Nathalie Fromin

Molecular detection of anammox bacteria in terrestrial ecosystems: distribution and diversity

Anaerobic oxidation of ammonium (anammox) is recognized as an important process in the marine nitrogen cycle yet nothing is known about the distribution, diversity and activity of anammox bacteria in the terrestrial realm. In this study, we report on the detection of anammox sequences of Candidatus ‘Brocadia’, ‘Kuenenia’, ‘Scalindua’ and ‘Jettenia’ in marshes, lakeshores, a contaminated porous aquifer, permafrost soil, agricultural soil and in samples associated with nitrophilic or nitrogen-fixing plants. This suggests a higher diversity of anammox bacteria in terrestrial than in marine ecosystems and could be a consequence of the larger variety of suitable niches in soils. Anammox bacteria were not ubiquitously present but were only detected in certain soil types and at particular depths, thus reflecting specific ecological requirements. As opposed to marine water column habitats where Candidatus ‘Scalindua’ dominates anammox guilds, ‘Kuenenia’ and ‘Brocadia’ appear to be the most common representatives in terrestrial environments.

Secretion activity of white lupin’s cluster roots influences bacterial abundance, function and community structure

White lupin (Lupinus albus L. cv. Amiga) reacts to phosphate deficiency by producing cluster roots which exude large amounts of organic acids. The detailed knowledge of the excretion physiology of the different root parts makes it a good model plant to study plant-bacteria interaction. Since the effect of the organic acid exudation by cluster roots on the rhizosphere microflora is still poorly understood, we investigated the abundance, diversity and functions of bacteria associated with the cluster roots of white lupin, with special emphasis on the influence of root proximity (comparing root, rhizosphere soil and bulk soil fractions) and cluster root growth stages, which are characterized by different excretion activities. Plants were grown for five weeks in microcosms, in the presence of low phosphate concentrations, on acidic sand inoculated with a soil suspension from a lupin field. Plate counts showed that bacterial abundance decreased at the stage where the cluster root excretes high amounts of citrate and protons. In vitro tests on isolates showed that the frequencies of auxin producers were highest in juvenile and mature cluster roots and significantly decreased in senescent cluster roots. However, no significant difference in the frequency of auxin producers was found between cluster and non cluster roots. The diversity and structure of bacterial communities were investigated by DGGE of 16S rDNA and 16S rRNA. The diversity and community structure were mostly influenced by root proximity and, to a lesser extent, by cluster root stage. The richness of bacterial communities decreased with root proximity, whereas the proportion of active populations increased. The high citrate and proton excretion occurring at the mature stage of cluster roots had a strong impact on the structure and richness of the bacterial communities, both in the root and in the rhizosphere soil.

Functional diversity of terrestrial microbial decomposers and their substrates.

The relationship between biodiversity and biogeochemical processes gained much interest in light of the rapidly decreasing biodiversity worldwide. In this article, we discuss the current status, challenges and prospects of functional concepts to plant litter diversity and microbial decomposer diversity. We also evaluate whether these concepts permit a better understanding of how biodiversity is linked to litter decomposition as a key ecosystem process influencing carbon and nutrient cycles. Based on a literature survey, we show that plant litter and microbial diversity matters for decomposition, but that considering numbers of taxonomic units appears overall as little relevant and less useful than functional diversity. However, despite easily available functional litter traits and the well-established theoretical framework for functional litter diversity, the impact of functional litter diversity on decomposition is not yet well enough explored. Defining functional diversity of microorganisms remains one of the biggest challenges for functional approaches to microbial diversity. Recent developments in microarray and metagenomics technology offer promising possibilities in the assessment of the functional structure of microbial communities. This might allow significant progress in measuring functional microbial diversity and ultimately in our ability to predict consequences of biodiversity loss in the decomposer system for biogeochemical processes.

Interactive effects of C, N and P fertilization on soil microbial community structure and function in an Amazonian rain forest

Summary1. Resource control over abundance, structure and functional diversity of soil microbial com-munities is a key determinant of soil processes and related ecosystem functioning. Copiotroph-ic organisms tend to be found in environments which are rich in nutrients, particularly carbon,in contrast to oligotrophs, which survive in much lower carbon concentrations.2. We hypothesized that microbial biomass, activity and community structure in nutrient-poorsoils of an Amazonian rain forest are limited by multiple elements in interaction. We testedthis hypothesis with a fertilization experiment by adding C (as cellulose), N (as urea) and P (asphosphate) in all possible combinations to a total of 40 plots of an undisturbed tropical forestin French Guiana.3. After 2 years of fertilization, we measured a 47% higher biomass, a 21% increase in sub-strate-induced respiration rate and a 5-fold higher rate of decomposition of cellulose paperdiscs of soil microbial communities that grew in P-fertilized plots compared to plots without Pfertilization. These responses were amplified with a simultaneous C fertilization suggesting Pand C colimitation of soil micro-organisms at our study site.4. Moreover, P fertilization modified microbial community structure (PLFAs) to a morecopiotrophic bacterial community indicated by a significant decrease in the Gram-positive : Gram-negative ratio. The Fungi : Bacteria ratio increased in N fertilized plots,suggesting that fungi are relatively more limited by N than bacteria. Changes in microbialcommunity structure did not affect rates of general processes such as glucose mineralizationand cellulose paper decomposition. In contrast, community level physiological profiles under Pfertilization combined with either C or N fertilization or both differed strongly from all othertreatments, indicating functionally different microbial communities.5. While P appears to be the most critical from the three major elements we manipulated, thestrongest effects were observed in combination with either supplementary C or N addition insupport of multiple element control on soil microbial functioning and community structure.6. We conclude that the soil microbial community in the studied tropical rain forest and the pro-cessesitdrivesisfinelytunedbytherelativeavailabilityinC,NandP.Anyshiftsintherelativeabun-dance of these key elements may affect spatial and temporal heterogeneity in microbial communitystructure,theirassociatedfunctionsandthedynamicsofCandnutrientsintropicalecosystems.Key-words: ecosystem functioning, functional significance, microbial community structure,multiple resource limitation, phospholipid fatty acids (PLFA), phosphorus, soil functioning,tropical forest

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.

Distinct Microbial Limitations in Litter and Underlying Soil Revealed by Carbon and Nutrient Fertilization in a Tropical Rainforest

Human-caused alterations of the carbon and nutrient cycles are expected to impact tropical ecosystems in the near future. Here we evaluated how a combined change in carbon (C), nitrogen (N) and phosphorus (P) availability affects soil and litter microbial respiration and litter decomposition in an undisturbed Amazonian rainforest in French Guiana. In a fully factorial C (as cellulose), N (as urea), and P (as phosphate) fertilization experiment we analyzed a total of 540 litterbag-soil pairs after a 158-day exposure in the field. Rates of substrate-induced respiration (SIR) measured in litter and litter mass loss were similarly affected by fertilization showing the strongest stimulation when N and P were added simultaneously. The stimulating NP effect on litter SIR increased considerably with increasing initial dissolved organic carbon (DOC) concentrations in litter, suggesting that the combined availability of N, P, and a labile C source has a particularly strong effect on microbial activity. Cellulose fertilization, however, did not further stimulate the NP effect. In contrast to litter SIR and litter mass loss, soil SIR was reduced with N fertilization and showed only a positive effect in response to P fertilization that was further enhanced with additional C fertilization. Our data suggest that increased nutrient enrichment in the studied Amazonian rainforest can considerably change microbial activity and litter decomposition, and that these effects differ between the litter layer and the underlying soil. Any resulting change in relative C and nutrient fluxes between the litter layer and the soil can have important consequences for biogeochemical cycles in tropical forest ecosystems.

Functional breadth and home-field advantage generate functional differences among soil microbial decomposers

In addition to the effect of litter quality (LQ) on decomposition, increasing evidence is demonstrating that carbon mineralization can be influenced by the past resource history, mainly through following two processes: (1) decomposer communities from recalcitrant litter environments may have a wider functional ability to decompose a wide range of litter species than those originating from richer environments, i.e., the functional breadth (FB) hypothesis; and/or (2) decomposer communities may be specialized towards the litter they most frequently encounter, i.e., the home-field advantage (HFA) hypothesis. Nevertheless, the functional dissimilarities among contrasting microbial communities, which are generated by the FB and the HFA, have rarely been simultaneously quantified in the same experiment, and their relative contributions over time have never been assessed. To test these hypotheses, we conducted a reciprocal transplant decomposition experiment under controlled conditions using litter and soil originating from four ecosystems along a land-use gradient (forest, plantation, grassland, and cropland) and one additional treatment using 13C-labelled flax litter allowing us to assess the priming effect (PE) in each ecosystem. We found substantial effects of LQ on carbon mineralization (more than two-thirds of the explained variance), whereas the contribution of the soil type was fairly low (less than one-tenth), suggesting that the contrasting soil microbial communities play only a minor role in regulating decomposition rates. Although the results on PE showed that we overestimated litter-derived CO2 fluxes, litter-microbe interactions contributed significantly to the unexplained variance observed in carbon mineralization models. The magnitudes of FB and HFA were relatively similar, but the directions of these mechanisms were sometimes opposite depending on the litter and soil types. FB and HFA estimates calculated on parietal sugar mass loss were positively correlated with those calculated on enzymatic activity, confirming the idea that the interaction between litter quality and microbial community structure may modify the trajectory of carbon mineralization via enzymatic synthesis. We conclude that although litter quality was the predominant factor controlling litter mineralization, the local microbial communities and interactions with their substrates can explain a small (< 5%) but noticeable portion of carbon fluxes.

C, N and P fertilization in an Amazonian rainforest supports stoichiometric dissimilarity as a driver of litter diversity effects on decomposition

Plant leaf litter generally decomposes faster as a group of different species than when individual species decompose alone, but underlying mechanisms of these diversity effects remain poorly understood. Because resource C : N : P stoichiometry (i.e. the ratios of these key elements) exhibits strong control on consumers, we supposed that stoichiometric dissimilarity of litter mixtures (i.e. the divergence in C : N : P ratios among species) improves resource complementarity to decomposers leading to faster mixture decomposition. We tested this hypothesis with: (i) a wide range of leaf litter mixtures of neotropical tree species varying in C : N : P dissimilarity, and (ii) a nutrient addition experiment (C, N and P) to create stoichiometric similarity. Litter mixtures decomposed in the field using two different types of litterbags allowing or preventing access to soil fauna. Litter mixture mass loss was higher than expected from species decomposing singly, especially in presence of soil fauna. With fauna, synergistic litter mixture effects increased with increasing stoichiometric dissimilarity of litter mixtures and this positive relationship disappeared with fertilizer addition. Our results indicate that litter stoichiometric dissimilarity drives mixture effects via the nutritional requirements of soil fauna. Incorporating ecological stoichiometry in biodiversity research allows refinement of the underlying mechanisms of how changing biodiversity affects ecosystem functioning.

Examination of Gould's modified S1 (mS1) selective medium and Angle's non-selective medium for describing the diversity of Pseudomonas spp. in soil and root environments

Abstract Studies on the diversity of environmental culturable Pseudomonas populations are dependent on the isolation procedure. This procedure includes the use of selective media which may influence the recovery of strains and thus the diversity described. In this study, we assessed the use of two agar isolation media for describing the diversity of soil- and root-inhabiting Pseudomonas associated with the perennial grass Molinia coerulea. A total of 382 Pseudomonas strains were recovered on either non-selective Angles medium, or on Goulds modified S1 (mS1) Pseudomonas-selective medium. Their diversity was assessed by restriction analysis of PCR (polymerase chain reaction)-amplified 16S-23S rDNA internal transcript spacer sequences. The comparison of mS1- and Angle-recovered populations showed that the use of mS1 selective medium led to an underestimation of both Pseudomonas counts and diversity, especially in the soil environment.

Diversity of leaf litter leachates from temperate forest trees and its consequences for soil microbial activity

Leaching of water-soluble compounds is a dominant process during the first stages of litter decomposition, providing the microorganisms in the underlying soil with an important source of labile carbon and nutrients. Leachate composition (quantity and quality) can vary considerably among different plant species, but its consequences for soil microbially-driven processes remains largely unexplored. Here, we evaluated the differences in leachate quantity and quality from freshly fallen leaf litter of widely distributed coniferous and deciduous broadleaf tree species of European temperate forests, and their effects on soil microbial responses in a microcosm experiment under controlled conditions. Leachates of broadleaf litter contained higher amounts of carbon and nitrogen available for microbes, but with substantially higher aromaticity than leachates from coniferous litter. A one-time leachate addition to soils immediately increased soil microbial respiration with longer lasting effects of deciduous broadleaf compared to coniferous litter leachates leading to a microbial community with an apparently more efficient use of carbon. When leachates of different species were mixed, the observed microbial responses differed in some cases from that expected based on soils to which leachates from single species were added. These non-additive effects were partly explained by the functional dissimilarity of leachate traits, suggesting complementary resources for microorganisms when leachates of different species are available. Our data show that species-specific litter-derived leachates of varying quantity and quality and their mixtures distinctly affect soil microorganisms. In forest ecosystems with recurrent leaf litter inputs from the same species, such leachate effects may determine soil processes also in the longer term, controlling biogeochemical cycling to an important degree.