Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2016, Vol. 51, № 1, p. 128-136.

UDC 631.46:579.64:632.937.12(470.23)

doi: 10.15389/agrobiology.2016.1.128eng

Acknowledgements:
Supported by the Ministry of Education and Sciences of the Russian Federation (Agreement number 14.604.21.0024, RFMEFI60414Х0024)

Bacillus thuringiensis STRAINS FROM NATURAL SOURCES
IN THE LENINGRAD REGION: ISOLATION AND IDENTIFICATION

V.P. Ermolova

All-Russian Research Institute for Agricultural Microbiology, Federal Agency of Scientific Organizations,
3, sh. Podbel’skogo, St. Petersburg, 196608 Russia,
e-mail Ermolovavalya1940@mail.ru

Received August 19, 2014

Recently crystal-forming bacilli of thuringiensis group are considered the main microbial producers of insecticides. For these bacilli the high adaptability is characteristic leading to wide distribution of these anaerobic spore-forming bacteria in nature. The same Bacillus thuringiensis  subspecies and variants were isolated on different continents regardless the presence and prevalence or absence of the host insects of this entomopathogen. In different countries and regions the researchers are searching for new B. thuringiensis isolates. In the paper the data are represented on B. thuringiensis isolation from natural substrates in the territory of Leningrad province. A total of 24 samples of soil, litter, water, silt, sick and died insects have been collected. The samples were cultivated on fish agar. Among more than 3,000 colonies, 62 ones with specific morphology were found. By microscopy with black aniline dye a total of 12 isolates of 62 isolates tested were found out to form both spores and differently shaped crystals of the endotoxin. The microorganisms were selected with regard to entomocidal and larvicidal activity and identified using H. De Barjac, A.A. Bonnefoi (1968) and O. Lysenko (1985) schemes. The investigation made it possible to classify isolates as B. thuringiensis of Н1 (var. thuringiensis, isolates №№ 12, 20, 40, 41), Н3a3b (var. kurstaki, isolates №№ 15, 29, 49) and Н14 (var. israelensis, isolates №№ 14, 25, 33, 38, 44) serovars. With regard to biological properties (production of acetyl methyl carbonate, lecithinase, pigment, b-exotoxin; pellicle in broth culture; sucrose, mannose, cellobiose, salicin fermentation; starch degradation; proteolytic activity) these isolates are close to standard strains. Isolates are characterized by high productivity, entomocidal and larvicidal activity and can be used as producers of biologicals against insects and larvae. In the isolates of BtH1, BtH3a3b and BtH14 serovars the titers varied as 2.429-2.78×109; 1.85×109-2.15×109 and 2.65×109-3.28×109 CFU/ml, respectively. The activity against Leptinotarsa decemlineata Say larvae in isolates №№ 12, 41 of the BtH1 serovar was the same as in standard strain BtH1 with LD50 at 0.19 %. Entomocidal activity of the isolates №№ 15, 29 and 49 of the BtH3a3b serovar expressed as LD50 for Ephestia kuehniella of the 2nd instar was 0.88; 0.82 and 0.92 %, respectively, while in the standard strain BtH3a3b the LD50 was 0.86 %. In the isolates №№ 33, 44 of the BtH14 serovar the titer was the same as in the standard strain, and the activity was even higher compared to the standard. In the isolates №№ 33, 44 the LD50 for the 4th instar Aedes aegypti larvae was 0.17×10-3 and 0.16×10-3 %, respectively, when in standard strain BtH14 it was 0.18×10-3 %. Thus, a total of 12 of the isolates which have been identified as B. thuringiensis are close to the type isolates on their biological characteristics and promising as producers of biologics with insecticidal action.

Keywords: Bacillus thuringiensis, isolation, identification.

 

Full article (Rus)

Full text (Eng)

 

REFERENCES

  1. Loginova L.G., Khraptsova G.I., Golovina M.G. Mikrobiologiya, 1976, 45(6): 1087-1091.
  2. Evdokimova G.A., Kislykh E.E., Mozgova N.P. Biologicheskaya aktivnost' pochv v usloviyakh agrotekhnogennogo zagryazneniya na Krainem Severe [Soil bioactivity at agrotechcnogenic pollution in the Far North]. Leningrad, 1984: 120.
  3. Quesada-Moraga E., Garcia-Tovar E., Valverde-Garcia P., Santiago-Alvarez C. Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain. Microbiol. Res., 2004, 159: 59-71 CrossRef
  4. Das J., Dangar T.K. Diversity of Bacillus thuringiensis in the rice field soils of different ecologies in India. Indian J. Microbiol., 2007, 47: 364-368 CrossRef
  5. Ramalakshmi A., Udayasuriyan V. Diversity of Bacillus thuringiensis isolated from Western Ghats of Tamil Nadu state, India. Curr. Microbiol., 2010, 61: 13-18 CrossRef
  6. Patel K.D., Bhanshali F., Ingle S.S. Diversity and characterization of Bacillus thuringiensis isolates from alluvial soils of Mahi River basin, India. J. Adv. Dev. Res., 2011, 2: 14-20.
  7. Chatterjee S.N., Bhattacharya T., Dangar T.K., Chandra G. Ecology and diversity of Bacillus thuringiensis in soil environment. African Journal of Biotechnology, 2007,  6(13): 1587-1591.
  8. Chen F.-C., Tsai M.-C., Peng C.-H., Chak K.-F. Dissection of cry gene profiles of Bacillus thuringiensis isolates in Taiwan. Curr. Microbiol., 2004, 48: 270-275 CrossRef
  9. Arrieta G., Espinoza A.M. Characterization of a Bacillus thuringiensis strain collection isolated from diverse Costa Rican Natural ecosystems. Rev. Biol. Trop., 2006, 54: 13-27 CrossRef
  10. Patyka V.F., Patyka T.I. Ekologiya Bacillus thuringiensis [Ecology of Bacillus thuringiensis]. Kiev, 2007.
  11. Al-Khamada A.D. Vestnik zashchity rastenii, 2009, 4: 54-62.
  12. Lee D.W., Je Y.H., Koh Y.H. Bacillus thuringiensis isolates from Korean forest environments. J. Asia Pac. Entomol., 2012, 15: 237-239 CrossRef
  13. 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. Biotechnol., 2013, 170: 58-66 CrossRef
  14. 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 a predominant agent]. St. Petersburg—Pushkin, 2009.
  15. Golovko A.E., Golyshin P.N., Ryabchenko N.F. Mikrobiologiya, 1993, 55(3): 104-110.
  16. Lee I.H., Je Y.H., Chang J.H., Roh J.Y., Oh H.W., Lee S.G., Shin S.C.,
    Boo K.S. Isolation and characterization of a Bacillus thuringiensis ssp. kurstaki strain toxic to Spodoptera exigua and Culex pipiens. Curr. Microbiol., 2001, 43: 284-287 CrossRef
  17. Li M.S., Je Y.H., Lee I.H., Chang J.H., Roh J.Y., Kim H.S., Oh H.W., Boo K.S. Isolation and characterization of a strain of Bacillus thuringiensis ssp. kurstaki containing a new δ-endotoxin gene. Curr. Microbiol., 2002, 45: 299-302 CrossRef
  18. Margalith Y., Ben-Dov E. Biological control by Bacillus thuringiensis subsp. israelensis. In: Insect pest management: techniques for environmental protection. J.E. Rechcigl, N.A. Rech-cigl (eds.). CRC Press, Boca Raton, FL, 2000: 243-301.
  19. 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: 1590-1595.
  20. Arango J.A., Romero M., Orduz S. Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera:Noctuidae). J. Appl. Microbiol., 2002, 92: 466-474 CrossRef
  21. Bai C., Vick A.B., Yi S-X. Characterization of a new Bacillus thuringiensis isolate highly active against Cochylis hospes. Curr. Microbiol., 2002, 44: 280-285 CrossRef
  22. 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 δ-endotoxin Cry1Ac. Curr. Microbiol., 2004, 48: 47-50 CrossRef
  23. 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. WorldJ. Microbiol. Biotechnol., 2004, 20: 749-753.
  24. Biopreparaty v sel'skom khozyaistve. Metodologiya i praktika primeneniya mikroorganizmov v rastenievodstve i kormoproizvodstve [Biologicals in agriculture. Approach and practice for usage of microorganisms in crop and silage production]. Moscow, 2005.
  25. Ermolova V.P., Kandybin N.V. Materialy Mezhdunarodnoi nauchno-prakticheskoi konferentsii «Biologicheskaya zashchita rastenii — osnova stabilizatsii agroekosistem» [Proc. Int. Conf. «Biocontrol in plant protection as a base for stable ecosystems». Issue 4]. Krasnodar, 2006, vypusk 4: 255-256.
  26. Kandybin N.V. Fundamental'nye i prikladnye issledovaniya mikrobiometoda zashchity rastenii ot vreditelei: sostoyanie i perspektivy. Materialy Mezhdunarodnoi nauchno-prakticheskoi konferentsii «Biologicheskaya zashchita rastenii — osnova stabilizatsii agroekosistem» [Proc. Int. Conf. «Biocontrol in plant protection as a base for stable ecosystems». Issue 4]. Krasnodar, 2006, vypusk 4: 32-44.
  27. Dave S.R., Dave R.H. Isolation and characterization of Bacillus thuringiensis for Acid red 119 dye decolourisation. Biores. Technol., 2009, 100: 249-253 CrossRef
  28. Patel K.D., Ingle S.S. Molecular characterization of novel serovars of Bacillus thuringiensis isolates from India. Indian J. Microbiol., 2012, 52(3): 332-336 CrossRef
  29. Khoury M.E., Azzouz H., Chavanieu A., Abdelmalak N., Chopineau J., Awad M.K. Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera:Pyralidae) larvae. Arch. Microbiol., 2014, 196(6): 435-444 CrossRef
  30. Mel'nikova E.A. V sbornike: Entomopatogennye bakterii i ikh rol' v zashchite rastenii [In: Entomopathogenic bacteria and their role on plant protection]. Novosibirsk, 1987: 118-131.
  31. Raddadi N., Cherif A., Ouzari H., Marzorati M., Brusetti L., Boudab-ous A., Daffonchio D. Bacillus thuringiensis beyond insect biocontrol: plant growth promotion and biosafety of polyvalent strains. Ann. Microbiol., 2007, 57(4): 481-494 CrossRef
  32. 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.
  33. De Barjac H., Bonnefoi A.A. Classification of strains of Bacillus thuringiensis Berlinerwith a key of their differentiation. Invert. Pathol., 1968, 11: 335-337.
  34. Lysenko O. Nonsporeforming bacteria pathogenic to insect: incidence and mechanisms. Am. Rev. Microbiol., 1985, 39: 217-224.
  35. Abbot W.S. A method for computing the effectiveness of an insecticide. J. Econ. Entomol., 1925, 18: 265-267.
  36. Labinskaya A.S. Mikrobiologiya s tekhnikoi mikrobiologicheskikh issledovanii [Microbiology and microbiological techniques]. Moscow, 1972: 139-142.
  37. Metodicheskie ukazaniya po primeneniyu i metodam kontrolya kachestva insektitsidnogo mikrobiologicheskogo sredstva «Baktitsid» [Guidelines for application and control of microbial insecticide Bakticid]. Moscow, 2001.
  38. Dospekhov B.A. Metodika polevogo opyta [Methods of field trials]. Moscow, 1973.

back