Influence of drought on competition between selected Rhizobium meliloti strains and naturalized soil rhizobia in alfalfa

Drought is an important environmental factor that can affect rhizobial competition and N₂ fixation. Three alfalfa (Medicago sativa L. and M. falcata L.) accessions were grown in pots containing soil from an irrigated (Soil 1) and a dryland (Soil 2) alfalfa field in northern Utah, USA. Mutants of three strains of Rhizobium meliloti Dang. from Pakistan (UL 136, UL 210, and UL 222) and a commercial rhizobial strain 102F51a were developed with various levels of resistance to streptomycin. Seeds inoculated with these individual streptomycin-resistant mutants were sown in the two soils containing naturalized rhizobial populations. Soils in the pots were maintained at −0.03, −0.5, and −1.0 MPa. After 10 weeks, plants were harvested and nodule isolates were cultured on agar medium with and without streptomycin to determine nodule occupancy (proportion of the nodules occupied by introduced rhizobial strains). Number of nodules, nodule occupancy, total plant dry weight, and shoot N were higher for Soil 1 than Soil 2. Number of nodules, plant dry weight, and shoot N decreased as drought increased from −0.03 to −1.0 MPa in the three alfalfa accessions. Rhizobial strains UL 136 and UL 222 were competitive with naturalized alfalfa rhizobia and were effective at symbiotic N₂ fixation under drought. These results suggest that nodulation, growth, and N₂ fixation in alfalfa can be improved by inoculation with competitive and drought-tolerant rhizobia and may be one economically feasible way to increase alfalfa production in water-limited environments. Joint contribution from USDA-ARS and the Utah Agric. Exp. Sta., Utah State Univ., Logan, UT 84322-4810, USA. Journal Paper No. 4931. Joint contribution from USDA-ARS and the Utah Agric. Exp. Sta., Utah State Univ., Logan, UT 84322-4810, USA. Journal Paper No. 4931.     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478     

Nitrogen-fixing characteristics of alfalfa cultivars nodulated by representatives of an indigenousRhizobium meliloti serogroup

Studies were conducted to evaluate whether field-grown cultivars of alfalfa (Medicago sativa L.) nodulate differentially with members of a soil population ofRhizobium meliloti, and to determine the influence of the dominant nodule occupants on N₂-dependent growth of the same cultivars under greenhouse conditions. Nodules were sampled from four replicate plots of Vernal, Anchor, and Saranac alfalfa, and the isolates analysed serologically. Results from agglutination tests identified serogroup 31 as a dominant nodule occupant. A significant cultivar effect was observed, with a greater and more consistent occupancy rate by serogroup 31 across the replicates on Vernal (60%) compared to Anchor (24%) or Saranac (36%). The symbiotic effectiveness of the parent isolate of serogroup 31 was evaluated on each cultivar over four successive harvests in a greenhouse study. Significant cultivar x N source interactions for herbage dry weight resulted following the second harvest. Of the three cultivars, only inoculated Vernal responded with an increase in shoot dry weight and N₂ assimilated relative to N supplemented plants between harvests two and three. In separate greenhouse experiments, field isolates of serogroup 31 from nodules on Vernal produced homogeneous, effective responses both on Vernal and Anchor. In contrast, serogroup 31 field isolates from Anchor nodules were highly heterogeneous in effectiveness on the parent host, with poorly effective isolates being substantially more effective on Vernal. The data indicate that attention should be given to the potential impact of the indigenousR. meliloti population upon cultivar ranking at specific field locations, and also to strain-cultivar idiosyncracies when carrying out physiological sutidies of regrowth characteristics.Technical Paper No. 8716 of the Oregon State University Agricultural Experiment Station.     

The effects of low root-zone temperature stress on two soybean (Glycine max) genotypes when combined with Bradyrhizobium strains of varying geographic origin

In areas with short growing seasons, poor early vegetative growth of soybean (Glycine max [L.] Merr.) is often attributed to the restrictive effect of cool soil conditions on nodulation and N₂-fixation by this subtropical grain legume. However, there are few studies regarding potential genetic variability of soybean and Bradyrhizobium japonicum genotypes for nodulation at cool root-zone temperatures (RZT). Experiments were conducted to (1) test for a threshold temperature for low RZT inhibition of soybean nodulation and (2) ascertain whether this threshold temperature response depends mainly on the micro- or macrosymbiont. In experiment 1 soybean seedlings (Glycine max [L.] Merr. cv. Maple Arrow) were inoculated with 1 ml of a log phase culture of B. japonicum strain 532C, H8 or H15 (the latter two strains were isolated from cold soils of Hokkaido, northern Japan) and maintained at either 16, 17.5, 19 or 25°C RZT. In experiment 2 seedlings of cv. Maple Arrow and a cold-tolerant Evans isoline were combined with strain 532C and two Hokkaido strains (H5, H30) at both 19 and 25°C RZT. Results indicated that N₂-fixation at 44 days after inoculation was substantially reduced (30–40%) by RZT as high as 19°C, due to development of less nodule mass and to a delay in the onset of N₂-fixation and a small decrease in the number of nodules formed. However, the number of nodules formed was sharply reduced and the time required for the first appearance of nodules was significantly delayed below an RZT of 17.5°C. Differences between cultivars for nodulation and N accumulation were apparent at 25°C, but were abolished by growth at 19°C, indicating that, in spite of differences in growth potential between the cultivars under optimum RZT, both cultivars were equally limited by low RZT. Differences between B. japonicum strains were consistent across temperatures and were largely attributable to higher rates of specific nodule activity recorded for strain 532C, which seemed well adapted to low RZT. These results suggest that the host plant mediates the sensitivity of N₂-fixation under low RZT and that inoculation with B. japonicum strains from cold environments is unlikely to enhance soybean N₂-fixation under cool soil conditions.     

Inoculation and production of container-grown red alder seedlings

Summary The inoculation ofAlnus rubra (red alder) withFrankia sp. can lead to a highly efficient symbiosis. Several factors contribute to the successful establishment of nitrogenfixing nodules: (1) quantity and quality ofFrankia inoculant; (2) time and method of inoculation; (3) nutritional status of the host plant.Frankia isolates were screened for their ability to nodulate and promote plant growth of container-grown red alder. Inoculations were performed on seedlings and seeds. Apparent differences in symbiotic performance could be seen when seeds or seedlings were inoculated. Plants inoculated at planting performed significantly better than those inoculated four weeks later in terms of shoot height, nodule number and shoot dry weight. If inoculation was delayed further, reduction in shoot height, nodule number and shoot dry weight resulted. The effect of fertilizer was also investigated with regard to providing optimal plant growth after inoculation. Plants receiving 1/5 Hoagland's solution minus nitrogen showed maximal plant growth with abundant nodulation. Plants receiving 1/5 Hoagland's solution with nitrogen showed excellent plant growth with significantly reduced nodulation.     

Rhizobium meliloti inoculation of alfalfa selected for tolerance to acid,aluminum-rich soils

Alfalfa (Medicago sativa L.) growth and nodulation in acid soil is reduced because the plant and its bacterial symbiontRhizobium meliloti cannot tolerate acid, aluminum-rich soil. A study was conducted to determine if a relatively acid-tolerant alfalfa germplasm combined with a relatively acid-tolerantR. meliloti strain could overcome these limitations. In a light room study, an acid-tolerant alfalfa germplasm inoculated with a more acid-tolerantR. meliloti strain produced greater top growth, nodule number and weight, and acetylene reduction values in an unlimed soil (pH 4.6) than the same germplasm inoculated with a relatively acid-sensitiveR. meliloti strain or an acid-sensitive germplasm inoculated with either a relatively acid-tolerant or acid-sensitiveR. meliloti strain.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2′,3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.

     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Effect of elevated temperature and water availability on CO2 exchange and nitrogen fixation of nodulated alfalfa plants

The aim of this study was to investigate the effects of predicted temperature increases and drought conditions of Mediterranean environments on N₂-fixing alfalfa plants. One-month-old plants inoculated with Sinorhizobium meliloti strain 102F78 were grown in growth chambers under different temperature (25/15 or 28/18 °C, day/night) and water availability (control or drought) regimes. Elevated temperature and drought reduced plant dry mass and leaf area, especially when both stresses were combined. The inhibitory effect of elevated temperature on plant growth was a consequence of decreased CO₂ and N₂ fixation rates. A photosynthetic decrease resulted from the inhibition of rubisco activity, probably associated with a lower activation state. An absence of differences in photosynthesis in relation to water availability suggests that drought decreased plant growth due to its negative effect on leaf area. Rising temperature and drought affected the nitrogen content negatively, although effects differed. Elevated temperature inhibited nodule activity drastically, whereas the inhibitory effect resulting from drought centred on nodule dry mass (DM) production. Plants exposed to a combination of elevated temperature and drought were the most negatively affected.     

Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L.

Screening of Rhizobium leguminosarum bv. phaseoli strains showed some that were able to nodulate common beans (Phaseolus vulgaris L.) at high temperatures (35 and 38°C/8 h/day). The nodulation ability was not related to the capability to grow or produce melanin-like pigment in culture media at high temperatures. However, nodules formed at high temperatures were ineffective and plants did not accumulate N in shoots. Two thermal shocks of 40°C/8 h/day at flowering time drastically decreased nitrogenase activity and nodule relative efficiency of plants otherwise grown at 28°C. Recovery of nitrogenase activity began only after seven days, when new nodules formed; total incorporation of N in tops did not recover for 2 weeks. Non-inoculated beans receiving mineral N were not affected by the thermal shock, and when growing continuously at 35 or 38°C had total N accumulated in shoots reduced by only 18%.     

Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N₂-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.     

Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti

Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR–RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR–RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N₂-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed.     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

A nodule endophytic plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> and its effects on growth,nodulation and metal uptake in <Emphasis Type="Italic">Medicago lupulina</Emphasis> under copper stress

The aim of this study was to determine the plant growth-promoting potential of the nodule endophytic Pseudomonas brassicacearum strain Zy-2-1 when used as a co-inoculant of Medicago lupulina with Sinorhizobium meliloti under copper (Cu) stress conditions. Strain Zy-2-1 was capable of producing ACC deaminase activity, IAA and siderophores, and was able to grow in the presence of Cu²⁺ up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 enhanced M. lupulina root fresh weight, total plant dry weight, number of nodules, nodule fresh weight and nitrogen content in the presence of 100 or 300 mg/kg Cu²⁺. In the presence of 500 mg/kg Cu²⁺, co-inoculation with S. meliloti and strain Zy-2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoculation alone. Furthermore, a higher amount of Cu accumulation in both shoots and roots and a higher level of Cu translocation to shoots were observed in co-inoculated plants. These results demonstrate that co-inoculation of M. lupulina with S. meliloti and P. brassicacearum Zy-2-1 improves plant growth, nitrogen nutrition and metal extraction potential. This can be of practical importance in the remediation of heavy metal-contaminated soils.     

The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa

Summary We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS^-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS^- mutants of R. meliloti 2011, which produced ineffective (Fix^-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Nodulation and growth ofPhaseolus vulgaris in solution culture

Summary Conditions and techniques for achieving good nodulation ofPhaseolus vulgaris L. in continuously aerated solution were developed from greenhouse experiments.If nodules had been established, their growth and activity and the growth of the plant were at least as good in solution culture as in gravel culture. Nodule formation was observed within 10 days of inoculation in small volumes of solution culture (1 liter). In large volumes (19 liters), similarly prompt nodulation occurred only if the plants were inoculated before or immediately after the seedlings were transferred to the solution from gravel or vermiculite; and the nodules were restricted to the roots that had been present at the time of transfer. Delayed inoculation, 2 days after transfer to large volume solutions, led to sparse nodulation observed only after 3 weeks. Delay or inhibition of nodulation in large volumes of solution could not be explained by failute of bacteria to colonize roots or by sparsity of root hairs.Nodule initiation in solution culture was severely inhibited at pH below 5.4. An additional problem in growing N₂-dependent bean in solution culture was the buildup of Cl^– to toxic levels in the plant in nitrate-free media, even at solution concentrations as low as 0.4 mM Cl^–. Daily addition of 0.5 to 1.0 mg N per plant delayed nodule growth and activity slightly, but increased plant growth and alleviated the severe N-deficiency that otherwise developed before the onset of N₂-fixation.     

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.