Nitrogen-fixing Bacillus sp. associated with Douglas-fir tuberculate ectomycorrhizae

Nitrogenase activities, measured by acetylene reduction, were detected under microaerophilic field conditions in Douglas-fir tuberculate ectomycorrhizae. Tuberculate ectomycorrhizae consist of densely packed clusters of ectomycorrhizal rootlets enclosed in a supplementary fungal peridium-like layer. Nitrogenase activity was primarily in the external layer and was greatly enhanced with added sucrose. The bacterium isolated, a nitrogen-fixing, spore-forming Bacillus sp., is an aerobe but requires anaerobic conditions for nitrogenase activity. Respiration in the tuberculate complex by the fungus, roots, and associated mycorrhizosphere microbes probably contributes to maintaining a microaerophilic niche where nitrogen fixation can take place. Water extracts of peridium or mycorrhizal root tips enhanced nitrogenase activity of this associative Bacillus sp., thereby indicating a close nutritional relationship between this bacterium and the tuberculate mycorrhizae. Thiamine more significantly enhanced bacterial nitrogenase activity than biotin; no activity was detected with p-aminobenzoic acid. Even though the levels ofnitrogenase activities in the tubercles in situ were low, as measured by the present methods, they may indicate a significant contribution to the nitrogen dynamics of these nitrogen-limited Douglas-fir forests over a long-term period.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

An indirect method for estimating 15N isotope fractionation during nitrogen fixation by a legume under field conditions

A new technique is proposed for measuring ¹⁵N isotope fractionation during N fixation that obviates some of the possible disadvantages of existing methods. Accurate calculation of N fixation by legumes using the ¹⁵N natural abundance technique requires a value for the isotopic composition of fixed N as an input. Isotopic fractionation in fixed N in legumes has usually been measured using N- free solution culture but results can vary with Rhizobium strain and growth conditions. The proposed method avoids these problems and can be used as an integral part of a field experiment for evaluating N fixation.The technique is essentially a process of adjusting values of ¹⁵ N for fixed N until % N fixation calculated by the ¹⁵N natural abundance method best matches % N fixation estimated by the ¹⁵N enrichment method. The use of high % N fixation values improves the sensitivity and reliability of the method.A field evaluation of this comparison technique using chickpea (Cicer arietinum L.) provided a ¹⁵N isotope fractionation factor (–2.37) for fixed N close to that obtained by N-free solution culture methods (–2.10). The availability of these two independent techniques allowed mutual corroboration of estimates of ¹⁵N isotope fractionation during N fixation.

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The decline in N2 fixation rate in common bean with the onset of pod-filling: Fact or artifact

It is not clear to what extent genetic, environmental and measurement factors are responsible for the commonly reported decline in nitrogenase activity with the onset of pod-filling in grain legumes. We address this question by observing nitrogenase activity and assimilate partitioning throughout the life span of an indeterminate variety of common bean (GN 1140) under controlled-environment and field conditions. Nitrogenase activity per plant was maintained well into pod-filling in GN 1140 under high-light conditions in growth cabinets. In contrast, plants exposed to a gradual reduction in light intensity during early reproductive growth had a decline in nitrogenase activity on a whole plant basis with the onset of pod-filling. However, the decline was due to an inability to maintain nodule growth, rather than a decrease in specific nitrogenase activity. Under field conditions, acetylene reduction assay of root crowns appeared to indicate a rapid decline in nitrogenase activity with the onset of pod-filling in GN 1140. This decline was not correlated with the water status of the soil or the plant. In contrast, acetylene reduction activity of root cores taken from outside the root crown region (non-crown) and N accumulation by above-ground biomass during pod-filling suggested that whole plant nitrogenase activity was maintained longer than that indicated by root crown assays. We conclude that although the occurrence of a decline in nitrogenase activity with the onset of pod-filling in grain legumes can be genetically determined, in many cases the decline can be the result of growing conditions and improper measurement techniques.     

Managing native and legume-fixed nitrogen in lowland rice-based cropping systems

Lowlands comprise 87% of the 145 M ha of world rice area. Lowland rice-based cropping systems are characterized by soil flooding during most of the rice growing season. Rainfall distribution, availability of irrigation water and prevailing temperatures determine when rice or other crops are grown. Nitrogen is the most required nutrient in lowland rice-based cropping systems. Reducing fertilizer N use in these cropping systems, while maintaining or enhancing crop output, is desirable from both environmental and economic perspectives. This may be possible by producing N on the land through legume biological nitrogen fixation (BNF), minimizing soil N losses, and by improved recycling of N through plant residues. At the end of a flooded rice crop, organic- and NH₄-N dominate in the soil, with negligible amounts of NO₃. Subsequent drying of the soil favors aerobic N transformations. Organic N mineralizes to NH₄, which is rapidly nitrified into NO₃. As a result, NO₃ accumulates in soil during the aerobic phase. Recent evidence indicates that large amounts of accumulated soil NO₃ may be lost from rice lowlands upon the flooding of aerobic soil for rice production. Plant uptake during the aerobic phase can conserve soil NO₃ from potential loss. Legumes grown during the aerobic phase additionally capture atmospheric N through BNF. The length of the nonflooded season, water availability, soil properties, and prevailing temperatures determine when and where legumes are, or can be, grown. The amount of N derived by legumes through BNF depends on the interaction of microbial, plant, and environmental determinants. Suitable legumes for lowland rice soils are those that can deplete soil NO₃ while deriving large amounts of N through BNF. Reducing soil N supply to the legume by suitable soil and crop management can increase BNF. Much of the N in legume biomass might be removed from the land in an economic crop produce. As biomass is removed, the likelihood of obtaining a positive soil N balance diminishes. Nonetheless, use of legumes rather than non-legumes is likely to contribute higher quantities of N to a subsequent rice crop. A whole-system approach to N management will be necessary to capture and effectively use soil and atmospheric sources of N in the lowland rice ecosystem.IRRI-NifTAL-IFDC joint contribution.     

A Photobacterium-like bacterium able to fix nitrogen

A Photobacterium-like bacterium isolated from the roots of eelgrass (Zostera marina) was shown to fix nitrogen under anaerobic conditions. Nitrogen fixation by Photobacterium spp. has not been reported previous to this.Abbreviation PHB Poly--hydroxybutyrate     

Soil and litter respiration rates in different microhabitats of a mixed oak-conifer forest and their control by edaphic conditions and substrate quality

Summary Seven isolates belonged toA. brasilense and 3 belonged toA. lipoferum. Isolates having more denitrifying capacity fixed less nitrogen in nitrogen free semi-solid malate medium. One strain ofA. lipoferum having high nitrogen fixing capacity with negative test for denitrification was tested as inoculant to supplement the nitrogen need of a wheat crop in field condition with different doses of N with and without the inoculant. While control without nitrogen yielded 1260 kg/ha the yield in inoculated treatment was 2070 kg/ha resulting in significant increase. In a treatment receiving 40 kg N/ha the grain yield was 2370 kg/ha as against yield of 3110 kg/ha in a similar treatment receiving fertiliser plus inoculant. Thus increase in yield was about 30%. Further the treatment receiving 80 kg N/ha yielded 2970 kg/ha as against yield of 4150 kg/ha in a treatment receiving inoculant alongwith the above dose of the fertiliser. Thus increase in yield due to application of inoculant was about 36%. Similarly, the uptake of N in different treatments was augmented due to inoculation of seeds with the culture.     

Competition of strains of Rhizobium of the cowpea group in two soil types

Summary Competition of five strains of Rhizobium of the cowpea group, onVigna radiata (L) Wilcjeck variety ML 5, was tested in loamy clay and loamy sand soils. Strains RM 6 and RM 5 were effective nodulators in loamy clay soil, and strains MNH, M 20 and RM 6 were effective nodulators in loamy sand soil. Strains RM 6 and MNH predominated nodule formation in loamy clay and loamy sand soils respectively.     

Field evaluation of N2-fixation and N-utilization by phaseolus bean varieties determined by15N isotope dilution*

Summary Differences in N₂-fixation byPhaseolus vulgaris bean cultivars were successfully evaluated in the field using¹⁵N isotope dilution technique with a non-fixing test crop of a different species (wheat). The Phaseolus cultivars could have been similarly ranked for N₂-fixation capacity from either seed yield or total nitrogen yield, but the isotope method provided a direct measure of N₂-fixation and made it possible to estimate the proportion of fixed to total nitrogen in the crop and in plant parts. Amounts of nitrogen fixed varied between 24.59 kg N/ha for the 60-day cultivar Goiano precoce to 64.91 kg N/ha for the 90-day cultivar Carioca. The per cent of plant nitrogen due to fixation was 57–68% for the 90-day cultivars and 37% for Goiano precoce (60-day cultivar). Fertilizer utilization was 17–30% of a 20 kg N/ha fertilizer application. 100 kg N/ha fertilizer application decreased N₂-fixation without suppressing it totally. Differences in yield between the highest yielding (Carioca) and the lowest (Moruna) 90-day cultivars were also due apparently to varietal differences in efficiency of conversion of nitrogen to economic matteri.e. seed, as well as to differences in capacity of genotypes for N₂-fixation. The work described here was in part supported by IAEA Research Contract No. RC/2084 UNDP/IAEA Project BRA/78/006     

The sarcoplasmic reticulum of an ultrafast lobster muscle: first evidence of a tubular configuration

The sarcoplasmic reticulum (SR) of the remarkably fast remotor muscle of lobster second antenna was investigated with regard to ultrastructure, calcium uptake and protein composition. The SR of this unique muscle dominates the volume of the cell. We were able to preserve the configuration of the SR in its native state simply by processing the muscle for electron microscopy at 0-4 degree C. For the first time the SR is seen as larger (0.1 micrometer diam.) tubules that crowd the space between myofibrils, Previous observations of this organelle have been reported that showed a rather unsatisfying discontinuous vesicular configuration of the SR. This report indicates that these structures were fixation artifacts. The uptake of calcium measured in a microsomal subfraction in the presence of ATP and oxalate does not differ significantly from similar experiments with vertebrate SR. However, gel electrophoresis of lobster SR demonstrate a single intrinsic Ca-ATPase protein of 100,000 mol, wt. with neither of the typical lower molecular weight proteins found in vertebrate SR.