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.

The boundaries of a nif gene cluster in Rhizobium meliloti were determined by Tn5 mutagenesis. These genes are clustered within a 14-to 15-kilobase (kb) region that includes the nitrogenase structural genes. Sequences within 10 kb on either side of this region are devoid of symbiotically essential-gene function. RNA blot analysis identified a 5-to 6-kb transcript that corresponds to the nitrogenase structural gene operon. The 5' end of this transcript and its polarity were determined by nuclease Si mapping. The 5' end of another niftranscript was also identified by nuclease SI mapping. The promoter regions for these two nif transcripts control transcription in divergent directions and are separated by 1.9 kb of symbiotically unessential DNA. One Tn5 insertion within the nitrogenase operon did not create a polar mutation. The implications of this finding and the overall emerging picture of the genetic organization of this nifregion are discussed.Gram-negative soil bacteria of the genus Rhizobium have the unique ability to form nitrogen-fixing nodules on the roots of leguminous plants. This is a complex process involving recognition and invasion of the appropriate legume by the bacteria, stimulation of plant cell division, multiplication and differentiation of bacteria within root cortical cells into morphologically altered forms called bacteroids, and the reduction of atmospheric nitrogen to ammonia, which is then assimilated by the plant (1, 2).

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhirobium meliloti isolated directly from soil and from nodules of Medicago sativn (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti o'btained directly from soil proved to be symbiotic (i.e. noddated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct I S genotypes of which 9,9 and 15 were from soil, M. alba and M. satina, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. satiaa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodalation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil. Segovia L, Pincro D, Palaaos R, Marthez-Romem E (1991) Genetic structure of a soil population of nonsymbiotic Rhbbium legumintmrum. Applied and Environmental Microbiology, 57, 426-433. Smith GE, Summers MD (1980) The bidirectional transfer of DNA and RNA to nitroceIlulose or diazobenzyloxymethyl paper. Analyticnl Biochemistry, 109,123-129. Snedecor CW, Cochran WG (1980) Statistical Methods, Iowa State University Press. SoberonChdvez G, Ndkra R, Olivera H, Segovia L (1986) Genetic rearrangements of a Rhizobium phnsmli symbiotic plasmid. Journal of Bacteriology, 167,487-491. SoberonChdvez G, Ndjera R (1989) Isolation from Soil of Rhiwbium leguminosnrum lacking symbiotic information. Canadian Journal of Microbiology, 35,464-468. Thurman NP, Bromfield ESP (1988) Effect of variation within and between Medicago and Melilotus species on the composition and dynamics of indigenous populations of Rhizqbium meliloti.Soil Biology and Biochemistry, 20,31-38. Vincent J M (1970) A Manual for the Practical Study of Root-nodule Bacteria I.B.P. Blackwell Scientific Publications, Oxford. Wheatcroft R, Watson RJ (1988) Distribution of insertion sequence ISRml in Rhizobium meliloti and other Gram-negative bacteria. Journal of Gcneml Microbiology, 134,113-121. Wheatcroft R, Bromfield ESP, Laberge S, Barran LR (1993) Species specific DNA probe and colony hybridization of Rhizobium rneliloti. In: New Horizons in Nitrogen Fixation (eds Palacios R, Mora J, Newton WE) p. 661. Kluwer, Bosto...

Thirty-three isolates of indigenous Rhizobium meliloti, either possessing cryptic plasmids that hybridize to probes for symbiotic genes or lacking a 1500-kb megaplasmid band in Eckhardt gels, were tested for infectivity on 10 legume species grown under axenic conditions. A previous study had shown that all but two isolates were symbiotically effective with Medicago sativa. All indigenous isolates and two reference strains of R. meliloti induced nodules which were symbiotically ineffective on Trigonella foenum-graecum (100% plants nodulated) and Phaseolus vulgaris (40 to 100% plants nodulated). Eighteen indigenous isolates of R. meliloti elicited ineffective nodules on Macroptilium atropurpureum (2 to 25% plants nodulated) and Leucaena leucocephala (11 to 75% plants nodulated). The identity of single colony nodule isolates from each R. meliloti inoculant and host combination was verified by phage typing and analysis of plasmid profiles; tests with subsamples of these isolates showed that all were capable of nodulating M. sativa. There was no apparent relationship between the host range of indigenous R. meliloti and either the presence of cryptic plasmids that hybridize to probes for symbiotic genes or the absence of a megaplasmid band in Eckhardt gels. The data suggest that nodulation promiscuity may be a relatively common characteristic of R. meliloti. Key words: host range, Rhizobium meliloti, Leucaena, Macroptilium, Phaseolus.

An assessment was made of the impact of temporal separation on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a single site that had no known history of alfalfa cultivation. Root nodules were sampled on six occasions over two seasons, and a total of 1620 isolates of S. meliloti were characterized on the basis of phage sensitivity using 16 typing phages. Plant infection tests indicated that symbiotic S. meliloti were deficient in the soil at the time of planting and that these bacteria were present at low density during the first season (<10(2)/g of soil); in the second season numbers increased markedly to about 10(5)/g of soil. Overall, 37 and 51 phage types, respectively, were encountered among the nodule isolates from M. sativa and M. alba. The data indicate significant temporal shifts in the frequency and diversity of types associated with the two legume species. Apparent temporal variation with respect to the frequency of types appeared largely unpredictable and was not attributable to any one sampling time. The results indicate an apparent reduction in phenotypic diversity over the course of the experiment. Differential host plant selection of specific types with respect to nodule occupancy was indicated by significant interactions between legume species and either the frequency or diversity of phage types. Isolates from M. sativa that were resistant to lysis by all typing phages (type 14) were unusual in that they were predominant on this host at all sampling times (between 53% and 82% nodule occupancy) and were relatively homogeneous on the basis of DNA hybridization with 98% of the isolates analysed sharing the same nod EFG hybridization profile. In contrast, those isolates from M. alba comprising type 14 were encountered at low total frequency (2%) and were genetically heterogeneous on the basis of Southern hybridization. The implications of the observed temporal and host plant variation for ecological studies are discussed.