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Organic waste generation in aquaculture worldwide requires circular economy approaches. The revalorization of wastes in the Chilean salmon industry could be a potential soil amendment. We investigated the thermo-drying process on salmon sludge, evaluating it as an alternative to reusing in horticulture. The chemical characterization of untreated sludge (wet sludges: WS) and dried sludges (DS) were evaluated with thermogravimetry, IR spectroscopy, and fluorescence. Additionally, the germination index of the DS was determined in L. sativa, R. sativus, and S. lycopersicum. The results show that DS reduces salinity, ammonium content, and pathogens compared to WS. During the thermic process, there were no significant decreases in organic matter, phosphorus, potassium, and humic and fulvic substances. The high salinity in DS inhibited seed germination. This research constituted a novel contribution to studying the DS as a strategy for horticultural production. In the same way, the drying technology for salmon sludge favored the management of wastes, contributing to the circular economy in both the agricultural and aquaculture sectors. However, we recommend considering that excessive incorporation of DS into germination substrates or direct application into the soil could affect the crop’s performance due to its high salinity.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Grapevine is one of the most important perennial fruit crops worldwide. Historically, vineyards were compatible with soil conservation practices and multitrophic biodiversity, but vineyards are now generally eroded and biologically impoverished, making them more susceptible to pests and diseases. However, the idiosyncrasy of the wine sector places wine growers in a unique position to lead the adoption of a range of sustainable management strategies and, thus, to pioneer a wider transformation of the agricultural sector. In this article, we provide an overview of nature-based management strategies that may be used for the regeneration of the functioning and biodiversity of vineyards and that may also lead to improved plant nutrition, grape berry quality and the suppression of pathogens and pests. These strategies include the use of microbial and nonmicrobial biostimulants, fertilization with organic amendments as well as foliar fertilization with nature-based products, the use of cover crops and the reintegration of livestock in vineyards, especially sheep. We will also pay special attention to the implementation of circular economy in the vineyard in relation to the previously mentioned management strategies and will also discuss the importance of considering all these aspects from a holistic and integrative perspective, rather than taking them into account as single factors. Assuming the integral role of soils in the functioning of agroecosystems, soils will be considered transversally across all sections. Finally, we will argue that the time is now ripe for innovation from the public and private sectors to contribute to the sustainable management of vineyards while maintaining, or even improving, the profit margin for farmers and winemakers.

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.

The utilization of compost to enhance plant productivity and symbiotic nitrogen fixation (SNF) has been recognized as an effective alternative to synthetic nitrogen fertilizers. This environmentally sustainable method is readily accessible to farmers. This study investigated the effect of olive pomace (“alperujo”, AL) compost on the nodulation and SNF of soybeans (Glycine max L.) and their natural symbiont (Bradyrhizobium diazoefficiens). For that, soybean plants were subjected to several doses of AL compost under controlled greenhouse conditions. At the end of the experiment, the dry weight of plant biomass (aerial part and roots), the number and fresh weight of nodules, and nitrogen and leghaemoglobin contents were analyzed. The application of AL compost significantly improved soybean growth, as demonstrated by an increase in both plant biomass and height. Furthermore, nodular leghaemoglobin content and nitrogen content were found to be enhanced by the addition of AL compost (7 and 40%, respectively), indicating an increase in nodule effectiveness and symbiotic efficiency. Our results provide clear evidence of the synergetic effect of AL compost on the soybean-B. diazoefficiens association, probably due to AL-compost improved soybean roots development, and rhizospheric organic matter and nutrients assimilation by rhizobia.

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

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

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.

En el siguiente trabajo se presentan los resultados de una experiencia de compostaje de la fracción orgánica procedente de recogida selectiva (FORS) con la tecnología Gore©Cover realizada durante los meses de junio y septiembre de 2021. El compostaje se realizó sobre una pila trapezoidal de 20 m de longitud, 2,5 metros de ancho y alto, elaborada con un 10 % de compost maduro y 90 % de fracción FORS. Durante el proceso se controló la temperatura, siendo termófila durante más dedos meses. El compost obtenido fue ligeramente básico y con una salinidad elevada, típica de este tipo de materiales. Su contenido en nutrientes fue notable, en especial el nitrógeno, fósforo y potasio con valores de 2,63 ± 0,24 %, 0,98 ± 0,10 y 13,4 ± 0,8 g kg-1, respectivamente. El compost no presentó microorganismos patógenos y se catalogó como clase A.

En este trabajo se estudió el efecto del compost de alperujo sobre la nodulación en la simbiosis leguminosa-rizobio, concretamente entre plantas de soja (Glycine maxL.) y su simbionte natural (Bradyrhizobium diazoefficiens). Para eso, se añadieron varias dosis de compost de alperujo, el principal residuo orgánico de la industria extractiva del aceite de oliva, a plantas de soja cultivadas en condiciones controladas de invernadero durante 4 semanas. Al finalizar el experimento se determinó el peso seco de la biomasa vegetal (parte aérea y raíz), el número y peso fresco de los nódulos aparecidos, así como el contenido en leghemoglobina de los mismos. Los resultados obtenidos sugieren que el compost de alperujo mejora significativamente el crecimiento de las plantas al mejorar la eficiencia simbiótica de la asociación leguminosa-rizobio

En este trabajo se está realizando un experimento de co-compostaje de lodos procedentes de la industria avícola (LAV), con residuos de orujillo (OR), cenizas de orujillo (COR) y poda urbana(PU). Se fabricaron dos pilas con diferentes proporciones de los residuos anteriores en una planta de tratamiento de situada en Guadix, Granada. El objetivo del trabajo es estudiar el comportamiento de este tipo de residuos con respecto al compostaje, ya que es la mejor alternativa para su valorización, transformando los residuos orgánicos en productos fertilizantes de calidad agrícola. Se trata de un experimento que está durando más de 300días. No obstante, en los resultados de las muestras analizadas, se puede observar la evolución de algunos parámetros físico-químicos, que indican que el proceso se está desarrollando adecuadamente, aunque está siendo muy largo debido a la naturaleza recalcitrante de los residuos utilizados, que contienen materia orgánica más difícilmente biodegradable.

Current and continuing climate change in the Anthropocene epoch requires sustainable agricultural practices. Additionally, due to changing consumer preferences, organic approaches to cultivation are gaining popularity. The global market for organic grapes, grape products, and wine is growing. Biostimulant and biocontrol products are often applied in organic vineyards and can reduce the synthetic fertilizer, pesticide, and fungicide requirements of a vineyard. Plant growth promotion following application is also observed under a variety of challenging conditions associated with global warming. This paper reviews different groups of biostimulants and their effects on viticulture, including microorganisms, protein hydrolysates, humic acids, pyrogenic materials, and seaweed extracts. Of special interest are biostimulants with utility in protecting plants against the effects of climate change, including drought and heat stress. While many beneficial effects have been reported following the application of these materials, most studies lack a mechanistic explanation, and important parameters are often undefined (e.g., soil characteristics and nutrient availability). We recommend an increased study of the underlying mechanisms of these products to enable the selection of proper biostimulants, application methods, and dosage in viticulture. A detailed understanding of processes dictating beneficial effects in vineyards following application may allow for biostimulants with increased efficacy, uptake, and sustainability.

Nitrous oxide (NO) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of NO in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing NO emissions from agricultural soils.

Soybean (Glycine max.) is one of the most important legumes cultivated worldwide. Its productivity can be altered by some biotic and abiotic stresses like global warming, soil metal pollution or over-application of herbicides like paraquat (1,1’-dimethyl-4,4’-bipyridinium dichloride). In this study, the effect of oxidative stress produced by paraquat addition (0, 20, 50 and 100 µM) during plant growth on symbiotic nitrogen fixation (SNF) and functionality of Bradyrhizobium diazoefficiens-elicited soybean nodules were evaluated. Results showed that the 50 µM was the threshold that B. diazoefficiens can tolerate under free-living conditions. In symbiosis with soybean, the paraquat addition statistically reduced the shoot and root dry weight of soybean plants, and number and development of the nodules. SNF was negatively affected by paraquat, which reduced total nitrogen content and fixed nitrogen close to 50% when 100 µM was added. These effects were due to the impairment of nodule functionality and the increased oxidative status of the nodules, as revealed by the lower leghaemoglobin content and the higher lipid peroxidation in soybean nodules from paraquat-treated plants.

RNA-based high-throughput sequencing is a valuable tool in the discernment of the implication of metabolically active bacteria during composting. In this study, “alperujo” composting was used as microbial model for the elucidation of structure-function relationships with physicochemical transformation of the organic matter. DNA and RNA, subsequently retrotranscribed into cDNA, were isolated at the mesophilic, thermophilic and maturation phases. 16S rRNA gene was amplified by quantitative PCR (qPCR) and Illumina MiSeq platform to assess bacterial abundance and diversity, respectively. The results showed that the abundance of active bacteria assessed by qPCR was maximum at thermophilic phase, which confirm it as the most active stage of the process. Concerning diversity, Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the main phyla presented in composts. Concomitantly, three different behaviours were observed for bacterial dynamics: some genera decreased during the whole process meanwhile others proliferated only at thermophilic or maturation phase. Statistical correlation between physicochemical transformations of the organic matter and bacterial diversity revealed bacterial specialisation. This result indicated that specific groups of bacteria were only involved in the organic matter degradation during bio-oxidative phase or humification at maturation. Metabolic functions predictions confirmed that active bacteria were mainly involved in carbon (C) and nitrogen (N) cycles transformations, and pathogen reduction.

Irrigation and soil management can impact soil nitrous oxide (NO) emissions. Flood and sprinkler irrigation systems together with conventional tillage are the main practices used in the high yielding maize systems in Mediterranean Spain. The objective of this field work was to quantify the effect of the irrigation system (i.e., flood, F; and sprinkler, S) and the soil tillage system (i.e., conventional tillage, CT; no-tillage maintaining the maize stover, NTr; and no-tillage removing the maize stover, NT) on the NO emissions from the soil during 3 yr (2015, 2016, and 2017). S irrigation, with mean values of 1.35 kg NO-N ha yr throughout the 3 yr, obtained 42% lower NO emissions than F irrigation. On average of the three growing seasons, yield-scaled NO emissions by grain yield and by grain N uptake in F irrigation were two-fold higher than in S irrigation. Moreover, in one out of three growing seasons (2017), no-tillage systems (i.e., NTr and NT) showed greater yield-scaled NO emissions compared with CT. The higher maize grain yield with the S irrigation compared to F irrigation, as well as the lower NO emissions reported under S irrigation resulted in the reduction of the yield-scaled NO emissions. Our findings highlight the role of sprinkler irrigation decreasing NO emissions in comparison to flood irrigation in Mediterranean agroecosystems.

Aim: To analyse the diversity of nodule-forming bacteria isolated from Lupinus cosentinii naturally grown in the Maamora cork oak forest (Rabat, Morocco). Methods and Results: Of the 31 bacterial strains, four were selected based on their REP-PCR fingerprinting that were studied by sequencing and phylogenetic analysis of their 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes as well as the nodC symbiotic gene. The nearly complete 16S rRNA gene sequence of the four representative strains showed that they are related to Tunisian strains of genus Microvirga isolated from L. micranthus with nucleotide identity values ranging from 98·67 to 97·13%. The single and concatenated sequences of the 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes indicated that the L. cosentinii-isolated strains had 99·2–99·9% similarities with the Tunisian L. micranthus microsymbionts. The nodC gene phylogeny revealed that the Moroccan strains clustered in the newly described mediterranense symbiovar, and nodulation tests showed that they nodulated not only L. cosentinii but also L. angustifolius, L. luteus and L. albus. Conclusions: To the best of our knowledge, this is the first report concerning the isolation, molecular identification and phylogenetic diversity of L. cosentinii nodule-forming endosymbionts and of their description as members of the Microvirga genus. Significance and Impact of the Study: In this work, we show that Microvirga sp. can be isolated from root nodules of wild-grown L. cosentinii in Northeast Africa, that selected strains also nodulate L. angustifolius, L. luteus and L. albus, and that they belong to symbiovar mediterranense. In addition, our data support that the ability of Microvirga to nodulate lupines could be related to the soil pH, its geographical distribution being more widespread than expected.

Legume-rhizobia symbiotic associations have beneficial effects on food security and nutrition, health and climate change. Hypoxia induced by flooding produces nitric oxide (NO) in nodules from soybean plants cultivated in nitrate-containing soils. As NO is a strong inhibitor of nitrogenase expression and activity, this negatively impacts symbiotic nitrogen fixation in soybean and limits crop production. In Bradyrhizobium diazoefficiens, denitrification is the main process involved in NO formation by soybean flooded nodules. In addition to denitrification, nitrate assimilation is another source of NO in free-living B. diazoefficiens cells and a single domain hemoglobin (Bjgb) has been shown to have a role in NO detoxification during nitrate-dependent growth. However, the involvement of Bjgb in protecting nitrogenase against NO in soybean nodules remains unclear. In this work, we have investigated the effect of inoculation of soybean plants with a bjgb mutant on biological nitrogen fixation. By analyzing the proportion of N in shoots derived from N2-fixation using the 15N isotope dilution technique, we found that plants inoculated with the bjgb mutant strain had higher tolerance to flooding than those inoculated with the parental strain. Similarly, reduction of nitrogenase activity and nifH expression by flooding was less pronounced in bjgb than in WT nodules. These beneficial effects are probably due to the reduction of NO accumulation in bjgb flooded nodules compared to the wild-type nodules. This decrease is caused by an induction of expression and activity of the denitrifying NO reductase enzyme in bjgb bacteroids. As bjgb deficiency promotes NO-tolerance, the negative effect of NO on nitrogenase is partially prevented and thus demonstrates that inoculation of soybean plants with the B. diazoefficiens bjgb mutant confers protection of symbiotic nitrogen fixation during flooding.

Metagenomic and transcriptomic techniques applied to composting could increase our understanding of the overall microbial ecology and could help us to optimise operational conditions which are directly related with economic interest. In this study, the fungal diversity and abundance of two-phase olive mill waste (“alperujo”) composting was studied using Illumina MiSeq sequencing and quantitative PCR, respectively. The results showed an increase of the fungal diversity during the process, with Ascomycota being the predominant phylum. Penicillium was the main genera identified at the mesophilic and maturation phases, with Debaryomyces and Sarocladium at the thermophilic phase, respectively. The fungal abundance was increased during composting, which confirms their important role during thermophilic and maturation phases. Some Basidiomycota showed an increased during the process, which showed a positive correlation with the humification parameters. According to that, the genus Cystofilobasidium could be used as a potential fungal biomarker to assess alperujo compost maturation.

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name “vachelliae” is proposed.

Bio-waste is a biodegradable organic waste composed by garden and park cuttings, and food and kitchen residues from households and catering establishments. Composting is an effective biological process for its treatment and recycling, converting this organic waste into an organic amendment. In this research, some small-scale composting reactors have been design using recycled materials. Also, the biological process has been studied by monitoring the temperature, mass and volume evolution of the composting substrate during two months. Thermophilic temperature (50ºC) and a significant reduction of mass (17%) and volume (50%) of the bio-waste mixture was found during the process. The main conclusion was that small-scale composting reactors can be effectively used for the bio-waste treatment.

“Alperujo” compost was evaluated as an organic fertiliser for pepper growth under greenhouse conditions. Even though the total nitrogen applied was similar, plants only grown with composts experienced a development decline as compared to those grown with standard nutrient solution. This was perhaps because nitrogen from the compost was essentially organic, and not easily available for roots. When, alternatively, the compost was supplemented with nitrate, a synergetic effect was observed, favouring plant development and fruit yield, simultaneously with the increase of compost rates. Compost affected the oxidative metabolism of pepper plants by increasing their antioxidative enzyme activities catalase and superoxide dismutases and the non-enzymatic antioxidants ascorbate and glutathione. Overall, when nitrogen limitation occurred and only compost was used as fertiliser, an oxidative stress took place, whereas in plants grown with nitrate-supplemented compost it did not. Furthermore, these pepper plants experienced a yield increase and, more importantly, an enhancement of the ascorbate content.

Nitrous oxide (NO) is one of the three main biogenic greenhouse gases (GHGs) and agriculture represents close to 30 % of the total NO net emissions. In agricultural soils, NO is emitted by two main microbial processes, nitrification and denitrification, both of which can convert synthetic nitrogen fertilizer into NO. Legume-rhizobia symbiosis could be an effective and environmental-friendly alternative to nitrogen fertilization and hence, to mitigate soil NO emissions. However, legume crops also contribute to NO emissions. A better understanding of the environmental factors involved in the emission of NO from nodules would be instrumental for mitigating the release of this GHG gas. In this work, in vivo NO emissions from nodulated soybean roots in response to nitrate (0, 1, 2 and 4 mM) and flooding have been measured. To investigate the contribution of rhizobial denitrification in NO emission from nodules, plants were inoculated with B. japonicum USDA110 and napA and nosZ denitrification mutants. The results showed that nitrate was essential for NO emissions and its concentration enhanced NO fluxes showing a statistical linear correlation, being the highest NO fluxes obtained with 4 mM nitrate. When inoculated plants grown with 4 mM nitrate were subjected to flooding, a 150- and 830-fold induction of NO emission rates from USDA110 and nosZ nodulated roots, respectively, was observed compared to non-flooded plants, especially during long-term flooding. Under these conditions, NO emissions from detached nodules produced by the napA mutant were significantly lower (p < 0.05) than those produced by the wild-type strain (382 versus 1120 nmol NO h g NFW, respectively). In contrast, nodules from plants inoculated with the nosZ mutant accumulated statistically higher levels of NO compared to wild-type nodules (2522 versus nmol 1120 NO h g NFW, p < 0.05). These results demonstrate that flooding is an important environmental factor for NO emissions from soybean nodules and th