0–2.5). Photographs were taken after 6 days of growth at room temperature. Seeds were obtained from the suppliers listed by Pueppke & Broughton (1999). Seeds of Leucaena leucocephala and Vigna unguiculata were surface-sterilized, planted, and inoculated as described previously (Broughton & Dilworth, 1971; Lewin et al., 1990). Plants were harvested 6 weeks after inoculation. At harvest, the aerial portion of the plant was collected and weighted. The total number of active (pink) nodules and their fresh weight were determined. Stationary-phase bacterial cultures in TY were washed twice with 25 mM

phosphate buffer (pH 7.5) and equilibrated to an optical density of 0.7. Adhesion tests were performed on roots of 6-day-old L. leucocephala and V. unguiculata plants using an established procedure (Albareda et al., 2006). Results were expressed click here as colony-forming units (CFU)

per mg of root tissue. Bacterial strains carrying the promoter-pPROBE constructs were grown on TY agar plates supplemented with the appropriate antibiotics. Using sterile toothpicks, fresh colonies were transferred to sterile 8-tube strips containing 100 μL of GYM supplemented with 100 mM of NaCl. Cells were homogenized by repeatedly drawing through a fine pipette, and for each transcriptional assay, equal quantities of bacteria were used to inoculate 1 mL of GYM supplemented with 0, 25, or 100 mM NaCl in 96-deep well plates. The plates were incubated at 27 °C with shaking at 200 r.p.m. Optical density

(595 nm) and fluorescence (excitation filter at 485 nm and emission filter selleck at 535 nm) from 100 uL of cultures were recorded 48 h post-inoculation using a Plate CHAMELEON Multilabel Detection Platform (Hidex Oy, Turku, Finland). A minimum of three transcriptional assays were performed for each bacterial strain carrying the constructs. Optical density and fluorescence values were first corrected with the values obtained from the media alone. Corrected fluorescence values were then normalized to the average optical density. Leucaena GPX6 leucocephala and V. unguiculata seeds were surface-sterilized, germinated, and planted as described previously. Two-day-old seedlings were inoculated with NGR 234 derivatives containing pALQ27 or pHC60. Plants were harvested at different times post-inoculation and their roots screened with an epifluorescence microscope Leica DMIRE2 [Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland] using GFP filter cubes (excitation BP 470/40 nm; emission BP 525/50 nm). Images were recorded with a Leica DC300F digital camera. The nucleotide sequence from S. meliloti 1021 of the ndvB gene was used to search the genome of NGR234 (Schmeisser et al., 2009). A putative ndvB homolog was identified (NGR_c32910). The predicted cyclic glucan synthase protein of NGR234 shares 98% and 90% identity with NdvB proteins of S. fredii and S. meliloti 1021, respectively.