Lotus angustissimus is a promising forage legume increasingly used to improve naturalgrasslands in Uruguay. However, a specific rhizobial inoculant for this species has not yet been developed, and the identification of its autochthonous symbionts remains unknown. Instead, L. angustissimus is inoculated with Bradyrhizobium sp. U531 the commercial inoculum for Lotus subbiflorus. We evaluated the symbiotic efficiency of sixteen Bradyrhizobium strains isolated from L. angustissimus nodules in a site without inoculation for 15 years, revealing efficient soil rhizobia suitable for inoculant development. Under controlled conditions, GL3 and GL5 strains significantly exceeded the symbiotic efficiency of strain U531 in L. angustissimus. In L. subbiflorus, strain GL5 exceeded the symbiotic efficiency of strain U531. Genomic and phylogenetic analyses showed that strain GL3 belongs to the species Bradyrhizobium canariense BTA-1T, whereas strain GL5 is related to Bradyrhizobium nitroreducens TSA1T, although average nucleotide identity suggests that it represents a different species. Despite their taxonomic divergence, both strains share a high percentage of identity with the nodBC and nifHK genes of Bradyrhizobium sp. WSM471, suggesting horizontal gene transfer of the symbiotic loci. The presence of denitrification genes (nap, nor, and nos) and nitrate-dependent anoxic growth in both strains was also confirmed. These findings demonstrate, for the first time, that B. canariense GL3 can efficiently nodulate L. angustissimus and L. subbiflorus. Bradyrhizobium sp. GL5, related to B. nitroreducens TSA1T, is also reported to establish an efficient symbiosis with these legumes. Due to the high symbiotic efficiency of these strains in two hosts, they represent elite candidates for the development of a specific dual-purpose inoculum.

Proline metabolism has been associated with the induction of reactive oxygen species (ROS), antioxidant enzymes, and the control of cellular redox status. Moreover, proline accumulation is a highly evolutionarily conserved response to diverse abiotic stresses in plants. Thus, proline quantification has been helpful in abiotic stress research as a stress marker. The need for a reliable, fast, and simple method to detect proline in plant tissues is a powerful resource to imply the physiological status of plants under abiotic stress. This chapter summarizes the main strategies for proline extraction and quantification, highlighting their limitations and advantages, and recommends and details a specific protocol for proline extraction and quantification. The chapter provides a friendly version of this protocol with notes useful for researchers to perform the protocol.

In Uruguayan soils, populations of native and naturalized rhizobia nodulate white clover. These populations include efficient rhizobia but also parasitic strains, which compete for nodule occupancy and hinder optimal nitrogen fixation by the grassland. Nodulation competitiveness assays using gusA-tagged strains proved a high nodule occupancy by the inoculant strain U204, but this was lower than the strains with intermediate efficiencies, U268 and U1116. Clover biomass production only decreased when the parasitic strain UP3 was in a 99:1 ratio with U204, but not when UP3 was at equal or lower numbers than U204. Based on phylogenetic analyses, strains with different efficiencies did not cluster together, and U1116 grouped with the parasitic strains. Our results suggest symbiotic gene transfer from an effective strain to U1116, thereby improving its symbiotic efficiency. Genome sequencing of U268 and U204 strains allowed us to assign them to species Rhizobium redzepovicii, the first report of this species nodulating clover, and Rhizobium leguminosarun, respectively. We also report the presence of hrrP- and sapA-like genes in the genomes of WSM597, U204, and U268 strains, which are related to symbiotic efficiency in rhizobia. Interestingly, we report here chromosomally located hrrP-like genes.

Bioethanol production from sugarcane generates by-products such as vinasse, with a high content of organic matter and potassium. While vinasse can be used as a fertilizer, its effect on the soil and its microbiota is unknown. In this work, we evaluated the effect of systematic vinasse application on microbial communities of soils used for sugarcane cultivation. The assays were carried out in the field and controlled conditions using the same soil type previously irrigated only with water or with vinasse. In the field, soil samples were taken prior to summer irrigation and one month later during years. No significant differences were found in the abundance of most microbial populations nor in the microbial activity by the vinasse treatment. However, in some sampling dates, populations of phosphate solubilizing bacteria, Bacillus spp. and ammonifying bacteria were significantly lower in soil with systematic vinasse irrigation compared to the control. Under controlled conditions, the treatments included two doses of vinasse and a control without vinasse. The abundance of heterotrophic bacteria and fungi increased in both soils with vinasse addition compared to the control, and at 180 days an increase in ammonifying bacteria was observed. Although microbial populations remained higher with the vinasse treatments, their activity decreased over time, with more evident effects on soils without application history. According to our results, vinasse application could have temporary effects on microbial communities, depending on soil type and doses, so local research is needed to create technical standards that regulate the use of vinasse on agricultural soils.

Ensifer (syn. Sinorhizobium) meliloti U143 is an effective nitrogen-fixing strain isolated from Uruguayan soils. For decades, this strain has been used as an inoculant for different alfalfa cultivars. Here we report for the first time a characterization of the U143 elite strain that includes the preliminary genomic sequence, its annotation, and physiological parameters related to its symbiotic efficiency and nitrate respiration capacity. Through Illumina sequencing, the genome of the U143 strain was sequenced. The genome length was 6,801,966 bp, and it contained two megaplasmids, an average GC content of 62.15 %, and 6522 protein-coding sequences. In the symbiotic plasmid, we identified nap, nir, nor, and nos sequences that explain the ability of the U143 strain to respire NO3- in free-living and microaerobic conditions. Field assays performed in two locations for two years showed that alfalfa inoculated with the U143 strain produced 41 % more total shoot dry matter than the non-inoculated control, and between 61.3 % and 66.5 % of shoot N in alfalfa inoculated with strain U143 derived from nitrogen fixation.

This review presents a summary of the work done in Uruguay on the diversity and agricultural use of rhizobia, a group of soil bacteria able to establish symbiotic associations almost exclusively with legumes and fix atmospheric nitrogen. Uruguay has a long tradition regarding the use of microbial inoculants for agriculture. It is worth mentioning that since 1960, Uruguay has formally regulated the use of microorganisms in agriculture, being considered one of the pioneer countries where state policies on microbial inoculants have been implemented. In general, the vast majority of the work carried out in Uruguay has focused on the selection and characterization of promising bacteria to be used as biofertilizers. Therefore, the most studied symbiotic associations were those established with legumes of agricultural interest for the country, such as alfalfa, clover, lotus and soybean. The studies carried out with autochthonous alpha- and beta- rhizobia associated with diverse legume species are also considered, taking into account their ecological importance and the interest in knowing and preserving native germplasm.

In acid soils, Rhizobium favelukesii strains, known as Oregon-like strains, are a potential risk for alfalfa production given their parasitic behaviour. In this study, we isolated five parasitic strains (ORY1 to ORY5) from alfalfa nodules grown in Uruguayan acid soils, with a 99.7% and a 100% 16S rRNA gene sequence identity to R. favelukesii type strain of LUP83. The BOX profiles of the five isolates showed two different patterns, suggesting some diversity among these acid-tolerant isolates. The genome sequence analysis of R. favelukesii strains ORY1, LPU83, and Or191 showed that they have around 87.5% of common coding genes, including the symbiotic genes. Moreover, the phylogenetic analysis of ORY1 symbiotic genes nifH, nifD, nifK, nodA, nodB, and nodD were related to the symbiotic genes of E. meliloti. We teste ORY1 competitiveness by inoculating seeds with 99:1 and 1:99 ratios of ORY1::gusA/E. meliloti U143. In both treatments, ORY1::gusA occupied more than 50% of nodules, evidencing its high competitiveness. However, the aerial biomass in these treatments was remarkably different, suggesting that the nodules induced by the efficient strain are essential to provide enough N for optimal plant growth. These findings support the needing of inoculating in areas where inefficient strains are likely to be present. Finally, we found three genes that encode amino acid sequences for domains of M16 peptidases (with homology to bacterial hrrP and sapA genes), two of them were contiguous and located in an accessory plasmid, whereas the other one was a chromosomal gene. These genes are likely to be involved in the parasitic behaviour of ORY1 strain.

The B value is required to quantify the nitrogen derived from the atmosphere (%Ndfa) in the Rhizobium-legume symbiosis using the 15 N natural abundance method. When the B value of a particular specie is not known, one possibility Is to use as a proxy the B value of a specie from the same genus, but this can cause the estimate of %Ndfa to be inaccurate. In this work, we compared two methodologies for determining the B value of Crotalaria juncea, C. spectabilis, C. ochroleuca and Cajanus cajan , using soil as the substrate. One method involvedgrowing plants in soil and averaging the lowest δ 15 N values of plant shoots (B-minimum), while the other consisted in adding sucrose to soil to immobilize the mineral nitrogen (N-immobilized), and then averaging the shoot δ 15 N values of all plants. Results showed that B values of C. cajan and C. ochroleuca obtained using the N-immobilized method were up to 1‰ lower than those reported in the literature for these species. Therefore, we propose that, at least in these species, B values determined with the N-immobilized method should be used to estimate the%Ndfa.

Summer legume cover crops (CC) such as Crotalaria juncea, Crotalaria spectabilis, Crotalaria ochroleuca, and Cajanus cajan could offer diverse advantages for the environment and productive cropping systems. A low transpiration efficiency (TE) of CC can induce soil water content to levels that present a challenge for the subsequent crop. In a 75-day growth chamber experiment, using the natural abundance of 13C, 18O, and 15N we evaluated the TE and BNF under two soil water conditions. Our results showed that the four species tested are good candidates for their use as CC because they showed good results in terms of productivity parameters, TE, and BFN. Cajanus cajan had the highest TE, a high shoot dry matter production, and accumulated more N from BFN in the shoot than C. spectabilis, C. juncea, and C. ochroleuca. ∆18O increased under moderate water deficit and showed an inversely proportional relationship with the amount of transpired water, supporting the use of this isotopic indicator as a proxy for transpiration and stomatal conductance. For the isotopic parameters no interaction between the factors water regimen and species were found. We propose the mass ratio of nitrogen fixed by the volume of transpired water and the isotopic discrimination of 13C as useful indicators of drought fixing legumes tolerance.

Biological nitrogen fixation by the Rhizobium -legume symbiosis allows the conversion of atmospheric nitrogen into ammonia within root nodules mediated by the nitrogenase enzyme. Nitrogenase activity results in the evolution of hydrogen as a result of a side reaction intrinsic to the activity of this enzyme. Some rhizobia, and also other nitrogen fixers, induce a NiFe uptake hydrogenase (Hup) to recycle hydrogen produced by nitrogenase, thus improving the efficiency of the nitrogen fixation process. In this work we report the generation and symbiotic behavior of hydrogenase-positive Rhizobium leguminosarum and Mesorhizobium loti strains effective in vetch ( Vicia sativa) and birsfoot trefoil ( Lotus corniculatus ) forage crops, respectively. The ability of hydrogen recycling was transferred to these strains through the incorporation of hup minitransposon Tn HB100 , thus leading to full recycling of hydrogen in nodules. Inoculation of Vicia and Lotus plants with these engineered strains led to significant increases in the levels of nitrogen incorporated into the host legumes. The level of improvement of symbiotic performance was dependent on the recipient strain and also on the legume host. These results indicate that hydrogen recycling has the potential to improve symbiotic nitrogen fixation in forage plants.

Environmental and biotic stresses induce metabolic changes in plants which led to the accumulation of specific metabolites. For instance, the accumulation of gamma-aminobutyric acid (GABA) and proline is a conserved response of plants to a wide range of stresses. In particular, these amino acids accumulate the most under abiotic stress conditions and, to a lesser extent, under biotic stress conditions. Multiple shared roles have been assigned to these molecules, including osmoprotection, osmotic adjustment, carbon source, redox balance and antioxidant functions. For some of these roles, however, the physiological function of these molecules has been a matter of debate. Recent studies point to unexplored functions of these molecules. For instance, proline accumulation was suggested to contribute to sustaining photosynthesis under stress conditions and proline catabolism was suggested to be relevant to induce plant autophagy. Moreover, many enzymes of proline metabolism were suggested to be important for optimal response to biotic stress. This is even clearer for GABA, for which several mechanisms of action were already described to explain its capacity to protect the plant against pathogens. However, the role of GABA under biotic stress is highly dependent on the biotic stressor involved. Furthermore, GABA was recently demonstrated to enhance biotic stress tolerance when applied as a priming agent and its effect seems to be dependent on ethylene. Here we will summarize and integrate the current knowledge of GABA and proline accumulation in response to stress and discuss future perspectives.

In this study, we report the draft genome sequence of Bradyrhizobium sp. strain Oc8, a rhizobium isolated from Crotalaria ochroleuca,efficient in C. ochroleuca, C. juncea, C. spectabilis, and Cajanus cajan. The whole genome of the strain Oc8 contains 46 scaffolds, 8,283,342 bp, and 63.27% of GC content. Bradyrhizobium sp. Oc8 is an effective nitrogen-fixing bacterium with potential use as an inoculant for legumes used as cover crops and green manures.

EDITORIAL article Front. Agron., 23 November 2021 | https://doi.org/10.3389/fagro.2021.796717

The legume-rhizobia symbiosis is an important process in agriculture because it allows the biological nitrogen fixation (BNF) which contributes to increasing the levels of nitrogen in the soil. Nitric oxide (NO) is a small free radical molecule having diverse signaling roles in plants. Here we present and discuss evidence showing the role of NO during different stages of the legume-rhizobia interaction such as recognition, infection, nodule development, and nodule senescence. Although the mechanisms by which NO modulates this interaction are not fully understood, we discuss potential mechanisms including its interaction with cytokinin, auxin, and abscisic acid signaling pathways. In matures nodules, a more active metabolism of NO has been reported and both the plant and rhizobia participate in NO production and scavenging. Although NO has been shown to induce the expression of genes coding for NITROGENASE, controlling the levels of NO in mature nodules seems to be crucial as NO was shown to be a potent inhibitor of NITROGENASE activity, to induce nodule senescence, and reduce nitrogen assimilation. In this sense, LEGHEMOGLOBINS (Lbs) were shown to play an important role in the scavenging of NO and reactive nitrogen species (RNS), potentially more relevant in senescent nodules. Even though NO can reduce NITROGENASE activity, most reports have linked NO to positive effects on BNF. This can relate mainly to the regulation of the spatiotemporal distribution of NO which favors some effects over others. Another plausible explanation for this observation is that the negative effect of NO requires its direct interaction with NITROGENASE, whereas the positive effect of NO is related to its signaling function, which results in an amplifier effect. In the near future, it would be interesting to explore the role of environmental stress-induced NO in BNF.

The legume-rhizobia symbiosis is an important process in agriculture because it allows the biological nitrogen fixation (BNF) which contributes to increasing the levels of nitrogen in the soil. Nitric oxide (⋅NO) is a small free radical molecule having diverse signaling roles in plants. Here we present and discuss evidence showing the role of ⋅NO during different stages of the legume-rhizobia interaction such as recognition, infection, nodule development, and nodule senescence. Although the mechanisms by which ⋅NO modulates this interaction are not fully understood, we discuss potential mechanisms including its interaction with cytokinin, auxin, and abscisic acid signaling pathways. In matures nodules, a more active metabolism of ⋅NO has been reported and both the plant and rhizobia participate in ⋅NO production and scavenging. Although ⋅NO has been shown to induce the expression of genes coding for NITROGENASE, controlling the levels of ⋅NO in mature nodules seems to be crucial as ⋅NO was shown to be a potent inhibitor of NITROGENASE activity, to induce nodule senescence, and reduce nitrogen assimilation. In this sense, LEGHEMOGLOBINS (Lbs) were shown to play an important role in the scavenging of ⋅NO and reactive nitrogen species (RNS), potentially more relevant in senescent nodules. Even though ⋅NO can reduce NITROGENASE activity, most reports have linked ⋅NO to positive effects on BNF. This can relate mainly to the regulation of the spatiotemporal distribution of ⋅NO which favors some effects over others. Another plausible explanation for this observation is that the negative effect of ⋅NO requires its direct interaction with NITROGENASE, whereas the positive effect of ⋅NO is related to its signaling function, which results in an amplifier effect. In the near future, it would be interesting to explore the role of environmental stress-induced ⋅NO in BNF.

Crotalaria spectabilis and Crotalaria juncea are cover crops (CC) that are used in many different regions. Among the main attributes of these species are their high potential for biomass production and biological fixation of nitrogen (BNF). Attempting to maximize these attributes, while minimizing water consumption through high transpiration efficiency (TE), is a challenge in the design of sustainable agricultural rotations. In this study, the relationship between biomass productivity, BNF, and TE in C. spectabilis and C. juncea was evaluated. For this purpose, an experiment was carried out under controlled conditions without water limitations and using non-inoculated soil. BNF was determined by the natural abundance of 15N, while TE was estimated by several different methods, such as gravimetric or isotopic method (13C). C. juncea produced 42% less dry matter, fixed 28% less nitrogen from the air, and had 20% less TE than C. spectabilis. TE results in both species were consistent across methodologies. Under simulated environmental conditions of high temperature and non-limiting soil water content, C. spectabilis was a relatively more promising species than C. juncea to be used as CC.