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Pyrrolnitrin from Rhizospheric Serratia marcescens NCIM 5696: Optimization of Process Parameters Using Statistical Tools and Seed-Applied Bioprotectants for Vigna radiata (L.) Against Fusarium oxysporum MTCC 9913

  • Shraddha Pawar
  • Ambalal ChaudhariEmail author
Article
  • 19 Downloads

Abstract

The extensive use of chemical fungicide in the health and agriculture sectors has increased environmental concerns and promoted an extensive search for alternative bioactives from the microbial system. In the present study, two rhizospheric strains of Serratia spp. (TO-2 and TW-3) have been shown to secrete pyrrolnitrin (PRN) in the range of 11.35 to 35.97 μg ml−1 using MSG and MSD medium after 72 h under static and shake conditions, respectively, but thereafter marginally declined in 96 to 240 h. Alternative one variable assortment at a time (OVAT) for PRN secretion by TW-3 yielded 59.27 μg ml−1 using (gl−1) glycerol (20), monosodium glutamate (14), KH2PO4 (14), NH4Cl (3), Na2HPO4 (4), and MgSO4 (0.3) at pH 7, 120 rpm within 72 h. Further, the Placket–Burman Design (PBD) identified KH2PO4, glycerol, pH, and monosodium glutamate as significant variables and optimized by centered composite design. Accordingly, 3% glycerol, 1.72% KH2PO4, 1.1% monosodium glutamate, 0.4% Na2HPO4, 0.03% MgSO4, 0.05% FeSO4, and 0.01% ZnSO4 were found to enhance the yield of PRN to 96.54 μg ml−1 by TW-3 in 72 h, 120 rpm. Thus, the statistical tool employed in the present study showed a threefold hike in PRN secretion over the OVAT approach, thereby indicating the scope for more PRN production from rhizobacteria. Further, seed application of low PRN (30 μg ml−1) concentration in treatments I and II showed > 90% germination in the initial seed germination and pot assay with the Fusarium oxysporum challenge compared to the control. Also, various growth parameters calculated during 11 days of experiment were significantly increased compared to the negative control (seed + fungus) in both treatments. Thus, the application of PRN at a low concentration to seeds of Vigna radiata (L.) offered protection against the phytopathogenic F. oxysporum MTCC 9913 challenge, suggesting biocontrol activity potential for use in agriculture soils particularly salt-affected soil.

Keywords

Pyrrolnitrin (PRN) OVAT Placket Burman Design (PBD) Centered composite design (CCD) Vigna radiata (L.) 

Notes

Acknowledgments

The authors acknowledge the infrastructural grant through UGC-SAP-DRS (III) (University Grants Commission, New Delhi) and DST-FIST (Department of Science and Technology, New Delhi), Govt. of India, to the School of Life Sciences of this Kavayitri Bahinabai Chaudhari North Maharashtra University. Ms. Shraddha Pawar acknowledges the financial support through UGC-BSR fellowship and from University Grant Commission (UGC), New Delhi.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

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References

  1. 1.
    Arima, K., Imanaka, H., Kousaka, M., Fukuta, A., & Tamura, G. (1964). Pyrrolnitrin, a new antibiotic substance, produced by Pseudomonas. Agricultural and Biological Chemistry, 28(8), 575–576.CrossRefGoogle Scholar
  2. 2.
    Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965–977.CrossRefGoogle Scholar
  3. 3.
    Burkhead, K. D., Schisler, D. A., & Slininger, P. J. (1994). Pyrrolnitrin production by biological control agent Pseudomonas cepacia B37w in culture and in colonized wounds of potatoes. Applied and Environmental Microbiology, 60(6), 2031–2039.Google Scholar
  4. 4.
    Chernin, L., Brandis, A., Ismailov, Z., & Chet, I. (1996). Pyrrolnitrin production by an Enterobacter agglomerans strain with a broad spectrum of antagonistic activity towards fungal and bacterial phytopathogens. Current Microbiology, 32(4), 208–212.CrossRefGoogle Scholar
  5. 5.
    Da Silva, G. P., Mack, M., & Contiero, J. (2009). Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnology Advances, 27(1), 30–39.CrossRefGoogle Scholar
  6. 6.
    deSouza, J. T., & Raaijmakers, J. M. (2003). Polymorphisms within the prnD and pltC genes from pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp. FEMS Microbiology Ecology, 43(1), 21–34.CrossRefGoogle Scholar
  7. 7.
    El-Banna, N., & Winkelmann, G. (1998). Pyrrolnitrin from Burkholderia cepacia: antibiotic activity against fungi and novel activities against Streptomycetes. Journal of Applied Microbiology, 85(1), 69–78.CrossRefGoogle Scholar
  8. 8.
    Fernando, W. D., Nakkeeran, S., de Kievit, T., Poritsanos, N., Zhang, Y., Paulit, T. C., Li, Z., & Ramarathnam, R. (2007). March. Multiple mechanisms of biocontrol by Pseudomonas chlororaphis PA23 affect stem rot of canola caused by Sclerotinia sclerotiorum. In Proceedings of the 12 th International Rapeseed Congress p (pp. 26–30).Google Scholar
  9. 9.
    Gerth, K., Trowitzsch, W., Wray, V., Höfle, G., Irschik, H., & Reichenbach, H. (1982). Pyrrolnitrin from Myxococcus fulvus (myxobacterales). The Journal of Antibiotics, 35(8), 1101–1103.CrossRefGoogle Scholar
  10. 10.
    Gorman, M., & Lively, D. H. (1967). Pyrrolnitrin: a new mode of tryptophan metabolism. In Biosynthesis (pp. 433–438). Berlin: Springer.Google Scholar
  11. 11.
    Gunst, R. F. (1996). Response surface methodology: process and product optimization using designed experiments. Technometrics, 38(3), 284–286.CrossRefGoogle Scholar
  12. 12.
    Hamill, R., Elander, R., Mabe, J., & Gorman, M. (1967). Metabolism of tryptophan by Pseudomonas aureofaciens V: conversion of tryptophan to pyrrolnitrin. Antimicrobial Agents and Chemotherapy, 1967, 388–396.Google Scholar
  13. 13.
    Kader, M. A. (2005). A comparison of seed germination calculation formulae and the associated interpretation of resulting data. Journal and Proceedings. Royal Society, 138, 65–75.Google Scholar
  14. 14.
    Kang, B. R., Han, S. H., Zdor, R. E., Anderson, A. J., Spencer, M., Yang, K. Y., Kim, Y. H., Lee, M. C., Cho, B. H., & Kim, Y. C. (2007). Inhibition of seed germination and induction of systemic disease resistance by Pseudomonas chlororaphis O6 requires phenazine production regulated by the global regulator, gacS. Journal of Microbiology and Biotechnology, 17(4), 586–593.Google Scholar
  15. 15.
    Keum, Y. S., Lee, Y. J., Lee, Y. H., & Kim, J. H. (2009). Effects of nutrients on quorum signals and secondary metabolite productions of Burkholderia sp. O33. Journal of Microbiology and Biotechnology, 19(10), 1142–1149.Google Scholar
  16. 16.
    Keum, Y. S., Zhu, Y. Z., & Kim, J. H. (2011). Structure-inhibitory activity relationships of pyrrolnitrin analogues on its biosynthesis. Applied Microbiology and Biotechnology, 89(3), 781–789.CrossRefGoogle Scholar
  17. 17.
    Kopp, J., Slouka, C., Ulonska, S., Kager, J., Fricke, J., Spadiut, O., & Herwig, C. (2018). Impact of glycerol as carbon source onto specific sugar and inducer uptake rates and inclusion body productivity in E. coli BL21 (DE3). Bioengineering, 5(1), 1–15.CrossRefGoogle Scholar
  18. 18.
    Lee, J., Simurdiak, M., & Zhao, H. (2005). Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation. The Journal of Biological Chemistry, 280(44), 36719–36727.CrossRefGoogle Scholar
  19. 19.
    Liu, X., Bimerew, M., Ma, Y., Müller, H., Ovadis, M., Eberl, L., Berg, G., & Chernin, L. (2007). Quorum-sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica. FEMS Microbiology Letters, 270(2), 299–305.CrossRefGoogle Scholar
  20. 20.
    Nisr, R. B., Russell, M. A., Chrachri, A., Moody, A. J., & Gilpin, M. L. (2011). Effects of the microbial secondary metabolites pyrrolnitrin, phenazine and patulin on INS-1 rat pancreatic β-cells. FEMS Immunology and Medical Microbiology, 63(2), 217–227.CrossRefGoogle Scholar
  21. 21.
    Pawar, S., Chaudhari, A., Prabha, R., Shukla, R., & Singh, D. P. (2019). Microbial pyrrolnitrin: natural metabolite with immense practical utility. Biomolecules, 9 (manuscript accepted).Google Scholar
  22. 22.
    Roitman, J. N., Mahoney, N. E., Janisiewicz, W. J., & Benson, M. (1990). A new chlorinated phenylpyrrole antibiotic produced by the antifungal bacterium Pseudomonas cepacia. Journal of Agricultural and Food Chemistry, 38(2), 538–541.CrossRefGoogle Scholar
  23. 23.
    Ruzzini, A. C., & Clardy, J. (2016). Gene flow and molecular innovation in bacteria. Current Biology, 26(18), R859–R864.CrossRefGoogle Scholar
  24. 24.
    Sanchez, S., Chavez, A., Forero, A., Garcia-Huante, Y., Romero, A., Sanchez, M., Rocha, D., Sanchez, B., Avalos, M., Guzman-Trampe, S., & Rodriguez-Sanoja, R. (2010). Carbon source regulation of antibiotic production. The Journal of Antibiotics, 63(8), 442–459.CrossRefGoogle Scholar
  25. 25.
    Synek, V. (2008). Evaluation of the standard deviation from duplicate results. Accreditation and Quality Assurance, 13(6), 335–337.CrossRefGoogle Scholar
  26. 26.
    Thomson, A. J., & El-Kassaby, Y. A. (1993). Interpretation of seed-germination parameters. New Forests, 7(2), 123–132.CrossRefGoogle Scholar
  27. 27.
    van Pée, K. H., & James, M. L. (2000). Biosynthesis of pyrrolnitrin and other phenylpyrrole derivatives by bacteria. Natural Product Reports, 17(2), 157–164.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Life SciencesKavayitri Bahinabai Chaudhari North Maharashtra UniversityJalgaonIndia

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