Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Ability of symbiotic and non-symbiotic rhizospheric microflora of maize (Zea mays) to weather micas and to promote plant growth and plant nutrition

Summary Maize was grown under axenic conditions in laboratory devices, in a K⁺-deficient medium, where biotite was the K⁺ source. In different treatments plants were inoculated by symbiotic (Glomus mosseae) and/or non symbiotic microflora. In those treatments inoculated byGlomus mosseae, the percentage of roots infection after 7 weeks plant growth was 65%. Rhizospheric bacterial population was approximately 10⁸/g (dry weight). Endomycorrhizae stimulated growth and K uptake. Non-symbiotic microflora increased also plant growth but promoted much more biotite weathering and K uptake. Endomycorrhizae and more particularly non-symbiotic microflora increased also Ca and Mg absorption by plants. Possible mechanisms involved and implications in plant growth and pedogenesis are discussed.     

Studies on nodulated and mycorrhizal peanuts

Yields of fruits, plant size and chemical content of shoots, roots and seeds were higher in peanuts infected with Glomus mosseae. Infection with Glomus and Rhizobium stimulated nodulation and acetylene reduction rates. Dually infected plants given various phosphorus treatments, produced more fruits, dry matter, and nodules and contained more phosphorus and nitrogen compared with nodulated-only plants. Mycorrhizal infections were high in all treatments. Together these two endophytes contributed greatly to the vigour of the host.     

Grain yield of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) varieties is markedly increased by rhizobial inoculation and phosphorus application in Ethiopia

A field experiment was conducted to assess plant growth, symbiotic performance and grain yield of common bean in response to rhizobial incoculation and phosphorus application at Galalicha in Southern Ethiopia during the 2012 and 2013 cropping seasons under rain-fed conditions. The treatments consisted of 2 released common bean varieties (Hawassa Dume and Ibbado), 3 levels of Rhizobium inoculation (uninoculated, inoculated with strain HB-429 or GT-9) and 4 levels of phosphorus application (0, 10, 20 and 30 kg P ha^?1) using a split-split plot design with four replications. Here, phosphorus levels, Rhizobium inoculation and common bean varieties were assigned as main, sub- and sub-sub treatments, respectively. The results revealed marked varietal differences in plant growth, grain yield and symbiotic performance. Of the two common bean varieties studied, Hawassa Dume generally showed superior performance in most measured parameters in 2013. Rhizobium inoculation significantly (p?≤?0.05) increased plant growth, symbiotic performance and grain yield. Applying Rhizobium strain HB-429 to bean crop respectively increased plant growth, %Ndfa, amount of N-fixed and grain yield by 19, 17, 54 and 48% over uninoculated control. Similarly, the application of 20 kg P ha^?1 to bean plants respectively resulted in 36, 20, 96 and 143% increase in plant growth, %Ndfa, N-fixed and grain yield when compared to the control. These results clearly indicate that plant growth, symbiotic performance and grain yield of common bean can be significantly increased by Rhizobium inoculation and phosphorus fertilization in Ethiopia. Rhizobium inoculants are a cheaper source of nitrogen than chemical fertilizers and when combined with moderate phosphorus application can markedly increase grain yield for resource-poor farmers.     

Influence of mycorrhizae and Rhizobium on cytokinin content in drought-stressed alfalfa

The objective of this research was to study the growth responseto drought of arbuscular mycorrhizal and non-mycorrhizal alfalfa(Medicago sativacv. Aragn) in relation to leaf cytokinin levels.In the experiment, four treatments were used: (a) plants inoculatedwith Clomus fasciculatum (Taxter sensu Gerd.) Gerdemann andTrappe and Rhizobium meliloti 102 F51 strain, (MR); (b) plantsinoculated with only Rhizobium (RP); (c) plants inoculated withonly mycorrhizae (MN); and (d) plants non-inoculated (NP). Non-mycorrhizalplants were supplemented with phosphorus and nonnodulated oneswith nitrogen to achieve similar size in all treatments. Plantswere subjected to drought by withholding irrigation in a cyclicway. The effects of drought on growth, number of stems, degreeof senescence, and leaf cytokinin levels were measured. Results of identification of cytokinins showed that dihydrozeatinriboside (dHZR) and ortno-topolin riboside (oTR) were predominantin alfalfa leaves. Nonsymbiotic plants (NP) showed higher totalcytokinin concentrations (dHZR and oTR). Under drought, NP plantsshowed the largest percentage drop in cytokinins and lower numberof stems as well as increased degree of senescent leaf tissuerelative to control values. By contrast, stressed symbioticplants (RP, MN and MR) showed higher green leaf weight thannonsymbiotic ones (NP) due to delay of leaf senescence and maintenance(RP) or increase (MN, MR) of stem leaf cytokinin levels duringdrought. The relationships between growth and the different cytokininsare discussed, suggesting an important role of mycorrhizal symbiosisin maintaining cytokinin levels under drought. Key words: Alfalfa, arbuscular mycorrhizae, cytokinins, drought, leaf senescence     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Symbiotic efficiency and infectivity of an autochthonous arbuscular mycorrhizal Glomus sp. from saline soils and Glomus deserticola under salinity

 The purpose of this study was to compare the effect of salinity on the symbiotic efficiencies and mycelial infectivity of two arbuscular mycorrhizal fungi (AMF), one isolated from saline soils (Glomus sp.) and the other (Glomus deserticola) from nonsaline soils (belonging to the Estación Experimental del Zaidín collection). Lettuce plants inoculated with either of these two fungi or maintained as uninoculated controls were grown in soil with three salt concentrations (0.25, 0.50 or 0.75 g NaCl kg^–1 dry soil). Both AMF protected host plants against salinity. However, when the results of shoot dry weight and nutrient contents were expressed relative to the total length of mycorrhiza formed, it was found that both AMF differed in their symbiotic efficiencies. These differences were more evident at the two highest salt levels. Glomus sp.-colonized plants grew less and accumulated less N and P, whereas they formed a higher amount of mycorrhiza. The mechanism by which Glomus sp. protected plants from the detrimental effects of salt was based on the stimulation of root development, while the effects of G. deserticola were based on improved plant nutrition. The increase in salinity of soil decreased the hyphal growth and/or viability of Glomus sp. to a higher extent than those of G. deserticola since the mycelial network generated by G. deserticola was more infective than that of Glomus sp. Accepted: 8 September 2000     

Co-inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases P use efficiency for N2 fixation in the common bean (Phaseolus vulgaris L.) under P deficiency in hydroaeroponic culture

Common bean (Phaseolus vulgaris L.) genotypes CocoT and Flamingo were inoculated with Rhizobium tropici CIAT899 and Glomus intraradices (Schenck & Smith) and grown under sufficient versus deficient phosphorus supply for comparing the effects of double inoculation on growth, nodulation, mycorrhization of the roots, phosphorus use efficiency and total nitrogen. Although the double inoculation induced a significant increase in all parameters whatever the phosphorus supply in comparison to control, significant differences were found among genotypes and treatments. Nevertheless, the highest phosphorus use efficiency and plant total nitrogen were found under P deficiency in combination with arbuscular mycorrhizal fungi. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve symbiotic nitrogen fixation even under phosphorus deficiency.     

Gas exchange is related to the hormone balance in mycorrhizal or nitrogen-fixing alfalfa subjected to drought

The beneficial effect of mycorrhization on photosynthetic gas exchange of host plants under drought conditions could be related to factors other than changes in phosphorus nutrition and water uptake. Our objective was to study the influence of drought on phytohormones and gas exchange parameters in Medicago sativa L. cv. Aragón associated with or in the absence of arbuscular mycorrhizal (AM) fungi and/or nitrogen-fixing bacteria. Four treatments were used: (1) plants inoculated with Glomus fasciculatum (Taxter sensu Gerd.) Gerdemann and Trappe and Rhizobium meliloti 102 F51 strain (MR); (2) plants inoculated with only Rhizobium (R); (3) plants inoculated with only mycorrhizae (M); and (4) non-inoculated plants (N). When endophytes were well established, treatments received different levels of phosphorus and nitrogen in the nutrient solution in order to obtain plants similar in size. Sixty days after planting, plants were subjected to two cycles of drought and recovery. Midday leaf water potential (Ψ), CO₂ exchange rate (CER), leaf conductance (g_w) and transpiration (T), as well as leaf and root abscisic acid (ABA) and cytokinin concentrations were measured after the second drought period. Gas exchange parameters were determined by infrared gas analysis. Cytokinins and ABA levels in tissues were analysed by ELISA and HPLC, respectively. Nodulated R and MR plants had the lowest ABA concentrations in roots under well-watered conditions. Water stress increased ABA concentrations in leaves of N, R and MR plants, while ABA concentration in M plants did not change. The highest production of ABA under water deficit was in the roots of non-mycorrhizal plants. The ratio of ABA to cytokinin concentration strongly increased in leaves and roots of non-mycorrhizal plants under drought. By contrast, this ratio was lowered in roots of M plants and remained unchanged in leaves and roots of MR plants when stress was imposed. The highest leaf conductances and transpirational fluxes under well-watered conditions were those of nitrogen-fixing R and MR plants, but these results were not impaired with increased CO₂ exchange rates. Photosynthesis, leaf conductance and transpiration rates decreased in all treatments when stress was imposed, with the strongest decrease occurring in non-mycorrhizal plants. The relationships found between these gas exchange parameters and the hormone concentrations in stressed alfalfa tissues suggest that microsymbionts have an important role in the control of gas exchange of the host plant through hormone production in roots and the ABA/cytokinin balance in leaves. The most relevant effect of mycorrhizal fungi was observed under drought conditions.     

Extraradical mycelium of arbuscular mycorrhizal fungi radiating from large plants depresses the growth of nearby seedlings in a nutrient deficient substrate

The effect of arbuscular mycorrhiza (AM) on the interaction of large plants and seedlings in an early succession situation was investigated in a greenhouse experiment using compartmented rhizoboxes. Tripleurospermum inodorum, a highly mycorrhiza-responsive early coloniser of spoil banks, was cultivated either non-mycorrhizal or inoculated with AM fungi in the central compartment of the rhizoboxes. After two months, seedlings of T. inodorum or Sisymbrium loeselii, a non-host species colonising spoil banks simultaneously with T. inodorum, were planted in lateral compartments, which were colonised by the extraradical mycelium (ERM) of the pre-cultivated T. inodorum in the inoculated treatments. The experiment comprised the comparison of two AM fungal isolates and two substrates: spoil bank soil and a mixture of this soil with sand. As expected based on the low nutrient levels in the substrates, the pre-cultivated T. inodorum plants responded positively to mycorrhiza, the response being more pronounced in phosphorus uptake than in nitrogen uptake and growth. In contrast, the growth of the seedlings, both the host and the non-host species, was inhibited in the mycorrhizal treatments. Based on the phosphorus and nitrogen concentrations in the biomass of the experimental plants, this growth inhibition was attributed to nitrogen depletion in the lateral compartments by the ERM radiating from the central compartment. The results point to an important aspect of mycorrhizal effects on the coexistence of large plants and seedlings in nutrient deficient substrates.     

Symbiotic effectiveness of Hup+ Rhizobium,VAM fungi and phosphorus levels in relation to nitrogen fixation and plant growth ofCajanus cajan

Effect of hydrogen uptake positive (Hup⁺) strain ofRhizobium sp. (pigeon pea) and VAM fungus (Glomus fasciculatum) was studied on the symbiotic parameters of pigeon pea (Cajanus cajan) cv. AL-15 at various levels of phosphorus. The Hup⁺ Rhizobium strain showed more nodulation, plant biomass and plant nitrogen content than its Hup⁻ counterpart. VAM infection in pigeon pea roots helped in translocating phosphorus from the soil and improved nitrogen fixation. Similarly, addition of phosphorus was found to play a positive role in enhancing all these parameters. Dual inoculation of Hup⁺ Rhizobium strain and VAM significantly increased nodulation, nitrogenase activity, plant nitrogen and phosphorus content and plant biomass compared to single inoculation of either organism and dual inoculation with Hup⁻ and VAM fungus.     

The contributions of plant and bacteria genotypes in the growth and nitrogen accumulation of inoculated alfalfa

The symbiotic efficiency of each of 30 alfalfa (16 Medicago sativa and 14 M. varia) cultivars inoculated with 7 Rhizobium meliloti strains was studied in three field experiments. Two-factor analysis of variance of the obtained date demonstrated that the green mass yield and nitrogen accumulation depend on genotypes of both partners. The total contribution of plant and bacterial genotypes to the variation of green mass yield increased from 0–17% in the first year of alfalfa growth to 40–78% in the third year. The effect of the genotypic variability of the symbiotic partners was higher for N accumulation than for the green mass. There was a negative correlation between plant mass and N accumulation in the uninoculated plants with the relative (%) deviations of these parameters in the inoculated plants. In the experiments conducted in the Tashkent region the efficiency of the “alfalfa-R. meliloti” symbiosis was higher than in the experiment conducted in the Tumen region.     

Plant-Growth-Promoting Fungicide-Tolerant Rhizobium Improves Growth and Symbiotic Characteristics of Lentil (Lens esculentus) in Fungicide-Applied Soil

The goal of this study was to identify lentil-specific rhizobial strains with the ability to tolerate fungicide and synthesize plant growth regulators even in soils contaminated with fungicides. A fungicide-tolerant and plant-growth-promoting rhizobial strain was used to assess its impact on lentil grown in fungicide-treated soils. The tebuconazole-tolerant Rhizobium sp. strain MRL3 produced plant-growth-promoting substances when grown in the presence and the absence of tebuconazole. Tebuconazole at the recommended and two and three times the recommended doses decreased consistently the dry biomass, symbiotic properties, nutrient uptake, and seed yields of lentil plants. In contrast, the fungicide-tolerant strain MRL3 significantly increased the measured parameters when lentil was grown in soils treated with varying concentrations of tebuconazole compared to uninoculated plants. As an example, strain MRL3 with 100 μg tebuconazole/kg soil significantly increased the root nitrogen, shoot nitrogen, root phosphorus, shoot phosphorus, and seed yield by 31, 10, 41, 21, and 117%, respectively, compared to the uninoculated plants grown in soil treated solely with 100 μg tebuconazole/kg soil. In conclusion, the Rhizobium strain MRL3 may be applied as biofertilizer to enhance the performance of lentil plants in fungicide-applied soils.     

Effects of disease resistance genes on Rhizobium symbiosis in an annual legume

Summary The evolution of disease resistance in plants may be constrained if genes conferring resistance to pathogens interfere with plant responses toward other, nonpathogenic organisms. To test for such effects, we compared symbiotic nitrogen fixation in Amphicarpaea bracteata plants that differed at a major locus controlling resistance to the pathogen Synchytrium decipiens. Both resistant and susceptible plant genotypes nodulated successfully and grew significantly better in the presence of Rhizobium, although growth enhancement by Rhizobium was altered by different levels of nitrate fertilization. Plants homozygous for disease resistance achieved 2% higher growth than susceptible homozygotes across all treatments, but this difference was not significant. Resistant and susceptible plant genotypes did not differ in the mean number of nodules formed per plant or in nodule diameter. However, there was highly significant variation among replicate families within each disease resistance category for both nodulation characteristics. These results imply that genetic variation exists among A. bracteata plants both for diease resistance and for traits affecting symbiotic nitrogen fixation. However, there were no evident pleiotropic effects of disease resistance genes on the plant-Rhizobium symbiosis.     

Amino acid content of leguminous proteins as affected by genetic and nutritional factors. I.

Summary Tetraploid and diploid red clover have been cultivated in greenhouse under aseptic conditions. The plant cultures were given only one source of nitrogen: either they were given nitrate in the nutrient solution or they were inoculated withRhizobium for establishing symbiotic fixation.The amino acid composition of protein from the different series and from different parts of the plants was analysed by paper chromatography.There was a clear arginine reaction from the tetraploid clover material but no arginine at all was found in the diploid clover material.Plants fed on nitrate and those fed on symbiotically fixed nitrogen showed the same amino acid pattern as far as can be judged from the chromatograms. The hydrolysates from inoculated plants showed usually more intense ninhydrine reaction on the chromatograms than hydrolysates from not inoculated plants, although diluted to the same nitrogen content. For this fact we have so far no explanation.     

Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen assimilation in nodules of alfalfa (Medicago sativa L.)

Legumes acquire significant amounts of nitrogen for growth from symbiotic nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report describes the impact of specifically reducing nodule NADH-GOGAT activity on symbiotic performance of alfalfa (Medicago sativa L.). Four independent transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA plants were fertile and showed normal growth in non-symbiotic conditions. The NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic alterations - similar to primary transformants. Clonally propagated plants were inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype was analyzed 21 days post-inoculation. Nodules of each GA line had reduced NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was accompanied by comparable decreases in RNA and protein. Plants from the GA89 line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly altered symbiotic phenotype: concomitantly activities of key enzyme for carbon and nitrogen assimilation decreased; nodule amino acids and amides were reduced while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in fresh weight, and had a lower N content than control plants. Photosynthesis was also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules resulted in plants having impaired nitrogen assimilation and altered carbon/nitrogen metabolic flux.     

Partitioning of nitrogen from biological fixation and fertilizer in Phaseolus vulgaris

Nitrogen uptake, distribution and remobilization in the vegetative and reproductive parts of the plant were studied in bean (Phaseolus vulgaris L.) cultivars Negro Argel and Rio Tibagi inoculated with either Rhizobium strain C05 or 127 K-17. Greenhouse grown plants were supplied with 2.5 mg N (plant)⁻¹ day⁻¹ as KNO₃ or K¹⁵NO₃ and the relative contribution to total plant nitrogen of mineral and symbiotically fixed nitrogen was determined. Control plants included those entirely dependent on fixed nitrogen as well as uninoculated plants supplied with 10 mg N (plant)⁻¹ day⁻¹. No differences were observed between inoculated treatments in total nitrate reductase activity and in the amount of mineral nitrogen absorbed, but there were considerable differences in the contribution of fixed nitrogen. Nitrogen fixation supplied from 58 to 72% of the total nitrogen assimilated during the bean growth cycle and the symbiotic combinations fixed most of their nitrogen (66 to 78% of total nitrogen) after flowering. Maximum uptake of mineral nitrogen was in the 15-day-period between flowering and mid-podfill (47 to 58% of total mineral nitrogen). Nitrogen partitioning varied with Rhizobium strains, and inoculation with strain C05 increased the nitrogen harvest index of both cultivars. Applied mineral nitrogen had a variable effect and in cv. Negro Argel was more beneficial to vegetative growth, resulting in smaller nitrogen harvest indices. Seed yield was not increased by heavy nitrogen fertilization. In contrast, cv. Rio Tibagi always benefited from nitrogen applications. Among the various nitrogen sources supplying the grain, the most important one was the fixed nitrogen translocated directly from nodules or after a rapid transfer through leaves, representing from 60 to 64% of the total nitrogen incorporated into the seeds.     

Interaction between alfalfa cultivars and Rhizobium strains for nitrogen Fixation

Summary Two experiments were conducted in the greenhouse to study the interaction between alfalfa cultivars (Medicago sativa L. and M. falcata L.) and strains of Rhizobium meliloti Dang. for acetylene reduction rate, plant height and dry weights of shoot, root and whole plant. Fifteen alfalfa cultivars were inoculated with 10 strains of Rhizobium in Experiment I. Variance component analysis revealed that more than 30% of the total variance was due to alfalfa cultivars for acetylene reduction rate and 26% was accounted for by Rhizobium strains. More than 36% of the total variation was attributed to the interaction between alfalfa cultivars and Rhizobium strains for this character. Twenty-five host cultivars and 11 Rhizobium strains were included in Experiment II. The results also showed that the interaction of alfalfa cultivars and Rhizobium strains contributed the largest portion of the total variation for dry weights of shoot, root and whole plant and acetylene reduction rate. The results clearly demonstrated that the non-additive effects were the major component of variation for these characters associated with nitrogen fixation in alfalfa. Therefore, an effective way of improving nitrogen fixation in alfalfa is to select for a favourable combination of specific Rhizobium strains and alfalfa cultivars.     

Positive role of nodulation on the establishment ofRhizobium japonicum in subsequent crops of soybean

The influence of soybean nodulation on the establishment ofRhizobium japonicum inRhizobium-free soil was examined. Seeds of nodulating (Rj ₁) and nonnodulating (rj ₁) isolines of soybeans and four other crop species (cowpeas, mungbeans, corn, and alfalfa) were grown in field plots that were inoculated with a genetically marked strain ofRhizobium (strain I-110 ARS) and the following year nodulating soybeans were grown in these plots and were inoculated with a different genetically marked subline of the same strain (strain I-110 FN). The proportion of nodules containing strain I-110 ARS relative to strain I-110 FN was determined and interpreted as reflecting the relative numbers of the two genetically marked sublines in the soil. The results clearly demonstrate that nodulation with the specific host plant (soybeans) has a significant positive role in the establishment ofRhizobium inRhizobium-free soil and suggests that alfalfa plants diminish the establishment of soybean rhizobia in soil.     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.