Bacillus amyloliquefaciens UTB96, an effective biocontrol and aflatoxin- degrading bacterium

Document Type : Research Paper

Authors

1 Department of Plant Protection, University of Tehran, Karaj, Iran

2 Head of R&D Department, Nature Biotechnology Company (Biorun), Karaj, Iran

Abstract

Aspergillus flavus grows on a wide range of agricultural and food products and causes contamination with aflatoxin. One of the suitable methods for controlling aflatoxin is the use of biological agents, which is possible by the application of effective bacterial antagonists. The objectives of the present study were to isolate and characterize a superior biocontrol agent against the above-mentioned fungus on pistachio, to develop an industrial culture medium and to optimize its growth conditions for enhancing maximum biomass production for Bacillus amyloliquefaciens UTB96. According to the results, this strain was capable of significant growth inhibition of A. flavus and reducing aflatoxin concentration in the pistachio up to 98.38%. Moreover, its VOCs showed growth inhibition as well. The most effective environmental factors were screened using the Bremenplacket design, in terms of their effects on the biomass production of the antagonistic bacterium and optimizing the production of bacterial biomass in culture medium designed using the screened factors. The results showed that sugarcane molasses and corn steep could be used as the industrial sources of carbon (C) and nitrogen (N) in production at 10 and 2 g/l respectively. Optimum conditions for maximum production of biomass in this culture medium included pH=7, 30°C and C/N ratio 1:23. Using the optimized culture medium and conditions in a semi-industrial method, the amount of biomass was reached up to 0.17 g/l, and the antagonistic effects increased by 8%. Bacillus species used in this work shows some similarities with B. velezensis that needs to be validated by further molecular analysis.

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Abalos, A. 2002. Utilization of response surface methodology to optimize the culture media for the producion of rhamnolipids by Pseudomonas aeruginosa AT10. Journal of Chemistry and Technical Biotechnology, 77: 777-784.
Afsharmanesh, H. & Ahmadzadeh, M. 2016. The Iturin lipopeptides as key compounds in antagonism of Bacillus subtilis UTB96 toward Aspergillus flavus. Biological Control of Pest and Plant Diseases, 5 (1): 79-95.
Costa, E., Teixdo, N., Usall, J., Atares, E. & Vinas, I. 2001. Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products. Applied Microbiology and Biotechnology, 56: 367-371.
Cuero, R.G., Duffus, E., Osuji, G. & Pettit, R. 1991. Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents. Journal of Agriculture Science, 117: 165-169.
Chitarra, G.S., Breeuwer, P., Nout, M.J.R., van Aelst, A.C., Rombouts, F.M. & Abee, T. 2003. An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores. Applied Microbiology, 94: 159-166.
Eaton, D.L. & Groopman, J.D. 1994. The toxicology of aflatoxins. Academic Press, New York, 383-424.
Emmert, E.A.B. & Handelsman, J. 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbial Letter, 171 (1): 1-9.
Fiddaman, P.J. & Rossal, S. 1993. The production of antifungal volatiles by Bacillus subtilis. Journal Applied Bacteriology, 74: 119-126.
Fravel, D.R. 2005. Commercialization and implementation of biocontrol. Annual Review of Phytopathology, 43: 337-359.
Green, M.R. & Sambrook, J. 2012. Molecular cloning: A laboratory manual. Cold Springer Harbor Laboratory Press, New York, USA.
Guan, S., Ji, C., Zhou, T., Li, J., Ma, Q. & Niu, T. 2008. Aflatoxin B1 degradation by Stenotrophomonas maltophilia and other microbs selected using coumarin medium. International Journal of Molecular Science, 9: 1489-1503.
Hagedorn, C., Could, W.D. & Bradinelli, R.T. 1989. Rhizobacteria of cotton and their repression of seedling disease pathogens. Applied Environment Microbiology, 55: 2793-2797.
Khanafari, A., Soudi, H., Miraboulfathi, M. & Karamei Osboo, R. 2007. An In vitro Investigation of Aflatoxin B1 Biological Control by Lactobacillus plantarum, Plant Journal of Biological Science, 4 (3): 163-68.
Kimura, N. & Hirano, S. 1988. Inhibitory strains of Bacillus subtilis for growth and Aflatoxin- production of aflatoxigenic fungi. Agriculture Biology and Chemistry, 52 (5): 1173-1179.
Kumar, P., Mahato, D.K., Kamle, M., Mohanta, T.K. & Kang, S.G. 2016. Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management. Frontiers in Microbiology, 7: 2170.
Lewis, J.A. 1991. Formulation and delivery systems of biocontrol agents with emphasis on fungi. In: Keister, D.L., Cregan, P.B. (eds): The rhizosphere and plant growth. Kluwer, Rotterdam, 279-287.
Maurhofer, M., Keel, C., Haas, D. & Défago, G. 1995. Influence of plant species on disease suppression by Pseudomonas fluorescens strain CHAO with enhanced antibiotic production. Plant Pathology, 44: 40-50.
Ongena, M. & Jacques, P. 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 16 (3): 115-125.
Pestka, J.J. & Bondy, G.S. 1990. Alteration of immune function following dietary mycotoxin exposure. Canadian Journal of Physiology and Pharmacology, 68 (7): 1009-1016.
Praveen, V. 2008. Nutritional regulation of actinomycin-D production by a new isolate of Streptomyces sindenensis using statistical methods. Index Journal of Expression Biology, 46: 138-144.
Reddyl, K.R.N., Saritha, P., Reddy, C.S. & Muralidharan, K. 2009. Aflatoxin B1 producing potential of Aspergillus flavus strains isolated from stored rice grains. African Journal of Biotechnology, 8 (14): 3303-3308.
Ramnani, P. 2005. Concomitant production and downstream processing of alkaline protease and biosurfactant from Bacillus licheniformis RG1: bioformulation as detergent additive. Process Biochemistry, 40: 3352-3359.
Rodgers, P.B. 1989. Potential of bological control organisms as a source of antifungal compounds for agrochemical and pharmaceutical product development. Pesticide Science, 27: 155-164.
Rodrigues, L. 2006. Response surface optimization of the medium components for production of biosurfactants by probiotic bacteria. Process Biochemistry, 41: 1-10.
Schaad, N.W., Jonse, J.B., & Chun, W .2001. Laboratory guide for identification of plant pathogenic bacteria. 3rd edition, APS Press, St. Paul, Minn. Sen, R. & Swaminathan, T. 1997. Application of response-surface methodology to evaluate the optimum environmental conditions for the enhanced production of surfactin. Applied Microbiology and Biotechnology, 47: 358-363.
Sedaghat Telgerd, N. 2009. Biological control assay of Aspergillus flavus in pistachio by strains of A. flavus without toxin and Bacillus subtilis. Master thesis. The college of agriculture and natural resource of Tehran University, 76.
Shifa, H., Tasneem, Sh., Gopalakrishnan, C. & Velazhahan, R. 2016. Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1, Arch. of Phytopathology and Plant Protection, 49 (5-6): 137-148.
Sousa, C., Klainer, B., Lima, K. & Pinto, G. 2014. Biomass production from Bacillus sp. RAB9 using several carbon sources. BMC Proceedings, 8(4): 172.
Sreekumar, G. & Krishnan, S. 2010. Enhanced biomass production study on probiotic Bacillus subtilis SK09 by medium optimization using response surface methodology. African Journal of Biotechnology, 9 (45): 8078-8084.
Stein, T. 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56: 845-857.
Teniola, O.D., Addo, P.A., Brost, I.M., Farber, P., Jany, K.D., Alberts, J.F., Van Zyl, W.H., Steyn, P.S. & Holzapfel, W.H. 2005. Degradation of aflatoxin B1 by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp.nov. DSM 44556 (T). International Journal of Food Microbiology, 105: 111-117.
Tuan, N.A. & Houng, N.T. 2014. Optimization of the fermentation medium to receive the highest biomass yield by Bacillus subtilis Natto and the initial test of Nattokinase yield. IOSR Journal of Engineering invention, 4 (12): 35-40.
Wright, S.J. & Thompson, R.J. 1985. Bacillus volatiles antagonize cyanobacteria. FEMS Microbial Letter, 30: 263-267.
Zhang, J. & Greasham, R. 1999. Chemically defined media for commercial fermentations. Applied Microbiology and Biotechnology, 51: 407-421.
Zabriskie, D.W., Armiger, W.B., Phillips, D.H. & Albano, P.A. 1980. Traders’ guide to fermentation medium formulation. Traders Protein, Memphis.