Effect of Lake Trophic Status and Rooted Macrophytes on Community Composition and Abundance of Ammonia-Oxidizing Prokaryotes in Freshwater Sediments

Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species Littorella uniflora, Juncus bulbosus, and Myriophyllum alterniflorum were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (amoA) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting amoA, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with M. alterniflorum (0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of L. uniflora and J. bulbosus. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like L. uniflora and J. bulbosus, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.Oxygen release from the roots of macrophyte species such as Littorella uniflora (L.) Asch. (shore weed), Lobelia dortmanna L. (water lobelia), and Glyceria maxima (Hartm.) Holmb. (reed sweet grass) stimulates nitrification and coupled nitrification-denitrification in the rhizosphere compared to that in unvegetated sediment (2, 36, 40). These interactions are of high ecological relevance especially in oligotrophic systems, since enhanced nitrogen loss due to rhizosphere-associated denitrification can retard natural eutrophication and succession of plant communities (1). While the microbial communities involved in coupled nitrification-denitrification have been well studied in rice paddy soils (7, 11), less information is available for natural freshwater sediments, especially those from oligotrophic lakes (2, 26).The first key step of coupled nitrification-denitrification, the oxidation of ammonia to nitrite, is catalyzed by two groups of prokaryotes—the ammonia-oxidizing bacteria (AOB) (24) and the only recently recognized ammonia-oxidizing archaea (AOA) (22). For both groups, the gene encoding the alpha-subunit of ammonia monooxygenase (amoA) has been widely used as a functional marker to analyze their community compositions (15, 25); recent studies demonstrated the ubiquity of AOA and their predominance over AOB in a broad range of environments (32, 38). AOA, but not AOB, were also strongly enriched in the rhizosphere of the freshwater macrophyte Littorella uniflora in a mesotrophic Danish lake, suggesting that AOA were primarily responsible for increased rates of nitrification in the rhizosphere of this plant species (19). Moreover, ammonia oxidizer communities differed between rhizosphere and unvegetated sediment, indicating a plant-specific effect on AOA and AOB community composition. The objectives of this study were therefore to test whether (i) AOA generally predominate over AOB in freshwater sediments and especially in macrophyte rhizospheres and (ii) macrophytes have species-specific effects on abundance and community composition of AOA and AOB in rhizosphere sediments and on root surfaces.To address these questions, two shallow heathland pools and five lakes in Denmark and Germany, ranging from low-pH and dystrophic sites to neutral-pH and oligotrophic and mesotrophic sites, were chosen, and three macrophyte species—Littorella uniflora, Juncus bulbosus L. (bulbous rush), and Myriophyllum alterniflorum DC. (alternate water milfoil)—were selected as model systems. These plant species differ in nitrogen nutrition, extent of radial oxygen loss, and lifestyle, presumably resulting in differential, plant species-specific effects on rhizosphere- and root-associated AOA and AOB communities. L. uniflora prefers nitrate as the nitrogen source, while J. bulbosus prefers ammonium (41, 45); oxygen release is high to moderate from the roots of L. uniflora and J. bulbosus (9, 12) but is minor from the roots of M. alterniflorum (M. Herrmann, P. Stief, and A. Schramm, unpublished results); L. uniflora and J. bulbosus remain photosynthetically active throughout the year, while only the below-ground parts of M. alterniflorum are retained during winter.Rhizosphere sediments and roots from each plant species were sampled from three different sites per species, and unvegetated sediment was obtained from all seven sites. The comparison of samples from these different sites and compartments (rhizosphere, root surface, unvegetated sediment) allowed an evaluation of the importance of plant species relative to that of environmental conditions related to lake trophic status and pH on ammonia oxidizer communities.     

Genetic Characterization of the Nitrate Reducing Community Based on <Emphasis Type="Italic">narG</Emphasis> Nucleotide Sequence Analysis

The ability of facultative anerobes to respire nitrate has been ascribed mainly to the activity of a membrane-bound nitrate reductase encoded by the narGHJI operon. Respiratory nitrate reduction is the first step of the denitrification pathway, which is considered as an important soil process since it contributes to the global cycling of nitrogen. In this study, we employed direct PCR, cloning, and sequencing of narG gene fragments to determine the diversity of nitrate-reducing bacteria occurring in soil and in the maize rhizosphere. Libraries containing 727 clones in total were screened by restriction fragment analysis. Phylogenetic analysis of 128 narG sequences separated the clone families into two main groups that represent the Gram-positive and Gram-negative nitrate-reducing bacteria. Novel narG lineages that branch distinctly from all currently known membrane bound nitrate-reductase encoding genes were detected within the Gram-negative branch. All together, our results revealed a more complex nitrate-reducing community than did previous culture-based studies. A significant and consistent shift in the relative abundance of the nitrate-reducing groups within this functional community was detected in the maize rhizosphere. Thus a substantially higher abundance of the dominant clone family and a lower diversity index were observed in the rhizosphere compared to the unplanted soil, suggesting that a bacterial group has been specifically selected within the nitrate-reducing community. Furthermore, restriction fragment length polymorphism analysis of cloned narG gene fragments proved to be a powerful tool in evaluating the structure and the diversity of the nitrate-reducing community and community shifts therein.     

Chironomus plumosus larvae increase fluxes of denitrification products and diversity of nitrate-reducing bacteria in freshwater sediment

Benthic invertebrates affect microbial processes and communities in freshwater sediment by enhancing sediment-water solute fluxes and by grazing on bacteria. Using microcosms, the effects of larvae of the widespread midge Chironomus plumosus on the efflux of denitrification products (N₂O and N₂ + N₂O) and the diversity and abundance of nitrate- and nitrous-oxide-reducing bacteria were investigated. Additionally, the diversity of actively nitrate- and nitrous-oxide-reducing bacteria was analyzed in the larval gut. The presence of larvae increased the total effluxes of N₂O and N₂ + N₂O up to 8.6- and 4.2-fold, respectively, which was mostly due to stimulation of sedimentary denitrification; incomplete denitrification in the guts accounted for up to 20% of the N₂O efflux. Phylotype richness of the nitrate reductase gene narG was significantly higher in sediment with than without larvae. In the gut, 47 narG phylotypes were found expressed, which may contribute to higher phylotype richness in colonized sediment. In contrast, phylotype richness of the nitrous oxide reductase gene nosZ was unaffected by the presence of larvae and very few nosZ phylotypes were expressed in the gut. Gene abundance of neither narG, nor nosZ was different in sediments with and without larvae. Hence, C. plumosus increases activity and diversity, but not overall abundance of nitrate-reducing bacteria, probably by providing additional ecological niches in its burrow and gut.     

Impact of acidification and eutrophication on macrophyte communities in soft waters. II. Experimental studies

In The Netherlands, there has been a dramatic decline during the last 30 years in the number of stands belonging to the phytosociological alliance Littorellion. Generally, the communities classified within this alliance occur in poorly buffered, oligotrophic waters, with very low phosphate, nitrogen and carbon dioxide levels in the water layer and considerably higher nutrient levels in the sediment. The plant species dominating these communities are isoetids such as Litoorella uniflora (L.) Aschers., Lobelia dortmanna L. and Isoetes lacustris L., which show various adaptations to make successful growth possible under these conditions.Field observations showed that the water where Littorella uniflora had disappeared or strongly decreased could be divided into two groups. A major group (77%) was characterized by the presence of submerged Juncus bulbosus L. and/or Sphagnum species. These water appeared to be strongly acidified (pH < 4.5) and had increased nitrogen levels with ammonium as the dominant N-source. Within this group, the waters with luxuriant growth of Juncus bulbosus and/or Sphagnum spp. had strongly increased carbon dioxide levels in both sediment and water.Different types of experiments proved causal relationships between the observed changes in macrophytes and the changed physico-chemical parameters. Ecophysiological experiments showed that Juncus bulbosus lacks the typical adaptations of the isoetid plant species, i.e. it uses very low amounts of sediment-CO₂ and releases only a little oxygen from the roots. However, Juncus bulbosus is more able than Littorella uniflora to use CO₂ from the water layer. From the nutrient-uptake experiments, the decreased nitrate and increased ammonium levels seem to be favourable to Juncus bulbosus. The culture experiments clearly demonstrated that the biomass of Juncus bulbosus only increased strongly when the sediment was poorly buffered and the pH of water was low. When combining factors like CO₂ enrichment of the sediment, with and without phosphate, and/or ammonium enrichment of the water in the culture experiments, it is clearly shown that phosphate and/or ammonium enrichment without CO₂ enrichment do not lead to an increase in biomass of Juncus bulbosus. Therefore, it is obvious that the changes in the macrophyte community can be ascribed primarily to changes in the carbon budget as a result of acidification.A minor group of waters (23%) was characterized by the absence of submerged Juncus bulbosus and/or Sphagnum spp. In most of these waters, submerged plant species occurred, such as Myriophyllum alterniflorum DC or non-rooted species such as Riccia fluitans L. These waters were not acidified, and generally had an increased alkalinity and higher nitrogen and phosphate levels of sediment and/or water. Culture experiments showed that phosphate enrichment of the sediment alone leads to luxuriant growth of submerged macrophyte species such as Myriophyllum alterniflorum, whereas phosphate enrichment of both sediment and water leads to mass development of non-rooted plant species such as Riccia fluitans.     

Impact of acidification and eutrophication on macrophyte communities in soft waters in The Netherlands I. Field observations

During the last decades a strong decline has been noticed in the number of waters dominated by “Littorellion” species, mostly isoetids such as Lobelia dortmanna L., Isoetes lacustris L. and Littorella uniflora (L.) Aschers. Sixty-eight waters, which were known to be dominated by L. uniflora after 1950 were investigated. In 1980, L. uniflora appeared to be absent or to have strongly decreased in 53 (78%) of the waters. In 41 of them, Littorella had been replaced by submerged Juncus bulbosus L. and/or Sphagnum spp. These changes seem to have been caused by changed inorganic carbon budgets as a consequence of acidification.In the remaining 12 waters, eutrophication of the water and/or sediment seems to be responsible for the changes in the plant communities. Enrichment with phosphate of the mineral sediment alone, leads to luxurious growth of submerged, rooted macrophyte species such as Myriophyllum alterniflorum DC and Ranunculus peltatus Schrank, whereas phosphate-enrichment of both sediment and water leads to luxurious growth of pleustophytes such as Riccia fluitans L. and Lemna minor L. in small, shallow waters, and to plankton bloom and luxurious growth of epiphytes in larger, deeper waters.In these cases light limitation seems to be responsible for the disappearance or decline of the “Littorellion” species.     

Acetogenic and Sulfate-Reducing Bacteria Inhabiting the Rhizoplane and Deep Cortex Cells of the Sea Grass Halodule wrightii

Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants. Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses Halodule wrightii and Thalassia testudinum compared to an adjacent unvegetated sediment. Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments. Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free H. wrightii roots. This suggests that the associated anaerobes are able to tolerate exposure to oxygen. To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed ³³P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of Acetobacterium, and (iv) species of Desulfovibrio. Microautoradiography revealed intercellular colonization of the roots by Acetobacterium and Desulfovibrio species. Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells. Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens. Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.     

Genome-Derived Criteria for Assigning Environmental narG and nosZ Sequences to Operational Taxonomic Units of Nitrate Reducers

Ninety percent of cultured bacterial nitrate reducers with a 16S rRNA gene similarity of ≥97% had a narG or nosZ similarity of ≥67% or ≥80%, respectively, suggesting that 67% and 80% could be used as standardized, conservative threshold similarity values for narG and nosZ, respectively (i.e., any two sequences that are less similar than the threshold similarity value have a very high probability of belonging to different species), for estimating species-level operational taxonomic units. Genus-level tree topologies of narG and nosZ were generally similar to those of the corresponding 16S rRNA genes. Although some genomes contained multiple copies of narG, recent horizontal gene transfer of narG was not apparent.Nitrate reducers (i.e., both dissimilatory nitrate reducers and denitrifiers) reduce nitrate to nitrite, which can then be reduced to ammonium by dissimilatory nitrate reducers or sequentially reduced to nitric oxide, nitrous oxide, and dinitrogen by denitrifiers (29). narG codes for the alpha subunit of the dissimilatory nitrate reductase, which reduces nitrate to nitrite and is thus common to both dissimilatory nitrate reducers and denitrifiers (29). nosZ codes for nitrous oxide reductase, which reduces nitrous oxide to dinitrogen and is common to denitrifiers but not dissimilatory nitrate reducers (29). Both narG and nosZ are commonly used as gene markers for community level analysis of nitrate reducers (2, 8, 9, 16, 18, 19, 20, 25). However, standardized criteria for assigning environmental narG and nosZ sequences to operational taxonomic units (OTUs) are required so that diverse data sets on nitrate-reducing communities can be normalized. The widespread ability of bacteria and archaea to denitrify (29) complicates the development of such criteria for genes involved in denitrification. Some closely related narG and closely related nosZ genes occur in distantly related taxa, and narG or nosZ phylogenies do not always reflect 16S rRNA phylogenies (17). However, nosZ-based phylogenies in general have a high degree of congruency with 16S rRNA gene-based phylogenies (3, 10, 30), and recent horizontal gene transfer of nosZ seems unlikely (10), indicating that denitrifier structural genes might be used for estimating the species-level novelty, as well as species-level diversity, of denitrifiers in environmental samples. The limited amount of data on horizontal gene transfer of narG (4, 24) identifies a need to extend such an approach to this gene. The limited number of studies that have compared 16S rRNA with narG or nosZ phylogenies accentuates the need for a more thorough analysis of the phylogenetic relatedness of these three genes (3, 4, 7). Thus, the main objectives of this study were to (i) resolve criteria for standardizing OTU assignment of environmental narG and nosZ sequences, (ii) determine whether those criteria can be used as indicators of novel species, and (iii) investigate the impact of horizontal gene transfer on narG.     

Effect of transient oxic conditions on the composition of the nitrate-reducing community from the rhizosphere of Typha angustifolia

Within a nitrate-reducing bacterial community, a niche differentiation between denitrifying and nitrate ammonifying bacteria may be determinated by a complex of environmental parameters, such as the availability of carbon, nitrate, and oxygen. Hence, oxygen- and carbon-releasing aerenchymatous plants may affect the composition of the nitrate-reducing community in waterlogged sediment. The composition of the nitrate-reducing community in the rhizosphere of the aerenchymatous plant species Typha angustifolia was compared with the community in nonrhizospheric sediment. All three functional groups (NO₂ ^– accumulators, N₂O producers, and presumed NH₄ ⁺ producers) were present at both sites with an ratio of 36:45:12 and 43:22:18 for nonrhizospheric and rhizospheric sediments, respectively. Most of the isolated were gram-negative, and approximately 50% of these strains demonstrated an obligatory oxidative metabolism.In the absence of nitrate, Enterobacteriaceae (belonging to the NO₂ ^– accumulating group) became dominant during enrichment of bacteria from the rhizosphere of T. angustifolia in a chemostat with glycerol (20 mM) as substrate, both under strictly anoxic and transient oxic conditions. Addition of nitrate to the chemostats led to the predominance of denitrifying pseudomonads, irrespective of the presence or absence of oxygen. However, in the presence of nitrate under anoxic conditions, enterobacteria persisted in the medium together with pseudomonads.It was concluded that oxidative bacteria such as pseudomonads are the better competitors for limiting amounts of glycerol, provided oxygen or nitrate is present. In the absence of these electron acceptors, fermentative bacteria become dominant.     

Comparison of fungal communities among ten macrophyte rhizospheres

The fungal community composition, size and several physico-chemical properties were individually investigated in ten macrophyte rhizospheric substrates using nested PCR-denaturing gradient gel electrophoresis and soil chemical methods. Results indicated that both Dothideomycetes and Sordariomycetes were dominant fungi in macrophyte rhizospheric substrates, and denitrifying fungi (Fusarium graminearum) was found in nine of ten macrophyte rhizospheres. Fungal Shannon-Wiener diversity index (H) and richness (S) in Thalia dealbata, Typha latifolia, Iris hexagona and Hemerocallis aurantiaca rhizospheres were higher than those in other six rhizospheres. Fungal number and biomass were 1.91 × 10³ CFUs g^?1 dw and 1.53 μg ergosterol g^?1 dw in Iris pseudacor rhizosphere, and were greater than in other nine rhizospheres. The correlation analysis showed that fungal number and biomass significantly and positively correlated to total soil phosphorus, while fungal H and S were significantly and negatively correlated to total organic carbon. The principal components analysis (PCA) showed that the fungal community significantly divided ten macrophyte rhizospheres into four groups, showing the significant difference of fungal communities among ten rhizospheric substrates. The current study revealed for the first time the importance of rhizospheric fungal community in distinguishing macrophyte rhizospheres, thus will undoubtedly widen our insight into fungal communities in aquatic rhizospheres.     

Competition between isoetids and invading elodeids at different concentrations of aquatic carbon dioxide

1. The growth of submerged macrophytes in softwater lakes is often assumed to be carbon limited. Isoetid species are well adapted to grow at low carbon availability and therefore commonly dominate the submerged macrophyte vegetation in softwater lakes. In many such lakes, however, large‐scale invasions of fast‐growing elodeid species, replacing the isoetid vegetation, have been observed. 2. In a laboratory experiment, we tested how rising aquatic carbon availability, in interaction with different densities of the isoetid Littorella uniflora, affected the growth (and thereby the potential invasion success) of the elodeid Myriophyllum alterniflorum. For this purpose, the growth of M. alterniflorum was determined at a combination of three concentrations of dissolved CO₂ (15, 90, 200 μmol L^?1) and three densities of L. uniflora (0, 553, 1775 plants m^?2). 3. At an ambient CO₂ of 15 μmol L^?1, M. alterniflorum could not sustain itself, whereas at raised CO₂ concentrations, growth became positive and increased with higher CO₂ availability. 4. The presence of L. uniflora, independent of its density, reduced the growth of M. alterniflorum by 50%. Whether this is related to nutrient availability or other factors is not clear. 5. Despite the growth reduction of M. alterniflorum by L. uniflora, at CO₂ ≥90 μmol L^?1, L. uniflora was still overgrown by M. alterniflorum. This may imply that, in field situations, M. alterniflorum can invade softwater systems with relatively high CO₂ availability, even in the presence of dense stands of L. uniflora.     

Nitrate respiration in Pseudomonas stutzeri A15 and its involvement in rice and wheat root colonization

Unlike most bacteria, the nitrogen-fixing rice-associated Pseudomonas stutzeri A15 disposes of three different nitrate reductases that enable conversion of nitrate to nitrite through three physiologically distinct processes, called nitrate assimilation, nitrate respiration and nitrate dissimilation. To study the role of nitrate respiration in rhizosphere fitness, a Pseudomonas stutzeri narG mutant was constructed and characterized by assessing its growth characteristics and whole-cell nitrate reductase activity in different oxygen tensions. Unexpectedly, the Pseudomonas stutzeri A15 narG mutant appeared to be a better root colonizer, outcompeting the wild type strain in a wheat and rice hydroponic system.     

Nitrogen‐cycling bacteria and archaea in the carbonate sediment of a coral reef

In the coarse‐grained carbonate sediments of coral reefs, advective porewater flow and the respiration of organic matter establish redox zones that are the scene of microbially mediated transformations of N compounds. To investigate the geobiology of N cycling in reef sediments, the benthic microbiota of Checker Reef in Kaneohe Bay, Hawaii, were surveyed for candidate nitrate reducers, ammonifying nitrite reducers, aerobic and anaerobic ammonia oxidizers (anammox) by identifying phylotypes of their key metabolic genes (napA, narG, nrfA, amoA) and ribotypes (unique RNA sequences) of anammox‐like 16S rRNA. Putative proteobacteria with the catalytic potential for nitrate reduction were identified in oxic, interfacial and anoxic habitats. The estimated richness of napA (≥202 in anoxic sediment) and narG (≥373 and ≥441 in oxic and interfacial sediment, respectively) indicates a diverse guild of nitrate reducers. The guild of nrfA hosts in interfacial reef sediment was dominated by Vibrio species. The identified members of the aerobic ammonium oxidizing guild (amoA hosts) were Crenarchaeota or close relatives of Nitrosomonadales. Putative anammox bacteria were detected in the RNA pool of Checker Reef sediment. More than half of these ribotypes show ≥90% identity with homologous sequences of Scalindua spp., while no evidence was found for members of the genera Brocadia or Kuenenia. In addition to exploring the diversity of these four nitrogen‐cycling microbial guilds in coral reef sediments, the abundances of aerobic ammonium oxidizers (amoA), nitrite oxidizers (nxrAB), ammonifying nitrite reducers (nrfA) and denitrifiers (nosZ) were estimated using real‐time PCR. Representatives of all targeted guilds were detected, suggesting that most processes of the biogeochemical N cycle can be catalyzed by the benthic microbiota of tropical coral reefs.     

Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5α and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 μM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 μM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

Waterfowl grazing effects on submerged macrophytes in a shallow mediterranean lake

Waterfowl exclusion cages were set up in Sentiz Lake, an eutrophic shallow lake in León (NW of Spain) in order to determine the role of waterfowl herbivory on macrophyte biomass and species composition. Total macrophyte biomass was high during the study (250 g DW m⁻² in summer). The macrophyte community was mainly formed by Myriophyllum alterniflorum (95% cover), Ceratophyllum demersum (5%) and Potamogeton gramineus (<0.5%). High densities of co-occurring coots (Fulica atra; 24 ind/ha) and ducks (Anas penelope, A. strepera and A. platyrhynchos; 18 ind/ha) did not have a significant effect on macrophyte biomass in the lake. There were no statistical differences between total biomass inside and outside the exclosures, although plant biomass reached a higher value inside the cages than in the lake. Biomass species composition was significantly different inside and outside exclosures; C. demersum was more abundant in the cages than in the lake. P. gramineus, almost absent in the lake, became co-dominant with M. alterniflorum in some exclosures. The detailed study of M. alterniflorum flower buds in summer showed significant herbivory by coots. Flower bud abundance was lower in the lake (35% lower in June; 85% lower in July) than under waterfowl exclusion. The effect of waterfowl on macrophyte biomass in Mediterranean wetlands seems to be negligible as compared to effects identified in northern European lakes. Apart from an important role in dispersal, waterfowl in Mediterranean areas have a strong qualitative effect on the structure of plant communities by selecting most palatable species or their reproductive structures.     

Acetogenic and sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cells of the sea grass Halodule wrightii.

Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants. Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses Halodule wrightii and Thalassia testudinum compared to an adjacent unvegetated sediment. Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments. Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free H. wrightii roots. This suggests that the associated anaerobes are able to tolerate exposure to oxygen. To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed (33)P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of Acetobacterium, and (iv) species of Desulfovibrio. Microautoradiography revealed intercellular colonization of the roots by Acetobacterium and Desulfovibrio species. Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells. Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens. Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.     

Abundance and activity of nitrate reducers in an arable soil are more affected by temporal variation and soil depth than by elevated atmospheric [CO2

Elevated atmospheric carbon dioxide concentrations ([CO(2) ]) might change the abundance and the function of soil microorganisms in the depth profile of agricultural soils by plant-mediated reactions. The seasonal pattern of abundance and activity of nitrate-reducing bacteria was studied in a Mini-FACE experiment planted with oilseed rape (Brassica napus). Three depths (0-10, 10-20 and 20-30 cm) were sampled. Analyses of the abundances of total (16S rRNA gene) and nitrate-reducing bacteria (narG, napA) revealed strong influences of sampling date and depth, but no [CO(2)] effects. Abundance and activity of nitrate reducers were higher in the top soil layer and decreased with depth but were not related to extractable amounts of nitrogen and carbon in soil. Dry periods reduced abundances of total and nitrate-reducing bacteria, whereas the potential activity of the nitrate reductase enzyme was not affected. Enzyme activity was only weakly correlated to the abundance of nitrate-reducing bacteria but was related to NH(4) (+) and NO(3) (-) concentrations. Our results suggest that in contrast to the observed pronounced seasonal changes, the elevation of atmospheric [CO(2) ] has only a marginal impact on nitrate reducers in the investigated arable ecosystem.     

Frequency and Diversity of Nitrate Reductase Genes among Nitrate-Dissimilating Pseudomonas in the Rhizosphere of Perennial Grasses Grown in Field Conditions

A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO₂ content (pCO₂) at the Swiss FACE (Free Air CO₂ Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil–associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas. The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO₂ in the rhizosphere of L. perenne. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO₂ treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO₂. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both narG and napA sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO₂ treatment.     

海草根际微生物与海草植株的互作效应

【目的】本文以三亚湾泰来草根际沉积物为主要研究对象,研究室内培养条件下泰来草根际沉积物微生物对于高温处理和海草定殖的响应。【方法】通过对培养过程中水体物理化学参数(如pH、溶解氧、磷酸盐、硝酸盐、亚硝酸盐和铵盐)的监测以分析环境因子的变化;16S rRNA扩增子测序研究微生物群落结构的动态响应;通过荧光定量分析16SrRNA基因丰度变化。【结果】研究表明高温处理组在培养35 d后海水中磷酸盐、硝酸盐、亚硝酸盐和铵盐含量以及pH均要高于模拟原位环境的对照组,高温处理组根际沉积物微生物丰度在培养过程中呈现先上升后降低的趋势,同时,高温处理组根际微生物中初始阶段由厚壁菌门(32.4%)、变形菌门(22.92%)和梭杆菌门(27.21%)占据优势,培养一段时间后,厚壁菌门和梭杆菌门大幅度减少,逐渐被蓝细菌门和放线菌门所替代,最终由变形菌门(51.1%)占据主导地位,其中,属于硫还原细菌的脱硫杆菌科(Desulfobacteraceae)和硫氧化细菌的螺杆菌科(Helicobacteraceae)的细菌丰度不断提高。【结论】揭示了海草的定殖会提高高温处理后沉积物的多样性,并塑造和改善其根际沉积物微生物群落组成。     

Impact of Plant Species and Site on Rhizosphere-Associated Fungi Antagonistic to Verticillium dahliae Kleb.

Fungi with antagonistic activity toward plant pathogens play an essential role in plant growth and health. To analyze the effects of the plant species and the site on the abundance and composition of fungi with antagonistic activity toward Verticillium dahliae, fungi were isolated from oilseed rape and strawberry rhizosphere and bulk soil from three different locations in Germany over two growing seasons. A total of 4,320 microfungi screened for in vitro antagonism toward Verticillium resulted in 911 active isolates. This high proportion of fungi antagonistic toward the pathogen V. dahliae was found for bulk and rhizosphere soil at all sites. A plant- and site-dependent specificity of the composition of antagonistic morphotypes and their genotypic diversity was found. The strawberry rhizosphere was characterized by preferential occurrence of Penicillium and Paecilomyces isolates and low numbers of morphotypes (n = 31) and species (n = 13), while Monographella isolates were most frequently obtained from the rhizosphere of oilseed rape, for which higher numbers of morphotypes (n = 41) and species (n = 17) were found. Trichoderma strains displayed high diversity in all soils, but a high degree of plant specificity was shown by BOX-PCR fingerprints. The diversity of rhizosphere-associated antagonists was lower than that of antagonists in bulk soil, suggesting that some fungi were specifically enriched in each rhizosphere. A broad spectrum of new Verticillium antagonists was identified, and the implications of the data for biocontrol applications are discussed.     

Seasonal regulation of nitrification in a rooted macrophyte (Vallisneria spiralis L.) meadow under eutrophic conditions

Variable oxygen release from the root of macrophytes growing in ammonium-rich organic substrates can stimulate the process of nitrification. To verify this hypothesis, we performed seasonal measurements of potential nitrification activity in sediments with and without the perennial submersed plant Vallisneria spiralis L. (Hydrocharitaceae). Pore water and sediment features were simultaneously considered in order to provide insights into the regulation of the process. Results demonstrated a significant effect of season and plant presence on potential nitrification activity, with higher rates in winter and lower rates in summer. Vegetated sediment displayed lower pore water ammonium, but always higher potential nitrification activity compared to the unvegetated substrate, regardless the season. Nitrification activity was strongly correlated with pore water redox status, which were affected by both season and plant presence. Along its annual cycle V. spiralis promoted more oxidized conditions in the rhizosphere likely due to elevated radial oxygen loss and the consequent maintenance of a larger nitrifying community. These outcomes confirm the results of a limited number of studies that demonstrated how sediment biogeochemistry may be controlled by plant-released oxygen also in organic-rich systems.