Indirect ELISA technique The indirect ELISA technique, modified from Kishinevsky and Maoz [55], was tested here for its ability to identify Cyclopia rhizobia under both glasshouse and field conditions. In the indirect ELISA method, the this website antigen is adsorbed, followed by the application of purified primary antibody and a single secondary antibody-conjugate. The antibody-conjugate (usually goat anti-rabbit conjugate) is commercially available and can be used in conjunction with a number of strain-specific antibody preparations. The

method is simpler, but has lower analytical sensitivity than the direct method [55, 56]. Production of strain-specific primary antibodies The four test strains used in this study were grown in a defined broth medium containing 0.5 g K2HPO4, 0.2 g MgSO4.7H20, Seliciclib nmr 0.1 g NaCl, 0.5 g KHPO4 and 10 g mannitol in 1 l distilled water53 and incubated at 20°C to obtain 0.4 OD600. To remove exopolysaccharides (produced in large quantities by strains UCT44b and UCT61a), the bacterial cells were washed three times by repeated centrifugation in phosphate-buffered saline (PBS) solution. The final sediment was suspended in 10 ml saline solution (150 mM NaCl) to a final concentration of > 109 CFU ml-1. Antibodies were prepared against each test strain using adult New Zealand White rabbits. The rabbits were bled prior to inoculation to assess their pre-inoculation antibody levels.

One rabbit was used for each test strain and was injected with the appropriate antigen according to the following protocol: Day 1: 0.5 ml intramuscular injections into each hind leg (with equal parts Freund’s complete adjuvant mixed prior to injection); Day 14: 1 ml intravenous injection; Day 21: 1 ml intravenous injection; Day 28: 1 ml intravenous injection; Day 35: trial bleed to check antiserum titre; Day 37: bleed by cardiac puncture after 0.15 ml intravenous acetylpromazine (sedative) injection. Intravenous

injections and trial bleeds were done via the marginal ear vein. Collected blood was incubated for 1 h at 37°C to facilitate clotting and then held at 4°C overnight to Cyclin-dependent kinase 3 extrude serum. The serum was removed, centrifuged to remove residual cells and stored at -20°C in 0.5 ml aliquots. Antiserum titres were tested using the long agglutination test of Vincent [52]. No precipitation reactions occurred with the pre-inoculation sera, but strong agglutinations occurred with the test antisera. Antisera agglutination titres were 1:600, 1:200, 1:400 and 1:500 for strains PPRICI3, UCT40a, UCT44b and UCT61a, respectively. Antigen preparation from roots nodules Cyclopia maculata seedlings were grown on nutrient-agar slants in individual sterile tubes. After three weeks of growth, the tubes were inoculated with test strains using three replicate tubes per strain and three uninoculated tubes as a negative control.