Experiments on nitrogen fixation by nodulated lucerne

Summary Field experiments at five sites (two on clay loam, two on a sandy soil and one on chalky loam) compared dry-matter yields and nitrogen contents of lucerne and ryegrass. These tested Rhizobium inoculation and fertilizer treatments in an experimental design proposed by Vincent and Nutman (IBP 69(66), amended 57/68). The crop and inoculation treatments (using large inocula of effective or ineffective strains) were set out in strips across each site to minimise contamination, and the fertilizer treatments (nitrogen, limeetc.) distributed at random within strips. This proved satisfactory and allowed valid statistical analysis. The massive inoculation with the ineffective strain suppressed the few naturally occurring effectiveR. meliloti at some of the sites, and provided a basis of comparison for assessing the amounts of nitrogen fixed by the native strains and by the introduced effective strains. However, it did so only during the first year because the introduced ineffective strains were overgrown by effective strains in the second year. Hence, ryegrass without nitrogen fertilizer was a better measure of the amounts of nitrogen obtainable from the soil. Estimates of fixation ranged from 0 to more than 300 kg N ha⁻¹ at the different sites, treatment comparisons indicating the main factors limiting fixation. Parallel experiments were done using a soil core technique described by Vincent. These gave very similar results to the field trials in their first years. Studies of the changes in the populations ofR. meliloti in the field trial plots and soil cores gave information on the effects of crop and fertilizers on numbers and effectiveness of isolates and was useful in interpreting the results.     

The influence of the phosphorus and nitrogen nutrition of pea plants on the growth of their progeny

Summary In glasshouse experiments with peas grown in nutrient cultures, seed from phosphorus-deficient plants contained much lower phosphorus concentrations than seed from plants not deficient in this element, but differences in the supply of mineral nitrogen to the plants (which were nodulated) had little effect on the chemical composition of the seed.Lower haulm weights and pea yields were obtained from sowings of low-phosphorus as compared with high-phosphorus seed when the plants were grown in cultures deficient in phosphorus. In cultures not deficient in phosphorus, there were no such differences between the performance of the seed.In field experiments carried out on a fertile soil, low-phosphorus seeds gave 20 to 25 per cent lower yields of both haulms and peas than were obtained from sowings of high-phosphorus seed.It appeared that the differences in the performance of the seeds weer related to differences in the concentration of phosphorus they contained rather than to any other variation in their composition or size.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: Host-induced ineffectivity inPisum sativum ecotype fulvum

Summary Pisum sativum ecotype fulvum forms ineffective nodules with Rhizobium strains, isolated from effective nodules of the cultivated pea in Europe. Rhizobium strains isolated from nodules of fulvum peas in Israel are fully effective on this host plant, but in association with the cultivated pea they induce nodules of poor N₂-fixing activity. The distribution of these fulvum-specific Rhizobium strains is restricted to regions where the fulvum pea occurs naturally. Rhizobium strains from other geographical regions induce mainly ineffective, or partially effective nodules on fulvum plants.A wide genetic variation, with regard to symbiotic response to a standard set of Rhizobium strains, was demonstrated in the fulvum plants collected in Israel. Based on variation in N₂-fixation three groups of plants can be distinguished. These plants offer the possibility for the study of host-genetic control on symbiotic nitrogen fixation.     

Soil P-status and cultivar maturity effects on pea-Rhizobium symbiosis

In a green-house experiment, five cultivars of Pisum sativum L. grown on soils from 10 different locations in Tunisia, showed significant differences in nodulation, shoot dry matter (shDM) yield and shoot nitrogen content (shNC). The effect of soil on biological nitrogen fixation, as evidenced by the number and weight of nodules, was mainly attributable to the available phosphorus content. Cate-Nelson ANOVA analysis established a critical value of soil test phosphorus (STP) of 20 mg P kg^–1 soil for nodule weight and number for the majority of cultivars. Within cultivars, nodulation varied with maturation period and was correlated with shoot NC. Thus, the overall interaction of soil-P content and cultivar-maturation period were correlated positively with nodulation and to symbiotic effectiveness of strains of Rhizobium leguminosarum bv. viceae indigenous to these soils. Based on an antibiotic susceptibility test and main variable factor analysis of the data obtained, 70 isolates of Rhizobia that nodulate pea, obtained from soils from agricultural sites throughout Tunisia, were identified as belonging to 18 distinct strains. These classes were identified on the basis of symbiotic efficiency parameters (shoot DM yield and shoot NC) as: ineffective (33 isolates), moderately effective (27 isolates), and efficient strains (10 isolates). This study shows that the Mateur site, an agricultural area for millennia in the northern region of Tunisia, harbors rhizobial strains that are highly efficient in fixing N₂ with peas. These results also indicate the importance of strain-cultivar interrelationships and specificity.     

Effect of Arsenic on Nodulation and Nitrogen Fixation of Blackgram (<Emphasis Type="Italic">Vigna mungo</Emphasis>)

Rhizobium–legume symbiotic interaction is an efficient model system for soil remediation and reclamation. We earlier isolated an arsenic (As) (2.8 mM arsenate) tolerant and symbiotically effective Rhizobium strain, VMA301 from Vigna mungo and in this study we further characterized its efficacy for arsenic removal from the soil and its nitrogen fixation capacity. Although nodule formation is delayed in plants with As-treated composite when the inoculum was prepared without arsenic in culture medium, whereas it attains the significant number of nodules compare to plant grown in As-free soil when the inoculum was prepared with arsenic supplemented medium. Arsenic accumulation was higher in roots than root nodules. Nitrogenase activity is reduced to almost 2 fold in plants with As-treated soil but not abolished. These results suggest that this strain, VMA301, has been able to establish an effective symbiotic interaction in V. mungo in As-contaminated soil and can perform dual role of arsenic bioremediation as well as soil nitrogen improvement.     

A comparison of the fluorescent ELISA and antibiotic resistance identification techniques for use in ecological experiments with Rhizobium trifolii

The fluorescent ELISA technique for the identification of bacteria was compared with antibiotic resistant mutants as marker systems for use with Rhizobium trifolii in root nodules and in soil. With an effective(CP3B) and an ineffective (R4) strain as a mixed 1:1 inoculum, there was a highly significant correlation ( P < 0.001) between the two techniques when the plants were grown at pH 5.5 when the majority of nodules were inhabited by the effective strain. At pH 6.5, where the ineffective strain predominated in the nodules, there was no correlation. The reason was that 85% of R4 nodules had volumes less than 0.1 mm₃ with bacterial numbers obviously below the necessary threshold for detection using the serological method. Both methods were efficient at enumerating rhizobia from soils although the recovery rate from a brown earth soil was significantly higher than from a peat soil. Fluorescent ELISA was able to detect rhizobia at 8.0 times 10₅ cells/ml soil suspension (1 g soil to 10 ml water) in the brown earth soil and at 2.0 times 10₅ cells/ml in the peat soil. The results are discussed in terms of the limitations of both techniques in ecological studies.     

Fitness variation among host species and the paradox of ineffective rhizobia

Legumes can preferentially select beneficial rhizobial symbionts and sanction ineffective strains that fail to fix nitrogen. Yet paradoxically, rhizobial populations vary from highly beneficial to ineffective in natural and agricultural soils. Classic models of symbiosis focus on the single dimension of symbiont cost‐benefit to sympatric hosts, but fail to explain the widespread persistence of ineffective rhizobia. Here, we test a novel framework predicting that spatio‐temporal and community dynamics can maintain ineffective strains in rhizobial populations. We used clonal and multistrain inoculations and quantitative culturing to investigate the relative fitness of four focal Bradyrhizobium strains varying from effective to ineffective on Acmispon strigosus. We found that an ineffective Bradyrhizobium strain can be sanctioned by its native A. strigosus host across the host's range, forming fewer and smaller nodules compared to beneficial strains. But the same ineffective Bradyrhizobium strain exhibits a nearly opposite pattern on the broadly sympatric host Acmispon wrangelianus, forming large nodules in both clonal and multistrain inoculations. These data suggest that community‐level effects could favour the persistence of ineffective rhizobia and contribute to variation in symbiotic nitrogen fixation.     

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.     

The effect of oxamyl on the growth of peas attacked by pea cyst nematode

Oxamyl treatment at drilling greatly increased the growth of the plants, the yield and quality of the peas harvested, in soil infested by small or large numbers of Heterodera goettingiana. Treated plants were continuing to set pods when non-treated plants were senescing. Rhizobium nodulation, sap nitrate and tissue nitrogen content were increased in treated plants. Oxamyl applied to growing plants reduced later nematode invasion and enabled root nodulation to proceed with an increase in tissue nitrogen but no increase in yield. Oxamyl applied at drilling in April was inactive against invasion by H. goettingiana in June, which enabled the nematode to multiply equally in treated and non-treated plots. The most economic treatments were at the smallest rates, giving the greatest increase in yield/kg of oxamyl applied.     

The effect of nitrogen source on levels of amino acids in peas

Summary When nitrogen fixation in peas was partially replaced by nitrate assimilation as the source of nitrogen, an increase was found in the amount of soluble nitrogen that could be extracted from the fruits of the plants, and within this soluble fraction, increases were found in the levels of some of the acidic amino acids. Levels of protein amino acids in the peas were generally unaffected by the type of nitrogen source except for the level of aspartic acid which was about 20 per cent lower in peas supplied with nitrate. No differences were found in the proportions of the essential amino acids.     

Field observations on nitrogen catch crops. III. Transfer of nitrogen to the succeeding main crop

In temperate climates with a precipitation surplus during autumn and winter, nitrogen (N) catch crops can help to reduce nitrogen losses from cropping systems by absorbing nitrogen from the soil and transfer it to a following main crop. In two field experiments the catch crop species winter rye (Secale cereale) and forage rape (Brassica napus ssp. oleifera (Metzg.) Sinsk) or oil radish (Raphanus sativus spp. oleiferus (DC.) Metzg.) were planted end of August and 3 weeks later with a non-limiting supply of N and zero-N controls. In the next spring catch crops were incorporated into the soil. In Expt 1, N transfer was measured as (i) the N uptake of a potato test crop, grown with zero and 12.5 g m^–2 N applied, and (ii) the increase in soil mineral N (0–30 cm) in uncropped soil covered with polythene film. In Expt 2, N transfer was measured as the increase in soil mineral N in covered cylinders placed in uncropped soil (in situ incubation). Subsidiary laboratory incubations were performed in Expt 2. In Expt 1, the apparent recovery in potato of fertilizer N (R _f) was 0.56. The recovery in potato of N mineralized from 'native' N pools other than catch crop material (R _n) ranged from 0.43 to 0.51, depending on the value assumed for the depth of N extraction by potato roots. The average recovery in potato of incorporated catch crop N (R _c) was 0.34. Expressed as `fertilizer N replacement factor' (F _r) the latter was 0.61 (i.e. 1 kg of N in catch crop material counts for 0.61 kg fertilizer N). Under the film in Expt 1 the fraction net mineralization of incorporated catch crop N (M _n) was 0.36 on August 11 and 0.43 on October 18. In Expt 2, the average value of M _n was 0.31, which was lower than in Expt 1 and probably associated with the drier soil in Expt 2. In the laboratory incubations (20°C) M _n showed values up to 0.54 after 84 days with the largest rates of change in mineralization occuring early after the start of the incubation. In conjunction with literature data it is concluded that cultivation of nitrogen catch crops shows promise as a means to reduce N input and N losses in temperate climates with wet winters.     

酸性旱地红壤无机氮同化菌株筛选及其全基因组分析

微生物执行的无机氮同化作用可固定施入土壤后未被作物直接吸收的化学氮肥,有效减少化学氮肥损失、降低环境氮素污染风险。土壤无机氮同化作用不是由大量冗余微生物共同执行的,而是由一小部分功能微生物优先执行。【目的】对酸性旱地红壤中的优势无机氮同化细菌进行富集、菌株分离鉴定及全基因组测序,并明确菌株在土壤中的氮同化能力,为酸性土壤化学氮肥应用及其转化过程研究提供菌株资源和理论依据。【方法】在酸性旱地红壤中添加KNO3或(NH4)2SO4作为无机氮源,以葡萄糖作为碳源,在好氧条件下进行富集预培养,采用稀释分离法筛选出优势无机氮同化细菌菌株;将菌株回接至土壤中从而验证其无机氮同化能力,并通过全基因组测序分析菌株的氮素代谢途径及相关功能基因。【结果】酸性旱地红壤经富集预培养一周后,优势无机氮同化微生物的16SrRNA基因相对丰度从0.20%–0.94%增长至20.2%–30.2%;分离筛选后得到的3株优势无机氮同化细菌菌株,鉴定为伯克霍尔德氏菌(Burkholderia sp.) M6-3、索状芽孢杆菌(Bacillus funiculus) M2-4和节杆菌(Arthrobacter sp.) M7...     

Competition ofRhizobium strains in nodule-formation

Summary By the use of a sterile culture system experiments have been carried out with peas in order to elucidate to what extent inoculation with an ineffective bacterial strain effects the ability of an effective strain to produce further nodulation. It has been established that effective strains, when applied to the plant after the formation of the first nodules by an ineffective strain, altogether fail to form nodules or the nodulation is delayed very much. Differences are noted in this respect between different bacterial strains. The results can be explained easiest by assuming that the strain first entering the roots causes an immunity in the plant against later infections. Other possibilities for explanation are also discussed. The conception that the resistance would be due to the saturation of roots with nodules formed by the first strain which would inhibit further nodulation is not in accordance with the results recorded.A method has been introduced for cultivation of plants under sterile conditions by dividing the roots into two or more culture flasks. This method is of great value when the nutrient uptake of plants is elucidated. Successive inoculation with ineffective and effective strains gave varying results by this technique. The experiments seem to imply that if only a part of the root system is inoculated with an ineffective strain, immunity does not regularly occur against later infections by effective strains. This suggests that the immunity is a somewhat local phenomenon.     

A mathematical approach towards nitrogen cycling in arable land inCajanus cajan field

Summary A balance sheet of nitrogen has been presented for an arable land in aCajanus cajan field. Quantitative inventory and transfer rates are suggested for several compartments. On the basis of data, it has been concluded that due to microbial activities appreciable nitrogen was returned to the available nitrogen pool by means of root and litter disappearance. Twenty-five percent of the total soil nitrogen has been taken up by fine roots, out of which 63.39 percent nitrogen locked up in the form of organic nitrogen and the rest of nitrogen (36.61%) was being added to the soil which governed the soil fertility. Transfer matrix of process model for the nitrogen transformation explaining component transferred from one compartment to another compartment and groups responsible for this transformation are dealt with. By means of several proposed equations the amount of nitrogen in soil at different intervals of time can be predicted. The quantifications of microbial transformations and their role in nitrogen turnover has been exploited. A high nitrogen content in the original material was found to promote decomposition.     

Role of uranium speciation in the uptake and translocation of uranium by plants

Uranium (U) uptake and translocation by plants was characterized using a computer speciation model to develop a nutrient culture system that provided U as a single predominant species in solution. A hydroponic uptake study determined that at pH 5.0, the uranyl (UO²⁺2) cation was more readily taken up and translocated by peas (Pisum sativum) than the hydroxyl and carbonate U complexes present in the solution at pH 6.0 and 8.0, respectively. A subsequent experiment tested the extent to which various monocot and dicot species take up and translocate the uranyl cation. Of the species screened, tepary bean (Phaseolus acutifolius) and red beet (Beta vulgaris) were the species showing the greatest accumulation of U. IN addition to providing fundamental information regarding U uptake by plants, the results obtained also have implications for the phytoremediation of U-contaminated soils. The initial characterization of U uptake by peas suggested that in the field, a soil pH of <5.5 would be required in order to provide U in the most plant-available form. A pot study using U-contaminated soil was therefore conducted to assess the extent to which two soil amendments, HEDTA and citric acid, were capable of acidifying the soil, increasing U solubility, and enhancing U uptake by red beet. Of these two amendments, only citric acid proved effective, decreasing the soil pH to 5.0 and increasing U accumulation by a factor of 14. The results of this pot study provide a basis for the development of an effective phytoremediation strategy for U-contaminated soils.     

Acetylene reduction by effective and ineffective clover and soybean root nodules

Summary Acetylene was reduced to ethylene by effective white clover nodules and by fully and partially effective intact nodules, nodule homogenates, and bacteroids of soybeans. Succinate and several amino acids markedly stimulated the reduction by effective soybean bacteroids, but the stimulation was slight with partially effective bacteroids. Acetylene metabolism by effective soybean bacteroids was also enhanced by excretions of in vitro-grown Rhizobium japonicum, excretions of bacteria derived from effective and ineffective nodules, and the soluble fraction from these nodules. Inhibitors of nitrogen fixation were not found in ineffective nodules. Ineffective soybean and white clover nodules and homogenates or isolated bacteria from ineffective soybean nodules did not reduce acetylene. Additions of succinate, amino acids, the soluble fraction of effective nodules, or excretions of effective bacteroids or of in vitro-grown cells of an effective R. japonicum strain did not promote nitrogen fixation by bacterial cells obtained from ineffective soybean nodules.     

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.     

Symbiotic effectiveness of Bradyrhizobium strains isolated from Parasponia and tropical legumes on Parasponia host species

The symbiotic effectiveness of Bradyrhizobium strains isolated from three species of Parasponia and from legumes were compared on Parasponia grown in Leonard-jars. Effectiveness of each symbiotic association was estimated from dry weight and total nitrogen of shoots and nodules of plants grown on medium free of combined nitrogen. Twenty strains isolated from three species of Parasponia were found to vary in their effectiveness on P. andersonii, the least effective fixing one fifth of the nitrogen of the most effective strains. The outcome of the symbiosis was not associated with the host source of the test strain. P. andersonii, P. rugosa and P. rigida responded differently to a selection of seven strains of Parasponia Bradyrhizobium; some strains were either ineffective or fully effective on each host, while others varied in their symbiotic performance. P. andersonii fixed significantly (P < 0.001) larger quantities of nitrogen than either P. rugosa or P. rigida with p. rigida being the least effective. In contrast to Bradyrhizobium strains from Parasponia spp. which formed nodules rapidly (within 11–20 days), nine strains isolated from legumes required between 25 and 74 days to form partially effective nodules. The thre Parasponia species formed relatively large quantities of nodule tissue relative to the amount of nitrogen fixed and shoot dry matter produced. The Bradyrhizobium isolated from Parasponia plants growing in Papua New Guinea soils could be grouped together on the basis of their infection characteristics on Parasponia and legumes.     

Field experiments on nitrogen fixation by nodulated legumes

Summary Phosphate increased nitrogen uptake by lucerne appreciably on a saline soil. Nitrogenous fertiliser or inoculation with an effective strain ofRhizobium meliloti did not increase the yield significantly. In soils where indigenousRhizobium japonicum was absent inoculation increased soybean yields and the additional fixed nitrogen removed by soybeans amounted to 40 to 120 kg ha⁻¹. Gram and groundnut also responded to Rhizobium inoculation in field trials.     

Modelling partitioning between structure and storage in sugar beet: Effects of drought and soil nitrogen

In the UK sugar beet is grown on contrasting soils that vary both in their nutritional status and water-holding capacities. Water shortage and low nitrogen reduce canopy growth and dry matter production, which is compensated in part by an increase in the fraction of assimilates partitioned to storage. Conversely, high nitrogen and ample water encourage growth of the canopy, increase assimilation of carbon dioxide but reduce the proportion of assimilates stored as sugar. This paper sets out to examine simple relationships between sugar yield, total dry matter and soil nitrogen in rain-fed and irrigated sugar beet crops (Beta vulgaris L.) from 46 field experiments spanning 12 years and a range of soil types, in order to improve prediction of sugar yields. Two partitioning functions were fitted to the data. The first represents a useful alternative formulation of the allometric growth function that overcomes some of the difficulties in the interpretation of the parameters. This model adequately described the seasonal progress of sugar yield (Y) in relation to total dry matter (W), but it was difficult to postulate biological mechanisms as to how the parameters should vary in relation to varying soil nitrogen or to drought. The second partitioning function, given by Y = W − (1/k) log(1 + kW), also described the data well, but had the more useful parameter, k, the decay rate of the fraction of assimilates partitioned to structural matter. This was shown to be greater in crops which had experienced significant drought and was inversely proportional to the amount of nitrogen taken up by the crops. Relationships between k and amounts of nitrogen fertilizer applied and/or amounts of residual nitrogen in the soil at sowing, however, were more variable. These could be improved by additionally taking account of soil type and rainfall following nitrogen fertilizer application in late spring. The models are a useful extension to yield forecasting models because they provide a simple means of estimating sugar yield from total dry matter in relation to factors that affect partitioning of assimilates such as drought and soil nitrogen availability. This revised version was published online in June 2006 with corrections to the Cover Date.