Semiselective medium for isolation of Rhizobium leguminosarum from soils

A medium which controlled the growth of soil bacteria without inhibiting Rhizobium leguminosarum strains and other fast- and slow- growing rhizobia was developed. This medium, MNBP, included antibiotics (penicillin G and bacitracin) which inhibit the growth of Gram-positive bacteria, fungicides (cycloheximide and benomyl) and also pentachloronitrobenzene in order to control actinomycetes. Colony counts of total bacteria from different soils were reduced by 88–95% on this medium, compared to those obtained on a nutrient-rich agar medium, MGYF, commonly used for culture of rhizobia. The numbers of both Gram-positive and Gram-negative bacteria were lowered on medium MNBP. When used in conjunction with a complementary selection step (inability to grow on Luria-Bertoni agar medium) and a specific identification method (colony blot hybridization with a biovar-specific DNA probe), this medium allowed the isolation of a population of 66 R. leguminosarum bv. viciae from a soil sample. The effectiveness of the isolation procedure was found to be satisfactory because the number of R. leguminosarum bv. viciae isolated agreed with that estimated by indirect plant infection counts.