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Title: 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

item BROMFIELD, E - Agriculture And Agri-Food Canada
item TAMBONG, J - Agriculture And Agri-Food Canada
item CLOUTIER, S - Agriculture And Agri-Food Canada
item PREVOST, D - Agriculture And Agri-Food Canada
item LAGURERRE, G - Institut National De La Recherche Agronomique (INRA)
item Van Berkum, Peter
item TRAN THI, T - Agriculture And Agri-Food Canada
item ASSABGUI, R - Agriculture And Agri-Food Canada
item BARRAN, L - Agriculture And Agri-Food Canada

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2009
Publication Date: 3/5/2010
Citation: Bromfield, E.S., Tambong, J.T., Cloutier, S., Prevost, D., Lagurerre, G., Van Berkum, P.B., Tran Thi, T.V., Assabgui, R., Barran, L.R. 2010. 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. Microbiology. 156:505-520.

Interpretive Summary: Certain crops, referred to a Legumes which include beans, peas and clovers, require bacteria to be associated with their roots in order to take nitrogen from the air for crop growth via a process called nitrogen fixation. Many of these crops originally came from other countries. So the problem is how to find the best bacteria to help these plants grow since the bacteria that evolved with the crops did not necessarily come to North America when the crops were introduced. In this work we wanted to find out if bacteria would associate with alfalfa and sweet clover if these plants were grown for two years in a Canadian field where these crops had not been cultivated before. In the first year very few bacteria were detected, indicating that for the most part the bacteria were not present. In the second year bacteria were detected indicating that they had been introduced with the seeds during the first year of the experiment. These bacteria were very different from each other, but the one common feature among all them was that they were very poor in producing nitrogen for the crop plants. Our result indicates the necessity to provide the correct bacteria at planting so that the crop is able to grow and produce. This information will be valuable to companies who sell these bacteria for crops. Also, ecologists will find this information useful in their studies to determine how parts of the soil environment can change when crop plants are grown.

Technical Abstract: 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 Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA)and protein encoding (atpD and recA) genes, Southern hybridization and phenotypic characteristics. Among phage resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency c. 68%) but was recovered infrequently (c.1%) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes nodulated and 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. Phyllobacterium sp. 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. To our knowledge, this is the first report 47 of the occurrence of E. medicae in North America.