Variation among common-bean accessions (Phaseolus vulgaris L.) from the Iberian Peninsula for N2-dependent growth and phosphorus requirement

The common-bean often faces phosphorus deficiency in soils where it is grown. Such a deficiency is a major limitation to yield improvement, especially as the common-bean depends upon nitrogen fixation. Screening for symbiotic nitrogen fixation under phosphorus deficiency was performed with 33 common-bean accessions representing the diversity of 15 European market classes from the Iberian Peninsula. These accessions were inoculated withRhizobium tropici CIAT899 and grown in an aerated nitrogen-free nutrient solution at deficientversus sufficient phosphorous supplies (75 vs. 250 μmol plan⁻¹ week⁻¹) in a glasshouse. A large variability in N₂-dependent growth under P deficiency was found with most tolerance to P deficiency among late type IV Andean landraces, with the exception of 3 early type I Andean landraces. From this screening four contrasting landraces were selected for their high efficiency in the use of P for their symbiotic N nutrition, and compared with the cultivar Linex in fields of a reference production area over 3 years. The landraces from the Iberian Peninsula expressed a higher growth than the cultivar Linex, although they showed a lower nodulation. We hypothesize that the identified P tolerance among Iberian Peninsula accessions may be useful for improving symbiotic nitrogen fixation in the common-bean when growth is limited by available soil-P and could contribute to sustainable farming systems by reducing farmers’ dependence on fertilizers.     

Climatic and hydrological aspects of the Hula restoration project

Drying the Hula marshes (northern Israel) in the 1950s caused changes in sub-surface hydrology that severely compromised agricultural sustainability of the Hula Valley peatlands. The complex dynamics between climatic and hydrological regimes produced widely fluctuating elevations in the water table, leading to deterioration of the peat soils through enhanced aerobic decomposition, wind erosion, and underground fires. The year-round intensive and diverse cropping systems used in 1960s were gradually replaced in the 1980s by extensive winter forage crops, leaving the peat uncropped and exposed to increased deterioration in the dry season. The Hula Restoration Project was initiated in 1993 to moderate some of these after-effects of the draining of the Hula marshes. The project includes a 90-km network of water-level regulating supply and drainage canals and a 100-ha lake and a barrier to underground water flow from the lake to cultivated areas. We followed changes in underground water levels for the five years before and the three years after re-flooding of the valley in May 1994. Prior to re- flooding, water tables in the area fluctuated 2–4 m annually, depending on location and microrelief and there was a strong north to south flow of water. After re-flooding the lake initially had a strong influence on water table elevation in the adjacent areas, which later stabilized. The underground barrier shifted the predominant water flow westward toward a north-south canal near the edge of the valley and then southward along the canal. The restoration project has been successful up to now, there are few underground fires since re-flooding, and all agricultural lands can be cultivated al year long.     

Atmospheric nitrogen fixation by hydrocarbon-oxidizing bacteria

Microorganisms have been found which concomitantly convert hydrocarbons, selected naphthenic acids, and atmospheric nitrogen into cellular substance. Bacteria are included in the genera Pseudomonas, Mycobacterium, and Azotobacter. Carbon sources utilized include the hydrocarbons methane, n-butane, n-tetradecane, toluene, and a naphthenic acid, cyclohexane-carboxylate. Uptake of isotopic nitrogen was employed as a criterion of nitrogen fixation. The results indicate a rather wide prevalence in nature of hydrocarbon-oxidizing bacteria capable of fixing atmospheric nitrogen. Their occurrence helps explain the high concentration of organic nitrogen commonly found in soils exposed to gas leakage from pipelines or natural-gas seeps, and suggests further consideration of the possibility of applying selected petroleum residua to soils in order to increase the agricultural potential by nitrogen-fixing processes.     

Contribution from nitrogen fixation (acetylene reduction) to the nitrogen budget of Lake Tohopekaliga (Florida)

Nitrogen fixation, as assayed by the acetylene reduction technique, provided 44% of the input of nitrogen to a lake in central Florida (Lake Tohopekaliga) during 1984. Ninety-four percent of the lake total fixation was found in the water column and associated with Anabaena spp. The lake-wide average nitrogen fixation rate of 5.7 g N/m²-yr amounted to a mass loading of 497 metric tons of nitrogen for the year, and is one of the highest nitrogen fixation rates reported.     

Herbaceous Wetlands of the Yucatan Peninsula: Communities at Extreme Ends of Environmental Gradients

Distribution of wetland communities and growth characteristics of dominant macrophyte species were studied in the SE and NW parts of the Yucatan Peninsula. Plant, water and soil samples were collected from 40 marshes ranging from permanently flooded to seasonally drying. Marsh soils were identified as either peats, mark or clays with many intergrades. Water quality ranged from mesosaline (chloride or sulfate dominated) to fresh (carbonate dominated). Nutrient concentrations, specifically phoshorus, were low. The dominant species were Typha domingensis (TD), Cladium jamaicense (CJ), Eleocharis cellulosa (EC), E. interstincta (EI), and algal mats composed mostly of blue green algae (BG). EC and BG dominate marshes with marly soils and high salinities. C3 grows well on peaty soils and is well adapted to extremely low phosphorus concentrations and occasional drying. TD dominates nutrient rich marshes with deep water and clayey soils. The species differ significantly in their growth, death and decomposition rates. All these rates are highest for TD, followed closely by EC, and slowest for CJ.     

Stable isotope variation of a highly heterogeneous shallow freshwater system

Food web structure is well known to vary widely among ecosystems. Recent research indicates that there can be a high degree of spatial heterogeneity within ecosystems as well. Xochimilco is a small heterogeneous freshwater system that has been transformed into a network of canals, small lakes, and wetlands. Located within Mexico City, this ecosystem has been intensively managed and highly impacted for more than 50 years. This system receives urban and agricultural runoff, with resulting impacts on water quality. The aquatic community is dominated by exotics such as carp (Cyprinus carpio) and tilapia (Oreocrhomis niloticus), though the system still supports endemic species such as the aquatic salamander, axolotl (Ambystoma mexicanum), and crayfish (Cambarellus montezumae), which are both endangered. In this study, we used carbon and nitrogen stable isotopes for the whole food web and gut content analysis from the exotic fishes to describe food web structure in different canals within Xochimilco. There were significant isotopic differences among canals. These differences may result from isotopic baseline differences as well as differences in actual food web structure: both are related to local spatial variation in water quality driven by nutrient inputs and exotic fishes. Within-ecosystem variability is likely to be seen in other perturbed shallow systems as well, and should be explicitly considered in future food web studies.     

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

Summary Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N₂ into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.     

Nitrogen dynamics in an Alaskan salt marsh following spring use by geese

Lesser snow geese (Anser caerulescens caerulescens) and Canada geese (Branta canadensis) use several salt marshes in Cook Inlet, Alaska, as stopover areas for brief periods during spring migration. We investigated the effects of geese on nitrogen cycling processes in Susitna Flats, one of the marshes. We compared net nitrogen mineralization, organic nitrogen pools and production in buried bags, nitrogen fixation by cyanobacteria, and soil and litter characteristics on grazed plots versus paired plots that had been exclosed from grazing for 3 years. Grazed areas had higher rates of net nitrogen mineralization in the spring and there was no effect of grazing on organic nitrogen availability. The increased mineralization rates in grazed plots could not be accounted for by alteration of litter quality, litter quantity, microclimate, or root biomass, which were not different between grazed and exclosed plots. In addition, fecal input was very slight in the year that we studied nitrogen cycling. We propose that trampling had two effects that could account for greater nitrogen availability in grazed areas: litter incorporation into soil, resulting in increased rates of decomposition and mineralization of litter material, and greater rates of nitrogen fixation by cyanobacteria on bare, trampled soils. A path analysis indicated that litter incorporation by trampling played a primary role in the nitrogen dynamics of the system, with nitrogen fixation secondary, and that fecal input was of little importance.     

Role of legumes in providing N for sustainable tropical pasture systems

Forage legumes have long been lauded for their ability to fix atmospheric nitrogen and contribute to the sustainability of agricultural production systems. However despite the benefits they bring in terms of increased herbage and animal production they are not widely used in temperate or tropical regions. In this review the amounts of biological nitrogen fixation (BNF) needed to sustain the soil-plant-animal system are discussed and related to the amounts fixed in tropical pastures. The data suggest that tropical forage legumes have the capacity to meet the requirements to balance the N cycle of grazed pastures. The actual amounts required will depend on the rate of pasture utilization and the efficiency of recycling via litter, excreta and internal remobilization. The efficiency of nitrogen fixation (% of legume N derived from fixation) is usually high in tropical pastures (>80%) and is unlikely to be affected by inorganic soil N in the absence of N fertilizer. Thus an estimate of the amoutns of N fixed could be obtained from simple estimates of legume biomass provided tissue levels of other nutrients such as phosphorus and potassium are adequate. Key factors for the achievement of sustainable grass/legume pastures include the selection of appropriate germplasm adapted to the particular environment and the judicious use of fertilizers such as phosphorus, potassium, calcium, magnesium and sulphur on acid infertile soils typical of the sub-humid and humid tropics. The main constraints to the widespread adoption of forage legumes include a lack of legume persistence, the presence of anti-quality factors such as tannins, variable Bradyrhizobium requirements and lack of acceptability by farmers. Strategies for the alleviation of these constrainst are discussed. Forage legumes can be used to recuperate degraded soils via their ability to improve the physical, chemical and biological properties of soils and these benefits could be of particular use for small-scale resource-poor farmers. The incorporation of forage legumes into agropastoral systems is discussed as an environmentally and economically attractive means to encourage the widespread adoption of legumes in the humid tropics.     

Phosphorus regulation of nitrogen fixation in a traditional Mexican agroecosystem

Although nitrogen is considered to be the nutrient that most commonly limits production of natural and managed terrestrial ecosystems, I propose that phosphorus may regulate productivity in many continuously cultivated agroecosystems that do not receive applications of synthetic fertilizers. One way P may limit agroecosystem productivity is by controlling nitrogen fixation of legume crops, thus affecting nitrogen availability in the overall agroecosystem. I tested this hypothesis in two studies by examining the effect of phosphorus nutrition on nitrogen fixation of alfalfa in traditional Mexican agroecosystems. All farms used in the research relied on alfalfa as the primary nitrogen source for maize cultivation and other crops, and had minimal or no reliance on synthetic fertilizers.In one study, I used the natural abundance of¹⁵N to estimate nitrogen fixation in five alfalfa plots with soils representing a wide range of P fertility. I found a correlation of r = 0.85 between foliage P concentrations and nitrogen fixation in the alfalfa plots. Mean nitrogen fixation in alfalfa plots ranged between 232–555 kg ha^–1 yr^–1 as estimated by the¹⁵N-natural abundance method.In a second study, I sampled soils from alfalfa plots on traditional farms located in 5 different physiographic regions of Mexico. Half of each soil sample was augmented with phosphorus in a greenhouse experiment. I grew alfalfa on the fertilized and unfertilized soils from each site and then determined nitrogenase activity (acetylene reduction) of the Rhizobium on the plant roots. Nitrogenase activity increased in the alfalfa grown on all soils with added phosphorus, with two of the five differences being statistically significant at P < 0.01, 0 and one at P < 0.05. Foliage P concentrations and nitrogenase activity were 0 positively correlated (r = 0.81,P < 0.01).0     

Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel 15N2-DNA stable isotope probing (5N2-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from 15N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from 15N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that 15N2-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

The contribution of Rhizobium meliloti to soil denitrification

The ability of Rhizobium meliloti cells to denitrify in soils under several conditions was tested. All the strains tested were able to remove large amounts of N-NO₃ ^- from soils. Both water filled pore space above 36% and temperatures above 20°C greatly increased nitrogen losses. However, even with optimal conditions for denitrification and the highest rhizobial populations found in agricultural soils, the contribution of Rhizobium to the total denitrification was virtually negligible as compared to other soil microorganisms.To whom correspondence should be addressed     

Nodulation and nitrogen fixation in Purshia: inoculation responses and species comparisons

Summary Nitrogen fixing trees and shrubs may be useful in revegetation efforts. Speculation that insufficient endophyte populations in surface soils may limit non-leguminous symbiotic nitrogen fixation in marginal land was explored.Purshia tridentata andP. glandulosa seedlings were grown in greenhouse trials using ten soils from nativePurshia sites. Treatments include a control, an inoculated treatment, and six mmole nitrogen amendment. When inoculated with aP.tridentata crushed nodule inoculum, two of five non-nodulating soils and three sparsely nodulating soils produced well nodulated plants. Inoculation also increased nodule mass, total nitrogen, nitrogen content and shoot dry mass in plants from some of the soils. Of the three soils failing to produce nodulated plants when inoculated, one produced plants that responded well to nitrogen additions but failed to nodulate under low nitrogen conditions; another produced severely stunted plants indicating nutritional limitations on the host; and the third produced plants that were not nitrogen deficient. An application of nitrogen completely suppressed nodulation in all but one soil. The twoPurshia species were similar in nodulation, nitrogen fixation and growth, although important exceptions exist that indicate species may differ in adaptability to certain soil conditions.     

A function of cyanobacterial mats in phosphorus-limited tropical wetlands

Cyanobacterial mats are important components of oligotrophic wetland ecosystems in the limestone-based regions of the Caribbean. Our goals were to: (1) Estimate the biomass and primary production of cyanobacterial mats, quantify the extent of nitrogen fixation and measure the activity of alkaline phosphatase (APA) in representative marshes of northern Belize; (2) Record changes in these variables following nutrient additions. The mat biomass ranged from 200 to 700 g m^–2 AFDM, with the epipelon contributing up to 87% of the total. Tissue nitrogen was similar in all marshes (1.1–1.5%), while tissue phosphorus was extremely low (0.0055–0.0129%) and well correlated with the N:P ratio in water. Nitrogen fixation expressed as nitrogenase activity was high in some marshes (17.5 nmol C₂H₄ cm^–2 h^–1) and low (< 5 nmol C₂H₄ cm^–2 h^–1) in others depending mainly on the proportion of heterocyst-forming cyanobacteria (Nostocales, Stigonematales) in the mat. Alkaline phosphatase activity was positively correlated with the N:P ratio of the mat. Experimental addition of phosphorus resulted in significant increase in primary production and nitrogen fixation while it suppressed the APA activity. The presented data clearly showed that oligotrophic marshes of northern Belize are strongly P limited. Increased input of phosphorus would profoundly change their structure and functions.     

Symbiotic nitrogen fixation of Rhizobium (Galega) in acid soils,and its survival in soil under acid and cold stress

Summary Goat's rue (Galega orientalis) is a potential perennial forage legume for northern temperate acid soils. Greenhouse experiments were performed to compare symbiotically nitrogen fixing goat's rue with plants receiving mineral nitrogen in five different acid soils. Soil acidity had the same effect on yields of symbiotically grown plants as on plants receiving mineral nitrogen, suggesting that the acid sensitivity of the symbiosis was not limiting plant growth, even under very acidic conditions. The survival of an antibiotic resistant Rhizobium (Galega) strain in acid soil and freezing conditions was also studied. The survival of the bacteria was not affected at 15°C, when the pH of the soil (measured in 0.01M CaCl₂) was 5.2 or 4.9. In pH 3.4, and after freezing to –5°C, the population density decreased from 3×10⁸ to 1×10⁵/g in a few weeks. It is concluded that goat's rue, its symbiotic nitrogen fixation and R. (Galega) are tolerant of moderately acid agricultural soils, but that harsh winters may reduce bacterial numbers in the soil.     

Microdiversity of Culturable Diazotrophs from the Rhizoplanes of the Salt Marsh Grasses Spartina alterniflora and Juncus roemerianus

Abstract Salt marshes dominated by Spartina alterniflora (smooth cordgrass) are among the most productive ecosystems known, despite nitrogen limitation. Rhizoplane/rhizosphere diazotrophy (nitrogen fixation) serves as a significant source of combined nitrogen in these systems. Several recent studies have demonstrated remarkable physiological and phylogenetic macro- and microdiversity within this important functional group of organisms. However, the ecological significance of this diversity is presently unknown. The physiological characteristics of the culturable, oxygen-utilizing fraction of the rhizoplane diazotroph assemblages from Spartina alterniflora and from another salt marsh grass, the black needle rush Juncus roemerianus, were examined in combination with an assessment of the phylogenetic relatedness by whole genome DNA–DNA hybridization. Analysis of substrate utilization data permitted quantitative evaluation of fully cross-hybridizing strain groups and physiological clusters. Phylogenetically related strains, defined by DNA homology ≥90% relative to the positive control, displayed extensive physiological diversity. Seven bootstrap-supported physiological clusters, composed largely of phylogenetically dissimilar strains, showed similar utilization patterns for at least one class of ecologically relevant substrates (carbohydrates, carboxylic acids, or amino acids). These diazotrophs appear to be physiologically adapted for utilization of specific substrates or classes of substrates, lending support to diazotrophic functional redundancy. Microenvironmental heterogeneity is credited for promoting this diversity by selecting for physiologically specialized diazotroph populations to occupy defined niches in situ. One outcome of this physiological diversity is maintenance of a crucial environmental function (nitrogen fixation) over a broad range of environmental conditions. Received: 15 October 1999; Accepted: 28 December 1999; Online Publication: 25 April 2000     

Nitrogen fixation in rice systems: state of knowledge and future prospects

Rice is the most important cereal crop. In the next three decades, the world will need to produce about 60% more rice than today's global production to feed the extra billion people. Nitrogen is the major nutrient limiting rice production. Development of fertilizer-responsive varieties in the Green Revolution, coupled with the realization by farmers of the importance of nitrogen, has led to high rates of N fertilizer use on rice. Increased future demand for rice will entail increased application of fertilizer N. Awareness is growing, however, that such an increase in agricultural production needs to be achieved without endangering the environment. To achieve food security through sustainable agriculture, the requirement for fixed nitrogen must increasingly met by biological nitrogen fixation (BNF) rather than by using nitrogen fixed industrially. It is thus imperative to improve existing BNF systems and develop N₂-fixing non-leguminous crops such as rice. Here we review the potentials and constraints of conventional BNF systems in rice agriculture, as well as the prospects of achieving in planta nitrogen fixation in rice.     

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.     

Tracing sources and pathways of dissolved nitrate in forest and river ecosystems using high-resolution isotopic techniques: a review

Nitrogen inputs into stream and river ecosystems, and the factors influencing those inputs, are important for various ecological and environmental concerns. Reliable information on where and how nitrogen compounds flow into aquatic ecosystems is indispensable to understanding the nutrient status of these ecosystems. Such information should include the biogeochemical mechanisms and hydrological controls of nutrient leaching into rivers from terrestrial systems such as forests, agricultural fields, and urbanized areas. Advancements in stable isotopomer measurements over the past two decades have expanded the variety of target substances and the precision with which they can be investigated. The high-throughput microbial denitrifier method allows for simultaneous measurement of nitrogen and oxygen isotope ratios and can provide high-resolution spatiotemporal information on both nitrate sources and biogeochemical processes. Although advanced techniques of stable isotope analysis have been used extensively to detect sources and estimate the relative contributions of multi-source systems in various rivers, there are still new horizons in investigating nitrogen transformations. For example, stable isotopes of oxygen (¹⁸O and ¹⁷O) occurring in nitrate due to atmospheric deposition can be used as natural tracers for evaluating internal nitrogen cycling; these isotopes are distinct from the oxygen within microbially generated nitrate in soils and water bodies. Another future challenge is improved use of nitrous oxide isotopomers in evaluating the relative contributions of nitrification and denitrification. Such analysis could provide insight into the nitrogen transformation that occurs under redox conditions at the boundary between terrestrial and aquatic systems, where nitrification and denitrification often occur simultaneously in soil and aquatic environments.     

Nitrogen and phosphorus dynamics and retention in ecotones of Lake Titicaca,Bolivia/Peru

We are studying present conditions and consequences of material movement from land to water in the Lake Titicaca basin, and how fluxes are affected by human activities. The principal objective of this research is to describe and explain the variability in the Andean Altiplano of (a) water, nutrient and sediment fluxes from land and (b) composition, nutrient limitation and other important features of nearshore lake communities, and compare the effects of different agricultural practices (especially traditional and modern) on these factors. We are focusing on a comparison of the impacts of two forms of agriculture in this region: ancient raised fields currently under rehabilitation, and flat pastures and fields, which are more common. Results of the first year of study indicate there is substantial variability in nitrogen and phosphorus dynamics in relation to ecotone complexity (simple vs. intermediate vs. complex). Raised field sites have the beneficial effect of reducing high available nutrient concentrations (nitrate and soluble reactive phosphorus) and sediment load (measured as turbidity) as the water passes through them enroute to the lake. Aquatic vegetation (algae and macrophytes) reflect well ambient total nitrogen and phosphorus concentrations. Experimental nutrient limitation bioassays indicate that nitrogen is the most important limiting nutrient, though there is important spatial variability within the landscape, and phosphorus as well as nitrogen can be limiting.