneficial features of the strains should be preserved. In this article, the problems of preserving beneficial features of insecticidal bacteria Bacillus thuringiensis var. israelensis (BTH14) are discussed. This strain is effective as the pest control agent against larvae of mosquitoes, midges and rice and champignons mosquitoes. Different methods and time of storage of various BTH14 strains are shown: 266/2 on meat-peptone agar (MPA) and in corpse of mosquitoes for one year, in sodium chloride crystals for one and a half year; 71 on MPA without reseeding for one year and for two years with reseeding every six months; 87a by cryopreservation for ten years; 87, 404, 19/43 as lyophilized bacteria for 28 years; 7-1/23, 71/82, 19/1 in sodium chloride crystals for 27 years. Culture of BTH14 strain was grown on MPA slants at 28-30 °C for 5-7 days until reaching the complete formation of spores and endotoxin crystals. Microscopic analysis was carried out with aniline black dye. Morphological analysis of colonies was performed with colony-purified BTH14. When BtH14 was stored by the lyophilization method, the spore culture in a tube on a slant MPA was washed with 5 ml of a 20 % NaCl solution. Then 0.5 ml of the resulting suspension with a titer of 107-108 CFU/ml was added with a Pasteur pipette into glass ampoules, covered with a sterile swab, then sterile stopper and frozen in a cold bath at a temperature of -22 °C for 1 hour, dried at -45 °C for 23 hours, sealed under vacuum over a gas burner and stored in a refrigerator at 3-5 °C. When using the BtH14 storage method in NaCl crystals, 5 ml of 0.9 % saline was added to a tube with spore culture on slant MPA, resuspended, and 0.5 ml of suspension was added to sterile tubes, covered with cotton-gauze stoppers and stored at room temperature. When BtH14 was stored by cryopreservation, the spore culture of BtH14 was suspended in meat-peptone broth (MBP) with 10 % glycerol. The resulting suspensions (200 μl each) were poured into cryovials and stored at -80 °C. The BtH14 titer and larvicidal activity for Aedes aegypti mosquitoes were measured once or twice per year. The results showed that the culture of the 266/2 strain after a year of storage in the corpses of mosquitoes Culex pipiens molestus or on MPA dissociated with the formation of 0.8 and 1.6 % of the IV S form morphotype which lost activity against A. aegypti mosquitoes. The titer of the spores and the larvicidity of the 71 strain were at the initial level after one year of storage on MPA in tubes with paraffinized plugs when reseeded every 6 months. These indicators decreased, respectively, by 12 and 16 % in a year and by 25-27 % after 2 years of storage. Cryopreservation of the 87a strain provided stability of titer and larvicidal activity after 10 years. Thus, the initial titer and larvicidal activity expressed as LC50 for A. aegypti mosquitoes were 2.74×109 CFU/ml and 0.178×10-3 %, respectively. After 6 and 10 years, they corresponded to the following indicators: 2.82×109 CFU/ml and 0.19×10-3 %; 2.72×109 CFU/ml and 0.18×10-3 %. The BtH14 strains 7-1/23, 71/82, and 19/1 were stored in NaCl crystals. After 27 years of storage, their titers and LC50 for A. aegypti mosquitoes varied within the range of 3.12×109-3.52×109 CFU/ml and 0.135×10-3-0.150×10-3 % as compared to the initial values that were 3.98×109-4.29×109 CFU/ml and 0.10×10-3-0.11×10-3 %, respectively. The 87, 404, and 19/43 strains were stored by the method of freeze drying. After 28 years, their titers and larvicidal LC50 for A. aegypti mosquitoes remained within 3.32×109-3.68×109 CFU/ml and 0.11×10-3-0.14×10-3 % as compared to the initial values 3.86×109-4.45×109 CFU/ml and 0.087×10-3-0.103×10-3 %, respectively. Thus, the best indicators for preservation of valuable properties of BtH14 were obtained when stored in a lyophilized state, in NaCl crystals and using cryopreservation.

Keywords: Bacillus thuringiensis var. israelensis (BtH14), titer, storage, larvicidal activity.

 

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REFERENCES

  1. Kandybin N.V., Patyka T.I., Ermolova V.P., Patyka V.F. Mikrobiokontrol' chislennosti nasekomykh i ego dominanta Bacillus thuringiensis [Microbiocontrol of insects and Bacillus thuringiensis as the dominant].  St. Petersburg—Pushkin, 2009 (in Russ.).
  2. Tikhonovich I.A., Kozhemyakov A.P., Chebotar' V.K., Kruglov Yu.V., Kandybin N.V., Laptev G.Yu. Biopreparaty v sel'skom khozyaistve. Metodologiya i praktika primeneniya mikroorganizmov v rastenievodstve i kormoproizvodstve [Methodology and practice of using microorganisms in plant growing and fodder production]. Moscow, 2005 (in Russ.).
  3. Lednev G.R., Novikova I.I. V sbornike: Biologicheskie sredstva zashchity rastenii, tekhnologii ikh izgotovleniya i primeneniya [In: Biologicals for plant protection, technologies for their production and use]. St. Petersburg, 2005: 261-272 (in Russ.).
  4. Danilov L.G. V sbornike: Biologicheskie sredstva zashchity rastenii, tekhnologii ikh izgotovleniya i primeneniya [In: Biologicals for plant protection, technologies for their production and use]. St. Petersburg, 2005: 282-293 (in Russ.).
  5. Polanczyk R.A., Piresda Silva R.F., Fiuza L.M. Effectiveness of Bacillus thuringiensis against Spodoptera frugipera (Lepidoptera: Noctuidae). Brazil. J. Microbiol., 2000, 31: 165-167 CrossRef
  6. Zhong C.H., Ellar D.J., Bishop., Johnson., Lin S.S., Hart E.R. Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders. J. Invertebr. Pathol., 2000, 76: 131-134 CrossRef
  7. De Maagd R.A., Bravo A., Crickmore N. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet., 2001, 17: 193-199 CrossRef
  8. Siegel J.P. The mammalian safety Bacillus thuringiensis based insecticides. J. Invertebr. Pathol., 2001, 77: 13-21 CrossRef
  9. Haidar R., Deschamps A., Roudet J., Calvo-Garrido C., Bruez E., Rey P., Fermaud M. Multi-organ screening of efficient bacterial control agents against two major pathogens of grapevine. Biol. Control, 2016, 92: 55-65 CrossRef
  10. Ermolova V.P. Bacillus thuringiensis strains from natural sources in the Leningrad region: isolation and identification. Agricultural Biology, 2016, 51(1): 128-136 CrossRef
  11. Raimondo S., Pauley T.K., Butler L. Potential impacts of Bacillus thuringiensis var. kurstakion five salamander species in West Virginia. Northeast. Nat., 2003, 10(1): 25-38 CrossRef
  12. Choi Y.S., Cho E.S., Je Y.H., Roh J.Y., Chang J.H., Li M.S., Seo S.J., Sohn H.D., Jin B.R. Isolation and characterization of a strain of Bacillus thuringiensis subsp. morrisoni PG-14 encoding d-endotoxin Cry1Ac. Curr. Microbiol., 2004, 48: 47-50 CrossRef
  13. Armengol G., Hernandez J., Velez J.G., Orduz S. Long-lasting effects of a Bacillus thuringiensis serovar israelensis experimental tablet formulation for Aedes aegypti (Diptera: Culicidae) control. J. Econ. Entomol., 2006, 99(5): 1590-1595 CrossRef
  14. Al-Momani F., Obeidat M., Saasoun I., Mequam M. Serotyping of Bacillus thuringiensis isolates their distribution in different Jordanian habitats and pathogenecity in Drosophila melanogaster. World Journal of Microbiology and Biotechnology, 2004, 20: 749-753 CrossRef
  15. Shrestha A., Sultana R., Chae J.-C., Kim K., Lee K.-J. Bacillus thuringiensis C-25 which is rich in cell wall degrading enzymes efficiently control lettuce drop caused by Sclerotinia minor. Eur. J. Plant Pathol., 2015, 142(3): 577-589 CrossRef
  16. Lacey L.A., Grywaczet D., Shapiro-Ilan D., Frutos R., Brownbridge M., Goettel M.S. Insect pathogens as biological control agents: back to the future. J. Invertebr. Pathol., 2015, 132: 1-41 CrossRef
  17. Eswarapriya B., Gopalsamy B., Kameswari B., Meera R., Devi P. Insecticidal activity of Bacillus thuringiensis IBt-15 strain against Plutella xycostella. Int. J. PharmTech. Res., 2010, 2(3): 2048-2053.
  18. Patel K.D., Bhanshali F.C., Ingle S.S. Diversity and characterization of Bacillus thuringiensis isolates from alluvial soils of Mahi river basin, India. Journal of Advances in Developmental Research, 2011, 2: 14-20.
  19. Rakhmanin YU.A., Novikov S.M., Rumyantsev G.I., Ivanov S.I. Gigiena i sanitariya, 2006, 5: 10-13 (in Russ.).
  20. Raddadi N., Cherif A., Ouzari H., Marzorati M., Brusetti L., Boudabous A., Daffonchio D. Bacillus thuringiensis beyond insect biocontrol: plant growth promotion and biosafety of polyvalent strains. Ann. Microbiol., 2007, 57(4): 481-494 CrossRef
  21. Narayanasamy P. Mechanisms of action of bacterial biological control agents. In: Biological management of diseases of crops. Progress in Biological Control, V. 15. Springer, Dordrecht, 2013: 295-429 CrossRef
  22. Patel K.D., Bhanshali F.C., Chaudhary A.V., Ingle S.S. A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types. Appl. Biochem. Biotech., 2013, 170: 58-66 CrossRef
  23. Raddadi N., Cherif A., Ouzari H., Marzorarl M., Brusetti L., Boudabous A., Daffonchio D. Bacillus thuringiensis beyond insect biocontrol: plant growth promotion and biosafety of polyvalent strains. Ann. Microbiol., 2007, 57(4): 481-494 CrossRef
  24. Speck M. L., Cowman R.A. Preservation of lactic streptococci at low temperatures. In: Culture collections of microorganisms. Proceedings of the International Conference on Culture Collections, Tokyo. H. Iizuka, T. Hasegawa (eds.). University Park Press, Baltimore, 1970: 241-250.
  25. Smirnov O.V., Grishechkina S.D. Vestnik zashchity rastenii, 2009, 1: 26-34 (in Russ.).
  26. Smirnoff U.A. The formation of crystals in Bacillus thuringiensis var thuringiensis Berliner before sporulation of temperature inculcation. J. Insect Pathol., 1965, 2: 242-250.
  27. Labinskaya A.S. Mikrobiologiya s tekhnikoi mikrobiologicheskikh issledovanii [Microbiology with the technique of microbiological research]. Moscow, 1972: 139-142 (in Russ.).
  28. Safronova V., Tikonovich I. Automated cryobank of microorganisms: Unigue possibilities for long-term authorized depositing of commercial microbial strains. In: Microbes in applied research: Current advances and challenges. A. Mendez-Vilas (ed.). World Scientific Publishing Co., 2012: 331-334 CrossRef
  29. Dospekhov B.A. Metodika polevogo opyta [Methods of field trials]. Moscow, 1973 (in Russ.).
  30. Safronova V.I., Sazanova A.L., Kuznetsova I.G., Popova Zh.P., Grishechkina S.D., Ermolova V.P., Andronov E.E. Whole genome sequencing and the comparative analysis of housekeeping locuses and virulence genes from the commercial strains of Bacillus thuringiensis with insecticidal activity. Agricultural Biology, 2015, 50(3): 332-338 CrossRef

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