Susceptibility of alfalfa (Medicago saliva L. cv. Aragon) nodules and leaves to water stress has been investigated. Nodule acetylene reduction activity (ARA), leaf CO2 exchange rate (CER) as well as soluble protein, proline and total soluble sugar (TSS) contents were determined during drought. Water status was estimated as water potential (Ψw) and Relative water content (RWC) of the respective tissues. Maximum rates of ARA required higher Ψw than CER. Nodules had lower RWC for a given Ψw than leaves. Water stress reduced soluble protein content in both tissues; however, the decline in soluble protein content was detected at greater Ψw in nodules than in leaves. Proline and TSS increased in leaves and nodules, and again the threshold Ψw triggering such accumulation was higher in nodule tissues. Oior results suggest that alfalfa nodules are more susceptible to water shortage than leaves. Effects of accumulated TSS and proline upon leaf and nodule physiology are discussed in relation to protein stability (proline), pH control (proline) and osmotic adjustment (proiine and TSS). The TSS accumulation induced by water stress suggests that substrate shortage would not be the primary effect of drought on nodule activity.

Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativd) plants

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

In performing phenotypic characterization tests, the organisms used for the inoculation of test media should be from fresh transfers and in good physiological condition. The methods described in this chapter are based primarily on methods developed for the characterization and identification of organisms which have usually been isolated on nutrient-rich media. While zoologists may be content to classify all insects or all vertebrates, prokaryote systematics generally deals with all prokaryotes and in an evolutionary context attempts to extend this study back to the origin of life itself. To test for acetamide hydrolysis, streak the surface of an acetamide agar slant with a sample from a dilute suspension. Incubate for up to 7 days. Care should be taken to ensure that the basal medium to which the carbohydrates are added does not contain fermentable or oxidizable sugars, and control media without any added carbohydrates should always be included during testing of cultures. Methanogenic members of the Archaea also fluoresce under UV light. Some facultative anaerobes, such as Escherichia coli and Staphylococcus aureus, grow aerobically by respiration with oxygen and grow anaerobically by fermentation.

Phenotypic Characterization and the Principles of Comparative Systematics

Human activities have clearly caused dramatic alterations of the terrestrial nitrogen cycle, and analyses of the extent and effects of such changes are now common in the scientific literature. However, any attempt to evaluate N cycling processes within ecosystems, as well as anthropogenic influences on the N cycle, requires an understanding of the magnitude of inputs via biological nitrogen fixation (BNF). Although there have been many studies addressing the microbiology, physiology, and magnitude of N fixation at local scales, there are very few estimates of BNF over large scales. We utilized >100 preexisting published estimates of BNF to generate biome- and global-level estimates of biological N fixation. We also used net primary productivity (NPP) and evapotranspiration (ET) estimates from the Century terrestrial ecosystem model to examine global relationships between these variables and BNF as well as to compare observed and Century-modeled BNF. Our data-based estimates showed a strong positive relationship between ecosystem ET and BNF, and our analyses suggest that while the model's simple relationships for BNF predict broad scale patterns, they do not capture much of the variability or magnitude of published rates. Patterns of BNF were also similar to patterns of ecosystem NPP. Our “best estimate” of potential nitrogen fixation by natural ecosystems is ∼195 Tg N yr−1, with a range of 100–290 Tg N yr−1. Although these estimates do not account for the decrease in natural N fixation due to cultivation, this would not dramatically alter our estimate, as the greatest reductions in area have occurred in systems characterized by relatively low rates of N fixation (e.g., grasslands). Although our estimate of BNF in natural ecosystems is similar to previously published estimates of terrestrial BNF, we believe that this study provides a more documented, constrained estimate of this important flux.

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Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems

Publisher Summary This chapter describes the various aspects of cereal-legume intercropping systems Intercropping is the growing of two or more crop species simultaneously in the same field during a growing season The intercropping of legumes with cereals offers scope for developing energy-efficient and sustainable agriculture The main types of intercropping include mixed intercropping, row intercropping, and strip intercropping Crop combinations differ with geographical location and there may be intercropping of tree crops, intercropping of tree and field crops, or intercropping of field crops Combinations of crops are determined primarily by the length of the growing season and the adaptation of crops to particular environments Different indices have been suggested for evaluating productivity and efficiency per unit area of land of cereal-legume intercrop systems These include comparisons of absolute yields, protein yields, caloric equivalent, and in economic terms, gross returns from intercrops and sole crops Differences in competitive ability affect the relative performance of component crops and thus the land equivalent ratio values of different cereal-legume intercrop systems It is found that phosphorus is a major nutrient that determines the production potential of most grain legumes usually intercropped with cereals

Cereal–Legume Intercropping Systems

1. Characteristics and methodology of the C2H2-C2H4 assay forin situ measurement of N2 fixation are outlined. 2. Electron micrographic analysis of the developmental morphology of the natural soybean symbiosis and C2H2-C2H4 analysis indicate that increasing N2-fixing activity from 12–35 days of age is accompanied by an increase in bacteroid number per cell, bacteroid number per vesicle and inclusions per bacteroid. The mole ratio of leghemoglobin to nitrogenase also increases from 50 to a relatively constant plateau of 500 to 1500 during this period. The quantitative validity of the C2H2-C2H4 assay as a measure of N2 fixation during a complete growth cycle of soybeans on nitrogen-free medium is demonstrated by Σ (C2H2→C2H4)×28/3 values which are 75–95% of the values determined for N2 fixed by Kjeldahl analyses. 3. A technique for the establishment of the first callus N2-fixing symbiosis in mixed cultures ofRhizobium legume provides a defined experimental system for exploration of legume symbiosis. N2-fixing activity is about 1% of the natural system and is influenced by exogenous auxins and cytokinins. Morphology, including infection threads and vesicle enclosed bacteroids, is similar to the nodule system. 4. N2-fixing activity of field-grown soybeans, including varieties which differed in flowering characteristics and maturity dates, and of peanuts was determined biweekly with the C2H2-C2H4 assay. Activity extended from nodule initiation to senescence and correlated with the nitrogen demands of the plant and in most cases >90% of the N2 fixed during the 60–70 day period of fruit formation and maturation. A logarithmic relationship between N2-fixing activity and age, and N2 fixed and age was demonstrated as a fundamental characteristic of these annual symbionts,i.e. log N2 fixed =k(t−t
 0), wheret
 0 is age at activity initiation. The resultant parameters: 1) age at activity initiation, 2) calculated rate of daily increase (7–9% for soybeans and 7–10% for peanuts), 3) age at end of logarithmic phase (about 80 days for soybeans), and 4) total N2 fixed (about 250 mg per soybean plant) are useful bases for evaluation of environmental, bacterial and host effects on N2 fixation. Various N fertilizers applied at planting and flowering inhibited N2 fixation of soybeans by decreasing the rate of daily increase. 5. Physical and chemical characteristics of nitrogenase, including those of crystalline Mo-Fe protein, reactions of nitrogenase, and model studies are consistent with a proposed mechanism. 6. Potential utilities of N2 fixation research include increased food protein production via initially enhanced N2 fixation of legumes such as soybeans and eventually extension of N2-fixing symbioses to non-legumes and new chemistry of N2, including the direct incorporation of aerial N2 into important organic compounds.

Biological nitrogen fixation: A key to world protein

A symbiotic nitrogen-fixing relationship has been established in culture between soybean cells and the bacterium Rhizobium.

Establishment of symbiosis between Rhizobium and plant cells in vitro.

OUR earlier communication1 presented evidence that important biological consequences accrue from the presence of stable chemical products of radiolysis which are derived from sugar in the ambient culture media of plant cells or tissue explants. These effects became evident because they neutralized the effect of growth stimulatory substances which induce cell division and cause otherwise quiescent cells to grow rapidly. Thus the potency of the effect of radiated sugar could be assayed. We also showed that growth in the presence of the radiolysis products of sugar may result in abnormal mitotic and meiotic chromosomes in other types of material (root tips and pollen mother cells).

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R. D. Holsten

Papers

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Biological nitrogen fixation: A key to world protein

Establishment of symbiosis between Rhizobium and plant cells in vitro.

Direct and Indirect Effects of Radiation: the Radiolysis of Sugar