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This study looked at the synergistic effect of Pseudomonas alkylphenolica PF9 and Sinorhizobium meliloti Rm41 on the Moroccan alfalfa population (Oued Lmaleh) grown under symbiotic nitrogen fixation and limited phosphorus (P) availability. The experiment was conducted in a growth chamber and after two weeks of sowing, the young seedlings were inoculated with Sinorhizobium meliloti Rm41 alone or combined with a suspension of Pseudomonas alkylphenolica PF9. Then, the seedlings were submitted to limited available P (insoluble P using Ca3HPO4) versus a soluble P form (KH2PO4) at a final concentration of 250 μmol P·plant-1·week-1. After two months of P stress, the experiment was evaluated through some agro-physiological and biochemical parameters. The results indicated that the inoculation of alfalfa plants with Sinorhizobium strain alone or combined with Pseudomonas strain significantly (p

International audience; Modern intensive agricultural systems generally focus on the productivity of monocultures. They are characterized by a low diversity of crops, with uniform and symmetrical planting layouts. They largely rely on the utilization of chemical inputs. They are widely denounced for their negative environmental impacts. In this context, the ecological intensification framework proposes the exploitation of biodiversity in order to better achieve such ecosystem services and soil conservation. Intercropping, i.e., the simultaneous growth of two or more crops mixed in the same field, appears to have the potentialities to improve the productivity, resilience capacity, and ecological sustainability of agroecosystems through the intensification of such positive interactions between plants as facilitation and niche complementarity. Cereal-legume intercropping turns out to be effective in low-N agroecosystems, since legumes have the ability to fix atmospheric nitrogen via their symbiosis with rhizobia. This fixed N, in turn, benefits the cereal through various ecological processes. The objective of the project is to improve the benefit of legumes for intercropped cereals in low-input agroecosystems through the management of plant-plant and plant-microbe interactions. The nitrogen-fixing symbiosis requires phosphorus and iron to be efficient. While these nutrients are prone to be lacking in N-limited agroecosystems, as is the case in Mediterranean agroecosystems, plant-plant interactions and rhizobacteria and mycorrhiza interactions seem to play an important role in their acquisition and efficient utilization. We propose the development of a participatory research project in four Mediterranean agroecosystems. Agronomic and environmental diagnosis will be performed in the field to assess N and P biogeochemical cycles, as well as Fe availability, in combination with the plant performances and the diversity of soil microorganisms. Molecular identification of soil microorganisms from the most productive sites will be done and research of genes for tolerance to Fe- and P-deficiencies will be realized. Glasshouse experiments involving various cultivars of cereals and legumes, as well as the previously identified microorganisms, will be done in order to disentangle the various mechanisms of nutrient acquisition, sharing, and transfer between plants. Other experiments will assess the effects of cereal-legume-microbe interactions on the development and architecture of the plant root systems and root hair development. The lines of research are integrated with a strategy of functional ecology on plant-microbe-soil interactions in the agroecosystems of Gabes (Tunisia), Boumedfaa (Algeria), Beni Mellal (Morocco), and Thessaloniki (Greece). Using multidisciplinary and innovative approaches, the program will provide novel knowledge and understanding of agroecosystem management for food production.

International audience; Phosphorus (P) deficiency is a critical environmental constraint that affects the growth and development of several legume crops that are usually cultivated in semi-arid regions and marginal areas. P-deficiency is known to be a significant limitation for symbiotic nitrogen fixation (SNF), and variability in SNF is strongly interlinked with the concentrations of inorganic phosphate (Pi) in the nodules. To cope with P-deficiency, legumes trigger various adaptive responses, including the induction and secretion of acid phosphatases (APases) and phytases, maintenance of Pi homeostasis in nodules and other organs, and improvement of oxygen consumption per unit of nodule mass. While the induction of phytases is a universal response to P-deficiency in higher plants, the physiological role of these enzymes in nodules of N2-fixing legume is crucial but still not fully understood. Understanding the role of APases and phytases activities in response to environmental constraints may be important in developing strategies to improve the tolerance of the nodulated legume to low-P availability. In this review, we summarize and discuss recent advances in the understanding of the expression of phytases within the nodules of legumes under P-deficiency and address whether this expression might influence the nodule respiration and contribute to adaptation of N2 fixing legumes to low-P environments. Finally, we will provide perspectives on future directions for research in this field.

International audience; Low phosphorus (P) availability is a primary factor that limits the production of common bean (Phaseolus vulgaris L.) in many parts of the world, especially when plant growth depends on N2 fixation. In order to understand how common bean copes with this nutritional constraint, two recombinant inbred lines (RILs) of common bean namely RILs 115 and 147 were studied in the field conditions during three growing seasons from 2012 to 2014. At flowering stage, plants were harvested and analyzed for their nodulation, growth, P content and yield. Results showed that for RIL115, the nodulation (43%), shoot (28%) and root growth (32%) was higher than for RIL147 in a low-P availability soil whatever the growing season. In addition, RIL115 had better growth and efficiency in use of P for the rhizobial symbiosis (7.29 mg nodule dry weight mg−1 nodule P) about twice higher may be involved in tolerance to low-P, and to be a useful marker for field data. At harvest, the RIL115 showed higher grain yield (1146 kg ha−1) than the RIL147 (1045 kg ha−1). It is concluded that the genotype of RIL 115 had a higher yield and could best adapt to low-P availability under field condition.

In this work, we studied the essential oils (EOs) obtained by hydrodistillation and lipophilic fractions extrated by carbon dioxide supercritical fluid extraction (SFE-CO2) from Teucrium polium (Lamiaceae). EOs and SFE-CO2 extracts were analyzed by GC and GC-TOFMS. EOs showed qualitative differences compared with SFE-CO2 extracts, though in both products germacrene D was the most abundant component. Notably a new EO chemotype, characterzed by germacrene D, β-eudesmol, shyobunol, and δ-cadinene, was reported. EOs along with SFE-CO2 extracts, plant powder, and solid residue after SFE were evaluated for total phenolic content (TPC) and antioxdiant activity by DPPH, FRAP, and ORAC assays. The raw plant material was the most active, followed by the solid residue after SFE-CO2 extraction. On the other hand, EO and SFE-CO2 extracts displayed weak activity. These results showed that the antioxidant compounds of T. polium are of hydrophilic nature and are not extracted or retained in the plant material after SFE-CO2 extraction.

A total of 160 samples of 13 types of fresh fruits and vegetables from domestic production and import were analysed to detect the presence of pesticide residues. Analysis was performed by multi-residual extraction followed by gas chromatography-mass spectrometry. In 42.5% of the tested samples, no residues were found and 12.5% of samples contained pesticide residues above maximum residue limits. Risk assessment for long-term exposure was done for all pesticides detected in this study. Except chlorpyrifos and lambda-cyhalothrin, exposure to pesticides from vegetables and fruits was below 1% of the acceptable daily intake. Short-term exposure assessment revealed that in seven pesticide/commodity combinations, including three pesticides (chlorpyrifos, deltamethrin and lambda-cyhalothrin), the acute reference dose had been exceeded.

International audience; While increased P-hydrolysing acid phosphatases (APase) activity in bean nodules is well documented under phosphorus (P) limitation, gene expression and subcellular localization patterns within the N2-fixing nodule tissues are poorly understood. The aim of this research was to track the enzyme activity along with the intra-nodular localization of fructose-1,6-bisphosphatase (FBPase), and its contribution to P use efficiency (PUE) under symbiotic nitrogen fixation (SNF) in Phaseolus vulgaris. The FBPase transcript were localized in situ using RT-PCR and the protein activity was measured in nodules of two contrasting recombinant inbred lines (RILs) of P. vulgaris, namely RILs 115 (P-efficient) and 147 (P-inefficient), that were grown under sufficient versus deficient P supply. Under P-deficiency, higher FBPase transcript fluorescence was found in the inner cortex as compared to the infected zone of RIL115. In addition, both the specific FBPase and total APase enzyme activities significantly increased in both RILs, but to a more significant extent in RIL115 as compared to RIL147. Furthermore, the increased FBPase activity in nodules of RIL115 positively correlated with higher use efficiency of both the rhizobial symbiosis (23%) and P for SNF (14% calculated as the ratio of N2 fixed per nodule total P content). It is concluded that the abundant tissue-specific localized FBPase transcript along with induced enzymatic activity provides evidence of a specific tolerance mechanism where N2-fixing nodules overexpress under P-deficiency conditions. Such a mechanism would maximise the intra-nodular inorganic P fraction necessary to compensate for large amount of P needed during the SNF process.

International audience; Phosphorus (P) deficiency initiates a myriad of transcriptional, biochemical and physiological responses stimulating either the root’s extracellular abilities to acquire soil P in the rhizosphere or optimize its intracellular use efficiency and allocation through all plant organs. Enhancing activity of acid phosphatase (APase) to acquire and remobilize Pi from organic P compounds is one important strategy for improving plant P nutrition. The release of APase to the rhizosphere is a typical and almost universal P-starvation response in higher plants. However, relatively little is known about the functions of intracellular APase in legume nodules. The aim of this review was to track the enzyme activity along with the intra-nodular localization of APase, and its contribution in the rhizobial symbiosis tolerance to P-deficiency. Our findings have revealed that expression of APase and phytases genes and activities of the corresponding enzymes were positively correlated with increases both of the P use efficiency for N2 fixation and nodule O2 permeability in the rhizobial symbiosis with legumes. The induced enzyme activity and the marked transcripts localization of APase and phytase in nodule cortex would control nodule respiration and contribute to adaptation of nodulated legumes to low-P availability. Thus, the increase of APase and phytase activities in legume nodules supports a physiological role of these enzymes in the regulation of nitrogenase activity in connection with the nodule-P status, and opens up a new scenario for a better understanding of the regulation of N2 fixation in legumes.

Legumes improve agricultural sustainability through symbiotic dinitrogen (N2) fixation which constitutes a major input into agroecosystems and may provide an ecologically acceptable complement or substitute for mineral nitrogen fertilizers. However, low soil nutrient availability, notably phosphorus (P), is among the most nutrient limitations for legumes whose sensitivity to P deficiency has been attributed to low soil P availability and higher P requirements during the symbiotic N2 fixation process. In response to P deficiency, plants use various adaptive strategies to improve soil P availability and their uptake efficiency, which involves modifications in nodulated-root architecture, rhizosphere acidification, and induction of genes involved in P use efficiency such as high-affinity P transporters and P-hydrolyzing phosphatases enzymes. This chapter reports numerous legume tolerance strategies to P deficiency that link morphological, physiological, and molecular responses. Stimulation of the root’s extracellular potentialities to improve solubilization and acquisition of the rhizosphere soil P as well as optimization of intracellular use efficiency and allocation of P has been described. Coincident with most knowledge on legume performance under P deficiency, exploration of biotic factors with synergistic and complementary interactions for the benefit of both plants and soil microorganisms is increasingly adopted. A holistic understanding of the key mechanisms underlying legume tolerance to abiotic constraints will be valuable for strategies to improve symbiotic N2 fixation and sustainable agriculture in a world of increasing population and declining renewable resources.

UE 0398 Domaine expérimental Melgueil équipe DIASCOPE; International audience; Low P availability in the soil is a major constraint to legume production, and efforts are being made to identify legume genotypes with tolerance to low P and greater phosphorus use efficiency (PUE) for N2 fixation. Common bean (Phaseolus vulgaris L.) genotypes vary in their adaptation to low-P soils. To investigate to what extent this variation may be related to PUE for N2 fixation, six recombinant inbred lines (RILs) of common bean contrasting in PUE for symbiotic N2 fixation, namely RILs 147, 115, 104, 83, 34, and 29 were studied in the field conditions during four growing seasons from 2011 to 2015. We collected biomass (aboveground and belowground) at flowering stage, and both samples were analyzed P content. Results indicate that low-P availability in the soil significantly limited plant growth, nodulation, and N2 fixation for all common bean RILs though with highest extent for common bean RILs 147, 83, and 29, whereas in this soil, common bean RILs 34 and 104 showed highest PUE for their N2 fixation. The genotypes with high PUE for N2 fixation showed greater mycorrhizal-plant symbiosis activity rate than the genotypes with low PUE for N2 fixation. We also found a significant correlation between nodule biomass and nodule P content for all common bean RILs. We conclude that quantifying PUE for N2 fixation may contribute to characterize the level of adaptation of N2–fixing legumes to low-P soils

International audience; Common bean (Phaseolus vulgaris L.) has the capacity to fix atmospheric N-2 into the biosphere through its aptitude to establish a symbiosis with soil rhizobia. In order to search for environmental constraints that might limit this symbiosis a nodular diagnosis was performed in eighteen field sites chosen with farmers of the Setif agro-ecosystem. Common bean was used as a model grain-legume with six recombinant inbred lines (RILs) and one local genotype Djadida. At flowering stage, the biomass of plants and nodules was determined by excavating 20 cm in depth and around the root-system of ten plants per genotype and per site. The results indicate a large spatial variation in nodulation and growth between genotypes, and the distribution of soils in four soil clusters, based on physico-chemical properties. The inhibition of nodulation of all genotypes in soil of clusters A and B was associated with high residual soil mineral nitrogen (2.23 +/- 0.49 g kg(-1) soil). The low nodulation of all genotypes in the phosphorus (P) deficient soil of cluster C (6.73 +/- 3.63 mg kg(-1) soil) was partly compensated by increasing their efficiency in use of the rhizobial symbiosis (13%), estimated by the slope of the regression model of shoot biomass as a function of nodule biomass. Interestingly, significant correlations were found between nodulation of all genotypes and Olsen-P content in soils of clusters C (R-2 = 0.97, P < 0.001) and D (R-2 = 0.94, P < 0.05). It is concluded that the RILs selected for their efficient use of P for symbiotic nitrogen fixation show the highest nodulation and growth and that the nodular diagnosis can be used to assess the growth response of N-2-dependent grain-legume to soils with low availabilities of N and P.

In order to better understand how mixed crop cultures mitigate stressful conditions, this study aims to highlight the beneficial effect of the intercropping legume-cereal in enhancing soil phosphorus (P) availability for plant growth and productivity in a P-deficient soil of a northern Algerian agroecosystem. To address this question, common bean (Phaseolus vulgaris L. cv. El Djadida) and maize (Zea mays L. cv. Filou), were grown as sole- and inter-crops in two experimental sites; S1 (P-deficient) and S2 (P-sufficient) during two growing seasons (2011 and 2012). Growth, nodulation and grain yield were assessed and correlated with the rhizosphere soil P availability. Results showed that P availability significantly increased in the rhizosphere of both species, especially in intercropping under the P-deficient soil conditions. This increase was associated with high efficiency in use of the rhizobial symbiosis (high correlation between plant biomass and nodulation), plant growth and resource (nitrogen (N) and P) use efficiency as indicated by higher land equivalent ratio (LER > 1) and N nutrition index (for maize) in intercropping over sole cropping treatments. Moreover, the rhizosphere P availability and nodule biomass were positively correlated (r2 = 0.71, p < 0.01 and r2 = 0.62, p < 0.01) in the intercropped common bean grown in the P-deficient soil during 2011 and 2012. The increased P availability presumably improved biomass and grain yield in intercropping, though it mainly enhanced grain yield in intercropped maize. Our findings suggest that modification in the intercropped common bean rhizosphere-induced parameters facilitated P uptake, plant biomass and grain yield for the intercropped maize under P-deficiency conditions.

To understand the relationship between phosphorus use efficiency (PUE) and respiration for symbiotic nitrogen fixation (SNF) in legume nodules, six recombinant inbred lines of common bean (RIL Phaseolus vulgaris L.), contrasting in PUE for SNF, were inoculated with Rhizobium tropici CIAT899, and grown under hydroaeroponic culture with sufficient versus deficient P supply (250 versus 75 μmol P plant−1 week−1). At the flowering stage, the biomass of plants and phytase activity in nodules were analyzed after measuring O2 uptake by nodulated roots. Our results show that the P-deficiency significantly increased the phytase activity in nodules of all RILs though with highest extent for RILs 147, 29 and 83 (ca 45%). This increase in phytase activity was associated with an increase in nodule respiration (ca 22%) and in use of the rhizobial symbiosis (ca 21%). A significant correlation was found under P-deficiency between nodule O2 permeability and phytase activity in nodules for RILs 104, 34 and 115. This observation is to our knowledge the first description of a correlation between O2 permeability and phytase activity of a legume nodule. It is concluded that the variation of phytase activity in nodules can increase the internal utilization of P and might be involved in the regulation of nodule permeability for the respiration linked with SNF and the adaptation to P-deficiency.

Although isotopic discrimination processes during nitrogen (N) transformations influence the outcome of (15)N based quantification of N2 fixation in legumes, little attention has been given to the effects of genotypic variability and environmental constraints such as phosphorus (P) deficiency, on discrimination against (15)N during N2 fixation. In this study, six Phaseolus vulgaris recombinant inbred lines (RILs), i.e. RILs 115, 104, 34 (P deficiency tolerant) and 147, 83, 70 (P deficiency sensitive), were inoculated with Rhizobium tropici CIAT899, and hydroaeroponically grown with P-sufficient (250 μmol P plant(-1) week(-1)) versus P-deficient (75 μmol P plant(-1) week(-1)) supply. Two harvests were done at 15 (before nodule functioning) and 42 (flowering stage) days after transplanting. Nodulation, plant biomass, P and N contents, and the ratios of (15)N over total N content ((15)N/Nt) for shoots, roots and nodules were determined. The results showed lower (15)N/Nt in shoots than in roots, both being much lower than in nodules. P deficiency caused a larger decrease in (15)N/Nt in shoots (-0.18%) than in nodules (-0.11%) for all of the genotypes, and the decrease in shoots was greatest for RILs 34 (-0.33%) and 104 (-0.25%). Nodule (15)N/Nt was significantly related to both the quantity of N2 fixed (R(2)=0.96***) and the P content of nodules (R(2)=0.66*). We conclude that the discrimination against (15)N in the legume N2-fixing symbiosis of common bean with R. tropici CIAT899 is affected by P nutrition and plant genotype, and that the (15)N/Nt in nodules may be used to screen for genotypic variation in P use efficiency for N2 fixation.

Although the role of phosphatases and antioxidant enzymes have been documented in phosphorus (P) deficiency tolerance, gene expression differences in the nodules of nitrogen fixing legumes should also affect tolerance to this soil constraint. In this study, root nodules were induced by Rhizobium tropici CIAT899 in two Phaseolus vulgaris recombinant inbred lines (RIL); RIL115 (low P-tolerant) and RIL147 (low P-sensitive) under hydroaeroponic culture with sufficient versus deficient P supply. Trehalose 6-P phosphatase and ascorbate peroxidase transcripts were localized within nodules in which O-2 permeability was measured. Results indicate that differential tissues-specific expression of trehalose 6-P phosphatase and ascorbate peroxidase transcripts within nodules was detected particularly in infected zone and cortical cells. Under P-deficiency, trehalose 6-P phosphatase transcript was increased and mainly localized in infected zone and outer cortex of RIL115 as compared to RIL147. Ascorbate peroxidase transcript was highly expressed under P-sufficiency in the infected zone, inner cortex and vascular traces of RIL115 rather than RIL147. In addition, significant correlations were found between nodule O-2 permeability and both peroxidase (r = 0.66*) and trehalose 6-P phosphatase enzyme activities (r = 0.79*) under sufficient and deficient P conditions, respectively. The present findings suggest that the tissue-specific localized trehalose 6-P phosphatase and ascorbate peroxidase transcripts of infected cells and nodule cortex are involved in nitrogen fixation efficiency and are likely to play a role in nodule respiration and adaptation to P-deficiency.

Phosphorus is an essential nutrient for rhizobial symbioses to convert N-2 into NH4 usable for N nutrition in legumes and N cycle in ecosystems. This N-2 fixation process occurs in nodules with a high energy cost. Phytate is the major storage form of P and accounts for more than 50 % of the total P in seeds of cereals and legumes. The phytases, a group of enzymes widely distributed in plant and microorganisms, are able to hydrolyze a variety of inositol phosphates. Recently, phytase activity was discovered in nodules. However, the gene expression localization and its role in N-2-fixing nodules are still unknown. In this work, two recombinant inbred lines (RILs) of common bean (Phaseolus vulgaris L.), selected as contrasting for N-2 fixation under P deficiency, namely RILs 115 (P-efficient) and 147 (P-inefficient) were inoculated with Rhizobium tropici CIAT 899, and grown under hydroaeroponic conditions with sufficient versus deficient P supply. With in situ RT-PCR methodology, we found that phytase transcripts were particularly abundant in the nodule cortex and infected zone of both RILs. Under P deficiency, phytase transcripts were significantly more abundant for RIL115 than for RIL147, and more in the outer cortex than in the infected zone. Additionally, the high expression of phytase among nodule tissues for the P-deficient RIL115 was associated with an increase in phytase (33 %) and phosphatase (49 %) activities and efficiency in use of the rhizobial symbiosis (34 %). It is argued that phytase activity in nodules would contribute to the adaptation of the rhizobia-legume symbiosis to low-P environments.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Although previous studies on N-2-fixing legumes have demonstrated the contribution of acid phosphatases to their phosphorus (P) use efficiency under P-deficient growth conditions, localization of these enzymes in bean nodules has not been demonstrated. In this study, phosphoenol pyruvate phosphatase (PEPase) gene transcripts were localized within the nodule tissues of two recombinant inbred lines, RIL115 (P-deficiency tolerant) and RIL147 (P-deficiency sensitive), of Phaseolus vulgaris. Nodules were induced by Rhizobium tropici CIAT899 under hydroaeroponic conditions with a sufficient versus a deficient P supply. The results indicated that PEPase transcripts were particularly abundant in the nodule infected zone and cortex of both RILs. Analysis of fluorescence intensity indicated that nodule PEPase was induced under conditions of P deficiency to a significantly higher extent in RIL147 than in RIL115, and more in the inner cortex (91%) than in the outer cortex (71%) or the infected zone (79%). In addition, a significant increase (39%) in PEPase enzyme activity in the P-deficient RIL147 correlated with an increase (58%) in the efficiency of use in rhizobial symbiosis. It was concluded that nodule PEPase is upregulated under conditions of P deficiency in the P-deficiency-sensitive RIL147, and that this gene may contribute to adaptation of rhizobial symbiosis to low-P environments.