Effects of low temperatures on nitrogenase activity in sainfoin (Onobrychis viciifolia) nodulated by arctic rhizobia

The temperate forage legume sainfoin (Onobrychis viciifolia) is readly nodulated by rhizobia isolated from arctic legumes (Astragalus and Oxytropis species). We have investigated the effects of low temperatures on nitrogenase activity in sainfoin nodulated by arctic and temperate (homologous) rhizobia. At low temperatures, nitrogenase activity of arctic rhizobia measured either with detached nodules or with whole plants, was higher than that of temperate rhizobia. At 5°C and 10°C, nitrogenase activity values of arctic rhizobia represented 12% and 33% of those measured at 20°C, while lower values of 3.7% and 22.4% were observed with temperate rhizobia. This cold adaptation was also reflected on bacterial growth where, at 5°C and 10°C, arctic rhizobia showed a shorter doubling time and synthesized more protein than temperate rhizobia.     

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia)

Summary Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif ^r showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.     

Influence of rhizobia,azotobacter and blue-green algae on N content and yield of rice

Summary Inoculation with root-nodule bacteria had favourable influence on N-uptake and yield of wheat. Since waterlogged root region of rice permits higher nitrogenase activity a pot culture experiment was conducted using same nine strains of rhizobia,Azotobacter chroococcum and bluegreen algae as inoculants.R. leguminosarum in combination with 50 kg N ha⁻¹;R. japonicum and a strain of rhizobium isolated from moong bean increased the yield of paddy cv. Pusa-33. On the other hand an adverse effect of bacterial inoculation and of applied N was observed in case of Azotobacter, and rhizobia isolated from green gram, cicer, soyabean and clover. The importance of plant type, growth conditions and application of inorganic N in determining the success of plant-rhizobial associations is emphasised.     

Amount of nitrogen fixed by forage,pasture and grain legumes in Cyprus,estimated by the A-value and a modified difference method

The increasing need for protein at low cost has created a need to evaluate the biological nitrogen fixing potential of legumes in Cyprus. In field studies which were conducted over the growing years of 1982–3 and 1983–4, legumes which are traditionally grown in the country were evaluated for dry matter and nitrogen yield and biological nitrogen fixation (BNF). The legumes studied were medic (Medicago truncatula Gearth), ochrus vetch (Lathyrus ochrus L.), bitter vetch (Vicia ervilia L.) and faba bean (Vicia faba L. var major) in the first year and in addition chickpea (Cicer arietinum L.), woollypod vetch (Vicia dasycarpa Ten.) and tickbean (Vicia faba L. var minor) in the second year. Using the A-value method with barley and oats as reference crops, nitrogen (N) fixed by the various legumes in the first year was 30–50% and from 55–67% of total N yield for the two reference crops, respectively. In the second year the estimates of N fixed ranged from 70 to 80% with similar results obtained for the two reference crops barley and ryegrass. However, in the second year chickpea, which had limited nodulation, fixed only 40% of its N yield. Estimates of nitrogen from the atmosphere (Ndfa) obtained by the difference method (DM) were 10 to 14% lower than those from the A-value method. These results were obtained after correcting for the amount of N derived from the applied fertilizer. The two methods were highly correlated (r=0.98) for estimates of amount of BNF. The rates of N₂ fixation of uninoculated legumes which are nodulated by the indigenous populations of Rhizobium in Cyprus are comparable to those of legumes inoculated with selected strains of Rhizobium in other countries. An exception was the amount of N fixed by chickpea. The appearance of the first nodules at late stages of growth may be the reason for the low BNF of this crop.     

Ecological roles of arbuscular mycorrhizal fungi in two wild legume plants

Two wild legume plants,Glycine soja andCassia mimosoides var.nomame, and a cultivated plant, soybean (Glycine max), were employed for a study of triple symbiosis with an inoculum ofScutellispora heterogama harvested from natural soils and an inoculum of their own rhizobial cells. The dry weight, colonization of arbuscular mycorrhizal fungus, nodule formation and N₂-fixation activity were estimated as the parameters of triple symbiosis. The two wild legume plants showed greater growth with colonization of arbuscular mycorrhizae than with nodulation, whereas the cultivated legume showed more nodulation than colonization of arbuscular mycorrhizae. Moreover,S. heterogama appeared to stimulate the triple symbiosis for the wild legume plants. The results suggested that spores ofS. heterogama are important in disturbed soils in Korea.     

Manipulation of rhizobia microflora for improving legume productivity and soil fertility: A critical assessment

Inputs of biologically fixed nitrogen derived from the symbiotic relationship between legumes and their root-nodule bacteria into terrestrial ecosystems amount to at least 70 million metric tons per year. It is obvious that this enormous quantity will need to be augmented as the world's population increases and as the natural resources that supply fertilizer nitrogen diminish. This objective will be achieved through the development of superior legume varieties, improvement in agronomic practice, and increased efficiency of the nitrogen fixation process itself by better management of the symbiotic relationship between plant and bacteria. This paper considers ways and means by which populations of root-nodule bacteria, established and introduced, can be manipulated ecologically, agronomically, edaphically and genetically to improve legume productivity and, as a consequence, soil fertility.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Identification of nitrogen-fixing enterobacteria from living Sassafras (Atherosperma moschatum Labill.) trees

Nitrogen-fixing enterobacteria (Enterobacter agglomerans and Citrobacter freundii) were commonly found associated with the microflora of stained Sassafras (Atherosperma moschatum Labill.) in forests of SE Tasmania. However, their populations never exceeded 3× 10⁴ cells g⁻¹ dry wood and comprised at most 3% of the total bacterial flora. Bacterial colonization of the wood appeared to coincide with that by non-hymenocetous fungi: bacteria were never isolated in significant numbers from wood not infected with fungi. The contribution of the N₂-fixing flora to the N economy of the habitat is considered to be negligible.     

Algal nitrogen fixation on temperate arable fields

Summary N₂-fixation by algae on the Broadbalk continuous wheat experiment was measured over a two year period using the acetylene reduction technique. The plots studied receive spring fertilizer treatments including farmyard manure and combinations of nitrochalk and Na, P, K and Mg which have remained much the same since the experiment started in 1843.Nitrogen applied at 196 kg ha^–1 in spring suppressed algal N₂-fixation until late in the season but at lower levels (48 kg N ha^–1) the denser plant canopy increased both surface moisture and fixation. Herbicide treatment decreased fixation on plots of moderate nutritional status early in the season but had little effect on unfertilised plots where weed cover was sparse. On plots where weed and crop cover was very dense herbicide treatment increased fixation in August.Algal N₂-ase activity, assayed by C₂H₂ reduction, continued throughout the night at a rate which averaged 33% of the midday value. Laboratory experiments indicate that dark fixation is very temperature sensitive and this value may represent a maximum. Algal crust in the field dried to 4.5–6.8% H₂O content became active 3 1/2 h after rewetting and reached a steady state after 7 h which represented only 6–22% of that at the previous maximum suggesting that many cells had been killed.In a year with average rainfall algae on plots receiving 48 kg N ha^–1 were estimated to fix 25–28 kg N ha^–1 and plots without fertiliser 13–19 kg N ha^–1. Algal fixation appeared to make a substantial contribution to the continuing fertility of unfertilised plots.     

Molecular phylogeny and ecogeography of Onobrychis viciifolia Scop. (Fabaceae) based on nrDNA ITS sequences and genomic ISSR fingerprinting

In this study 40 specimens from 8 representative Iranian populations of Onobrychis viciifolia SCOP. were collected from their natural habitats. The specimens were biometrically assessed using 43 quantitative and 15 qualitative morphological characters. At each sample site we recorded ecogeographical variables. In order to evaluate the difference between the variation due to phenotypic responses and genetic adaptation, we generated nucleotide sequence data from the internal transcribed spacer of the nuclear rDNA genes (ITS) and carried out genomic fingerprints using inter‐simple sequence repeat PCR (ISSR). Cluster analysis of morphological characters divided the eight populations into three major groups. Leaf length, leaflet length and width, calyx length, plant height, and stem length were introduced as diagnostic characters for three different morphological groups. Furthermore, canonical correspondence analysis (CCA) of ecogeographical data showed correlations between morphological variations and ecogeographical factors. Clay%, saturation percentage (SP), total neutralizing value (TNV%), texture, minimum temperature, and geographic separation were the main environmental variables associated with morphological groups of O. viciifolia. ISSR analysis revealed three main groups which are in correspondence with morphological groups, indicating that the three morphological groups have been shaped by genetic and phenotypic responses to environmental conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of sulphur nutrition on growth and nitrogen fixation of pea (Pisum sativum L.)

A S-deficient soil was used in pot experiments to investigate the effects of S addition on growth and N₂-fixation in pea (Pisum sativum L.). Addition of 100 mg S pot⁻¹ increased seed yield by more than 2-fold. Numbers of pods formed were the most sensitive yield component affected by S deficiency. Sulphur addition also increased the concentration of N in leaves and stems, and the total content of N in the shoots. The amounts of N fixed by pea were determined at four growth stages from stem elongation to maturity, using the ¹⁵N dilution technique. Sulphur addition doubled the amount of N fixed at all growth stages. In contrast, leaf chlorophyll content and shoot dry weight were increased significantly by S addition only after the flowering and pod fill stage, respectively. Pea roots were found to have high concentrations of S, reaching approximately 10 mg g⁻¹ dry weight and being 2.6–4.4 times the S concentration in the shoots under S-sufficient conditions. These results suggest that roots/nodules of pea have a high demand for S, and that N₂-fixation is very sensitive to S deficiency. The effects of S deficiency on pea growth were likely to be caused by the shortage of N, due to decreased N₂-fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

Rhizobial ecology of the woody legumePsorothamnus spinosus in a Sonoran Desert Arroyo

Psorothamnus spinosus seedlings were selected at random and excavated from 10×30 m quadrats along a 270 m transect in a southern Californian desert arroyo. Forty-six of the fifty seedlings excavated (92%) were nodulated. The mean number of nodules/plant was 7.0±3.6. Maximum rooting depth was 40 cm, and most (58.8%) of the nodules were found between 15 and 30 cm depth. The mean root/shoot ratio was 1.4±0.4. The rhizobial population density between 10 to 40 cm depth across all quadrats was non-detectable using a plant infection, most probable number method. All 70 isolates from field nodules were fast-growingRhizobium spp. Two colony morphologies were observed: wet, translucent, and wet, opaque. All isolates tested were effective on bothP. spinosus and another desert woody legume, mesquite (Prosopis glandulosa). Many isolates had a high relative efficiency, and seven of eight efficient isolates had uptake-hydrogenase activity. Root stimulation of the low rhizobial population found in the arroyo and subsequent root nodulation may be an important factor in seedling establishment by woody legumes in N-limited desert arroyo systems.     

Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar phaseoli isolated from soils of Rwanda

We have isolated 48 strains of Rhizobium leguminosarum biovar phaseoli from nodules of Phaseolus vulgaris L. cultivated on 32 different soils at 22 various locations in Rwanda, Central Africa. The symbiotic effectiveness of the strains was appraised in the greenhouse by measuring shoots dry matter and total plant nitrogen content after six weeks of growth. Of the strains tested 19%, 58% and 23% were rated very effective, effective and ineffective, respectively. A very significant correlation (r=0.96, P<0.01) was observed between shoots dry matter and total N content. By using the total nitrogen balance method, it was estimated that in the presence of a very effective strain, up to 86% of the N present in the shoots comes from N₂ fixation. No significant correlations were observed between the symbiotic effectiveness of the strains and the pH of the soils from which they originated, the tolerance of the strains to acidity or their ability to produce organic acids. The nine very effective strains selected were highly competitive against two ineffective strains with the two P. vulgaris cultivars Rubona-5 and Kiryumukwe.Contribution no 367, Station de recherches, Agriculture Canada.Contribution no 367, Station de recherches, Agriculture Canada.     

Pesticidal effect on soybean-rhizobia symbiosis

Summary Relative compatibility of selected pesticides at two levels of application (recommended rate and 5× or 10 ×) with soybean-rhizobia symbiosis was tested in pot culture experiments using a prepared peat inoculant.PCNB, carboxin and carboxin+captan at recommended level were innocuous to growth, nodulation, N₂-fixation and total N content of shoot. Carboxin and carboxin+captan but not PCNB at 10 times recommended level proved detrimental to nodulation and N₂-fixation. Carbaryl and malathion at recommended level had no adverse effect but at 10 times recommended level severely reduced N₂-fixation but not other parameters. Acephate, diazinon and toxaphene at both levels reduced N₂-fixation and total N content but not growth and nodulation. All five herbicides used at recommended and 5 times recommended level adversely affected nodulation and N₂-fixation. Glyphosate proved least toxic to all parameters. 2,4-DB at recommended level was less harmful to nodulation and N₂-fixation than trifluralin, alachlor and metribuzin.     

Occurrence of effective nitrogen-scavenging bacteria in the rhizosphere of kallar grass

Bacteria occurring in high numbers on the rhizoplane of kallar grass grown at a natural site in Pakistan were effective scavengers of traces of combined nitrogen from the atmosphere. Bacteria grew under appropriate conditions in nitrogen-free semi-solid malate medium in the form of a typical subsurface pellicle which resulted in a significant nitrogen gain in the medium within 3 to 4 days of incubation; this could be also measured by¹⁵N-dilution. Bacteria grew and incorporated nitrogen under an atmosphere containing NH₃ and N₂O. A rapid and strong binding of strain W1 to roots of kallar grass grown in hydroponic culture was found by using a³²P-tracer technique. We obtained no evidence for diazotrophy of our strains because they failed to grow on nitrogen-free media when gases of high purity were used. No¹⁵N₂ was incorporated when bacteria were grown on¹⁵N₂ although a nitrogen gain was found, no acetylene reduction was observed and no homology with DNA containing sequences ofnifHDK structural genes for the nitrogenase components fromKlebsiella pneumoniae were detected. Owing to close contact of these bacteria with roots of kallar grass, utilization of scavenged nitrogen by the plant may have to be taken into account.     

Discovery of the heliobacteria

The first photosynthetic bacterium obtained in pure culture wasRhodospirillum rubrum, isolated by Erwin Esmarch in 1887. The organism appeared to be an aerobic heterotroph, and Esmarch was unaware of its photosynthetic capability. The overall general characteristics of a number of major species of photosynthetic bacteria were described by Molisch and van Niel before 1945. Subsequently, our knowledge of the anoxygenic phototrophs increased greatly through the systematic study of numerous new species isolated from enrichment cultures in which capacity for anaerobic (and anoxygenic) growth with light as the energy source was a primary selective factor. A further refinement of the enrichment technique required ability to use N₂ as the sole source of nitrogen for growth under anaerobic photosynthetic conditions, and this led to the isolation of additional new species, including the heliobacteria. The first recognition of the heliobacteria was facilitated by serendipity, which was a significant factor in a number of other researches on photosynthetic bacteria (Gest 1992).