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.     

Associative effect of cellulolytic fungi andAzospirillum lipoferum on yield and nitrogen uptake by wheat

The associative effect of cellulolytic fungi, such asAspergillus awamori andA. niger, with the nitrogen fixer,Azospirillum lipoferum was studied in a soil amended with rice straw. All the inoculants gave significantly higher grain and straw yield and nitrogen uptake by wheat crop than did the uninoculated treatment. The doubling of chemical nitrogen dose significantly increased the yield and nitrogen uptake. It was observed thatA awamori performed significantly better followed byA. niger andA. lipoferum. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum. Another experiment was conducted in the subsequent year in soil amended with and without rice straw using cellulolytic culture eitherA. awamori orSclerotium rolfsii, andA. lipoferum. Application of straw in soil significantly reduced the yield and N-uptake by wheat crop as compared to the controls. All the inoculants exceptS. rolfsii gave significantly higher grain yield. However, N-uptake by grain was significantly increased only by combined inoculation ofA. lipoferum and either one of the cellulolytic fungi. Similar trends on yield and N-uptake of straw due to inoculants were observed. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum followed byA. awamori alone on grain yield and only combined inoculants on N-uptake by the crop.     

Effect of Rhizobium inoculation methodologies on nodulation and growth of Leucaena leucocephala

The aim of this study was to evaluate the effect of five methods of Rhizobium inoculum application on nodulation and nitrogen fixation in Leucaena leucocephala seedlings cultivated for 6 months in the greenhouse. Plants inoculated with alginate beads were significantly more developed and more nodulated than plants inoculated with the other methodologies used.     

Effect of inoculation ofAzospirillum lipoferum on nitrogen fixation in rhizosphere soil,their association with root,yield and nitrogen uptake by mustard (Brassica juncea)

Summary A microplot field experiment was conducted in the presence or absence of P and N application to evaluate the influence of the seed inoculation of mustard (cv. Baruna T₅₉) withAzospirillum lipoferum on N₂-fixation in rhizosphere, association of the bacteria with the roots and grain yield and N uptake. Inoculation significantly increased the N content in rhizosphere soil particularly at early stage (40 days) of plant growth, which was accompanied by the increased association of the bacteria (A. lipoferum) in rhizosphere soil, root surface washing and surface-sterilized macerated root. A significant increase in grain yield and N uptake was also observed due to inoculation. Application of P particularly at the 20 kg. ha^–1 level further enhanced the beneficial effect ofAzospirillum lipoferum inoculation, while N addition markedly reduced such an effect.     

Nitrogen limitations to field bean productivity: A comparison of combined nitrogen applications with Rhizobium inoculation

Summary Nitrogen limitations to the yield of a field crop ofVicia faba have been examined. Application of nitrogen totalling 560 kg/ha increased dry matter yield at flowering by 674 kg/ha (32%) and grain yield at final harvest by 1.6 tonnes/ha (24%). Attempts to reduce nitrogen limitations by replacing the native rhizobia with strains ofRhizobium leguminosarum selected for high rates of nitrogen fixation were unsuccessful but the introduction of poor rhizobia reduced grain yield. The reasons for this and the implications of the results for crop improvement are discussed.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Dual inoculation withRhizobium sp. andGlomus fasciculatum enhances nodulation,yield and nitrogen fixation in chickpea (Cicer arietinum Linn.)

Summary Seed inoculation with Rhizobium and soil inoculation withGlomus fasciculatum increased nodulation, nitrogen and phosphorus concentration in plants and yield of chickpea (Cicer arietinum) var. BG 212 in pots containing unsterilized soil especially with 50kgP₂O₅ ha⁻¹ in the form of superphosphate. Inoculation with Rhizobium orG. fasciculatum separately or in combination significantly increased the N₂ fixed in straw and grain than uninoculated controls as determined by¹⁵N atom percent excess of plants grown in soil amended with labelled ammonium sulphate (¹⁵NH₄)₂SO₄) at the rate of 20kg N ha⁻¹. These increases were most pronounced when P was applied at 50kgP₂O₅ ha⁻¹.     

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

Differences between cereal crop cultivars in root-associated nitrogen fixation,possible causes of variable yield response to seed inoculation

Large differences in N₂-ase activity with fractions of active plants from 3–67% and maximal activities from 3–35 nmol C₂H₄.h^-1 were found between sterile, Azospirillum-inoculated seedlings of 14 German cereal cultivars. Examples of similar cultivar differences in gnotobiotic or unsterile cereals in response to Azospirillum inoculation, in root exudation and the specificity of bacteria-root interactions are reviewed. As possible causes of yield responses to bacterial seed inoculation N₂-fixation, plant growth regulating metabolites and bacterial interaction with root pathogens are discussed. The need for suitable screening methods to select and breed cultivars with desirable responses to beneficial rhizosphere bacteria is pointed out.     

Yield increases in spring wheat (Triticum aestivum L.) inoculated withAzospirillum lipoferum under greenhouse and field conditions of a temperate region

Summary Spring wheat (Triticum aestivum var. Arkas) was associated withAzospirillum lipoferum under greenhouse and field conditions of a temperate region. Controls were treated with autoclaved bacteria. The soils used were: sand, sandy loam, and a peat-clay mixture. In experiments run over a period of three years, there were increases in grain yield, N-yield of the grains, and 1000 grain weight.Depending from environmental conditions, increase changed from year to year, and within one given year. There was, however, no experiment without positive response to the inoculation. Highest grain yield increase (70%) was found on sand supplemented with P and K only, but up to 32% were also obtained on peat-clay soil containing 0.28% total N. Under greenhouse conditions, one third of technical N-fertilizer could be saved by bacterial activities. With high probability the effects observed have been at least partly due to bacterial N₂-fixation, because the N-yield of the grains was increased (up to 33%), and the most pronounced response was found on sand without any N-fertilizer added.     

Associative effect of Rhizobium and phosphate-solubilizing bacteria on the yield and nutrient uptake of chickpea

Combined inoculation of Rhizobium and ‘Phosphate-solubilizing’Pseudomonas striata orBacillus polymyxa with and without added chemical fertilizer on chickpea yield and nutrient content was studied under greenhouse conditions. While the single inoculation of Rhizobium increased the nodulation and nitrogenase activity, the ‘phosphate-solubilizers’ increased the available phosphorus content of the soil. Combined inoculation of Rhizobium andP. striata orB. polymyxa increased the above parameters and also the dry matter content, the grain yield and nitrogen and phosphorus uptake significantly over the uninoculated control. The inoculation effects were more pronounced in the presence of added fertilizers. The possibilities of saving half the dose of N and replacing superphosphate with rockphosphate and inoculation with ‘phosphate-solubilizers’ are discussed.     

Associative effect ofAzospirilium brasilense withRhizobium japonicum on nodulation and yield of soybean (Glycine max)

Summary Azospirillum was associated with nodules of soybean. In general, seed inoculation with a broth culture ofAzospirillum brasilense alone significantly increased nodulation and grain yield of soybean grown in pots in unsterilized soil with different levels of urea ranging from 0 to 80 kg N/ha. This trend was significantly reproducible in a second experiment when a carrier based inoculant of the bacterium was used for seed inoculation.Inoculation withRhizobium japonicum andA. brasilense in combination generally increased grain yield in both the experiments, although the data were not significant.     

Composted sawdust as a carrierfor Bradyrhizobium,Rhizobium and Azospirillum in crop inoculation

Sawdust was composted by inoculation with a cellulose-decomposing fungus (Cephalosporium sp.) and an N₂-fixing bacterium (Azospirillum brasilense). The product was investigated as a possible carrier for Bradyrhizobium, Rhizobium and Azospirillum. The simple technology and composition of the carrier supported good growth and survival of the investigated strains. Yield increases following crop inoculation with the carrier containing the Bradyrhizobium/Rhizobium/Azospirillum mixture were observed with soybean (34–62%), groundnuts (4–39%), lucerne (24–82%) and a grass mixture of bird's foot trefoil and ryegrass (20–21%).     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

Studies on the nodulation of strain-specific resistant pea lines using single,mixed and delayed inoculation by Rhizobium leguminosarum

Symbiotic interactions between peas and Rhizobium leguminosarum were investigated by inoculating four pea lines, three of which are strain-specific resistant to the European strain 311d, with various combinations of two strains of Rhizobium, 311d and Tom⁺⁺. The strains were almost equally good to infect the susceptible European cultivar Hero when added singly inoculated. After mixed inoculation (1:1 proportion) strain analysis by ELISA revealed that the nodules were preferentially formed by 311d, although some Tom⁺⁺ nodules were also found mainly on the upper part of the root. Our conclusion is that Tom⁺⁺ is less compatible in comparison with 311d. In addition, we found that as the Hero plants emerged, they were becoming more resistant towards infection with not adapted bacteria. The strain-specific resistant lines from Afghanistan belong to two different systems: Afgh. I, completely resistant to 311d and highly nodulating with Tom⁺⁺, and Afgh. III, incompletely resistant to 311d and poorly nodulating with Tom⁺⁺. Mixed inoculations resulted in nodule depressions, as compared to single inoculations with Tom⁺⁺ ranging from 87% to 14%. The ability of 311d to block infection sites on the roots were found to depend on the degree of symbiotic adaptation between Afgh. I and Tom⁺⁺, respectively Afgh. III and Tom⁺⁺. Strain analysis after double strain inoculation of Afgh. I plants revealed that some nodules were induced by strain 311d. Thus, the presence of Tom⁺⁺ in this case influences the degree of host resistance. However, in Afgh. III plants the resistance towards nodulation were unaffected by the presence of Tom⁺⁺. We suggest that the degree of symbiotic adaptation may change the barrier of resistance towards infection.     

Selection of Rhizobium leguminosarum bv. viceae strains for inoculation of Pisum sativum L. cultivars: Analysis of symbiotic efficiency and nodulation competitiveness

From an analysis of 481 Rhizobium leguminosarum bv. viceae strains with 7 pea cultivars in pot and field experiments, we demonstrated that effective strains could be isolated from a rich medium-acid grey forest soil of the Oröl area (Central region of the European part of Russia) but not from a poor acid podzolic soil of the St. Petersburg area (North-West Russia). The proportion of the isolates significantly increasing N accumulation in pea plants (10.2%) is higher than that of strains increasing the shoot dry mass (4.6%) in the pot experiments. The mean values of the increase for N accumulation (33.8%) upon inoculation are also higher than for shoot mass (27.0%) in these experiments. N accumulation in the inoculated pea plants in the pot experiments was significantly correlated with seed yield and seed N accumulation in field experiments, while for shoot dry mass these correlations were either weak or not significant. Two-factor analysis of variance demonstrated that the contribution of plant cultivars to the variation of the major symbiotic efficiency parameters is higher (30.8–31.6%) and contributions of cultivar-strain specificity is lower (5.4–8.8%) than the contributions of strain genotypes (13.4–14.9%). We identified an ineffective R. leguminosarum bv. viceae strain 50 which can be used as a tester for assessing the nodulation competitiveness of the effective strains by an indirect method (analysis of dry mass and N accumulation in pea plants inoculated with the mixture of the tested effective strains and the tester strain). The relative competitive ability (RCA) determined by this method was 75.7–82.8% for strain 52 but only 10.5–13.8% for strain 250a; this difference was confirmed by a direct method (use of the streptomycin-resistant mutants). Results of screening of the diverse collection of 53 effective R. leguminosarum bv. viceae strains by the indirect method permits us to divide them into 3 groups (32 high-competitive, 10 medium-competitive and 11 low-competitive strains) but reveals no correlation between the competitiveness and symbiotic efficiency. N accumulation in the pea shoots is demonstrated to be a much more suitable criterion than the shoot mass for selection either of the highly-effective or of highly-competitive (by the indirect estimation) R. leguminosarum bv. viceae strains in the pot experiments.     

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.     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring     

施氮量对不同类型花生品种衰老特性和产量的影响

为了探讨花生高产的适宜施氮量,在大田高产条件下,以珍珠豆型花生品种白沙1016和普通型花生品种花育17为材料,研究了施氮量对不同类型花生品种衰老特性和产量的影响。结果表明,两花生品种叶片叶绿素含量和光合速率、SOD和CAT活性均随着施氮量的增加而增加,MDA含量随施氮量的增加而降低,只是白沙1016品种在施氮超过135kg/hm2后上述指标增加或降低不显著,说明增施氮肥可以延缓花生叶片的衰老。在一定施氮量范围内,两花生品种有效荚果数随着施氮量的增加而增加,千克果数随着施氮量的增加而降低,导致荚果产量随着施氮量的增加而增加(珍珠豆型花生品种白沙1016施氮在0-90kg/hm2、普通型花生品种花育17在0-135kg/hm2范围内),但是超过此范围后再增加施氮量反而导致有效荚果数下降、千克果数增多、荚果产量下降。