Isolation and Characterization of Bacillus sp with In-vitro Antagonistic Activity against Fusarium oxysporum from Rhizosphere of Tomato

Authors
P. Prashar 1
N. Kapoor 2
S. Sachdeva 1
1 Department of Biotechnology, Faculty of Engineering and Technology, Mana Rachna International University, Faridabad, India.
2 School of Sciences, Indira Gandhi National Open University, New Delhi, India.
Abstract
The present investigation was carried out to isolate and characterize antagonistic bacteria against wilt causing fungal pathogen i.e. Fusarium oxysporum, from the rhizosphere of tomato. Fifty-six bacterial strains were isolated from the rhizosphere soil samples of healthy tomato fields, collected from different locations of Faridabad district, Haryana, India. Out of these, ten isolates were found to be antagonistic against the tested fungal pathogen i.e. Fusarium oxysporum, under in-vitro conditions. On the basis of percentage inhibition of radial growth of Fusarium oxysporum, isolate TNAM5 was found to be the most effective antagonistic rhizobacteria. Based on its morphological and biochemical properties along with 16s rRNA sequence analysis, it was identified as a Bacillus sp., with close nucleotide identity to Bacillus subtilis group. Average percentage inhibition given by this isolate was 47.77% and it was found to produce diffusible and volatile antifungal metabolites along with hydrogen cyanide and ammonia. Effect of physiological parameters on the growth and antagonistic behavior of the potential isolates was also examined. Optimal conditions for antagonistic activity were found to be 28ºC and pH 6.5, while the maximum growth was observed at 35ºC and pH 7.0. However, increase in salinity did not significantly affect (P> 0.05) the antagonistic behavior or growth of the isolates and they were found to withstand NaCl concentration up to 8.0% (w/v). The present study, hence, provides a potential biocontrol agent for Fusarium oxysporum, however, field studies of this isolate as soil inoculant in tomato are required in order to establish its actual performance.

Keywords

1. Adebayo, O. S. and Ekpo, E. J. A. 2004. Efficacy of Fungal and Bacterial Biocontrol Organisms for the Control of Fusarium Wilt of Tomato. Nig. J. Hortic. Sci., 9: 63-68.
2. Akhtar, M. S., Shakkel, U. and Siddqui, Z. A. 2010. Biocontrol of Fusarium Wilt by Bacillus pumilus, Pseudomonas, Alcaligenes, and Rhizobium sp. on Lentil. Turk. J. Biol., 34: 1-7.
3. Aneja, K. R. 2003. Experiments in Microbiology, Plant Pathology and Biotechnology. Fourth Edition, New Age International Publishers, New Delhi.
4. Anjaiah, V., Thakur, R. P. and Koedamn, N. 2006. Evaluation of Bacteria and Trichoderma for Biocontrol of Pre-harvest Seed Infection by Aspergillus flavus in Groundnut. Biocontrol. Sci. Tech., 16(4): 431- 436.
5. Asaka, O. and Shoda, M. 1996. Biocontrol of Rhizoctonia solani Damping-off of Tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol., 62: 4081-4085.
6. Bashan, Y., Holguin, G. and Lifshitz, R. 1993. Isolation and Characterization of Plant Growth-Promoting Rhizobacteria. In: "Methods in Plant Molecular Biology and Biotechnology", (Eds): Glick, B. R. and Thompson, J. E.. CRC Press, Boca Raton, Florida, PP. 331-345.
7. Cappuccino, J. and Sherman, N. 2010. Microbiology: A Laboratory Manual. Ninth Edition, Benjamin/Cummings Publishing, CA, PP???
8. Chung, W. C., Wu, R. S., Hsu, C. P., Huang, H. C. and Huang, J. W. 2011. Application of Antagonistic Rhizobacteria for Control of Fusarium Seedling Blight and Basal Rot of Lily. Australasian Plant Pathol., 40(3): 269-276.
9. Dunlap, C. A., Schisler, D.A., Price, N.P. and Vaughan, S.F. 2011. Cyclic Lipopeptide Profile of Three Bacillus Strains; Antagonist of Fusarium Headblight. J. Microbiol., 49:603-609.
10. Egamberdiyeva, D. and Khandakar, R. I. 2008. Salt-tolerant Rhizobacteria: Plant Growth Promoting Traits and Physiological Characterization within Ecologically Stressed Environments. In: "Plant-bacteria Interactions: Strategies and Techniques to Promote Plant Growth" (Eds.): Ahmad, I., Pichtel, J. and Hayat, S.. Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim, Germany, PP. 257-282.
11. Fiddaman, P. J. and Rossall, S. 1993. The Production of Antifungal Volatiles by Bacillus subtilis. J. Appl. Bacteriol., 74: 119-126.
12. Gupta, C. D., Dubey, R. C., Kang, S. C. and Maheshwari, D. K. 2001. Antibiotic Mediated Necrotrophic Effect of Pseudomonas GRC2 against Two Fungal Plant Pathogens. Curr. Sci., 81: 91-94.
13. Hanssen, I. M., Lapidot, M. and Thomma, B. P. H. J. 2010. Emerging Viral Diseases of Tomato Crops. Mol. Plant-Microbe Interact., 23(5): 539–548.
14. Holt, J.G., Kreig, W. R., Sneath, P. H. A., Staley, J. T. and William, S. T. 1994. Bergey’s Manual of Determinative Bacteriology. Ninth Edition, Williams and Wilkins, Baltimore, MD.
15. Karimi, K., Amini, J., Harighi, B. and Bahramnejad, B. 2012. Evaluation of Biocontrol Potential of Pseudomonas and Bacillus spp. against Fusarium Wilt of Chickpea. Aus. J. Crop Sci., 6: 695-703.
16. Kumar, N. J. 2007. Ground Water Information Booklet Faridabad District, Haryana. Central Ground Water Board, Ministry of Water Resources, Government of India.
17. Kumar, R. S., Ayyadurai, N., Pandiaraja, P., Reddy, A. V., Venkateswarlu, Y., Prakash, O. and Sakthivel, N. 2005. Characterization of Antifungal Metabolite Produced by a New Strain Pseudomonas aeruginosa pupa3 that Exhibits Broad Spectrum Antifungal Activity and Biofertilizing Traits. J. Appl. Microbiol., 98: 145-154.
18. Landa, B. B., Navas-Cortesb, J. A. and Jimenez-Diaz, R. M. 2004. Influence of Temperature on Plant–rhizobacteria Interactions Related to Biocontrol Potential for Suppression of Fusarium Wilt of Chickpea. Plant Pathol., 53:341-352.
19. Larkin, R. P. and Fravel, D. R. 1998. Efficacy of Various Fungal and Bacterial Biocontrol Organisms for Control of Fusarium Wilt of Tomato. Plant Dis., 82: 1022-1028.
20. Lee, C. H., Kempf, H. J., Lim. Y. and Cho, Y. H. 2000. Biocontrol Activity of Pseudomonas cepacia AF2001 and Anthelmintic Activity of Its Novel Metabolite, Cepacidine A. J. Microbiol. Biotechnol., 10: 568-571.
21. Lee, K. J., Kamala-Kannan, S., Sub, H. S., Seong, H. S. and Lee, G. W. 2008. Biological control of Phytophthora Blight in Red Pepper (Capsicum annuum L.) Using Bacillus subtilis. World J. Microbiol. Biotechnol., 24:1139-1145.
22. Matthews, G., Wiles, T. and Baleguel, P. 2003. A Survey of Pesticide Application in Cameroon. Crop Prot., 22: 707-714.
23. Montealegre, J. R., Reyes, R., Pérez, L. M., Herrera, R,, Silva, P. and Besoain, X. 2003. Selection of Bioantagonistic Bacteria to Be Used in Biological Control of Rhizoctonia solani in Tomato. Electron. J. Biotechn., 6(2): 115-127.
24. Ngowi, A. V. F., Mbise, T. J., Ijani, A. S. M., London, L. and Ajayi. O. C. 2007. Smallholder Vegetable Farmers in Northern Tanzania: Pesticides Use Practices, Perceptions, Cost and Health Effects. Crop Prot., 26: 1617-1624.
25. Ntow, W .J., Gijzen, H. J., Kelderman, P. and Drechsel, P. 2006. Farmer Perceptions and Pesticide Use Practices in Vegetable Production in Ghana. Pest Manag. Sci., 62: 356-365.
26. Ploetz, R. C. 2005. Panama Disease: An Old Nemesis Rears Its Ugly Head. Part 2. The Cavendish Era and Beyond. Apsnet Features 1005. (http://www.apsnet.org/online/feature/panama).
27. Sahu, G. K. and. Sindhu, S. S. 2011. Disease Control and Plant Growth Promotion of Green Gram by Siderophore Producing Pseudomonas sp. Res. J. Microbiol., 6: 735-749.
28. Sargaonkar, A. P., Gupta, A. and Devotta, S. 2008. Mutivariate Analysis of Groundwater Resources in Ganga-Yamuna Basin (India). J. Enviorn. Sci. Engg., 50(3): 215:222.
29. Siddiqui, Z. A. and Akhtar, M. S. 2007. Biocontrol of a Chickpea Root-rot Disease Complex with Phosphate-solubilizing Microorganisms. J. Plant Pathol., 89(1):67-77.
30. Smith, I. M., Dunez, J., Phillips, D. H., Lelliott, R. A. and Archer, S. A. 1988. European Handbook of Plant Diseases. Blackwell Scientific Publications, Oxford.
31. Vincent, J. M. 1947. Distortion of Fungal Hyphae in the Presence of Certain Inhibitors. Nature, 150: 850.
32. Wang, J., Liu, J., Chen, H. and Yao, J. 2007. Characterization of Fusarium gram- inearum Inhibitory Lipopeptide from Bacillus subtilis IB. Appl. Microbiol. Biotechnol., 76: 889-894.
33. Wei, G., Kloepper, J. W. and Sadik, T. 1991. Induction of Systemic Resistance of Cucumber to Colletotrichum orbiculare by Selected Strains of Plant Growth Promoting Rhizobacteria. Phytopathol., 81: 1508-1512.
34. Yildiz, H. N., Altinok, H. H. and Dikilitas, M. 2012. Screening of Rhizobacteria against Fusarium oxysporum f. sp. melongenae, the Causal Agent of Wilt Disease of Eggplant. Afr. J. Microbiol. Res., 6(15): 3700-3706.
Journal of Agricultural Science and Technology
Volume 15, Issue 7
November and December 2013
Pages 1501-1512