Title

Production of two lipopeptide antibiotics by Bacillus subtilis in the rhizosphere

Date of Completion

January 2009

Keywords

Agriculture, Soil Science|Chemistry, Agricultural

Degree

Ph.D.

Abstract

Biological control agents like Bacillus subtilis offer an alternative and supplement to synthetic pesticides. Antibiotic production by biocontrol strains of B. subtilis can play a major role in plant disease suppression. Our current understanding of B. subtilis antibiosis comes from culture media measurements of antibiotic production and in vitro suppression of pathogens. Quantifying the antibiotic metabolite chemistry of B. subtilis biofilms growing on root surfaces provides a more accurate understanding of in planta antibiotic production. An analytical method based on solid-phase extraction (SPE) with high-performance liquid chromatography (HPLC) and electrospray ionization mass spectroscopy (ESI-MS) has been developed to quantify antibiotics produced by B. subtilis growing on plant roots. Cucumber (Cucumis sativus ) was grown in composted soil and potting media inoculated with B. subtilis strain QST 713 (AgraQuest, USA). Pythium ultimum , which causes damping-off, crown and root rots in cucumber, was used as the test pathogen. Two important B. subtilis antibiotics, surfactin and iturin A, were extracted from root and rhizosphere soil using acidified organic solvents followed by cleaning and concentration using SPE. HPLC and ESI-MS were used to measure surfactin and iturin A. In healthy plants, rhizosphere concentrations of both antibiotics increased with plant age, with surfactin concentrations tripling between 15 and 43 days, and iturin concentrations increasing 20-fold over the same growth period. In diseased roots, surfactin concentrations were 4 to 10 times greater than controls, but iturin concentrations declined with the infection severity. ^