Centre de Microbiologie du Sol et de l'Environnement

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.

Soils are the product of the activities of plants, which supply organic matter and play a pivotal role in weathering rocks and minerals. Many plant species have a distinct ecological amplitude that shows restriction to specific soil types. In the numerous interactions between plants and soil, microorganisms also play a key role. Here we review the existing literature on interactions between plants, microorganisms and soils, and include considerations of evolutionary time scales, where possible. Some of these interactions involve intricate systems of communication, which in the case of symbioses such as the arbuscular mycorrhizal symbiosis are several hundreds of millions years old; others involve the release of exudates from roots, and other products of rhizodeposition that are used as substrates for soil microorganisms. The possible reasons for the survival value of this loss of carbon over tens or hundreds of millions of years of evolution of higher plants are discussed, taking a cost-benefit approach. Co-evolution of plants and rhizosphere microorganisms is discussed, in the light of known ecological interactions between various partners in terrestrial ecosystems. Finally, the role of higher plants, especially deep-rooted plants and associated microorganisms in the weathering of rocks and minerals, ultimately contributing to pedogenesis, is addressed. We show that rhizosphere processes in the long run are central to biogeochemical cycles, soil formation and Earth history. Major anticipated discoveries will enhance our basic understanding and allow applications of new knowledge to deal with nutrient deficiencies, pests and diseases, and the challenges of increasing global food production and agroecosystem productivity in an environmentally responsible manner.

Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.

The silicate rock weathering followed by the formation of carbonate rocks in the ocean, transfers CO 2 from the atmosphere to the lithosphere. This CO 2 uptake plays a major role in the regulation of atmospheric CO 2 concentrations at the geologic timescale and is mainly controlled by the chemical properties of rocks. This leads us to develop the first world lithological map with a grid resolution of 1° × 1°. This paper analyzes the spatial distribution of the six main rock types by latitude, continents, and ocean drainage basins and for 49 large river basins. Coupling our digital map with the GEM‐CO2 model, we have also calculated the amount of atmospheric/soil CO 2 consumed by rock weathering and alkalinity river transport to the ocean. Among all silicate rocks, shales and basalts appear to have a significant influence on the amount of CO 2 uptake by chemical weathering.

The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaselous vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.

Statistical relationships were established between the fate of C and N from 47 types of crop residues and their biochemical characteristics during a soil incubation at 15°C. The incubations were carried out under nonlimiting N in order to differentiate the effects of biochemical characteristics of residues from those of soil N availability. Depending on the residue, the apparent mineralization of residue C after 168 d varied from 330 to 670 g kg −1 of added C. Mineralization kinetics were described using a two‐compartment decomposition model that decomposes according to first‐order kinetics. Amounts of C mineralized after 7 d and the decomposition rate coefficient of the labile fraction were related mainly to the soluble C forms of the residue. No statistical relationship was established between the N concentration of residues and their decomposition in the soil. The incorporation of crop residues into soil led to various soil mineral N dynamics. Two residues caused net N mineralization from the time of their incorporation, whereas all the others induced net N immobilization (1–33 g N kg −1 of added C). After 168 d, only residues with a C/N ratio <24 induced a surplus of mineral N compared with the control soil. The mineral N dynamics were related mainly to the organic N concentration of the residues and to their C/N ratio. At the start of incubation, these dynamics were also influenced by the presence of polyphenols in the plant tissues. Finally, this study showed the need to include the biochemical quality of crop residues in any C and N transformation models that describe decomposition. In contrast, the N concentration or C/N ratio of the residues are sufficient to predict the net effects of crop residues on soil mineral N dynamics.

Fusarium oxysporum is well represented among the rhizosphere microflora. While all strains exist saprophytically, some are well-known for inducing wilt or root rots on plants whereas others are considered as nonpathogenic. Several methods based on phenotypic and genetic traits have been developed to characterize F. oxysporum strains. Results showed the great diversity affecting the soil-borne populations of F. oxysporum. In suppressive soils, interactions between pathogenic and nonpathogenic strains result in the control of the disease. Therefore nonpathogenic strains are developed as biocontrol agents. The nonpathogenic F. oxysporum strains show several modes of action contributing to their biocontrol capacity. They are able to compete for nutrients in the soil, affecting the rate of chlamydospore germination of the pathogen. They can also compete for infection sites on the root, and can trigger plant defence reactions, inducing systemic resistance. These mechanisms are more or less important depending on the strain. The nonpathogenic F. oxysporum are easy to mass produce and formulate, but application conditions for biocontrol efficacy under field conditions have still to be determined.

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5' end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.

Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa.

Summary Plant diseases induced by soil‐borne plant pathogens are among the most difficult to control. In the absence of effective chemical control methods, there is renewed interest in biological control based on application of populations of antagonistic micro‐organisms. In addition to Pseudomonas spp. and Trichoderma spp., which are the two most widely studied groups of biological control agents, the protective strains of Fusarium oxysporum represent an original model. These protective strains of F. oxysporum can be used to control wilt induced by pathogenic strains of the same species . Exploring the mechanisms involved in the protective capability of these strains is not only necessary for their development as commercial biocontrol agents but raises many basic questions related to the determinism of pathogenicity versus biocontrol capacity in the F. oxysporum species complex. In this paper, current knowledge regarding the interaction between the plant and the protective strains is reviewed in comparison with interactions between the plant and pathogenic strains. The success of biological control depends not only on plant–microbial interactions but also on the ecological fitness of the biological control agents. Contents Summary 529 I. Biological control of plant diseases: state of the art 530 II. Main modes of action of biological control agents 530 III. The protective strains of F. oxysporum: an unexplored model 532 IV. Future directions for the study of the protective capacity of strains of F. oxysporum 539 V. How to make biological control successful in the field? 540 References 541

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO3)2, (NH4)2SO4, and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH4)2SO4 and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO3)2 or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.

Thirty-one strains of two new genomic species (genomic species 1 and 2) of rhizobia isolated from root nodules of Phaseolus vulgaris and originating from various locations in France were compared with reference strains of rhizobia by performing a numerical analysis of 64 phenotypic features. Each genomic species formed a distinct phenon and was separated from the other rhizobial species. A comparison of the complete 16S rRNA gene sequences of a representative of genomic species 1 (strain R602spT) and a representative of genomic species 2 (strain H152T) with the sequences of other rhizobia and related bacteria revealed that each genomic species formed a lineage independent of the lineages formed by the previously recognized species of rhizobia. Genomic species 1 clustered with the species that include the bean-nodulating rhizobia, Rhizobium leguminosarum, Rhizobium etli, and Rhizobium tropici, and branched with unclassified rhizobial strain OK50, which was isolated from root nodules of Pterocarpus klemmei in Japan. Genomic species 2 was distantly related to all other Rhizobium species and related taxa, and the most closely related organisms were Rhizobium galegae and several Agrobacterium species. On the basis of the results of phenotypic and phylogenetic analyses and genotypic data previously published and reviewed in this paper, two new species of the genus Rhizobium, Rhizobium gallicum and Rhizobium giardinii, are proposed for genomic species 1 and 2, respectively. Each species could be divided in two subgroups on the basis of symbiotic characteristics, as shown by phenotypic (host range and nitrogen fixation effectiveness) and genotypic data. For each species, one subgroup had the same symbiotic characteristics as R. leguminosarum biovar phaseoli and R. etli biovar phaseoli. The other subgroup had a species-specific symbiotic phenotype and genotype. Therefore, we propose that each species should be subdivided into two biovars, as follows: R. gallicum biovar gallicum and R. gallicum biovar phaseoli; and R. giardinii biovar giardinii and R. giardinii biovar phaseoli.

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.

ABSTRACT The function of nitric oxide (NO), a gaseous free radical emitted by many plants, is incompletely understood. In the present study the hypothesis that NO generation, like that of the reactive oxygen species, occurs as a general response to different environmental cues was tested. Leaf peels and mesophyll cell suspensions of Nicotiana tabacum cv. Xanthi were loaded with the NO‐specific fluorophore, diaminofluorescein, and subjected to an abiotic stressor. Light stress and mechanical injury had no apparent effect on NO production. In contrast, high temperatures, hyperosmotic stress, salinity and epi‐illumination in a microscope all led to rapid surges in NO‐induced fluorescence. The fluorescence originated from cells of the palisade mesophyll and across all epidermal cell types, including guard cells, subsidiary cells, and long and short trichomes. Fluorescence was evident first in the plastids, then in the nucleus and finally throughout the cytosol. Nicotiana plumbaginifolia cell suspensions expressing the calcium reporter aequorin provided evidence that, under hyperosmotic stress, NO participates in the elevation of free Ca 2+ in the cytoplasm. The physiological significance of NO production in response to abiotic stressors is discussed.

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1/FF390. This in silico analysis of the specificity of FR1/FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1/FF390 for Fungi was validated in vitro by cloning--sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.

Land-use practices aiming at increasing agro-ecosystem sustainability, e.g. no-till systems and use of temporary grasslands, have been developed in cropping areas, but their environmental benefits could be counterbalanced by increased N2O emissions produced, in particular during denitrification. Modelling denitrification in this context is thus of major importance. However, to what extent can changes in denitrification be predicted by representing the denitrifying community as a black box, i.e. without an adequate representation of the biological characteristics (abundance and composition) of this community, remains unclear. We analysed the effect of changes in land uses on denitrifiers for two different agricultural systems: (i) crop/grassland conversion and (ii) cessation/application of tillage. We surveyed potential denitrification (PD), the abundance and genetic structure of denitrifiers (nitrite reducers), and soil environmental conditions. N2O emissions were also measured during periods of several days on control plots. Time-integrated N2O emissions and PD were well correlated among all control plots. Changes in PD were partly due to changes in denitrifier abundance but were not related to changes in the structure of the denitrifier community. Using multiple regression analysis, we showed that changes in PD were more related to changes in soil environmental conditions than in denitrifier abundance. Soil organic carbon explained 81% of the variance observed for PD at the crop/temporary grassland site, whereas soil organic carbon, water-filled pore space and nitrate explained 92% of PD variance at the till/no-till site, without any residual effect of denitrifier abundance. Soil environmental conditions influenced PD by modifying the specific activity of denitrifiers, and to a lesser extent by promoting a build-up of denitrifiers. Our results show that an accurate simulation of carbon, oxygen and nitrate availability to denitrifiers is more important than an accurate simulation of denitrifier abundance and community structure to adequately understand and predict changes in PD in response to land-use changes.

Assessing soil microbial community structure by the use of molecular techniques requires a satisfactory sampling strategy that takes into account the high microbial diversity and the heterogeneous distribution of microorganisms in the soil matrix. The influence of the sample size of three different soil types (sand, silt and clay soils) on the DNA yield and analysis of bacterial and fungal community structure were investigated. Six sample sizes from 0.125 g to 4 g were evaluated. The genetic community structure was assessed by automated ribosomal intergenic spacer analysis (A-RISA fingerprint). Variations between bacterial (B-ARISA) and fungal (F-ARISA) community structure were quantified by using principal component analysis (PCA). DNA yields were positively correlated with the sample size for the sandy and silty soils, suggesting an influence of the sample size on DNA recovery, whereas no correlation was observed in the clay soil. B-ARISA was shown to be consistent between the different sample sizes for each soil type indicating that the sampling procedure has no influence on the assessment of bacterial community structure. On the contrary for F-ARISA profiles, strong variations were observed between replicates of the smaller samples (<1 g). Principal component analysis analysis revealed that sampling aliquots of soil > or =1 g are required to obtain robust and reproducible fingerprinting analysis of the genetic structure of fungal communities. However, the smallest samples could be adequate for the detection of minor populations masked by dominant ones in larger samples. The sampling strategy should therefore be different according to the objectives: rather large soil samples (> or =1 g) for a global description of the genetic community structure, or a large number of small soil samples for a more complete inventory of microbial diversity.

Aim The spatial organization of soil microbial communities on large scales and the identification of environmental factors structuring their distribution have been little investigated. The overall objective of this study was to determine the spatial patterning of microbial biomass in soils over a wide extent and to rank the environmental filters most influencing this distribution. Location French territory using the French Soil Quality Monitoring Network. This network covers the entire French territory and soils were sampled at 2150 sites along a systematic grid. Methods The soil DNA extracted from all these soils was expressed in terms of soil molecular microbial biomass and related to other soil and land-use data over French territory. Results This study provides the first extensive map of microbial biomass and reveals the heterogeneous and spatially structured distribution of this biomass on the scale of France. The main factors driving biomass distribution are the physico-chemical properties of the soil (texture, pH and total organic carbon) as well as land use. Soils from land used for intensive agriculture, especially monoculture and vineyards, exhibited the smallest biomass pools. Interestingly, factors known to influence the large-scale distribution of macroorganisms, such as climatic factors, were not identified as important drivers for microbial communities. Main conclusions Microbial abundance is spatially structured and dependent on local filters such as soil characteristics and land use but is relatively independent of global filters such as climatic factors or the presence of natural barriers. Our study confirms that the biogeography of microorganisms differs fundamentally from the biogeography of ‘macroorganisms’ and that soil management can have significant large-scale effects.

Taking into account the strong iron competition in the rhizosphere and the high affinity of pyoverdines for Fe(III), these molecules are expected to interfere with the iron nutrition of plants, as they do with rhizospheric microbes. The impact of Fe-pyoverdine on iron content of Arabidopsis thaliana was compared with that of Fe-EDTA. Iron chelated to pyoverdine was incorporated in a more efficient way than when chelated to EDTA, leading to increased plant growth of the wild type. A transgenic line of A. thaliana overexpressing ferritin showed a higher iron content than the wild type when supplemented with Fe-EDTA but a lower iron content when supplemented with Fe-pyoverdine despite its increased reductase activity, suggesting that this activity was not involved in the iron uptake from pyoverdine. A mutant knock-out iron transporter IRT1 showed lower iron and chlorophyll contents when supplemented with Fe-EDTA than the wild type but not when supplemented with Fe-pyoverdine, indicating that, in contrast to iron from EDTA, iron from pyoverdine was not incorporated through the IRT1 transporter. Altogether these data suggest that iron from Fe-pyoverdine was not incorporated in planta through the strategy I, which is based on reductase activity and IRT1 transporter. This is supported by the presence of pyoverdine in planta as shown by enzyme-linked immunosorbent assay and by tracing 15N of 15N-pyoverdine.