Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation

1 Agriculture and Agri-Food Canada, Ottawa, Ontario K1A OC6, Canada 2 Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Quebec G1V 2J3, Canada 3 INRA, USC 1242 Symbioses Tropicales et Méditerranéennes, F-34398 Montpellier, France 4 USDA ARS, Bldg 006, BARC-West, 10300 Baltimore Ave, Beltsville, MD 20705, USA Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer , Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding ( atpD and recA ) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency 68 %) but was recovered infrequently ( 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris , but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium . A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species ‘ Sinorhizobium morelense ’. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha . The ecological and practical implications of the findings are discussed. Correspondence E. S. P. Bromfield eden.bromfield{at}agr.gc.ca Abbreviations: PT, phage type; RE, relative effectiveness Supplementary material is available with the online version of this paper.