Investigation of Bacillus subtilis as a Biopesticide Against Botrytis cinerea

Author

Kenneth K. Ng, California Polytechnic State University, San Luis ObispoFollow

DOI: https://doi.org/10.15368/theses.2012.35
Available at: https://digitalcommons.calpoly.edu/theses/717

Date of Award

4-2012

Degree Name

MS in Engineering - Biochemical Engineering

Department/Program

Biomedical and General Engineering

Advisor

Nirupam Pal

Abstract

The objective of this thesis was to investigate BiOWiSH^TM-Aqua, a commercial dry solid formulation containing a consortium of bacteria and yeast, as a biopesticide for treatment of Botrytis cinerea, a gray mold that affects strawberries. BiOWiSH^TM-Aqua was compared with another commercial product specifically used as a fungicide and bacteriocide, Serenade® Garden Disease Control Spray (concentrated Bacillus subtilis strain QST 713). Both laboratory tests as well as in vivo lab tests were conducted. BiOWiSH^TM-Aqua results varied widely from plate to plate, regardless of experimental conditions. In some of these plates, inhibition zones were observed around colonies from BiOWiSH^TM-Aqua, indicating efficacy. The organism responsible for the inhibition zones of B. cinerea growth was isolated from BiOWiSH^TM-Aqua, and 16s rRNA analysis identified this culture as a strain of B. subtilis. This strain was designated as B. subtilis ssp. KLB. The B. subtilis KLB concentration required to completely inhibit B. cinerea was 9.1x10⁴ CFU/mL when B. subtilis KLB was inoculated 48 hours before B. cinerea, 1.3x10⁵ CFU/mL at 24 hours, and 3.2x10⁶ CFU/mL when both were inoculated at the same time. Various preliminary experiments using B. subtilis KLB were also conducted to investigate its economic feasibility, to characterize the organism, and to test its post-harvest in vivo viability. B. subtilis KLB cell concentration was 1.6x10⁹ CFU/mL in a bioreactor with LB at the end of the log growth phase. B. subtilis KLB achieved cell concentrations as high as 5x10⁹ CFU/mL in shake flasks with food-grade tapioca as a carbon source. Inoculation of B. subtilis KLB on post-harvest strawberries did not have an effect on Botrytis infection rates compared to the negative control. These various experiments were the first step in research to potentially produce B. subtilis KLB on a commercial scale.