Symbiotic Characteristics of Rhizobium leguminosarum bv. trifolii Isolates Which Represent Major and Minor Nodule-Occupying Chromosomal Types of Field-Grown Subclover (Trifolium subterraneum L.)

The symbiotic effectiveness and nodulation competitiveness of Rhizobium leguminosarum bv. trifolii soil isolates were evaluated under nonsoil greenhouse conditions. The isolates which we used represented both major and minor nodule-occupying chromosomal types (electrophoretic types [ETs]) recovered from field-grown subclover (Trifolium subterraneum L.). Isolates representing four ETs (ETs 2, 3, 7, and 8) that were highly successful field nodule occupants fixed between 2- and 10-fold less nitrogen and produced lower herbage dry weights and first-harvest herbage protein concentrations than isolates that were minor nodule occupants of field-grown plants. Despite their equivalent levels of abundance in nodules on field-grown subclover plants, ET 2 and 3 isolates exhibited different competitive nodulation potentials under nonsoil greenhouse conditions. ET 3 isolates generally occupied more subclover nodules than isolates belonging to other ETs when the isolates were mixed in 1:1 inoculant ratios and inoculated onto seedlings. In contrast, ET 2 isolates were less successful at nodulating under these conditions. In many cases, ET 2 isolates required a numerical advantage of at least 6:1 to 11:1 to occupy significantly more nodules than their competitors. We identified highly effective isolates that were as competitive as the ET 3 isolates despite representing serotypes that were rarely recovered from nodules of field-grown plants. When one of the suboptimally effective isolates (ET2-1) competed with an effective and competitive isolate (ET31-5) at several different inoculant ratios, the percentages of nodules occupied by the former increased as its numerical advantage increased. Although subclover yields declined as nodule occupancy by ET2-1 increased, surprisingly, this occurred at inoculant ratios at which large percentages of nodules were still occupied by ET31-5.     

Genetic Structure of Rhizobium leguminosarum biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities

An investigation was carried out to determine the genetic structure in soil populations of Rhizobium leguminosarum bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of R. leguminosarum bv. trifolii (AT and CT) and two populations of R. leguminosarum bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the Rhizobium populations (<10² to >10⁵/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of R. leguminosarum bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local Vicia hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.     

Genotypic and Phenotypic Comparisons of Chromosomal Types within an Indigenous Soil Population of Rhizobium leguminosarum bv. trifolii

The relative genetic similarities of 200 isolates of Rhizobium leguminosarum bv. trifolii recovered from an Oregon soil were determined at 13 enzyme loci by multilocus enzyme electrophoresis (MLEE). These isolates represented 13 antigenically distinct serotypes recovered from nodules formed on various clover species. The MLEE-derived levels of relatedness among isolates of R. leguminosarum bv. trifolii were found to be in good agreement with the levels of relatedness established by using repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the PCR technique and with levels of relatedness from previously published DNA reassociation studies. BIOLOG substrate utilization patterns showed that isolates within an electrophoretic type (ET) were phenotypically more similar to each other than to isolates of other ETs. The soil isolates were represented by 53 ETs which could be clustered into seven groups (groups B, E, G, H1, H2, I, and J). Evidence for multilocus structure within the population was obtained, and group B was identified as the primary creator of the disequilibrium. Of 75 isolates belonging to the nodule-dominant serotype AS6 complex, 72 were found in group B. Isolates WS2-01 and WS2-02 representing nodule-dominant serotypes recovered from subclover grown at another Oregon site were also found in group B. Isolates representing the most numerous ETs in group B (ETs 2 and 3) were either suboptimally effective or completely ineffective at fixing nitrogen on six different clover species. Another four groups of isolates (groups A, C, D, and F) were identified when 32 strains of diverse origins were analyzed by MLEE and incorporated into the cluster analysis. Group A was most dissimilar in comparisons with other groups and contained strain USDA 2124 (T24), which produces trifolitoxin and has unique symbiotic characteristics.     

Genetic Structure of Acetobacter diazotrophicus Populations and Identification of a New Genetically Distant Group

A total of 55 isolates of Acetobacter diazotrophicus recovered from diverse sucrose-rich host plants and from mealybugs associated with sugarcane plants were characterized by the electrophoretic mobilities of 12 metabolic enzymes. We identified seven different electrophoretic types (ETs), six of which are closely related within a genetic distance of 0.195 and exhibit high DNA-DNA homology. The seventh ET was largely divergent, separated at a genetic distance of 0.53, and had only 54% DNA homology to the reference strain. Strains corresponding to ET 7 could represent a distinct nitrogen-fixing species of the genus Acetobacter. More genetic diversity was found in isolates from Brazil than in those from Mexico, probably due to the very different crop nitrogen fertilization levels used.     

Genetic diversity of rhizobia from Leucaena leucocephala nodules in Mexican soils

Leucaena species are leguminous plants native to Mexico. Using two L. leucocephala cultivars grown in different soils, we obtained 150 isolates from the nodules. Twelve rDNA types were identified which clustered into groups corresponding to Mesorhizobium, Rhizobium , and Sinorhizobium by restriction fragment length polymorphism (RFLP) of amplified 16S rRNA genes. Types 2, 4, 5, 6, 10, 11, and 12 were distinct from all the defined species. Others had patterns indistinguishable from some recognized species. Most of the isolates corresponded to Sinorhizobium . Forty-one electrophoretic types (ETs) were identified among the isolates based on the different combinations of electrophoretic patterns of 13 metabolic enzymes. ETs were clustered into groups in general agreement with the rDNA types. Diverse plasmid patterns were obtained among the isolates, but common plasmids were observed among most isolates within rDNA types 5, 10, and 11. The symbiotic plasmids were identified among most of the isolates, except for the Mesorhizobium isolates. The affinities of host cultivars for different rhizobial groups and the impact of soil cultivation on the soil populations of rhizobia were analysed from the estimation of isolation frequencies and diversity. The results showed differences in rhizobial populations in cultivated and uncultivated soils and also differences in rhizobia trapped by L. leucocephala cv. Cunningham or Peruvian.     

Genetic Structure and Symbiotic Characteristics of a Bradyrhizobium Population Recovered from a Pasture Soil

We examined the genetic structure and symbiotic characteristics of Bradyrhizobium isolates recovered from four legume species (Lupinus albus [white lupine], Lupinus angustifolius [blue lupine], Ornithopus compressus [yellow serradella], and Macroptilium atropurpureum [sirato]) grown in an Oregon soil. We established that multilocus enzyme electrophoresis (MLEE) can provide insights into the genetic relatedness among Bradyrhizobium strains by showing a positive correlation (r² = ≥0.90) between the relatedness of Bradyrhizobium japonicum strains determined by MLEE at 13 enzyme loci and that determined by other workers using either DNA-DNA hybridization or DNA sequence divergence estimates. MLEE identified 17 electrophoretic types (ETs) among 95 Bradyrhizobium isolates recovered from the four hosts. Although the overall genetic diversity among the ETs (H = 0.69) is one of the largest measured to date in a local population of any soilborne bacterial species, there was no evidence of multilocus structure (linkage disequilibrium) within the population. The majority of the isolates (73%) were represented by two closely related ETs (2 and 3) which dominated the root nodules of white lupine, serradella, and siratro. In contrast, ET1 dominated nodules of blue lupine. Although representative isolates from all of the 17 ETs nodulated siratro, white lupine, blue lupine, and big trefoil (Lotus pedunculatus), they were either completely ineffective or poorly effective at fixing nitrogen on these hosts. Despite the widespread use of serradella as a surrogate host for lupine-nodulating bradyrhizobia, 7 of the 17 ETs did not nodulate this host, and the remaining 10 ETs were ineffective at fixing nitrogen.     

Effect of Acidity on the Composition of an Indigenous Soil Population of Rhizobium trifolii Found in Nodules of Trifolium subterraneum L.

Acidity affected which members of an indigenous soil population of Rhizobium trifolii nodulated Trifolium subterraneum L. cv. Mt. Barker. In three experiments involving plants grown either in mineral salts agar adjusted to pH 4.8 or 6.8 and inoculated with a soil suspension or grown directly in samples of unamended soil (pH 4.8) or soil amended with CaCO₃ (pH 6.4), 121 of 151 isolates of R. trifolii were placed into four serogroups. Seventy-nine of these isolates were placed into two serogroups (6 and 36) whose nodulating ability was affected by the pH of the plant root environment. Representatives of serogroup 6 occupied the greatest percentage of the nodules at the low pH in both mineral salts agar (77%) and in unlimed soil (47 and 57%). The same serogroup was a minor nodule occupant at the higher pH in mineral salts agar (0%) and in limed soil (0 and 10%). In contrast, serogroup 36 was virtually absent in nodules formed at the low pH, whereas it was the dominant serogroup at the higher pH in both mineral salts agar (32%) and in limed soil (35 and 49%). Despite the isolates from within each serogroup being antigenically identical, separation of cellular proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed four and six different gel types within serogroups 6 and 36, respectively. Isolates represented by one or two gel types dominated the contribution of each serogroup to the nodule population. Further evidence for differences between isolates within each gel type were revealed from measurements of symbiotic effectiveness.     

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.     

Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers

Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mimosine, a Toxin Present in Leguminous Trees (Leucaena spp.), Induces a Mimosine-Degrading Enzyme Activity in Some Rhizobium Strains

Thirty-seven Rhizobium isolates obtained from the nodules of leguminous trees (Leucaena spp.) were selected on the basis of their ability to catabolize mimosine, a toxin found in large quantities in the seeds, foliage, and roots of plants of the genera Leucaena and Mimosa. A new medium containing mimosine as the sole source of carbon and nitrogen was used for selection. The enzymes of the mimosine catabolic pathway were inducible and were present in the soluble fraction of the cell extract of induced cells. On the basis of a comparison of the growth rates of Rhizobium strains on general carbon and nitrogen sources versus mimosine, the toxin appears to be converted mostly to biomass and carbon dioxide. Most isolates able to grow on mimosine as a source of carbon and nitrogen are also able to utilize 3-hydroxy-4-pyridone, a toxic intermediate of mimosine degradation in other organisms.     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.     

Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD , a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii , R. leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii [11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover ( Trifolium repens ) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare.     

Bacterial Biosynthesis of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase and Indole-3-Acetic Acid (IAA) as Endophytic Preferential Selection Traits by Rice Plant Seedlings

In this study, bacteria were isolated from the rhizosphere and inside the roots and nodules of berseem clover plants grown in the field in Iran. Two hundred isolates were obtained from the rhizosphere (120 isolates), interior roots (57 isolates), and nodules (23 isolates) of clover plants grown in rotation with rice plants. Production of chitinase, pectinase, cellulase, siderophore, salicylic acid, hydrogen cyanide, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, solubilization of phosphate, antifungal activity against various rice plant pathogen fungi, N₂ fixation, and colonization assay on rice seedlings by these strains was evaluated and compared (endophytic isolates vs. rhizosphere bacteria). The results showed both the number and the ability of plant growth-promoting (PGP) traits were different between endophytic and rhizosphere isolates. A higher percentage of endophytic isolates were positive for production of IAA, ACC deaminase, and siderophore than rhizosphere isolates. Therefore, it is suggested that clover plant may shape its own associated microbial community and act as filters for endophyte communities, and rhizosphere isolates with different (PGP) traits. We also studied the PGP effect of the most promising endophytic and rhizosphere isolates on rice seedlings. A significant relationship among IAA and ACC deaminase production, the size of root colonization, and plant growth (root elongation) in comparison with siderophore production and phosphate solubilization for the isolates was observed. The best bacterial isolates (one endophytic isolate and one rhizosphere isolate), based on their ability to promote rice growth and colonize rice roots, were identified. Based on 16S rDNA sequence analysis, the endophytic isolate CEN7 and the rhizosphere isolate CEN8 were closely related to Pseudomonas putida and Pseudomonas fluorescens, respectively. It seems that PGP trait production (such as IAA, ACC deaminase) may be required for endophytic and rhizosphere competence as compared to other PGP traits in rice seedlings under constant flooded conditions. The study also shows that the presence of diverse rhizobacteria with effective growth-promoting traits associated with clover plants may be used for sustainable crop management under field conditions.     

A Simple Technique of Studying Water Deficit Effects on Nitrogen Fixation in Nodules without Influencing the Whole Plant

Cowpea (Vigna unguiculata L. Walp cv C-152) plants were grown in a system in which watering was withheld from the soil zone containing nodules, while the plants were able to maintain normal water status. The system was developed in a pot by making two soil zones, an upper and a lower separated by a gravel column between these two zones. Plants extended their roots into the lower layer of soil and were able to absorb water. The dry matter accumulation, photosynthesis rate, and leaf area development of the plant were not affected when the upper soil zone was dried, but the water potential of the nodules was lower than in the nodules in fully irrigated pots. Nitrogenase activity in the nodules obtained from plants stressed in the upper zone only was lower than in nodules obtained from fully irrigated plants. The present technique is helpful in distinguishing the direct water stress effects on nitrogen fixation compared to those mediated via photosynthate availability.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

Influence of Soil and Nonsoil Environments on Nodulation by Rhizobium trifolii

Indigenous serotypes 1-01 and 2-02 of Rhizobium trifolii occupied similar percentages (18 to 23%) of root nodules on soil-grown subclover (Trifolium subterraneum L.) and were virtually absent (4.5%) from nodules of soil-grown white clover (Trifolium repens L.). In contrast (with the exception of one dilution [10⁻⁴]), serotype 1-01 occupied a substantial portion of nodules (16 to 40%) on white clover seedlings grown on mineral salts agar and exposed to samples of the same soil in the form of a 10-fold dilution series (10⁻¹ to 10⁻⁵). Under the latter conditions, occupancy of subclover nodules by 1-01 and of nodules of both plant species by 2-02 was consistent with the results obtained with soil-grown plants.     

Long-term effects of crop management on Rhizobium leguminosarum biovar viciae populations

Little is known about factors that affect the indigenous populations of rhizobia in soils. We compared the abundance, diversity and genetic structure of Rhizobium leguminosarum biovar viciae populations in soils under different crop managements, i.e., wheat and maize monocultures, crop rotation, and permanent grassland. Rhizobial populations were sampled from nodules of pea- or vetch plants grown in soils collected at three geographically distant sites in France, each site comprising a plot under long-term maize monoculture. Molecular characterization of isolates was performed by PCR-restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer as a neutral marker of the genomic background, and PCR-restriction fragment length 0polymorphism of a nodulation gene region, nodD, as a marker of the symbiotic function. The diversity, estimated by richness in types and Simpson's index, was consistently and remarkably lower in soils under maize monoculture than under the other soil managements at the three sites, except for the permanent grassland. The highest level of diversity was found under wheat monoculture. Nucleotide sequences of the main rDNA intergenic spacer types were determined and sequence analysis showed that the prevalent genotypes in the three maize fields were closely related. These results suggest that long-term maize monoculturing decreased the diversity of R. leguminosarum biovar viciae populations and favored a specific subgroup of genotypes, but the size of these populations was generally preserved. We also observed a shift in the distribution of the symbiotic genotypes within the populations under maize monoculture, but the diversity of the symbiotic genotypes was less affected than that of IGS types. The possible effect of such changes on biological nitrogen fixation remains unknown and this requires further investigation.     

Nodule distribution on the roots of actinorhizal Discaria trinervis (Rhamnaceae) growing in pots

Roots of actinorhizal plants can develop nitrogen-fixing nodules with actinomycetic bacteria of the genus Frankia. We aimed to know if unrestricted growth of roots in pots could influence the pattern of nodule development that we had previously observed for Discaria trinervis growing in pouches. Growth pouches, although being a space saving device convenient for the analysis of nodule development, do restrict root growth. Thus, the pattern of root nodule development was analysed in actinorhizal D. trinervis growing in pots with inert substrates. Inoculation of axenic seedlings growing in perlite resulted in clustering of nodules in a defined region of the taproot and upper lateral roots. When surface sterilized seeds were sown in pots containing vermiculite that had been previously inoculated with Frankia cells, nodules were again concentrated in defined portions of the main and lateral roots. Potted plants developed comparable numbers of nodules with respect to plants grown in pouches. However, a significant proportion of nodules appeared in lateral roots. As it was first inferred from field grown plants, these results confirm that D. trinervis plants growing in pots display the same autoregulatory mechanism for nodule formation that was previously observed in growth pouches.