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

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