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Aims: In Morocco, the Zaida abandoned mine is a major source of metallic trace elements that are harmful to the environment and human health. With the aim to use the heavy metal tolerant legume tree Robinia pseudoacacia in the phytostabilization of this mine’s tailings, we isolated and characterized its indigenous microsymbionts, and tested their effects on the plant growth under high Pb-acetate concentrations. Methods: Thus, 27 bacteria were isolated from nodules of the plant grown in the Zaida abandoned mine tailings, and we examined their tolerance to high concentrations of Pb-acetate in-vitro. Four strains were then selected for symbiotic, molecular, and phenotypic analyses, and we analyzed the effect of inoculation on the plant growth parameters under increasing Pb-acetate concentrations (in-vivo). Results: The isolated strains tolerate high lead concentrations. The Phylogenetic analyses of 16 S rRNA, five housekeeping genes (MLSA), and three symbiotic genes showed the affiliation of the strains with Ensifer meliloti sv. rigiduloides and E. kummerowiae sv. meliloti. Representative strains of both symbiovars tolerate 1100 µg ml Pb-acetate in-vitro and nodulate R. pseudoacacia even under 1200 µg ml of Pb-acetate. In all tests, the plant proline content increased as a response mechanism to increasing concentrations of Pb-acetate. Inoculation experiments under high concentrations of Pb-acetate showed that both strains improved the plant growth parameters and chlorophyll content compared to controls. Conclusion: Our results show that E. meliloti sv. rigiduloides strain RPZ12 improves plant growth under Pb stress conditions more than E. kummerowiae sv. meliloti strain RPZ17. Strain RPZ12 would be an excellent inoculum to use for the phytostabilization of Zaida mine tailings. This is the first description of symbiovar meliloti in E. kummerowiae.

Although the use of synthetic fertilizers has increased crop yields to feed a growing population, excess nitrogen fertilizer has resulted in nitrate contamination of surface and groundwater, soils, sediments, seas, and oceans. During the N cycle in nature, nitrification and denitrification are the only processes by which the ammonium formed after fixation of atmospheric N2 is transformed into nitrate, which is further reduced again to N2, which is released into the atmosphere. During these processes, nitrous oxide (N2O), a potent greenhouse gas involved in global climatic change, is formed. Legumes are, after cereals, the main source of food for the world population. These plants provide proteins, carbohydrates, minerals, vitamins, oils, fiber, and other compounds of high nutraceutical value and beneficial properties for health. Legumes possess the extraordinary ability to establish atmospheric N2-fixing symbiosis with soil bacteria known as rhizobia. A consequence of the symbiosis is the formation in the roots, sometimes in stems, of specialized structures, the nodules, where the fixation (reduction) of the inert compound N2 to form the biologically active compound ammonium (NH4 +) takes place. This ammonium is subsequently converted into other nitrogenous compounds that are transferred to the rest of the plant for use in the synthesis of amino acids, proteins, etc. This explains the high N content of the leguminous plants, the reason why these plants fertilize the soil where they are grown, and why they are used in crop rotation and in revegetation and reforestation programs. Rhizobia do not nitrify, and most of them lack, or do not express, the complete set of genes responsible for the synthesis or the enzymes involved in denitrification, among them, the nitrous oxide reductase enzyme (Nos) responsible for the reduction of N2O to N2. Since denitrification also occurs within the nodules, nitrate can be converted into N2. In rhizobia lacking the nitrous oxide reduc

Purpose: Alfalfa is one of the most valuable forage crops in temperate climate zones. Ensifer meliloti, the endosymbiont of alfalfa, contains all the denitrification genes but the capacity of alfalfa root nodules to produce nitrous oxide (NO) is not known. In this work, NO emissions as well as the influence of bacteroidal denitrification on nodulation competitiveness and NO release from alfalfa nodules has been investigated. Methods: Medicago sativa cv. Victoria plants were inoculated with E. meliloti 1021, a periplasmic nitrate reductase (Nap) defective mutant, a Nap overexpressing strain and a nitrous oxide reductase defective mutant. Plants were grown in the presence of different nitrate and copper treatments and subjected to flooding during one week before harvesting. NO production by the nodules was analysed by using gas chromatography. Methyl viologen-dependent nitrate reductase (MV-NR), nitrite reductase (MV-NIR) and nitrous oxide reductase (NOR) enzymatic activities were measured in isolated bacteroids. Results: Alfalfa root nodules produce NO in response to nitrate and flooding. Overexpression of Nap improved nodulation competitiveness and induced NO emissions from nodules. Copper is required for an effective symbiosis as well as triggered a reduction of NO production due to the induction of the NOR and a reduction of NIR activities in the bacteroids. Conclusion: Alfalfa root nodules emit NO. Nap is involved in nodulation competitiveness and in NO emissions by the nodules. Bacteroidal NOR and NIR activities are modulated by Cu and may be considered as effective targets for the mitigation strategies of NO emissions derived from alfalfa crops.

Retama dasycarpa is an endemic Retama species native to the cold semi-arid bioclimates of the High Atlas Mountains in Morocco. In this work, we analyzed the diversity of the microsymbionts nodulating this plant and their different phenotypic and symbiotic characteristics. Phylogenetic analysis of the 16S rRNA gene revealed that the tested isolates clustered in the Bradyrhizobium genus. Multilocus sequence analyses of four housekeeping genes (recA, gyrB, glnII and atpD) for 12 selected strains grouped them into four clusters close to B. lupini USDA 3051, B. frederickii CNPSo 3446, B. valentinum LmjM3 and B. retamae Ro19. The individual phylogenies of these core genes and the symbiotic genes nodC, nodA and nifH were congruent. These isolates showed a broad host range, being able to nodulate different legume hosts, such as R. sphaerocarpa, R. monosperma, Lupinus luteus, Cytisus grandiflorus and Chamaecytisus albidus, but not Phaseolus vulgaris or Glycine max. They all had a similar metabolic capacity, using the majority of the carbohydrates and amino acids tested as sole sources of carbon and nitrogen. Furthermore, out of the 12 selected strains, some displayed plant growth-promoting features, with six of them solubilizing phosphate and three of them producing siderophores. The present work provides, for the first time, a detailed description about the microsymbionts associated with the endemic legume R. dasycarpa.

Lagoon systems are often confined, and their waters are poorly renewed, which makes them vulnerable to pollutants’ accumulation. Here, the impact of different sources of anthropogenic contamination (domestic, urban, industrial, and agricultural) on the nitrate (NO) content, emission of the greenhouse gas nitrous oxide (NO), abundance of total bacterial archaeal, nitrifying, and denitrifying communities, and diversity and composition of bacterial communities in the sediments of the RAMSAR-protected Marchica lagoon (Nador, Morocco) was investigated. Six lake sites differing in NO concentration were selected. Wastewater coming from industrial activities results in the greatest concentration of NO in sediments and emissions of NO. Increased carbon to nitrogen content in sites near domestic activities resulted in an increase in the abundance of total bacterial and archaeal communities, as well as nitrification and denitrification genes, but low NO emissions due to a greater presence of microorganisms involved in NO production over those able to reduce NO. Significant differences in bacterial community composition between sites were observed, with the NO content being the main driver of these changes. Increased NO content in the sampling sites significantly reduced bacterial diversity. Bacterial genera involved in the degradation of organic and inorganic pollutants and nitrous oxide reduction, such as Robiginitalea, Symbiobacterium, Bacillus, Fusibacter, Neptunomonas, Colwellia, and Alteromonas, were the most abundant in the lagoon. The results suggest that the type of anthropogenic contamination directly impacts the nitrate content in the sediments of the Marchica lagoon, which determines variations in nitrous oxide emissions, nitrogen-cycling gene abundances, and bacterial diversity.

Ononis repens is a legume of the Fabaceae family with great ecological and medicinal importance that grows wild in the Maamora cork oak forest (Rabat, Morocco). The aim of this work was to analyze the diversity, phylogeny, and plant growth-promoting features of the plant root nodules microsymbionts. Out of twenty nodC-containing rhizobial bacteria, four strains, ORM6, ORM8.1, ORM 13.1, and ORM16, were selected for further molecular and phenotypic analyses. All four strains were able to metabolize a wide range of carbohydrates and amino acids as sole carbon and nitrogen sources, respectively, and grew in the presence of different pH, NaCl, and temperature conditions. They also solubilized inorganic phosphate and produced siderophores, and ORM16 was the only strain to produce indol acetic acid. The rrs gene sequence analysis showed that the four strains are members of the Mesorhizobium genus. The individual and concatenated sequences of the recA, glnII, and gyrB housekeeping genes revealed that the strains ORM6 and ORM 13.1 clustered with M. jarvisii ATCC 33,669 and M. intechii BD68 , with which they share 93.3 and 93.7% similarity, respectively; that the strain ORM8.1 affiliated with a group of which the strain M. shonense AC39a was the closest relative species with 84.3% similarity; and that the strain ORM16 clustered with M. australicum LMG24608, with 80.5% similarity. These results suggest that the four O. repens-isolated strains could belong to three putative new genospecies. All four strains nodulated their original host as well as Astragalus gombiformis and A. armatus, but only the strain ORM16 was able to form nodules on Vachellia gummifera. Analysis of the symbiotic nodC gene phylogenies showed that the strains ORM6, ORM8.1, ORM 13.1, and ORM16 formed a group apart from the known symbiovars in the genus Mesorhizobium, for which the name maamori is proposed.

High-mountain lakes and rivers are usually oligotrophic and strongly influenced by atmospheric transport processes. Thus, wet deposition of reactive N species (Nr), mainly in the form of nitrate (NO), is a major source of N input in these high-mountain ecosystems. Bacterial denitrifiers are thought to be largely responsible for reduction of NO to nitrous oxide (NO) and molecular dinitrogen (N) as main biological pathway of N removal in these ecosystems. Nitrifiers, through the oxidation of ammonium to NO, can also be a source of NO and NO. However, there is uncertainty regarding the abiotic and biotic factors controlling Nr elimination from aquatic ecosystems at different altitudes and seasons. We examined the efficiency of Nr removal as NO and N (total removal) or N only (clean removal) in a model lake and its downwater river ecosystem (Sierra Nevada, Spain) representative of Mediterranean high-mountain freshwater ecosystems along an altitudinal gradient during the warm period of the year. Denitrification activity and the abundance of nitrifiers and denitrifiers in sediments were measured at thaw, mid ice-free and late ice-free periods. We found the efficiency of total and clean removal of Nr increased from the downwater river to the high-mountain lake. Regardless of the location, the efficiency of total removal of Nr decreased over the ice-free period whereas that of clean removal of Nr peaked at mid ice-free period. The efficiency of total removal of Nr was mainly controlled by the abundance of archaeal nitrifiers and bacterial denitrifiers. Abiotic (ammonium and NO concentration) and biotic (mainly nosZI-type denitrifiers) factors drove changes in the efficiency of clean removal of Nr. Our results suggest that abiotic and biotic factors can control the efficiencies of Nr removal in Mediterranean high-mountain lakes and their downwater rivers, and that these efficiencies increase with altitude and vary over the ice-free period.

Resumen del poster presntado en: 15th European Nitrogen Fixation Conference (ENFC). Napóles Italia. 31 agosto - 3 septiembre (2023)

The Moroccan endemic Vachellia gummifera grows wild under extreme desert conditions. This plant could be used as an alternative fodder for goats, and camels, in order to protect the Argan forests against overgrazing in Central and Southwestern Moroccan semiarid areas. With the aim to improve the V. gummifera population's density in semiarid areas, we proposed its inoculation with performing plant growth-promoting bacteria. Hence, 500 bacteria were isolated from the plant rhizosphere. From these, 291 isolates were retained for plant growth-promoting (PGP) activities assessment. A total of 44 isolates showed the best phosphates solubilization potential, as well as siderophore and auxin production. The combination of REP-PCR (repetitive extragenic palindromic-polymerase chain reaction) fingerprinting, PGP activities, and phenotypic properties, allowed the selection of three strains for the inoculation experiments. The three selected strains' 16S rRNA sequencing showed that they are members of the Enterobacter and Pseudomonas genera. The inoculation with three strains had diverse effects on V. gummifera growth parameters. All single and combined inoculations improved the plant shoot weight by more than 200%, and the root length by up to 139%, while some combinations further improved protein and chlorophyll content, thereby improving the plant's forage value. The three selected strains constitute an effective inoculum for use in the arid and semiarid zones of southern Morocco.

Legumes can contribute to emissions of the potent greenhouse gas nitrous oxide (NO) directly by some rhizobia species that are able to denitrify under free-living conditions and in symbiotic association with the plant. In this study, the capacity of Phaseolus vulgaris-Rhizobium etli symbiosis to emit NO in response to nitrate (NO) has been demonstrated for the first time. We found that bacteroidal assimilatory nitrate reductase (NarB) as well as nitrite reductase (NirK) and nitric oxide reductase (cNor) denitrifying enzymes contribute to nitric oxide (NO) and NO formation in nodules. We also show that R. etli NarK is involved in NO extrusion and links NO reduction by NarB in the cytoplasm with NirK and cNor denitrification activities in the periplasm. The knowledge generated in this work will be instrumental for exploring strategies and sustainable practices in agricultural soil management to increase legume crop yield and mitigate greenhouse gas emissions.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.