Tight coupling of root-associated nitrogen fixation and plant photosynthesis in the salt marsh grass Spartina alterniflora and carbon dioxide enhancement of nitrogenase activity

The coupling of root-associated nitrogen fixation and plant photosynthesis was examined in the salt marsh grass Spartina alterniflora. In both field experiments and hydroponic assay chambers, nitrogen fixation associated with the roots was rapidly enhanced by stimulating plant photosynthesis. A kinetic analysis of acetylene reduction activity (ARA) showed that a five-to sixfold stimulation occurred within 10 to 60 min after the plant leaves were exposed to light or increased CO2 concentrations (with the light held constant). In field experiments, CO2 enrichment increased plant-associated ARA by 27%. Further evidence of the dependence of ARA on plant photosynthate was obtained when activity in excised roots was shown to decrease after young greenhouse plants were placed in the dark. Seasonal variation in the ARA of excised plant roots from field cores appears to be related to the annual cycle of net photosynthesis in S. alterniflora.     

Tight coupling of root-associated nitrogen fixation and plant photosynthesis in the salt marsh grass Spartina alterniflora and carbon dioxide enhancement of nitrogenase activity.

The coupling of root-associated nitrogen fixation and plant photosynthesis was examined in the salt marsh grass Spartina alterniflora. In both field experiments and hydroponic assay chambers, nitrogen fixation associated with the roots was rapidly enhanced by stimulating plant photosynthesis. A kinetic analysis of acetylene reduction activity (ARA) showed that a five-to sixfold stimulation occurred within 10 to 60 min after the plant leaves were exposed to light or increased CO2 concentrations (with the light held constant). In field experiments, CO2 enrichment increased plant-associated ARA by 27%. Further evidence of the dependence of ARA on plant photosynthate was obtained when activity in excised roots was shown to decrease after young greenhouse plants were placed in the dark. Seasonal variation in the ARA of excised plant roots from field cores appears to be related to the annual cycle of net photosynthesis in S. alterniflora.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora.

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

Evidence for NH4+ switch-off regulation of nitrogenase activity by bacteria in salt marsh sediments and roots of the grass Spartina alterniflora

The regulatory effect of NH4+ on nitrogen fixation in a Spartina alterniflora salt marsh was examined. Acetylene reduction activity (ARA) measured in situ was only partially inhibited by NH4+ in both the light and dark after 2 h. In vitro analysis of bulk sediment divided into sediment particles, live and dead roots, and rhizomes showed that microbes associated with sediment and dead roots have a great potential for anaerobic C2H2 reduction, but only if amended with a carbon source such as mannose. Only live roots had significant rates of ARA without an added carbon source. In sediment, N2-fixing mannose enrichment cultures could be distinguished from those enriched by lactate in that only the latter were rapidly inhibited by NH4+. Ammonia also inhibited ARA in dead and live roots and in surface-sterilized roots. The rate of this inhibition appeared to be too rapid to be attributed to the repression and subsequent dilution of nitrogenase. The kinetic characteristics of this inhibition and its prevention in root-associated microbes by methionine sulfoximine are consistent with the NH4+ switch-off-switch-on mechanism of nitrogenase regulation.     

Enumeration and Localization of N(2)-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N(2)-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N(2)-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N(2)-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N(2)-fixing bacteria recoverable from the rhizoplane, although high numbers of N(2)-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N(2) fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

Formyltetrahydrofolate synthetase sequences from salt marsh plant roots reveal a diversity of acetogenic bacteria and other bacterial functional groups

Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.     

Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.     

Molecular phylogenetic and biogeochemical studies of sulfate-reducing bacteria in the rhizosphere of spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO42- reduction rates and geochemical parameters. SO42- reduction was rapid in marsh sediments with rates up to 3.5 &mgr;mol ml-1 day-1. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO42- reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10(7) ml-1. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.     

Development and N2-Fixing Activity of the Benthic Microbial Community in Transplanted Spartina alterniflora Marshes in North Carolina

Growth and maturation of transplanted salt marshes is often limited by the availability of nitrogen (N). We examined the role of N₂-fixing benthic microbial assemblages (microalgae and associated bacteria) in two restored marshes (1-year-old and 6-year-old marsh) and a natural salt marsh in the Newport River Estuary, North Carolina. Benthic N₂ fixation (nitrogenase activity, NA), chlorophyll a (Chl a ) concentration, Spartina alterniflora (smooth cordgrass) stem counts, and sediment organic matter content were determined in the three marshes. Significant differences were observed between sites for both Chl a and NA. The 1-year-old marsh always exhibited the highest levels of NA and Chl a . Sediment organic matter content was lowest in the 1-year-old marsh (∼2%), intermediate in the 6-year-old marsh (∼5%), and highest in the natural marsh (∼10%). Carbon and nitrogen analyses were also performed on the 1-year-old marsh sediments, which were depleted in N. A positive correlation was observed between surface sediment N and Chl a . Remineralized, microbially derived N may provide growth-limiting inorganic N to Spartina transplants. N₂-fixing microbial assemblages in the 1-year-old marsh may also be an important food source for marsh infauna. Benthic N₂-fixing microbial assemblages play a key role in the N economy of restored salt marshes.     

Metabolism of low molecular weight organic compounds by sulfate-reducing bacteria in a Delaware salt marsh

Oxidation of acetate, lactate, pyruvate, and ethanol to CO₂ in anaerobic salt marsh sediments was rapid, with the oxidation rate being significantly inhibited (60–90% decrease) in the presence of 2 mM sodium molybdate, an inhibitor of sulfate-reducing bacteria (SRB). 2-Bromoethanesulfonic acid (BES), an inhibitor of methanogenic bacteria, generally had no effect on the oxidation rate. Acetate was the only intermediate product detected in the oxidation of lactate and ethanol. Competition studies with lactate, acetate, and ethanol indicated that the preferred order of substrate utilization was lactate, then acetate, then ethanol. The turnover times of these three compounds in salt marsh sediments via the combined CO₂ plus acetate pool was rapid (10–13 hours) with a two- to threefold increase in the turnover time in the presence of molybdate. These results strongly suggest that SRB play a major role in the terminal metabolism of low molecular weight organic compounds in anaerobic salt marsh sediment.     

Molecular characterization of sulfate-reducing bacteria in a New England salt marsh

Sulfate reduction, mediated by sulfate-reducing bacteria (SRB), is the dominant remineralization pathway in sediments of New England salt marshes. High sulfate reduction rates are associated with the rhizosphere of Spartina alterniflora when plants elongate aboveground. The growth process concurrently produces significant amounts of new rhizome material belowground and the plants leak dissolved organic compounds. This study investigated the diversity of SRB in a salt marsh over an annual growth cycle of S. alterniflora by exploring the diversity of a functional gene, dissimilatory sulfite reductase (dsrAB). Because the dsrAB gene is a key gene in the anaerobic sulfate-respiration pathway, it allows the identification of microorganisms responsible for sulfate reduction. Conserved dsrAB primers in polymerase chain reaction (PCR) generated full-length dsrAB amplicons for cloning and DNA sequence analysis. Nearly 80% of 380 clone sequences were similar to genes from Desulfosarcina and Desulfobacterium species within Desulfobacteraceae. This reinforces the hypothesis that complete oxidizers with high substrate versatility dominate the marsh. However, the phylotypes formed several clades that were distinct from cultured representatives, indicating a greater diversity of SRB than previously appreciated. Several dsrAB sequences were related to homologues from gram-positive, thermophilic and non-thermophilic Desulfotomaculum species. One dsrAB lineage formed a sister group to cultured members of the delta-proteobacterial group Syntrophobacteraceae. A deeply branching dsrAB lineage was not affiliated with genes from any cultured SRB. The sequence data from this study will allow for the design of probes or primers that can quantitatively assess the diverse range of sulfate reducers present in the environment.     

Microbial carbon monoxide consumption in salt marsh sediments

We have examined sediments from a fringing salt marsh in Maine to further understand marine CO metabolism, about which relatively little is known. Intact cores from the marsh emitted CO during dark oxic incubations, but emission rates were significantly higher during anoxic incubations, which provided evidence for simultaneous production and aerobic consumption in surface sediments. CO emission rates were also elevated when cores were exposed to light, which indicated that photochemical reactions play a role in CO production. A kinetic analysis of marsh surface sediments yielded an apparent K(m) of about 82 ppm, which exceeded values reported for well-aerated soils that consume atmospheric CO (65nM). Surface (0-0.2 cm depth interval) sediment slurries incubated under oxic conditions rapidly consumed CO, and methyl fluoride did not inhibit uptake, which indicated that neither ammonia nor methane oxidizers contributed to the observed activity. In contrast, aerobic CO uptake was inhibited by additions of readily available organic substrates (pyruvate, glucose and glycine), but not by cellulose. CO was also consumed by surface and sub-surface sediment slurries incubated under anaerobic conditions, but rates were less than during aerobic incubations. Molybdate and nitrate or nitrite, but not 2-bromoethanesulfonic acid, partially inhibited anaerobic uptake. These results suggest that sulfidogens and acetogens, but not dissimilatory nitrate reducers or methanogens, actively consume CO. Sediment-free plant roots also oxidized CO aerobically; rates for Spartina patens and Limonium carolinianum roots were significantly higher than rates for Spartina alterniflora roots. Thus plants may also impact CO cycling in estuarine environments.     

High overall diversity and dominance of microdiverse relationships in salt marsh sulphate-reducing bacteria

The biogeochemistry of North Atlantic salt marshes is characterized by the interplay between the marsh grass Spartina and sulphate-reducing bacteria (SRB), which mineralize the diverse carbon substrates provided by the plants. It was hypothesized that SRB populations display high diversity within the sediment as a result of the rich spatial and chemical structuring provided by Spartina roots. A 2000-member 16S rRNA gene library, prepared with delta-proteobacterial SRB-selective primers, was analysed for diversity patterns and phylogenetic relationships. Sequence clustering detected 348 16S rRNA sequence types (ribotypes) related to delta-proteobacterial SRB, and it was estimated that a total of 623 ribotypes were present in the library. Similarity clustering showed that approximately 46% of these sequences fell into groups with < 1% divergence; thus, microheterogeneity accounts for a large portion of the observable genetic diversity. Phylogenetic comparison revealed that sequences most frequently recovered were associated with the Desulfobacteriaceae and Desulfobulbaceae families. Sequences from the Desulfovibrionaceae family were also observed, but were infrequent. Over 80% of the delta-proteobacterial ribotypes clustered with cultured representatives of Desulfosarcina, Desulfococcus and Desulfobacterium genera, suggesting that complete oxidizers with high substrate versatility dominate. The large-scale approach demonstrates the co-existence of numerous SRB-like sequences and reveals an unexpected amount of microdiversity.     

Salt tolerance and osmotic adjustment of Spartina alterniflora (Poaceae) and the invasive M haplotype of Phragmites australis (Poaceae) along a salinity gradient

An invasive variety of Phragmites australis (Poaceae, common reed), the M haplotype, has been implicated in the spread of this species into North American salt marshes that are normally dominated by the salt marsh grass Spartina alterniflora (Poaceae, smooth cordgrass). In some European marshes, on the other hand, Spartina spp. derived from S. alterniflora have spread into brackish P. australis marshes. In both cases, the non-native grass is thought to degrade the habitat value of the marsh for wildlife, and it is important to understand the physiological processes that lead to these species replacements. We compared the growth, salt tolerance, and osmotic adjustment of M haplotype P. australis and S. alterniflora along a salinity gradient in greenhouse experiments. Spartina alterniflora produced new biomass up to 0.6 M NaCl, whereas P. australis did not grow well above 0.2 M NaCl. The greater salt tolerance of S. alterniflora compared with P. australis was due to its ability to use Na(+) for osmotic adjustment in the shoots. On the other hand, at low salinities P. australis produced more shoots per gram of rhizome tissue than did S. alterniflora. This study illustrates how ecophysiological differences can shift the competitive advantage from one species to another along a stress gradient. Phragmites australis is spreading into North American coastal marshes that are experiencing reduced salinities, while Spartina spp. are spreading into northern European brackish marshes that are experiencing increased salinities as land use patterns change on the two continents.     

不同发育时间的互花米草盐沼对大型底栖动物群落的影响

2004-2006年对长江口崇明东滩湿地芦苇(Phragmites australis)盐沼和不同发育时间的互花米草(Spartina alterniflora)盐沼的大型底栖动物群落特征进行分析研究.结果表明:互花米草盐沼发育初期,大型底栖动物群落以腹足类为主,物种丰富度(D=2.18)和多样性(H′=2.19)均低于芦苇盐沼(D=2.61, H′=2.29);随着时间的推移,互花米草与本地生物逐渐形成互动和稳定的格局,大型底栖动物群落组成中多毛类的种类逐渐上升(由3种变为6种),物种数和物种丰富度也上升,从而逐步形成新的大型底栖动物群落,物种丰富度(D=2.70)和多样性(H′=2.48)逐渐上升并高于芦苇盐沼(D=2.19, H′=2.09);从大型底栖动物群落的重新形成到稳定阶段,需要若干年的时间.     

福建漳江口红树林和盐沼湿地的多毛类动物群落

为了比较漳江口4种植物生境之间多毛类动物群落的差异性,2010年对漳江口潮间带秋茄、桐花树、白骨壤和互花米草4种植物生境的多毛类动物进行4个季度的定量取样.共获得15种多毛类动物,4个季度在4种植物生境中均出现三角洲双须虫、溪沙蚕、拟突齿沙蚕、凿贝才女虫、小头虫和加州中蚓虫.多毛类动物栖息密度、生物量、丰度指数、均匀度指数和多样性指数的季节变化不明显;但4种植物生境之间多毛类动物栖息密度、生物量、丰度指数、均匀度指数和多样性指数有显著差异,且互花米草生境与3种红树林生境之间多毛类动物优势种不同.Pearson相关分析表明,漳江口红树林和盐沼湿地除了多毛类物种数与泥温显著相关外,多毛类动物栖息密度、生物量、丰度指数、均匀度指数和多样性指数均与泥温、盐度、总有机碳、总氮无显著相关关系,其原因是漳江口4种植物生境多毛类动物常见种小头虫、加州中蚓虫和溪沙蚕均是广温、广盐及耐高有机质含量的种类.     

Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora

N(2) fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the alpha-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

Phototrophic N2 Fixation Suppressed by Activated Sulfate Reduction in Anoxic Rice Soil Slurries

Interactions between sulfate reduction (SR) and phototrophic nitrogenase activities were investigated in rice soil slurries mixed with rice straw. Activation of SR by adding exogenous sulfate suppressed acetylene-reducing activity (ARA) of the slurries, which was associated with phototrophic purple bacteria (PB) enumerated to 108-109 MPN g-1 dry weight (dw) soil. Adding 5 mm sodium molybdate, an inhibitor of SR, markedly increased ARA. However, in the slurries receiving both molybdate and exogenous sulfate, the effects declined simultaneously with partial recovery of SR. These results indicate outcompetition of sulfate-reducing bacteria (SRB) with PB in rice soil, when sulfate concentrations are high enough to support SR. The increasing effects of molybdate on ARA continued during the incubation in the sulfate-depleted condition, probably because of absence of SR and toxicity of molybdate to methanogenesis. Accordingly, stopping activities of the competitive microorganisms may be efficient to increase N2 fixation in rice soil.     

Quantification of ammonia-oxidizing bacteria and factors controlling nitrification in salt marsh sediments

To elucidate the geomicrobiological factors controlling nitrification in salt marsh sediments, a comprehensive approach involving sediment geochemistry, process rate measurements, and quantification of the genetic potential for nitrification was applied to three contrasting salt marsh habitats: areas colonized by the tall (TS) or short (SS) form of Spartina alterniflora and unvegetated creek banks (CBs). Nitrification and denitrification potential rates were strongly correlated with one another and with macrofaunal burrow abundance, indicating that coupled nitrification-denitrification was enhanced by macrofaunal burrowing activity. Ammonia monooxygenase (amoA) gene copy numbers were used to estimate the ammonia-oxidizing bacterial population size (5.6 x 10(4) to 1.3 x 10(6) g of wet sediment(-1)), which correlated with nitrification potentials and was 1 order of magnitude higher for TS and CB than for SS. TS and CB sediments also had higher Fe(III) content, higher Fe(III)-to-total reduced sulfur ratios, higher Fe(III) reduction rates, and lower dissolved sulfides than SS sediments. Iron(III) content and reduction rates were positively correlated with nitrification and denitrification potential and amoA gene copy number. Laboratory slurry incubations supported field data, confirming that increased amounts of Fe(III) relieved sulfide inhibition of nitrification. We propose that macrofaunal burrowing and high concentrations of Fe(III) stimulate nitrifying bacterial populations, and thus may increase nitrogen removal through coupled nitrification-denitrification in salt marsh sediments.     

Evidence for Coexistence of Two Distinct Functional Groups of Sulfate-Reducing Bacteria in Salt Marsh Sediment

Oxidation of acetate in salt marsh sediment was inhibited by the addition of fluoroacetate, and also by the addition of molybdate, an inhibitor of sulfate-reducing bacteria. Molybdate had no effect upon the metabolism of acetate in a freshwater sediment in the absence of sulfate. The inhibitory effect of molybdate on acetate turnover in the marine sediment seemed to be because of its inhibiting sulfate-reducing bacteria which oxidized acetate to carbon dioxide. Sulfide was not recovered from sediment in the presence of molybdate added as an inhibitor of sulfate-reducing bacteria, but sulfide was recovered quantitatively even in the presence of molybdate by the addition of the strong reducing agent titanium chloride before acidification of the sediment. Reduction of sulfate to sulfide by the sulfate-reducing bacteria in the sediment was only partially inhibited by fluoroacetate, but completely inhibited by molybdate addition. This was interpreted as showing the presence of two functional groups of sulfate-reducing bacteria—one group oxidizing acetate, and another group probably oxidizing hydrogen.