Rapid quantification of Bacillus subtilis antibiotics in the rhizosphere

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 vivo antibiotic production. An analytical method based on solid-phase extraction (SPE) with high-performance liquid chromatography (HPLC) and mass spectroscopy (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). 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 HPLC-MS were used to measure surfactin and iturin A. Rhizosphere concentrations of both antibiotics increased with plant age. For plants grown in peat-based potting media, surfactin concentrations increased from 9 μg g−1 root fresh weight (RFW) at 15 d to 30 μg g−1 RFW at 43 d. Iturin concentrations were 7 μg g−1 RFW at 15 d and 180 μg g−1 RFW at 43 d. In an initial field trial in a composted fine sandy loam, we demonstrated rhizosphere production of surfactin and iturin under competition and predation by the myriad macro- and microfauna existing in a fertile high-organic soil, with mature B. subtilis-inoculated cucumber roots yielding 33 μg g−1 RFW surfactin and 630 μg g−1 RFW iturin at 78 d.