Franck Poly

Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil

Several specific primers for the nifH gene were tested with different pure telluric N2-fixing strains. A PolF/PolR primer set provided successful amplification of 19 representative N2-fixing strains. Three restriction enzymes, HaeIII, NdeII and MnlI, chosen for restriction fragment length polymorphism (RFLP) analyses, were the most discriminating for the study of nifH gene diversity as they resulted in differences between strains at the species level. Amplification by selected primers and RFLP were applied to assess the genetic diversity of the nifH gene pool in soil. Pair soils, one under cultivation, the second under permanent pasture, were found to harbor a contrasting diversity of nifH genes. Pure strain profiles could not be recognized in the nifH soil patterns. Using the simple procedure described, it was shown that the structure of nitrogen fixers in soil was influenced by soil functioning.

Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability

ABSTRACT Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.

Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment.

Over the last decade, important advances in molecular biology led to the development of culture-independent approaches to describing bacterial communities. These new strategies, based on the analysis of DNA directly extracted from environmental samples, circumvent the steps of isolation and culturing of bacteria, which are known for their selectivity leading to a non-representative view of the extent of bacterial diversity. This review provides an overview of the potentials and limitations of some molecular approaches currently used in microbial ecology. Examples of applications to the study of indigenous soil microbial community illustrate the feasibility and the power of such approaches.

Comparison of nifH Gene Pools in Soils and Soil Microenvironments with Contrasting Properties

ABSTRACT The similarities and differences in the structures of thenifH gene pools of six different soils (Montrond, LCSA-p, Vernon, Dombes, LCSA-c, and Thysse Kaymor) and five soil fractions extracted from LCSA-c were studied. Bacterial DNA was directly extracted from the soils, and a region of thenifH gene was amplified by PCR and analyzed by restriction. Soils were selected on the basis of differences in soil management, plant cover, and major physicochemical properties. Microenvironments differed on the basis of the sizes of the constituent particles and the organic carbon and clay contents. Restriction profiles were subjected to principal-component analysis. We showed that the composition of the diazotrophic communities varied both on a large scale (among soils) and on a microscale (among microenvironments in LCSA-c soil). Soil management seemed to be the major parameter influencing differences in the nifH gene pool structure among soils by controlling inorganic nitrogen content and its variation. However, physicochemical parameters (texture and total C and N contents) were found to correlate with differences amongnifH gene pools on a microscale. We hypothesize that the observed nifH genetic structures resulted from the adaptation to fluctuating conditions (cultivated soil, forest soil, coarse fractions) or constant conditions (permanent pasture soil, fine fractions). We attempted to identify a specific band within the profile of the clay fraction by cloning and sequencing it and comparing it with the gene databases. Unexpectedly, the nifH sequences of the dominant bacteria were most similar to sequences of unidentified marine eubacteria.

Heterogeneous Cell Density and Genetic Structure of Bacterial Pools Associated with Various Soil Microenvironments as Determined by Enumeration and DNA Fingerprinting Approach (RISA)

A bstractThe cell density and the genetic structure of bacterial subcommunities (further named pools) present in the various microenvironments of a silt loam soil were investigated. The microenvironments were isolated first using a procedure of soil washes that separated bacteria located outside aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical fractionation was then applied to the inner part in order to separate bacteria located inside stable aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate fractions, and the dispersible day fraction). Bacterial densities measured by acridine orange direct counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative distribution of cells in soil. Bacteria were preferentially located in the inner part with 87.6% and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH bacteria, respectively. The rRNA intergenic spacer analysis (RISA) was used to study the genetic structure of the bacterial pools. Different fingerprints and consequently different genetic structures were observed between the unfractionated soil and the microenvironments, and also among the various microenvironments, giving evidence that some populations were specific to a given location in addition to the common populations of all the microenvironments. Cluster and multivariate analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate fractions to the whole soil community structure, as well as the clear distinction between the pool associated to the macroaggregate fractions and the pools associated to the microaggregate ones. Furthermore, these statistical analyses allowed us to ascertain the influence of the clay and organic matter content of microenvironments on the genetic structure relatedness between pools.

EFFECTS OF GRAZING ON MICROBIAL FUNCTIONAL GROUPS INVOLVED IN SOIL N DYNAMICS

Enhancement of soil nitrogen (N) cycling by grazing has been observed in many grassland ecosystems. However, whether grazing affects the activity only of the key microbial functional groups driving soil N dynamics or also affects the size (cell number) and/or composition of these groups remains largely unknown. We studied the enzyme activity, size, and composition of five soil microbial communities (total microbial and total bacterial communities, and three functional groups driving N dynamics: nitrifiers, denitrifiers, and free N2 fixers) in grassland sites experiencing contrasting sheep grazing regimes (one light grazing [LG] site and one intensive grazing [IG] site) at two topographical locations. Enzyme activity was determined by potential carbon mineralization, nitrification, denitrification, and N2 fixation assays. The size of each community (except N2 fixers) was measured by the most-probable-number technique. The composition of the total soil microbial community was characterized by phospholipid f...

Relative contributions of plant traits and soil microbial properties to mountain grassland ecosystem services.

Summary 1. Plant functional diversity and soil microbial community composition are tightly coupled. Changes in these interactions may influence ecosystem functioning. Links between plant functional diversity, soil microbial communities and ecosystem functioning have been demonstrated in experiments using plant monocultures and mixtures, using broad plant and microbial functional groups, but have not been examined in diverse natural plant communities. 2. We quantified the relative effects of plant and microbial functional properties on key ecosystem functions. We measured plant functional diversity, soil microbial community composition and parameters associated with nitrogen (N) cycling and key nutrient cycling processes at three grassland sites in different parts of Europe. Because plant structure and function strongly influence soil microbial communities, we determined relationships between ecosystem properties, plant traits and soil community characteristics following a sequential approach in which plant traits were fitted first, followed by the additional effects of soil micro-organisms. 3. We identified a continuum from standing green biomass and standing litter, linked mostly with plant traits, to potential N mineralization and potential leaching of soil inorganic N, linked mostly with microbial properties. Plant and microbial functional parameters were equally important in explaining % organic matter content in soil. A parallel continuum ran from plant height, linked with above-ground biomass, to plant quality effects captured by the leaf economics spectrum, which were linked with the recycling of carbon (C) and N. 4. More exploitative species (higher specific leaf area, leaf N concentrations and lower leaf dry matter content) and taller swards, along with soil microbial communities dominated by bacteria, with rapid microbial activities, were linked with greater fodder production, but poor C and N retention. Conversely, dominance by conservative species (with opposite traits) and soil microbial communities dominated by fungi, and bacteria with slow activities, were usually linked with low production, but greater soil C storage and N retention. 5. Synthesis – Grassland production, C sequestration and soil N retention are jointly related to plant and microbial functional traits. Managing grasslands for selected, or multiple, ecosystem services will thus require a consideration of the joint effects of plant and soil communities. Further understanding of the mechanisms that link plant and microbial functional traits is essential to achieve this.

Shifts between Nitrospira- and Nitrobacter-like nitrite oxidizers underlie the response of soil potential nitrite oxidation to changes in tillage practices

Despite their role in soil functioning, the ecology of nitrite-oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter- and Nitrospira-like NOB (by quantitative PCR) and the community structure of the Nitrobacter-like NOB (by PCR-DGGE and cloning-sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no-tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no-tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization--i.e. ammonification--measured by the 15N dilution technique) were also surveyed. PNO was always higher for the no-tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter-like NOB and was also strongly related to gross mineralization, a proxy of N-availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira-like NOB. Selection of a dominant population was observed under no-tillage, and PNO was loosely correlated to the community structure of Nitrobacter-like NOB. Our results demonstrate that Nitrobacter-like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira-like and Nitrobacter-like NOB and to a weaker extent by shifts of populations within Nitrobacter-like NOB.

Effects of aboveground grazing on coupling among nitrifier activity, abundance and community structure

The influence of switches in grassland management to or from grazing on the dynamics of nitrifier activity, as well as the abundance of ammonia-oxidizing bacteria, AOB and ammonia-oxidizing archeae, AOA, was analyzed for two years after changing management. Additionally community structure of AOB was surveyed. Four treatments were compared in mesocosms: grazing on previously grazed grassland (G-G); no grazing on ungrazed grassland (U-U); grazing on ungrazed grassland (U-G) and cessation of grazing on grazed grassland (G-U). Nitrifier activity and abundance were always higher for G-G than U-U treatments and AOB community structure differed between these treatments. AOA abundance was in the same range as AOB abundance and followed the same trend. Grazing led to a change in AOB community structure within <5 months and a subsequent (5–12 months) increase in nitrifier activity and abundance. In contrast, cessation of grazing led to a decrease in nitrifier activity and abundance within <5 months and to a later (5–12 months) change in AOB community structure. Activity in G-U and U-G was similar to that in U-U and G-G, respectively, after 12 months. Sequence analysis of 16S rRNA gene clones showed that AOB retrieved from soils fell within the Nitrosospira lineage and percentages of AOB related to known Nitrosospira groups were affected by grazing. These results demonstrate that AOB and AOA respond quickly to changes in management. The selection of nitrifiers adapted to novel environmental conditions was a prerequisite for nitrification enhancement in U-G, whereas nitrification decrease in G-U was likely due to a partial starvation and decrease in the abundance of nitrifiers initially present. The results also suggest that taxonomic affiliation does not fully infer functional traits of AOB.

Niche construction by the invasive Asian knotweeds (species complex Fallopia): impact on activity, abundance and community structure of denitrifiers and nitrifiers.

Big Asian knotweeds (Fallopia spp.) are among the most invasive plant species in north-western Europe. We suggest that their success is partially explained by biological and chemical niche construction. In this paper, we explored the microbial mechanisms by which the plant modifies the nitrogen cycle. We found that Fallopiaspp. decreased potential denitrification enzyme activity (DEA) by reducing soil moisture and reducing denitrifying bacteria density in the soil. The plant also reduced potential ammonia and nitrite oxydizing bacteria enzyme activities (respectively, AOEA and NOEA) in sites with high AOEA and NOEA in uninvaded situation. Modification of AOEA and NOEA were not correlated to modifications of the density of implicated bacteria. AOB and Nitrobacter-like NOB community genetic structures were significantly different in respectively two and three of the four tested sites while the genetic structure of denitrifying bacteria was not affected by invasion in none of the tested sites. Modification of nitrification and denitrification functioning in invaded soils could lead to reduced nitrogen loss from the ecosystem through nitrate leaching or volatilization of nitrous oxides and dinitrogen and could be considered as a niche construction mechanism of Fallopia.