7Dr. Gary Banowetz (USDA-ARS, Corvallis, OR, USA). Table 2 Bacteria that are sensitive to P. fluorescens SBW25 culture filtrate Test species Strain Zone size (cm2) Bacillus megaterium K2 15.3 ± 0.22 Dickeya dadantii X179 6.7 ± 0.29   1447 10.1 ± 0.57 Erwinia amylovora 153 13.5 ± 0.34 Pseudomonas syringae maculicola M4 12.2 ± 1.45   tomato DC3000 31.0 ± 0.97 The sizes of the zones of clearing produced in the lawns of bacteria

surrounding the central well containing the filtrate are indicated. Association of the antimicrobial activity of SBW25 culture H 89 in vitro filtrate with a ninhydrin-reactive BV-6 nmr compound The possibility that the antimicrobial activity of SBW25 culture filtrates was associated with the ninhydrin-reactive component of the filtrate was examined in additional fractionation studies. Preliminary experiments

determined that most of the ninhydrin-reactive compound from SBW25 culture filtrate was extracted from the dried culture filtrate solids by extraction with 85% ethanol. To determine if the antimicrobial activity of P. fluorescens SBW25 culture filtrate could be attributed to the ninhyrin-reactive component of the filtrate, aliquots of the 85% ethanol extract were fractionated on replicate cellulose TLC plates. One of the chromatograms was stained with ninhydrin, and the remaining cellulose plate was divided into twelve 1-cm zones that were then extracted with water. The resulting extracts were tested for antimicrobial activity in our standard assay. All of the antimicrobial activity towards BI 10773 cell line D. dadantii 1447 was coincident with the position of the ninhydrin-band on the replicate plate (Figure 2). Similar results were obtained with P. syringae pv. maculicola M4. Figure 2 The distribution of antimicrobial activity and ninhydrin-banding after TLC

fractionation of an 85% ethanol extract of dried culture filtrate from P. fluorescens SBW25. The 85% ethanol extract was prepared and applied to cellulose TLC plates as described in Methods. One of the developed plates was sprayed with ninhydrin (Figure 2A) and the replicate plate was divided into zones, as indicated, for removal and extraction of the cellulose. The aqueous extracts of the cellulose from each zone were assayed for antimicrobial activity Galactosylceramidase according to the standard assay described in the Methods section. The resulting antimicrobial activity against Dickeya dadantii (Figure 2B) was measured after 48 h. Zones without bars did not result in a cleared zone when assayed with either D. dadantii or P. syringae pv. maculicola M4. Purification of the ninhydrin-reactive component of SBW25 culture filtrate Purification of the ninhydrin-reactive compound from P. fluorescens SBW25 culture filtrate was undertaken by a modification of the strategy used by McPhail et al.[12] to purify FVG. SBW25 culture filtrate (840 mL) was taken to dryness in vacuo, and the dried solids were extracted with 85% ethanol.