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doi: 10.15389/agrobiology.2019.6.1281eng

UDC: 579.64:632.937.15

Acknowledgements:
Supported financially 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

 

INSECTICIDAL PROPERTIES OF Bacillus thuringiensis var. israelensis. II. COMPARATIVE MORPHOLOGICAL AND MOLECULAR GENETIC ANALYSIS OF THE CRYSTALLOGENIC AND ACRYSTALLOGENIC STRAINS

V.P. Ermolova, S.D. Grishechkina, M.E. Belousova, K.S. Antonets, A.A. Nizhnikov

All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail ermolovavalya1940@mail.ru, svetagrishechkina@mail.ru, m.belousova@arriam.ru, k.antonets@arriam.ru, a.nizhnikov@arriam.ru (✉ corresponding author)

ORCID:
Ermolova V.P. orcid.org/0000-0002-9473-8334
Antonets K.S. orcid.org/0000-0002-8575-2601
Grishechkina S.D. orcid.org/0000-0002-4877-705X
Nizhnikov A.A. orcid.org/0000-0002-8338-3494
Belousova M.V. orcid.org/0000-0002-2886-026X

Received July 19, 2019

 

Currently, the bacterium Bacillus thuringiensis var. israelensis represents a key agent for biological protection against dipteran species, which are harmful to livestock and crop production and transmit infectious diseases of economically important animals. The production strains can be obtained by isolation from natural resources, selection of previously used isolates, screening of genetic collections, and genetic or genomic engineering. The issue of preservation and control of practically valuable properties of strains is of high importance. Biologicals are of significant interest due to their substantial advantages over chemical pesticides and are considered in modern agricultural systems as environmentally and socially priority alternatives to agrochemicals. In the present work, we performed the first comprehensive comparative analysis of crystallogenic and acrystallogenic variants of Bacillus thuringiensis var. israelensis (BtH14) isolated after storage of the strain in different modes. For crystallogenic variants, genes encoding the target insecticidal toxins, Cry4 and Cry11, were detected by the polymerase chain reaction (PCR), and it was shown that the acrystallogenic variants are devoid of these genes. It was found that the culture fluid of crystallogenic variants is approximately 7000 times more active against the Aedes aegypti larvae than the same of acrystallogenic. The aim of this work was to compare the morphological, biochemical, technological, larvicidal properties of the crystal-forming and acrystallogenic variants of the strains of Ваcillus thuringiensis var. israelensis (BtH14) and testing for the presence of genes encoding Cry insecticidal toxins, which are key determinants of virulence. We studied the strains 404 and 87 stored for 28 years by freeze-drying, then 2 years in test tubes on canted fish agar (FA) with replanting every 6 months; the 7-1/23 strain stored for 28 years in crystals of NaCl, then 2 years in culture liquid (CL) at 3 °С. Bacterial strains were inoculated on Petri dishes to obtain separate colonies. On day 7 of growth, the 404/14, 87/21, 7-1/23-4 (crystal-forming) and 404/19, 87/33, 7-1/23-8 (acrystallogenic) variants were selected by microscopic analysis using aniline black dye. The differences in the colony morphology were not revealed: the colonies were flat, opaque, grayish-white, rough, rounded, the structure was fine-grained, and the consistency was viscous. The differences either in the morphology of the vegetative cultures, or in the main biochemical properties (the formation of acetylmethyl carbinol, lecithinase, the use of carbohydrates, the splitting of starch, etc.), or in the titer on the yeast-polysaccharide medium were not shown as well. The productivity of the 404/14, 87/21, 7-1/23-4 and 404/19, 87/33, 7-1/23-8 strains varied from 3.36×109 CFU/ml to 4.02×109 CFU/ml and from 3.74×;109 CFU/ml to 4.13×109 CFU/ml, respectively. The larvicidal activity of the crystal-forming variants, expressed in LC50 for L4 Aedes aegypti, was (0.12-0.16)×;10-3 %, while acrystallogenic variants were inactive within the standard dilutions (×10-3 %) 1.0; 0.5; 0.25; 0.125; 0.06. Only their 1 % suspension (7000-fold higher concentration) caused 22-39 % death of the Aedes larvae after 24 hours; the same concentration of active variants resulted in 100 % death in 15 minutes. It was established that cultural liquid of the acrystallogenic variants formed a precipitate and a supernatant layer after 12 hours, while the crystal-forming variants remained suspended. The investigated variants of BtH14 were analyzed for the presence of genes encoding insecticidal toxins. The results of the PCR analysis with the Bti-specific primers confirmed the belonging of the both crystal-forming and acrystallogenic variants to BtH14. It has been found that the 404/14, 87/21, 7-1/23-4 strains carry genes encoding the Сry4 and Cry11 insecticidal toxins, while 404/19, 87/33, 7-1/23-8 acrystallogenic variants are devoid of these genes agreeing with the absence of larvicidal activity against A. aegypti.

Keywords: Bacillus thuringiensis, culture liquid, larvicidal activity, insecticidal toxins, Cry4, Cry11.

 

REFERENCES

  1. Ben-Dov E. Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins. Toxins, 2014, 6(4): 1222-1243 CrossRef
  2. Akbaev M.Sh., Vodyanov A.N., Kosminkov N.E., Yatusevich A.I., Pashkin P.I., Vasilevich F.I. Parazitologiya i invazionnye bolezni zhivotnykh [Parasitology and invasive animal diseases]. Moscow, 2000 (in Russ.).
  3. Polanczyk R.A., Pires da Silva R.F., Fiuza L.M. Effectiveness of Bacillus thuringiensis against Spodoptera frugipera (LepidopteraNoctuida). Brazilian Journal of Microbiology, 2000, 31(3) 165-167 CrossRef
  4. Sessitsch A., Reiter B., Berg G. Endophytic bacterial communities of field grown potato plants and their plant growth promoting abilities. Canadian Journal of Microbiology, 2004, 50: 239-249 CrossRef
  5. Kandybin N.V., Patyka T.I., Ermolova V.P., Patyka V.F. Mikrobiokontrol' chislennosti nasekomykh i ego dominanta Bacillus thuringiensis [Insect microbiocontrol and its dominant Bacillus thuringiensis]. St. Petersburg—Pushkin, 2009 (in Russ.).
  6. Scherwinski K., Wolf A., Berg G. Assessing the risk of biological control agents on the indigenous microbial communities: Serratia plyuthica HRO-C48 and Streptomyces sp. HRO-71 as model bacteria. Biocontrol, 2006, 52: 87-112 CrossRef
  7. Lednev G.R., Novikova I.I. V sbornike: Biologicheskie sredstva zashchity rastenii, tekhnologii ikh izgotovleniya i primeneniya [In: Biological plant protection products, technologies for their manufacture and use]. St. Petersburg, 2005: 261-272 (in Russ.).
  8. Danilov L.G. Vestnik zashchity rastenii, 2018, 3: 38-42 (in Russ.).
  9. Lacey L.A., Grzywacz D., Shapiro-Ilan D.I., Frutos R., Brownbridge M., Goettel M.S. Insect pathogens as biological control agents: back to the future. Journal of Invertebrate Pathology, 2015, 132: 1-41 CrossRef
  10. Bravo A., Gill S.S., Soberon M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 2007, 49: 423-435 CrossRef
  11. Arglo-Filho R.C., Loguercio L.L. Bacillus thuringiensis in an environmental pathogen and host-specificity has developted as an adaptation to human-generated ecological nishes. Insects, 2014, 5(1): 62-91 CrossRef
  12. Mnif I., Ghribi D. Potential of bacterial derived biopesticides in pest management. Crop Protection, 2015, 77: 52-64 CrossRef
  13. Naidar R., Deschamps A., Roudet J., Calvo-Garrido C., Bruez E., Rey P., Fermaud M. Multiorgan screening of efficient bacterial control agents against two major pathogens of grapevine. Biological Control, 2016, 92: 55-65 CrossRef
  14. Heydari A., Pessaraki M. A review on biological control of fungal plant pathogens using microbial antagonists. Journal of Biological Sciences, 2010, 1(4): 273-290 CrossRef
  15. Akram W., Mahboob A., Jave d A. Bacillus thuringiensis strain 199 can induce systemic resistance in tomato against Fusarium wilt. European Journal of Microbiology and Immunology, 2013, 3: 275-280 CrossRef
  16. Choudhary D.K., Johri B.N. Interactions of Bacillus spp. and plants — with special reference to induced systemic resistance. Microbiological Research, 2009, 164(5): 493-513 CrossRef
  17. Kumar P., Dubey R.C., Mahshwari D.K. Bacillus strain isolated from rhizosphere showed plant growth promoting and antagonistic activity against phythopathogens. Microbiological Research, 2012, 167(8): 493-499 CrossRef
  18. 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. Annals of Microbiology, 2007, 57(4): 481-494 CrossRef
  19. Raymond B., Federici B.A. In defense of Bacillus thuringiensis, the safest and most successful microbial insecticide available to humanity — a response to EFSA. FEMS Microbiology Ecology, 2017, 93(7): fix084 CrossRef
  20. Al-Khamada A.D. Vestnik zashchity rastenii, 2009, 4: 54-62(in Russ.).
  21. Pane C., Villecco D., Campanile F., Zaccardelli M. Novel strains of Bacillus isolated from compost and compost-amended soils as biological control agents against soil-borne phytopathogenic fungi. Biocontrol Science and Technology, 2012, 22(12): 1373-1388 CrossRef
  22. Tao A., Pang F., Huang S., Yu G., Li B., Wang T. Characterization of endophytic Bacillus thuringiensis strains isolated from wheat plants as biocontrol agents against wheat flagsmut. Biocontrol Science and Technology, 2014, 24(8): 901-924 CrossRef
  23. The manual of biocontrol agents. British Protection Council Publication, Alton, V.K., 2014.
  24. 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
  25. 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. Current Microbiology, 2004, 48: 47-50 CrossRef
  26. Grishechkina S.D., Ermolova V.P., Kovalenko T.K., Antonets K.S., Belousova M.E., Yakhno V.V., Nizhnikov A.A. Polyfunctional properties of the Bacillus thuringiensis var. thuringiensis industrial strain 800/15. Agricultural Biology [Sel’skokhozyaistvennaya Biologiya], 2019, 54(3): 494-504 CrossRef
  27. Grishechkina S.D., Ermolova V.P., Romanova T.A., Nizhnikov A.A. Search for natural isolates of Bacillus thuringiensis for development of ecologically friendly biologicals. Agricultural Biology [Sel’skokhozyaistvennaya Biologiya], 2018, 53(5): 1062-1069 CrossRef
  28. Shrestha A., Sultana R., Chae J.-C., Kim K., Lee K.-J. Bacillus thuringiensis C-25 which is rich in sell wall degrading enzymes efficiently control lettuce drop caused by Sclerotinia minor. Eur. J. Plant Pathol., 2015, 142(3): 577-589 CrossRef
  29. Saber W.I.A. Ghoneem K.V., Al-Askar A.A., Rashad Y.M., Ali A.A., Rashad E.M. Chitinase production by Vacillus subtilis ATCC 11774 and its effect on biocontrol of Rhizoctonia disease of potato. Acta Biologica Hungarica, 2015, 66(4): 436-448 CrossRef
  30. Malovichko Y.V., Nizhnikov A.A., Antonets K.S. Repertoire of the Bacillus thuringiensis virulence factors unrelated to major classes of protein toxins and its role in specificity of host-pathogen interactions. Toxins, 2019, 11: e11060347 CrossRef
  31. Zhang M.-Y., Lovgren A., Low M.G., Landen R. Characterization of an avirulent pleiotropic of the insect pathogen Bacillus thuringiensis: reduced expression of flagellin and phosphlipases. Infection and Immunity, 1993, 61(12): 4947-4954.
  32. Lebenko E.V., Sekerina O.A., Chemerilova V.I. Mikrobiologiya, 2005, 74(1): 87-91 (in Russ.).
  33. Ermolova V.P., Grishechkina S.D., Nizhnikov A.A. Activity of insecticidal Bacillus thuringiensis var. israelensis strains stored by various methods. Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, 53(1): 201-208 CrossRef
  34. 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.
  35. Guidi V., Patocchi N., Lüthy, P., Tonolla M. Distribution of Bacillus thuringiensis subsp. israelensis in soil of a Swiss Wetland reserve after 22 years of mosquito control. Applied and Environmental Microbiology, 2011, 77(11): 3663-3668 CrossRef
  36. Hansen B.M., Hendriksen N.B. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Applied and Environmental Microbiology, 2001, 67(1): 185-189 CrossRef
  37. Dospekhov B.A. Metodika polevogo opyta [Methods of field trials]. Moscow, 1973 (in Russ.).
  38. Ben-Dov E., Zaritsky A., Dahan E., Barak Z., Sinai R., Manasherob R., Margalith Y. Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis. Applied and Environmental Microbiology, 1997, 63(12): 4883-4890.
  39. Schneider S., Hendriksen N.B., Melin P., Lundström J.O., Sundh I. Chromosome-directed PCR-based detection and quantification of Bacillus cereus group members with focus on B. thuringiensis serovar israelensis active against nematoceran larvae. Applied and Environmental Microbiology, 2015, 81(15), 4894-4903 CrossRef
  40. Bravo A., Sarabia S., Lopez L., Ontiveros H., Abarca C., Ortiz A., Quintero R. Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Applied and Environmental Microbiology, 1998, 64(12): 4965-4972.

 

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