Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Alfalfa Root Exudates and Compounds which Promote or Inhibit Induction of Rhizobium meliloti Nodulation Genes

Using a plate induction assay, we demonstrate that alfalfa exudes inducer of Rhizobium meliloti nodulation genes. The inducer is exuded from the infectible zone of the root, accumulates to at least 1 micromolar, and is not affected by 10 millimolar nitrate. No zones of inhibition are observed. A nodulation minus mutant line of alfalfa, MN-1008, exudes normal levels of inducer. R. meliloti grown in rich medium requires ten-fold higher concentrations of luteolin to achieve half-maximal induction as compared to cells grown in a minimal medium. Flavonoids other than luteolin are found to have activity in R. meliloti nodulation gene induction assays. The compounds apigenin and eriodictyol have activities two-fifths and one-seventh that of luteolin, respectively. Several of the flavonoids tested (morin = naringenin > kaempferol = chrysin > quercetin = fisetin = hesperitin) demonstrate antagonistic activity toward induction by luteolin. The most effective antagonist is the coumarin, umbelliferone.     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia

A study was conducted to determine whether colonization of legume roots and nodulation byRhizobium meliloti andBradyrhizobium japonicum could be enhanced by using inocula containing microorganisms that produce antibiotics suppressing soil or rhizosphere inhabitants but not the root-nodule bacteria. An antibiotic-producing strain of Pseudomonas and one of Bacillus were isolated, and mutants ofR. meliloti andB. japonicum sp. resistant to the antibiotics were used. The colonization of the alfalfa rhizosphere and nodulation byR. meliloti were enhanced by inoculation of soil withPseudomonas sp. in soil initially containing 2.7×10⁵ R. meliloti per g. The colonization of soybean roots byB. japonicum was enhanced by inoculating soil with three cell densities ofBacillus sp., and nodulation was stimulated byBacillus sp. added at two cell densities. In some tests, the dry weights of soybeans and seed yield increased as a result of these treatments, and co-inoculation with Bacillus also increased pod formation. Inoculation of seeds withBacillus sp. and the root-nodule bacterium enhanced nodulation of soybeans and alfalfa, but colonization byB. japonicum andR. meliloti was stimulated only during the early period of plant growth. Studies were also conducted withStreptomyces griseus and isolates ofR. meliloti andB. japonicum resistant to products of the actinomycete. Nodulation of alfalfa byR. meliloti was little or not affected by the actinomycete alone; however, both nodulation and colonization were enhanced if the soil was initially amended with chitin andS. griseus was also added. Chitin itself did not affectR. meliloti. Treatments of seeds with chitin orS. griseus alone did not enhance colonization of alfalfa roots byR. meliloti or soybean roots byB. japonicum, but the early colonization of the roots by both bacterial species was promoted if the seeds received both chitin andS. griseus; this treatment also increased nodulation and dry weights of alfalfa and soybeans and the N content of alfalfa. It is suggested that co-inoculation of legumes with antibiotic-producing microorganisms and root-nodule bacteria resistant to those antibiotics is a promising means of promoting nodulation and possibly nitrogen fixation.     

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3′,4′,5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin-7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots.     

Release and Modification of nod-Gene-Inducing Flavonoids from Alfalfa Seeds

Traces of luteolin, an important rhizobial nod gene inducer in Rhizobium meliloti, are released by alfalfa (Medicago sativa L.) seeds, but most luteolin in the seed exudate is conjugated as luteolin-7-O-glucoside (L7G). Processes affecting the production of luteolin from L7G in seed exudate are poorly understood. Results from this study establish that (a) seed coats are the primary source of flavonoids, including L7G, in seed exudate; (b) these flavonoids exist in seeds before imbibition; and (c) both the host plant and the symbiotic R. meliloti probably can hydrolyze L7G to luteolin. Glycolytic cleavage of L7G is promoted by glucosidase activity released from sterile seeds during the first 4 hours of imbibition. Thus, L7G from imbibing alfalfa seeds may serve as a source of the nod-gene-inducing luteolin and thereby facilitate root nodulation by R. meliloti.     

Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Alfalfa Controls Nodulation during the Onset of Rhizobium-induced Cortical Cell Division

The formation of first nodules inhibits subsequent nodulation in younger regions of alfalfa (Medicago sativa L.) roots by a feedback regulatory mechanism that controls nodule number systemically (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Following inoculation with wild-type Rhizobium meliloti, almost all infections associated with cortical cell division developed into mature nodules. While the distribution of Rhizobium- induced cell divisions closely paralleled the distribution of first emergent nodules, only 9 to 15% of total cell division foci failed to become functional nodules. Nodule formation was restricted to the primary root when plants were inoculated before lateral root emergence. Excision of these primary root nodules allowed nodules to reappear in lateral roots clustered around the location of the root tip at the time of nodule removal. Apparently, this region regained susceptibility to infection within the first hours after excision of primary nodules and suppression of nodulation was restored a day later probably due to the development of new infection foci. Our results suggest that alfalfa controls nodulation during the onset of cell division in the root cortex and not during infection development as in soybean.     

Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

Phytotoxin resistance ofRhizobium meliloti mutants

The efficiency of twoRhizobium meliloti mutants for nodulation and nitrogen fixation (acetylene reduction) under three concentrations ofp-coumaric and ferulic acids, was evaluated using a sand-culture medium. The effect of the test concentrations of both acids on the growth of alfalfa was also determined. The results revealed that the test mutants showed considerable resistance to the higher concentration of the test phytotoxins in terms of nodulation and nitrogen fixation, whereas seedling growth was inhibited by increased phytotoxin concentrations. The possible advantages of mutants resistance to the phytotoxins under field conditions are briefly discussed.     

The effect of combined and separate inoculation of alfalfa plants with <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N₂O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N₂O with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C₂H₄ nmol/flask · 24 h) and low denitrification activity (1.8 μmol N₂O/flask · 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.     

The Biological Activity of the Sinorhizobium meliloti Glucan

The study of the effect of periplasmic glucan isolated from the root-nodule bacterium Sinorhizobium meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity decreased by 2.5–11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10–39 and 12–27%, respectively.     

Receptor Site on Clover and Alfalfa Roots for Rhizobium

Sites on white clover and alfalfa roots that bind Rhizobium trifolii and R. meliloti capsular polysaccharides, respectively, were examined by fluorescence microscopy. Fluorescein isothiocyanate-labeled capsular material from R. trifolii bound specifically to root hairs of clover but not alfalfa. Binding was most intense at the root hair tips. Treatment of clover roots with 2-deoxyglucose (2-dG) prevented binding of R. trifolii capsular material to the roots. The sugar 2-dG enhanced the elution of clover root protein, which could bind to and specifically agglutinate R. trifolii but not R. meliloti or R. japonicum. The mild elution procedure left the roots intact. Agglutination of R. trifolii and passive hemagglutination of rabbit erythrocytes coated with the capsular material of R. trifolii were specifically inhibited by 2-dG. These results suggest that clover roots contain proteins that cross-link complementary polysaccharides on the surface of clover root hairs and infective R. trifolii through 2-dG-sensitive binding sites. Alfalfa root hairs were shown to specifically bind to a surface polysaccharide from R. meliloti.     

Identification of the sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other Rhizobia and Agrobacterium tumefaciens

A plasmid of 150 Mdal from Rhizobium leguminosarum RCC1001 was found to be a Sym plasmid (pSym1) carrying genes for root nodulation and nitrogen fixation on plants of the pea vetch cross-inoculation group. The plasmid was expressed not only in different R. leguminosarum and R. trifolii hosts, but also in Agrobacterium tumefaciens and R. meliloti, although in root nodules induced by A. tumefaciens and R. meliloti hosts no nitrogen was fixed. The host range for root nodule induction appeared to be determined by pSym1 and only included plants of the pea vetch cross-inoculation group; in contrast, the host range for the induction of root hair deformations, which was found also to be determined by pSym1 was less restricted and included besides plants of the pea vetch group in addition plants of the clover group. This corroborates previous findings that host specificity for nodulation and nitrogen fixation is exerted at a stage after the induction of root hair deformations.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.