Feth el Zahar Haichar

Plant host habitat and root exudates shape soil bacterial community structure

The rhizosphere is active and dynamic in which newly generated carbon, derived from root exudates, and ancient carbon, in soil organic matter (SOM), are available for microbial growth. Stable isotope probing (SIP) was used to determine bacterial communities assimilating each carbon source in the rhizosphere of four plant species. Wheat, maize, rape and barrel clover (Medicago truncatula) were grown separately in the same soil under 13CO2 (99% of atom 13C) and DNA extracted from rhizosphere soil was fractionated by isopycnic centrifugation. Bacteria-assimilating root exudates were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of 13C-DNA and root DNA, whereas those assimilating SOM were identified from 12C-DNA. Plant species root exudates significantly shaped rhizosphere bacterial community structure. Bacteria related to Sphingobacteriales and Myxococcus assimilated root exudates in colonizing roots of all four plants, whwereas bacteria related to Sphingomonadales utilized both carbon sources, and were identified in light, heavy and root compartment DNA. Sphingomonadales were specific to monocotyledons, whereas bacteria related to Enterobacter and Rhizobiales colonized all compartments of all four plants, used both fresh and ancient carbon and were considered as generalists. There was also evidence for an indirect important impact of root exudates, through stimulation of SOM assimilation by a diverse bacterial community.

Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots.

A specificity of Brassicaceous plants is the production of sulphur secondary metabolites called glucosinolates that can be hydrolysed into glucose and biocidal products. Among them, isothiocyanates are toxic to a wide range of microorganisms and particularly soil-borne pathogens. The aim of this study was to investigate the role of glucosinolates and their breakdown products as a factor of selection on rhizosphere microbial community associated with living Brassicaceae. We used a DNA-stable isotope probing approach to focus on the active microbial populations involved in root exudates degradation in rhizosphere. A transgenic Arabidopsis thaliana line producing an exogenous glucosinolate and the associated wild-type plant associated were grown under an enriched 13CO2 atmosphere in natural soil. DNA from the rhizospheric soil was separated by density gradient centrifugation. Bacterial (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Acidobacteria), Archaea and fungal community structures were analysed by DGGE fingerprints of amplified 16S and 18S rRNA gene sequences. Specific populations were characterized by sequencing DGGE fragments. Roots of the transgenic plant line presented an altered profile of glucosinolates and other minor additional modifications. These modifications significantly influenced microbial community on roots and active populations in the rhizosphere. Alphaproteobacteria, particularly Rhizobiaceae, and fungal communities were mainly impacted by these Brassicaceous metabolites, in both structure and composition. Our results showed that even a minor modification in plant root could have important repercussions for soil microbial communities.

Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites.

Previous studies on the effect of secondary metabolites on the functioning of rhizosphere microbial communities have often focused on aspects of the nitrogen (N) cycle but have overlooked biological denitrification inhibition (BDI), which can affect plant N-nutrition. Here, we investigated the BDI by the compounds of Fallopia spp., an invasive weed shown to be associated with a low potential denitrification of the soil. Fallopia spp. extracts were characterized by chromatographic analysis and were used to test the BDI effects on the metabolic and respiratory activities of denitrifying bacteria, under aerobic and anaerobic (denitrification) conditions. The BDI of Fallopia spp. extracts was tested on a complex soil community by measuring denitrification enzyme activity (DEA), substrate induced respiration (SIR), as well as abundances of denitrifiers and total bacteria. In 15 strains of denitrifying bacteria, extracts led to a greater BDI (92%) than respiration inhibition (50%). Anaerobic metabolic activity reduction was correlated with catechin concentrations and the BDI was dose dependent. In soil, extracts reduced the DEA/SIR ratio without affecting the denitrifiers: total bacteria ratio. We show that secondary metabolite(s) from Fallopia spp. inhibit denitrification. This provides new insight into plant-soil interactions and improves our understanding of a plants ability to shape microbial soil functioning.

Stable isotope probing of bacterial community structure and gene expression in the rhizosphere of Arabidopsis thaliana

The rhizosphere is an active compartment where plant and microorganisms establish a molecular dialogue. In this study, we analysed the impact of Arabidopsis thaliana on bacterial community structure and the expression of certain beneficial genes using DNA- and mRNA-SIP in the rhizosphere of plantlets grown under (13)CO(2) for 13, 21 and 27 days. DNA- and rRNA-SIP revealed changes in bacterial communities inhabiting the rhizosphere soil that were probably related to modification of root exudates, while root-colonizing populations were maintained over time suggesting their metabolic versatility and adaptation. The impact of the plant via root exudates on the expression of the noncoding RNAs rsmZ, acdS gene encoding 1-aminocyclopropane-1-carboxylate deaminase and nosZ gene encoding nitrous oxide reductase, in the root-adhering soil and on the roots of A. thaliana was determined using mRNA-SIP. Results showed that these genes were present and expressed by bacteria inhabiting roots and by those that derive nutrients from the breakdown of organic matter in soils or from root exudates. The expression of rsmZ under natural conditions indicates the importance of noncoding RNAs in bacterial adaptation to their ecological niches.

Stable isotope probing of carbon flow in the plant holobiont.

Microbial communities associated with a plant host, constituting a holobiont, affect the physiology and growth of the plant via metabolites that are mainly derived from their photosynthates. The structure and function of active microbial communities that assimilate root exudates can be tracked by using stable isotope probing (SIP) approaches. This article reviews results from ongoing SIP research in plant-microbe interactions, with a specific focus on investigating the fate of fresh and recalcitrant carbon in the rhizosphere with 13C enriched-root exudates, in addition to identifying key players in carbon cycling. Finally, we discuss new SIP applications that have the potential to identify novel enzymes implicated in rhizoremediation or plant genes dedicated to root exudation by combining SIP approaches and genome wide associations studies.

The effects of plant nutritional strategy on soil microbial denitrification activity through rhizosphere primary metabolites

The aim of this study was to determine (i) whether plant nutritional strategy affects the composition of primary metabolites exuded into the rhizosphere and (ii) the impact of exuded metabolites on denitrification activity in soil. We answered this question by analysing primary metabolite content extracted from the root-adhering soil (RAS) and the roots of three grasses representing different nutrient management strategies: conservative (Festuca paniculata), intermediate (Bromus erectus) and exploitative (Dactylis glomerata). We also investigated the impact of primary metabolites on soil microbial denitrification enzyme activity without carbon addition, comparing for each plant RAS and bulk soils. Our data show that plant nutritional strategy impacts on primary metabolite composition of root extracts or RAS. Further we show, for the first time, that RAS-extracted primary metabolites are probably better indicators to explain plant nutrient strategy than root-extracted ones. In addition, our results show that some primary metabolites present in the RAS were well correlated with soil microbial denitrification activity with positive relationships found between denitrification and the presence of some organic acids and negative ones with the presence of xylose. We demonstrated that the analysis of primary metabolites extracted from the RAS is probably more pertinent to evaluate the impact of plant on soil microbial community functioning.

Identification of B-type procyanidins in Fallopia spp. involved in biological denitrification inhibition.

Nitrogen (N) is considered as a main limiting factor in plant growth, and nitrogen losses through denitrification can be responsible for severe decreases in plant productivity. Recently, it was demonstrated that Fallopia spp. is responsible for biological denitrification inhibition (BDI) through the release of unknown secondary metabolites. Here, we investigate the secondary metabolites involved in the BDI of Fallopia spp. The antioxidant, protein precipitation capability of Fallopia spp. extracts was measured in relation to the aerobic respiration and denitrification of two bacteria (Gram positive and Gram negative). Proanthocyanidin concentrations were estimated. Proanthocyanidins in extracts were characterized by chromatographic analysis, purified and tested on the bacterial denitrification and aerobic respiration of two bacterial strains. The effect of commercial procyanidins on denitrification was tested on two different soil types. Denitrification and aerobic respiration inhibition were correlated with protein precipitation capacity and concentration of proanthocyanidins but not to antioxidant capacity. These proanthocyanidins were B-type procyanidins that inhibited denitrification more than the aerobic respiration of bacteria. In addition, procyanidins also inhibited soil microbial denitrification. We demonstrate that procyanidins are involved in the BDI of Fallopia spp. Our results pave the way to a better understanding of plant-microbe interactions and highlight future applications for a more sustainable agriculture.

Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI.

Plant host habitat and root exudates shape fungal diversity

The rhizospheric microbiome is clearly affected by plant species and certain of their functional traits. These functional traits allow plants to adapt to their environmental conditions by acquiring or conserving nutrients, thus defining different ecological resource-use plant strategies. In the present study, we investigated whether plants with one of the two nutrient-use strategies (conservative versus exploitative) could influence fungal communities involved in soil organic matter degradation and root exudate assimilation, as well as those colonizing root tissues. We applied a DNA-based, stable-isotope probing (DNA-SIP) approach to four grass species distributed along a gradient of plant nutrient resource strategies, ranging from conservative to exploitative species, and analyzed their associated mycobiota composition using a fungal internal transcribed spacer (ITS) and Glomeromycotina 18S rRNA gene metabarcoding approach. Our results demonstrated that fungal taxa associated with exploitative and conservative plants could be separated into two general categories according to their location: generalists, which are broadly distributed among plants from each strategy and represent the core mycobiota of soil organic matter degraders, root exudate consumers in the root-adhering soil, and root colonizers; and specialists, which are locally abundant in one species and more specifically involved in soil organic matter degradation or root exudate assimilation on the root-adhering soil and the root tissues. Interestingly, for arbuscular mycorrhizal fungi analysis, all plant roots were mainly colonized by Glomus species, whereas an increased diversity of Glomeromycotina genera was observed for the exploitative plant species Dactylis glomerata.