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 VX-680 cost antigen is adsorbed, followed by the application of purified primary antibody and a single secondary antibody-conjugate. The antibody-conjugate (usually goat anti-TSA HDAC 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, https://www.selleckchem.com/products/nsc-23766.html 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 the 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.