Symbiotic effectiveness of indigenous arctic rhizobia on a temperate forage legume: Sainfoin (Onobrychis viciifolia)

Sainfoin (Onobrychis viciifolia), a temperate perennial forage legume, can be nodulated by rhizobia isolated from 3 arctic legume species:Astragalus alpinus, oxytropis maydelliana andOxytropis arctobia. Arctic rhizobia, which are adapted to growth at low temperatures, may be useful in improving symbiotic nitrogen fixation during cold phases of the growing season, if they are effective on a temperate legume. In this study, we report on the symbiotic effectiveness of arctic rhizobia on sainfoin, as appraised by the total shoot dry matter yield obtained from 2 harvests. Under N-free conditions, 5 arctic strains at the first harvest and 8 at the second harvest were as effective as temperate standard strains. In the presence of 30 mgl⁻¹ NO₃-N, 7 arctic strains gave significantly higher yields than temperate strains at the second harvest. These results indicate that effective arctic rhizobia have a potential for use as inoculants on sainfoin. Contribution no 325 of Agriculture Canada Research Station a Sainte-Foy.     

Influence of Rhizobial inoculation on yield of wheat (Triticum aestivum L.)

Summary Soil + charcoal (1∶3) carrier based and liquid cultures of Rhizobia were used to inoculate wheat seed cv. HD2329. The plants received 100 kg N in two equal splits and 60 kg P₂O₅ and 40 kg K₂₀ ha⁻¹. Inoculation with rhizobia had little effect on grain yield of wheat. Significant increase in straw yield and N-uptake occurred due to inoculation. A comparison of results of a similar experiment conducted during 1983–84, showed that inoculation with the same strains of rhizobia and application 50 kg N ha⁻¹ as basal dressing, was more effective in increasing yield and N-uptake in wheat cv. HD2329. It appears reasonable to assume occurrence of nitrogen fixation by root nodule bacteria in rhizosphere of wheat.     

Inoculation of pea by application of Rhizobium in the planting furrow

Summary Selected streptomycin resistant strains ofRhizobium leguminosarum suspended in nutrient broth were added to the planting furrow immediately before the sowing of pea. The nodule occupancy by a strain isolated from Risø soil (Risø la) was increased from 74 to 90%, when the inoculum rate was increased from 3.7×10⁶ to 3.7×10⁸ cells per cm row. The experimental soil contained 10³ to 10⁴ cells ofR. leguminosarum per gram. An almost inefficient strain isolated from Risø soil (SV10) was less competitive with respect to nodulation on two pea cultivars than an efficient Risø strain (SV15) and an efficient non-Risø strain (R1045). The nodule occupancy by the introduced strains varied between pea cultivars.Irrespective of the generally high nodulation by the efficient strains introduced to the soil, the pea seed yield, compared to pea nodulated by the indigenous population, was not significantly increased. Neither were two commercial inoculants, applied in rates corresponding to 3 times the recommended rate, able to increase the yield. This suggests that the indigenous populations ofR. leguminosarum were sufficient in number and nitrogen fixing capacity to ensure an optimal pea crop. However, some inoculation treatments slightly increased the seed N concentration and total N accumulation, indicating that it may be possible to select or develop bacterial strains that may increase the yield.     

1H, 13C and 15N resonances of the AlgE62 subunit from Azotobacter vinelandii mannuronan C5-epimerase

The 17.7 kDa R2 module from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been isotopically labeled (¹³C,¹⁵N) and recombinantly expressed. Here we report the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R2.     

NMR assignments of 1H, 13C and 15N resonances of the C-terminal subunit from Azotobacter vinelandii mannuronan C5-epimerase 6 (AlgE6R3)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been ¹³C, ¹⁵N isotopically labelled and recombinantly expressed. We report here the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R3.     

Association between N2-fixing bacteria and pearl millet plants: Responses,mechanisms and persistence

Responses to inoculation with N₂-fixing bacteria were studied in relation to genotypic differences in pearl millet, effect of nitrogen levels, and FYM additions in India. In some experiments, inoculation increased mean grain yield up to 33% over the uninoculated control, whereas in the remaining 11 experiments there was no significant increase. Increased grain yields, >10% over the uninoculated controls were observed in 46% of the experiments withAzospirillum lipoferum (18.7% average increase) and withAzotobacter chroococcum (13.6% average increase). Yield increases were nil or reduced in three experiments withAzos. lipoferum and four experiments withAztb. chroococcum. In two experiments continued inoculation for two or three years resulted in increased grain, plant biomass yield, and N uptake. Interactions of bacterial cultures with cultivars or years were not observed. The counts of the inoculated strains increased two to three-fold when inoculation was continued for three years. Repeated inoculations increased the mean cumulative N uptake from season 1 to season 3 by 19 kg ha^–1. Repeated inoculations withAztb. chroococcum andAzos. lipoferum increased mean grain yield of a succeeding crop by 14.4% and 9.8%, respectively, over the uninoculated control. Inoculation increased the efficiency of N-assimilation by pearl millet. Marginal increase in nitrogenase activity, associated with the inoculated plants was observed during later stages of plant growth. Increased leaf nitrate reductase activity (NRA) was observed after inoculation with these bacteria. The responses to inoculation are mainly attributable to increased plant N assimilation which could be the effect of growth promoting substances secreated by the bacteria; and thus the contribution from BNF may be small.CRISAT, journal article 732.     

Molecular diversity,effectiveness and competitiveness of indigenous rhizobial population infecting mungbean <Emphasis Type="Italic">Vigna radiata</Emphasis> (L. Wilczek) under semi-arid conditions

Nodules from mungbean crop raised for the first time at Ram Dhan Singh (RDS) farm of Chaudhary Charan Singh (CCS) Haryana Agricultural University, Hisar were collected from 17 different locations. Twenty-five mungbean rhizobia were isolated and authenticated by plant infection test. DNA of all these rhizobia was extracted purified and amplified using enterobacterial repetitive intergenic consensus (ERIC) primers. All the mungbean rhizobial isolates were clustered into 4 groups at 65% of similarity and were further divided into 17 subclusters at 80% of similarity. All the 4 types of rhizobia were not present at any of the location and group 2 or 4 rhizobia were invariably present. Efficacy of these rhizobia in terms of nodulation, nitrogen uptake and chlorophyll a fluorescence was determined under pot culture conditions. Strain MB 307 showed maximum nitrogen uptake of 31.9 mg N plant⁻¹ followed by strain MB 1205, MB 1206(2), MB 308, MB 1524 and strain MB 1521 was found to be the least efficient in terms of N 2 fixation. Nodule occupancy by different rhizobia ranged from 5.5 to 40.3%. Most of the strains belonging to the 2nd group which clustered maximum number of strains were comparatively better competitors and formed 19.5–40.3% of the nodules and were also effective. Isolate MB 307, the most efficient strain, was found to have nodule occupancy of 31.5%. Such type of predominant, efficient and better competitor strains should be selected for enhancing nodule competitiveness.     

1H, 15N, 13C resonance assignment of the AlgE6R1 subunit from the Azotobacter vinelandii mannuronan C5-epimerase

Isotopically labelled, ¹³C/¹⁵N from of recombinant subunit of the first R-module from alginate C5-epimerase 6 (AlgE6R1) from Azotobacter vinelandii mannuronan C5-epimerase was produced. We report here the ¹H, ¹⁵N, ¹³C resonance assignment of this subunit from AlgE6 epimerase.     

Associative effect of inoculation of different strains of Azotobacter and homologous rhizobium on the yield of moong (Vigna radiata), soybean (Glycine max) and pea (Pisum sativum)

Summary It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.     

Arctic and subarctic soil populations of Rhizobium leguminosarum biovar trifolii nodulating three different clover species: Characterisation by diversity at chromosomal and symbiosis loci

Indigenous soil populations of Rhizobium leguminosarum biovar trifolii from Arctic and subarctic regions have been characterised with emphasis on chromosomal and symbiotic genes. Three clover species were used to trap rhizobia from soils along a latitudinal gradient from 78°N to 60°N in Norway. For the first time R. l. bv. trifolii was isolated from Svalbard at 78°N. Under the extreme conditions in the Arctic, rhizobia have survived as saprophytes and in symbiosis with clover legumes. The chromosomal diversity of the soil populations was mapped by rep-PCR. Separation of chromosomal types were strongly influenced by geographic origin. Symbiotic genes, the nodEF and nifDK IGS gene regions, were investigated by PCR-RFLP. The nifDK IGS were more conserved than the nodEF genes. Sym plasmids were widely distributed in different chromosomal types and across the latitudinal gradient.     

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Québec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha^-1 P-superphosphate, or with strain 68 plus 70 kg ha^-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha^-1 P-superphosphate, or with strain P31 plus 35 kg ha^-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.     

Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions

A pot experiment was conducted in the green house to investigate the establishment of phosphate solubilizing strains of Azotobacter chroococcum, including soil isolates and their mutants, in the rhizosphere and their effect on growth parameters and root biomass of three genetically divergent wheat cultivars (Triticum aestivum L.). Five fertilizer treatments were performed: Control, 90 kg N ha^—1, 90 kg N + 60 kg P₂O₅ ha^—1, 120 kg N ha^—1 and 120 kg N + 60 kg P₂O₅ ha^—1. Phosphate solubilizing and phytohormone producing parent soil isolates and mutant strains of A. chroococcum were isolated and selected by an enrichment method. In vitro phosphate solubilization and growth hormone production by mutant strains was increased compared with soil isolates. Seed inoculation of wheat varieties with P solubilizing and phytohormone producing A. chroococcum showed better response compared with controls. Mutant strains of A. chroococcum showed higher increase in grain (12.6%) and straw (11.4%) yield over control and their survival (12—14%) in the rhizosphere as compared to their parent soil isolate (P4). Mutant strain M37 performed better in all three varieties in terms of increase in grain yield (14.0%) and root biomass (11.4%) over control.     

Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 × 10⁴ g of soil^-1 for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha^-1 in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P ≤ 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil^-1 eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil^-1 were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil^-1. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N₂-fixing crops indicates a potential for improving inoculation technology, the N₂ fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.     

On the criteria for comparing the ability of compounds for retardation of nitrification in soil

Summary A field experiment was conducted with ADT-31 rice variety. Foliar spray ofAzotobacter chroococcum was applied on 15^th, 30^th and 45^th day after transplanting of rice crop. The foliar spray of Azotobacter culture significantly increased the grain and straw yield of rice crop.     

Effect of varying periods of inoculation ofAzolla pinnata R. Brown on its growth,nitrogen fixation and response to rice crop

Summary Inoculation of water fernAzolla pinnata R. Brown (Bangkok isolate) at the rate of 500kg fresh weight ha⁻¹ in rice fields at weekly intervals after planting in addition to 30 kg N ha⁻¹ as urea showed a decrease in its growth and N₂-fixation with delay in application. Use of Azolla up to 3 weeks after planting (WAP) during wet and 4 WAP during dry season produced significantly more grain yield than 30 kg N ha⁻¹, whereas its application upto one WAP produced more grain yield than 60 kg N ha⁻¹. Grain yield with Azolla applied at the time of planting was similar to that of 60 kg N treatment during the wet season. Higher grain yields in zero and one WAP Azolla treatments resulted due to increase in both number of panicles m⁻² and number of grains/panicle while the subsequent Azolla inoculations increased grain yield mainly by producing more number of grains/panicle. Dry matter and total N yields at maturity of rice crop were more with Azolla application upto 3 WAP during wet and 2 WAP during dry season while the reduction in sterility (%) was observed upto one WAP over 30 kg N ha⁻¹ during both seasons. Number of tillers m⁻² and dry matter production at maximum tillering and flowering were more than 30 kg N ha⁻¹ with the use of Azolla upto one WAP. Increased grain N yield was observed with the use of Azolla upto 4 WAP during two seasons whereas straw N yield increased upto one WAP during wet and 2 WAP during dry season.     

Survival and colonization of inoculated bacteria in kallar grass rhizosphere and quantification of N2-fixation

Two experiments have been conducted, one in semi-solid Hoagland nutrient medium and the other in shallow pots containing saline soil. N₂-fixing bacteria belonging toAzospirillum, Azotobacter, Klebsiella andEnterobacter were inoculated separately on kallar grass grown in semi-solid nutrient medium. It was shown that inoculation affects root proliferation and also results in¹⁵N isotopic dilution. The % Ndfa ranged from 47–70 whereas no significant effect on the total nitrogen uptake was observed. The bacterial colonization of the root surface and the presence of enteric bacteria inside the root hair cells is reported. In a soil pot experiment, non-N₂-fixingPolypogon monspeliensis was used as a reference plant (control). A treatment receiving a high rate of nitrogen was also used as a non-N₂-fixing control.¹⁵N-labelled ammonium sulphate at 20 kg N ha^–1 and 90 kg N ha^–1 was used. The % Ndfa in the aerial parts of kallar grass was 12–15 whenP. monspeliensis was used as reference plant whereas 37–39% Ndfa was estimated when the treatment receiving high nitrogen fertilizer was used as a non-N₂-fixing control. These investigations revealed some problems of methodology which are discussed.     

Nitrogen contributions from faba bean (Vicia faba L.) reliant on soil rhizobia or inoculation

Background and Aims

Understanding the impact of soil rhizobial populations and inoculant rhizobia in supplying sufficient nodulation is crucial to optimising N₂ fixation by legume crops. This study explored the impact of different rates of inoculant rhizobia and contrasting soil rhizobia on nodulation and N₂ fixation in faba bean (Vicia faba L.).

Methods

Faba beans were inoculated with one of seven rates of rhizobial inoculation, from no inoculant to 100 times the normal rate of inoculation, sown at two field sites, with or without soil rhizobia present, and their nodulation and N₂ fixation assessed.

Results

At the site without soil rhizobia, inoculation increased nodule number and increased N₂ fixation from 21 to 129 kg shoot N ha^?1, while N₂ fixation increased from 132 to 218 kg shoot N ha^?1 at the site with high background soil rhizobia. At the site without soil rhizobia, inoculation increased concentrations of shoot N from 14 to 24 mg g^?1, grain N from 32 to 45 mg g^?1, and grain yields by 1.0 Mg (metric tonne) ha^?1. Differences in nodulation influenced the contributions of fixed N to the system, which varied from the net removal of 20 kg N ha^?1 from the system in the absence of rhizobia, to a net maximum input of 199 kg N ha^?1 from legume shoot and root residues, after accounting for removal of N in grain harvest.

Conclusions

The impact of inoculation and soil rhizobia strongly influenced grain yield, grain N concentration and the potential contributions of legume cropping to soil N fertility. In soil with resident rhizobia, N₂ fixation was improved only with the highest inoculation rate.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Influence of analogue-resistant mutants ofAzotobacter chroococcum solubilizing phosphate on yield and quality of sunflower (Helianthus annuus)

Two phosphate-solubilizing analogue-resistant mutants ofAzotobacter chroococcum showed a positive interactive effect on sunflower (Helianthus annuus). Plant height, leaf area, seed mass, seed yield, protein and oil content increased significantly by inoculation withA. chroococcum mutants. Both levels of fertilizers applied (80N+30P₂O₅ and 80N+60P₂O₅ kg/hm² showed increased seed yield. Likewise, inoculation resulted in a significantly higher yield. Application ofP-solubilizingA. chroococcum strains with the lower fertilizer level showed no significant difference on plant growth parameters when compared with the higher fertilizer level, indicating that inoculation of P-solubilizingAzotobacter strains can reduce the necessary levels of fertilization from 60P₂O₅ to 30P₂O₅ kg/hm².     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.