SIGNIFICANCE OF BACTERIAL-ANABAENA (CYANOPHYCEAE) ASSOCIATIONS WITH RESPECT TO N2 FIXATION IN FRESHWATER1, 2

The functional aspects of specific associations between bluegreen algae and bacteria were investigated using both naturally occurring and cultured species of Anabaena. In take waters where bacteria were associated with Anabaena heterocysls, the bacteria exhibited a chemotactic response to a variety of amino acids and glucose. Earlier autoradiographic evidence that bacteria associated with heterocysts incorporate identical substrates indicates that associated bacteria probably benefit by utilizing algal excretion products. In return, the bacteria stimulate algal N₂fixation. The most likely mechanism explaining such stimulation appeared to be bacterial oxygen removal in microzones (< 3 μm diam) bordering heterocysts during periods of high ambient oxygen concentrations. In the presence of bacteria, Anabaena rapidly overcame nitrogenase- inhibiting concentrations of oxygen. Axenic cullures had more extensive nitrogenase inhibition, and took longer to recover in response to oxygenation. Algal-bacterial mutualism aids Anabaena in maintaining concurrent optimal N2 fixation and high photosynthetic rates in highly oxygenated surface waters.     

Seasonal fluctuations in the population of denitrifying and N2-fixing bacteria in an acid soil of a Norway spruce forest

The seasonal fluctuations in the concentration of cultured denitrifying and N₂-fixing bacteria were followed in an ammonium fertilised and a control soil of a Norway spruce forest near Villingen/Black Forest from December 1994 to August 1998. The horizontal distribution of bacteria in three layers was determined by the MPN-method and by molecular probing (colony hybridisation) using specific 0.4–0.7 kb DNA probes for denitrification steps (narG, nirS, nirK and nosZ) and for N₂-fixation (nifH). The data showed that highest bacterial counts and higher numbers of denitrifying and N₂-fixing bacteria were generally detectable in the upper (= 5 cm) soil layer and that their amount decreased with soil depth. The concentration of these cultured bacteria showed seasonal fluctuations with highest numbers in autumn/winter/early spring and with low counts in summer. Denitrifying and N₂-fixing bacteria amounted to less than 10% of the total number of cultured bacteria determined by the MPN-method. Fertilisation with ammonium did not cause a shift in the population of these bacteria. These findings were corroborated by hybridisation experiments with genomic DNA isolated from the different layers. Strongest DNA–DNA hybridisation band intensities were obtained in the upper soil layer and their intensities decreased with soil depth. Soil samples from Villingen assayed in the laboratory produced N₂O (in dependence of nitrate and C₂H₂ added to the vessels) and utilised this gas with higher activities in the assays with the fertilised soil. It is concluded that molecular techniques can successfully be applied for assessing seasonal fluctuations of bacterial populations in soil. Relative abundance of denitrifying and N₂-fixing bacteria can be determined from experiments with DNA isolated from soils. Attempts to transform these results to the total population of soil bacteria on a single cell basis are faced with many uncertainties.     

TRANSFER OF N2 AND CO2 FIXATION PRODUCTS FROM ANABAENA OSCILLARIOIDES TO ASSOCIATED BACTERIA DURING INORGANIC CARBON SUFFICIENCY AND DEFICIENCY1

Transfer of N₂ and CO₂ fixation products from the bloom forming blue-green alga, Anabaena oscillarioides Bory, to attached and free swimming bacteria is common during active growth of the former. Incubation with ¹⁵N₂ and ¹⁴CO₂ followed by size fractionation filtration reveals that: i) magnitudes of fixed N and C excretion, relative to N₂ and CO₂ fixation, are dictated by dissolved inorganic carbon (DIC) availability for A. oscillarioides photosynthetic production, ii) associated bacteria exhibit preferences for recently fixed excreted N compounds, iii) bacterial utilization of excreted N is independent of ambient light conditions, and iv) lag times between N₂ fixation and detectable bacterial assimilation of excreted fixed N compounds are ca. 1–2 h. Both ¹⁴NH₄Cl dilution and Hg(NH₃)₂ Cl₂ precipitation techniques indicate that NH₃ is a major excretion product from A. oscillarioides, particularly during DIC limited growth. Active N and C excretion and transfer to associated bacteria are features of viable A. oscillarioides filaments. Hence, transfer of these metabolites reflects complex mutualistic, and possibly symbiotic associations rather than solely signaling senescence.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Tomato Growth in Soil Amended with Sugar Mill By-Products Compost

Sugar mill by-products compost may be a good soil amendment to promote tomato (Lycopersicon esculentum L.) growth. In addition, the compost may further promote plant growth by inoculation with N₂-fixing bacteria. Compost from sugar-mill waste was prepared with and without the N₂-fixing bacteria, Azotobacter vinelandii, Beijerinckia derxii and Azospirillum sp. and incubated for 50 days. Each compost type was added to 10 kg of soil in pots at rates of 0, 15, and 45 g with and without fertilizer N at rates of 0, 0.75, and 1.54 g. A blanket application of P and K was applied to all pots. Shoot and root dry weights and N content of the whole plant was measured at 55 days. Dry weight of tomato shoots was increased by 40% by addition of fertilizer N and root weight was increased by 66%. Without fertilizer N the high rate of inoculated compost increased shoot growth 180% and uninoculated compost increased shoot growth 112%. For most treatments with and without fertilizer N, inoculated compost enhanced shoot growth and nitrogen content more than uninoculated compost. Root weights were nearly doubled by addition of either compost in comparison to the 0 N treatment. At the low rate of compost addition without fertilizer N, root weight was the same for uninoculated and inoculated compost but at the high rate of compost addition root weight was 32% higher for inoculated compost. The N₂-fixing bacteria colonized roots when inoculated compost was used. Sugar mill by-products compost proved to be an effective soil amendment for promoting the growth of tomato plants.     

Evolution of symbiotic bacteria within the extra- and intra-cellular plant compartments: experimental evidence and mathematical simulation (Mini-review)

Using the example of nodular legume-rhizobia symbiosis (LRS), we discuss the evolution in plant micro-symbionts of mutualistic traits that are apparently host-beneficial and therefore the products of inter-species evolution. These traits include: in planta activation of N₂ fixation machinery; exporting the products of nitrogenase reaction into the plant cells/tissues; and the terminal differentiation of bacteria into non-reproductive N₂-fixing bacteroids. It seems probable that such adaptive traits evolved by natural selection within the populations of endosymbiotic bacteria that colonize the extra- and intra-cellular compartments provided by the hosts (i.e., infection threads and symbiosomes). This evolution would occur under the impacts of group (inter-deme, kin) selection pressures induced by the partners’ metabolic and regulatory feedbacks that ensure the high activity of symbiotic N₂ fixation. These important feedbacks include: progressive allocation of C compounds into N₂-fixing nodules; maintenance of micro-aerobic intracellular environments that are indispensable for intensive N₂ fixation; and stringent control by the host over bacterial reproduction in planta. A computational simulation of the associated co-evolutionary processes reveals the trade-off between inter-species and individual species components of progressive and adaptive LRS evolution. This is expressed as a correlated increase of ecological efficiency, functional integrity and genotypic specificity of mutualistic symbiosis. Thus, the evolution of rhizobia in symbiosis may be represented by a progressive multi-level scenario based on increasing the dependency of bacteria on the host-provided nutrients accompanied by increasing complexity of the bacterial genomes and of the symbiosis-encoding gene networks.     

Evolution of legume-rhizobium symbiosis for an improved ecological efficiency and genotypic specificity of partner interactions

Mathematical simulation of the evolution of polymorphic legume-rhizobium symbiosis showed that co-evolution of the partners for an improved ecological efficiency of symbiosis is greatly stimulated when low-active N₂-fixing and non-N₂-fixing strains of nodule bacteria are prohibited from colonizing nodules. The results of analysis of the model were collated with the comparative morphology of the infection process in various legumes, and its was assumed that mechanisms controlling bacterial reproduction in nodules arose in early evolution of symbiosis in primitive legumes owing to a transition from mixed to clonal infection. The development of such mechanisms was associated with adaptively valuable macroevolutionary transformations of symbiosis and directed its microevolution towards a parallel increase in the specificity and efficiency of mutualism. The increase was due to a reorganization of selective processes in endosymbiotic bacterial populations, which was based on changes in their genetic and spatial structures and optimized metabolic feedbacks between the partners (preferential allocation of photosynthesis products to the most active N₂-fixing strains).     

Structural Diversity of Bacterial Communities Associated with Bloom-Forming Freshwater Cyanobacteria Differs According to the Cyanobacterial Genus

The factors and processes driving cyanobacterial blooms in eutrophic freshwater ecosystems have been extensively studied in the past decade. A growing number of these studies concern the direct or indirect interactions between cyanobacteria and heterotrophic bacteria. The presence of bacteria that are directly attached or immediately adjacent to cyanobacterial cells suggests that intense nutrient exchanges occur between these microorganisms. In order to determine if there is a specific association between cyanobacteria and bacteria, we compared the bacterial community composition during two cyanobacteria blooms of Anabaena (filamentous and N₂-fixing) and Microcystis (colonial and non-N₂ fixing) that occurred successively within the same lake. Using high-throughput sequencing, we revealed a clear distinction between associated and free-living communities and between cyanobacterial genera. The interactions between cyanobacteria and bacteria appeared to be based on dissolved organic matter degradation and on N recycling, both for N₂-fixing and non N₂-fixing cyanobacteria. Thus, the genus and potentially the species of cyanobacteria and its metabolic capacities appeared to select for the bacterial community in the phycosphere.     

Detoxification of mercury and organomercurials by nitrogen-fixing soil bacteria

Minimal inhibitory concentration values of HgCl₂ and 5 organomercurials were determined against 24 mercury-resistant N₂-fixing soil bacteria previously isolated from soil and identified in our laboratory. These bacterial strains also displayed multiple antibiotic resistant properties. Typical growth pattern of a highly mercury-resistantBeijerinckia sp (KDr₂) was studied in liquid broth supplemented with toxic levels of mercury compounds. Four bacterial strains were selected for determining their ability to volatilize mercury and their Hg-volatilizing capacity was different. Cell-free extracts prepared from overnight mercury-induced cells catalyzed Hg²⁺-induced NADPH oxidation. Specific activities of Hg²⁺-reductase which is capable of catalyzing conversion of Hg²⁺ →Hg(o) of 10 Hg-resistant bacterial strains are also reported.     

Nitrogen-Fixing (Acetylene Redution) Activity and Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland Rice

Nitrogen-fixing activity associated with different wetland rice varieties was measured at various growth stages by an in situ acetylene reduction method after the activities of blue-green algae (cyanobacteria) in the flood water and on the lower portion of the rice stem were eliminated. Nitrogen-fixing activities associated with rice varieties differed with plant growth stages. The activities increased with plant age, and the maximum was about at heading stage. The nitrogen fixed during the whole cropping period was estimated at 5.9 kg of N per ha for variety IR26 (7 days) and 4.8 kg of N per ha for variety IR36 (95 days). The population of aerobic heterotrophic N₂-fixing bacteria associated with rice roots and stems was determined by the most-probable-number method, using semisolid glucose-yeast extract and semisolid malate-yeast extract media. The addition of yeast extract to the glucose medium increased the number and activity of aerobic heterotrophic N₂-fixing bacteria. The glucose-yeast extract medium gave higher counts of aerobic N₂-fixing bacteria associated with rice roots than did the malate-yeast extract medium, on which Spirillum-like bacteria were usually observed. The lower portion of the rice stem was also inhabited by N₂-fixing bacteria and was an active site of N₂ fixation.     

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N₂-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions. In this paper we review some recent progress in this field, particularly at our institute in Rio de Janeiro, concerning specificity of selected Azospirillum strains in the infection of cereal roots and the promotion of responses in the host plants. The possible mechanisms of plant response are discussed including the possibility that plant growth substances or bacterial nitrate reductase are involved. The application of¹⁵N and N balance techniques to the quantification of plant associated BNF are considered and the possible strategies that may be adopted to further the understanding of true N₂-fixing plant/diazotroph associations. The recent discovery of many more plant-associated N₂-fixing bacteria suggests that further research in this area may eventually lead to the development of such associations with applications for agricultural productivity.     

Inoculation of forage grasses with N2-fixing Enterobacteriaceae

Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N₂-fixing bacteria. The most numerous N₂-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were conducted using 18 isolates of these bacteria to determine if a N₂-fixing association could be established between the bacteria and the grassesCynodon dactylon andPanicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N₂ were being fixed (>8kg N ha⁻¹). However, plant systems extrapolated to fix>8 kg N ha⁻¹ contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not be re-isolated from healthy grass roots indicating that the N₂-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors limiting colonization of grass roots.     

Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Frankia in the rhizosphere of nonhost plants: A comparison with root-associated N2-fixing Enterobacter,Klebsiella and Pseudomonas

Bacterial growth in the rhizosphere and resulting changes in plant growth parameters were studied in small aseptic seedlings of birch (Betula pendula and B. pubescens) and grasses (Poa pratensis and Festuca rubra). The seedlings were inoculated with three Frankia strains (Ai1a and Ag5b isolated from native Alnus root nodules and Ai17 from a root nodule induced by soil originating from a Betula pendula stand), and three associative N₂-fixing bacteria (Enterobacter agglomerans, Klebsiella pneumoniae and Pseudomonas sp., isolated from grass roots). Microscopic observations showed that all the Frankia strains were able to colonize and grow on the root surface of the plants tested without addition of an exogenous carbon source. No net growth of the associative N₂-fixers was observed in the rhizosphere, although inoculum viable counts were maintained over the experimental period. Changes in both the biomass and morphology of plant seedlings in response to bacterial inoculation were recorded, which were more dependent on the plant species than on the bacterial strain.     

INTERACTION OF NITROGEN FIXATION AND PHOSPHORUS LIMITATION IN APHANIZOMENON FLOS-AQUAE (CYANOPHYCEAE)1

The effect of simultaneous nitrogen fixation and phosphorus limitation on the physiological adaptation and growth performance of Aphanizomenon flos-aquae (L.) Ralfs PCC 7905 was studied in continuous culture. In the absence of ammonia, N₂ fixation occurred and the maximum growth rate (as determined in diluted batch cultures) was lower. However, no distinction could be made between the steady-state N uptake rates (based on cellular N contents) of N₂-fixing cells and cells grown with ammonia. At the higher dilution rates, the residual P concentration increased with increasing dilution rate, more so under N₂-fixing conditions, compared to the cultures grown in the presence of ammonia. More generally, the yield of biomass per consumed P, as the biomass concentration itself, decreased with increasing dilution rate, and both were lower under N₂-fixing conditions. The restricted biomass production under N₂-fixing conditions suggests that reduction of N loading may benefit lake restoration projects. The influence of N₂-fixation on the severity of P limitation is discussed in terms of metabolic control analysis. From the increase of the residual P concentration on switching from ammonium to N₂-fixing conditions, it is deduced that under N₂-fixing and P-limited conditions, control of growth is shared by N and P metabolism.     

Immunochemical Localization of Nitrogenase in Marine Trichodesmium Aggregates: Relationship to N2 Fixation Potential

Colonial aggregation among nonheterocystous filaments of the planktonic marine cyanobacterium Trichodesmium is known to enhance N₂ fixation, mediated by the O₂-sensitive enzyme complex nitrogenase. Expression of nitrogenase appears linked to the formation of O₂-depleted microzones within aggregated bacterium-associated colonies. While this implies a mechanism by which nonheterocystous N₂ fixation can take place in an oxygenated water column, both the location and regulation of the N₂-fixing apparatus remain unknown. We used an antinitrogenase polyclonal antibody together with postsection immunocolloidal gold staining and transmission electron microscopy to show that (i) virtually all Trichodesmium cells within a colony possessed nitrogenase, (ii) nitrogenase showed no clear intracellular localization, and (iii) certain associated bacteria contained nitrogenase. Our findings emphasize the critical role coloniality plays in regulating nitrogenase expression in nature. We interpret the potential for a large share of Trichodesmium cells to fix N₂ as an opportunistic response to the dynamic nature of the sea state; during quiescent conditions, aggregation and consequent expression of nitrogenase can proceed rapidly.     

Oxygen-Poor Microzones as Potential Sites of Microbial N2 Fixation in Nitrogen-Depleted Aerobic Marine Waters

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N₂. Bioassays aimed at identifying environmental factors controlling the development and proliferation of N₂ fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N₂ fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N₂ fixation. In all cases bacterial N₂ fixation occurred in association with surficial microenvironments or microzones. Since nitrogenase is oxygen labile, we hypothesized that the aggregates themselves protected their constituent microbes from O₂. Microelectrode O₂ profiles revealed that aggregates had lower internal O₂ tensions than surrounding waters. Tetrazolium salt (2,3,5-triphenyl-3-tetrazolium chloride) reduction revealed that patchy zones existed both within microbes and extracellularly in the mucilage surrounding microbes where free O₂ was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N₂ fixation is mediated by the availability of the appropriate types of reduced microzones. Organic carbon enrichment appears to serve as an energy and structural source for aggregate formation, both of which were required for eliciting N₂ fixation responses of these waters.     

The invasive Sorghum halepense harbors endophytic N2-fixing bacteria and alters soil biogeochemistry

Exotic plants invading new habitats frequently initiate broad changes in ecosystem functioning. Sorghum halepense is an invasive grass capable of growing in nitrogen (N)-poor tallgrass prairie soils that creates near monocultures in once phylogenetically diverse-communities. The biogeochemistry of soils invaded by S. halepense was compared to that of un-invaded native prairie soils. Invaded soils contained two to four times greater concentrations of alkaline metals, micronutrients, and essential plant nutrients than native prairie soils. The notable exception was Ca⁺², which was always significantly lower in invaded soils. The N-content of S. halepense above-ground biomass was 6.4 mg g^?1 (320 mg N plant^?1) and suggested a supplemental N source supporting plant growth. Altered soil biogeochemistry in invaded areas coupled with high above-ground biomass in N-poor soils suggested N₂-fixing activity associated with S. halepense. Nitrogenase activity of plant tissues indicated that N₂-fixation was occurring in, and largely restricted to, S. halepense rhizomes and roots. A culture approach was used to isolate these N₂-fixing bacteria from plant tissues, and 16S rRNA gene sequencing was used to identify these bacterial isolates. Nitrogenase activity of bacterial isolates indicated several were capable of N₂-fixation. In addition to N₂-fixation, other roles involved in promoting plant growth, namely mobilizing phosphorus and iron chelation, are known for closest matching relatives of the bacterial isolates identified in this work. Our results indicate that these plant growth-promoting bacteria may enhance the ability of S. halepense to invade and persist by altering fundamental ecosystem properties via significant changes in soil biogeochemistry.     

Fixation of Dinitrogen-15 Associated with Rice Plants

Rice plants (IR26 and Latisail) obtained at near heading stage from a wetland field were transferred to water culture and exposed to ¹⁵N₂ in a gas-tight growth chamber for 7 days to measure N₂-fixing activities associated with the rice. The activities measured varied from 6.5 to 11.6 μmol of N₂ fixed per hill per day. The outer leaf sheath had about 2.5 times higher N₂-fixing activities per unit weight than the root. Slight activities were also found in the basal node and inner leaf sheath. Wrapping basal parts of the stem with aluminum foil did not decrease the activities of N₂ fixation in these parts. Thus, the outer leaf sheath as well as the root are N₂-fixing sites in rice plants. N₂ fixation found in above-ground parts is not due to photoautotrophic organisms. Less than 10% of the fixed nitrogen was translocated from the fixing sites to the leaf blades and the young panicles.