All strains were maintained at −80°C in Luria-Bertani liquid medium (LB medium) [42] containing a final concentration of 15% (v/v) glycerol. Annual bluegrass seeds (Poa annua L.) were obtained from 1996 mid-Willamette Valley grass seed screenings and were provided by International Seeds, Halsey, OR, and by C and R Farm, Tangent, OR. Prior to use, the seeds were cleaned to remove straw and seeds of other species. Culture filtrate production Pseudomonas fluorescens cells were inoculated into the modified Pseudomonas Minimal Salts Medium (PMS medium) described by Banowetz et al. [10], and cultured and harvested as described in the same reference. To prepare culture

filtrates, the 7-day P. fluorescens cultures were centrifuged (3000 × g, 15 min), and the supernatant was passed through a bacteriological filter (Millipore GP Express Steritop, SAHA datasheet 0.22 μM pore size, Millipore, Billerica, MA). The resulting sterile culture filtrate

was stored at 4°C prior to use. Agar diffusion assays for antimicrobial activity To test the antimicrobial activity of P. fluorescens SBW25 filtrate, bacterial strains were grown overnight in LB medium (6 mL) at 28°C (except for Escherichia coli, which was grown at 37°C) with shaking (225 rpm). The following morning, the stationary phase bacterial suspensions were adjusted with sterile water to an optical density of 0.2 at 600 nm (or 0.8 in the case of E. coli) as measured with a Superspec 3000 (Biorad Inc., Hercules, CA). A 300-μL

aliquot of MLN4924 the diluted culture was spread onto the surface GNA12 of a 925 Minimal Medium plate (100 × 15 mm, containing 25 mL of medium). The 925 Minimal Medium [43] was prepared with the modifications described by Halgren et al.[25]. After spreading the bacterial lawn, central wells were punched in the agar with a No. 9 cork borer, and a 300-μL aliquot of SBW25 culture filtrate was dispensed into the well. The plates were incubated for 48 h at 28°C, examined, and scored. Zones of inhibition in the area adjacent to the well were quantified with Able Image Analyzer® software (MU Labs, Ljublijana, Slovenia). Three AZD8931 price replicate plates were prepared for each bacterial strain tested, and the experiment was repeated for any strain that appeared sensitive to the SBW25 filtrate. Germination arrest assays The ability of SBW25 culture filtrate to inhibit the germination of Poa annua seeds was tested according to the protocol described by Banowetz et al.[10]. Ethanol extraction of culture filtrate Measured volumes of P. fluorescens culture filtrate were taken to dryness in vacuo at a temperature ≤ 45°C. After evaporation, the dry solids were extracted three times (5 min per extraction) with 90% or 85% (v/v) ethanol as indicated. Each of the three extractions was performed by swirling the solids with a volume of ethanol solution equal to one-third of the original volume of culture filtrate.