Increased Nitrogen Fixation Research Articles

SiO2 nanoparticles can enhance nitrogen retention and reduce copper resistance genes during aerobic composting of swine manure

SiO2 nanoparticles (SiO2 NPs) are low-cost, environmentally friendly materials with significant potential to remove pollutants from complex environments. In this study, SiO2 NPs were used for the first time as an additive in aerobic composting to enhance nitrogen retention and reduce the expression of copper resistance genes. The addition of 0.5 g kg−1 SiO2 NPs effectively reduced nitrogen loss by 72.33 % by decreasing denitrification genes (nosZ, nirK, and napA) and increasing nitrogen fixation gene (nifH). The dominant factors affecting nitrification and denitrification genes were Firmicutes and C/N ratio. Additionally, SiO2 NPs decreased copper resistance genes by 28.96 % − 37.52 % in compost products. Copper resistance genes decreased most in the treatment with 0.5 g kg−1 SiO2 NPs. In summary, 0.5 g kg−1 SiO2 NPs have the potential to reduce copper resistance genes and enhance nitrogen retention during aerobic composting, which may be used to improve compost quality.

Effects of Phosphorus Fertilizer, Poultry Manure Applications with Bacillus megaterium M-3 Inoculation on Yield and Yield Components of Common Vetch (Vicia sativa)

Phosphorus is considered one of the essential elements for legumes as it increases nitrogen fixation due to nodulation. Hence, for high productivity in legumes, it should be added as inorganic or organic fertilizer to soils containing insufficient phosphorus. Especially in recent years, using inorganic and organic fertilizers together has gained importance in terms of sustainable agriculture, considering plant growth and soil and environmental health together. The effects of three different doses of phosphorus fertilizer (0, 50, 100 kg P2O5 ha-1), and two different doses of poultry manure (0, 3 t ha-1) applications with two different doses (B0 or B1) of phosphorus solubilizing bacteria (Bacillus megaterium M-3) inoculation on hay yield and yield components of common vetch were examined in this study. In fact, while the highest hay yield and crude protein rate were obtained by bacterial inoculation together with phosphorus fertilizer application, the effect of poultry manure application on yield and yield parameters was variable. Instead, the applications had no significant effect on the number of main branches, ADF and NDF rates. Consequently, the application of 100 kg P2O5 ha-1 together with Bacillus megaterium inoculation can be recommended for high hay yield in common vetch under irrigated conditions in areas with poor or medium soils in terms of phosphorus.

Analysis of promoter region and regulatory elements of Rhizobium giardinii DNA-binding response regulator A3AY_RS01 genes

BackgroundRhizobium giardinii has been demonstrated to colonize the roots of a variety of legume species, including common beans, and to increase nitrogen fixation. This suggests that Rhizobium giardinii might be a beneficial tool for sustainable agriculture by lowering dependency on synthetic nitrogen fertilizers and enhancing soil fertility. Understanding the regulatory components in the R. giardinii A3AY_RS01 genes might also lead to the creation of innovative ways for increasing the effectiveness of nitrogen fixation in other agriculturally important bacteria. Therefore, this study was aimed to predict regulatory element of R. giardinii DNA-binding response regulator A3AY_RS01 genes. ResultsThe locations for 19 % of the Transcriptional start site (TSSs) were within −300 bp relative to the start codon and ten candidate motifs were identified that are shared by at least 50 % of the R. giardinii A3AY_RS01 promoter input sequences from both strands. Motif 1 was revealed as the common promoter motif for all of R. giardinii A3AY_RS01 genes that serves as binding sites for TFs involved in the expression regulation of these genes. Hence, it was revealed that Motif 1 may serve as the binding site chiefly for Ferric uptake regulator (Fur) transcription factor family to regulate expression of A3AY_RS01 genes. High CpG density in the promoter than body regions were observed for most of the genes except for A3AY_RS0102950, A3AY_RS0120195 and A3AY_RS0131150 genes. Nonetheless, promoter areas were richer than body regions in both techniques. ConclusionsMV1 motif can serve as a binding site for the Fur transcription factor gene family in R. giardinii to regulate the expression of R. giardinii A3AY_RS01 genes. R. giardinii A3AY_RS01 genes are rich in CpG Islands, and play an important role in the regulation of the gene expression of nitrogen fixing in this bacterium.

Effect of nitrogen fixation enhancing type SEN1 gene on soybean growth

ABSTRACT It is well known that the growth of legumes is greatly affected not only by fertilizer nitrogen but also by fixed nitrogen by symbiosis with rhizobia. We have reported that a polymorphism in SEN1, which is essential for nitrogen fixation in Lotus japonicus, could be used to enhance nitrogen fixation and plant growth. In this study, we examined whether the same strategy could be applied to Glycine max. Sequencing of orthologs of the LjSEN1 gene in 38 soybean cultivars revealed that the polymorphism occurred at Glyma.08g076300. We defined the standard sequence that most of the cultivars retained as Peking type SEN1, and the mutated sequence found in Enrei as Enrei type SEN1. To investigate the effect of genotype differences, we generated near-isogenic lines by crossing Enrei with Fukuyutaka, which has Peking type SEN1, and analyzed their characteristics. The results showed that Enrei type enhanced nitrogen fixation activity and promoted plant growth. Since soybean SEN1 has been reported to be a homolog of vacuolar iron transporter-like protein, we investigated the iron content in the nodules. In the result, the iron content in the nodules of Enrei type was significantly higher than that of Peking type. Iron is known to be present in the active center of nitrogenase, and the high iron content may have contributed to the increased nitrogen fixation activity. These results suggest that Enrei type SEN1 is a nitrogen fixation enhancing gene and that introduction of Enrei type SEN1 may reduce nitrogen fertilizer application and increase yield.

Influence of Nutrient Regulation on Ricebean (Vigna umbellata (Thunb.) Ohwi and Ohashi) Growth and Dry Matter

Aims: Within the realm of sustainable agriculture and its attendant issues, there exists a compelling need for a viable approach to cropping systems that integrates pragmatic and economically sound crop management tactics. This is imperative to uphold land productivity and secure a lasting provision of crops for human consumption. Acknowledging the pivotal contribution of organic manure when combined with chemical fertilizers in crop cultivation and the safeguarding of soil well-being, a research inquiry was formulated for a noteworthy cropping sequence.
 Study Design: Randomized block design.
 Place and Duration: School of Agricultural Sciences, Nagaland University, Medziphema. Duration: 2019-2021.
 Methodology: The study took place from 2019 to 2021 at the School of Agricultural Sciences (SAS), Medziphema Campus in Nagaland. The primary aim was to assess the lasting impact of both manure and fertilizers on yield and soil condition in a ricebean system over this two-year duration. In the second week of April in both 2019 and 2020, Dhaincha (Sesbania aculeata), a green manure crop, was sown using seeds. After a 30-day decomposition period, this green manure was incorporated into the soil. Soil samples were collected from the upper 0-15 cm layer of each experimental plot and analyzed. The experimental field was laid out in randomized block design with three level of organic manures i.e., poultry manure (PM), pig manure (PGM) and farm yard manure (FYM) along with doses of inorganic fertilizers : 100% RDF, 75% RDF, 50% RDF. The treatment combinations included T1 : GM (Sesbania) + PM (0.7 t ha-1) + 100 % RDF; T2: GM (Sesbania) +PM (0.7 t ha-1) + 75 % RDF; T3: GM (Sesbania) + PM (0.7 t ha-1) + 50 % RDF ; T4: GM (Sesbania) + PGM (0.7 t ha-1) + 100 % RDF; T5: GM (Sesbania) +PGM (0.7 t ha-1) + 75% RDF; T6: GM (Sesbania) + PGM (0.7 t ha-1) + 50 % RDF; T7: GM (Sesbania) +FYM (4 t ha-1) + 100 % RDF; T8: GM (Sesbania) + FYM (4 t ha-1) + 75 % RDF; T9: GM (Sesbania) +FYM (4 t ha-1) + 50 % RDF.
 Results: In treatment T1, which involved the application of a blend of green manure and poultry manure at a rate of 0.7 tons per hectare, coupled with a full dose of RDF, exhibited outstanding outcomes in ricebean cultivation. This resulted in elevated seed yields, improved seed quality, sustained soil health, and maximized economic gains for farmers in the foothill conditions of Nagaland.
 Conclusion: The substantial enhancement in ricebean growth parameters can be credited to the rich nutrient content and beneficial micronutrients present in poultry manure. combining Recommended Dose of Fertilizers (RDF) with poultry manure, known for its ability to boost nodule formation in legumes, improved soil fertility, fostered beneficial microbial activity, and promoted legume health, ultimately leading to higher yields. In contrast, farmyard manure (FYM) outperformed pig manure due to its balanced nutrients, microbial activity, organic matter, and positive impact on soil quality, creating an optimal environment for plant growth. Poultry manure's capacity to stimulate nodule formation, enhance soil fertility, and support legume health resulted in increased nitrogen fixation and improved yields in leguminous crops, fostering early root development and robust growth in ricebean crops.

Genetic variation and genetic complexity of nodule occupancy in soybean inoculated with USDA110 and USDA123 rhizobium strains

BackgroundSymbiotic nitrogen fixation differs among Bradyrhizobium japonicum strains. Soybean inoculated with USDA123 has a lower yield than strains known to have high nitrogen fixation efficiency, such as USDA110. In the main soybean-producing area in the Midwest of the United States, USDA123 has a high nodule incidence in field-grown soybean and is competitive but inefficient in nitrogen fixation. In this study, a high-throughput system was developed to characterize nodule number among 1,321 Glycine max and 69 Glycine soja accessions single inoculated with USDA110 and USDA123.ResultsSeventy-three G. max accessions with significantly different nodule number of USDA110 and USDA123 were identified. After double inoculating 35 of the 73 accessions, it was observed that PI189939, PI317335, PI324187B, PI548461, PI562373, and PI628961 were occupied by USDA110 and double-strain nodules but not by USDA123 nodules alone. PI567624 was only occupied by USDA110 nodules, and PI507429 restricted all strains. Analysis showed that 35 loci were associated with nodule number in G. max when inoculated with strain USDA110 and 35 loci with USDA123. Twenty-three loci were identified in G. soja when inoculated with strain USDA110 and 34 with USDA123. Only four loci were common across two treatments, and each locus could only explain 0.8 to 1.5% of phenotypic variation.ConclusionsHigh-throughput phenotyping systems to characterize nodule number and occupancy were developed, and soybean germplasm restricting rhizobium strain USDA123 but preferring USDA110 was identified. The larger number of minor effects and a small few common loci controlling the nodule number indicated trait genetic complexity and strain-dependent nodulation restriction. The information from the present study will add to the development of cultivars that limit USDA123, thereby increasing nitrogen fixation efficiency and productivity.

Strigolactones repress nodule development and senescence in pea.

Strigolactones are a class of phytohormones that are involved in many different plant developmental processes, including the rhizobium-legume nodule symbiosis. Although both positive and negative effects of strigolactones on the number of nodules have been reported, the influence of strigolactones on nodule development is still unknown. Here, by means of the ramosus (rms) mutants of Pisum sativum (pea) cv Terese, we investigated the impact of strigolactone biosynthesis (rms1 and rms5) and signaling (rms3 and rms4) mutants on nodule growth. The rms mutants had more red, that is, functional, and larger nodules than the wild-type plants. Additionally, the increased nitrogen fixation and senescence zones with consequently reduced meristematic and infection zones indicated that the rms nodules developed faster than the wild-type nodules. An enhanced expression of the nodule zone-specific molecular markers for meristem activity and senescence supported the enlarged, fast maturing nodules. Interestingly, the master nodulation regulator, NODULE INCEPTION, NIN, was strongly induced in nodules of all rms mutants but not prior to inoculation. Determination of sugar levels with both bulk and spatial metabolomics in roots and nodules, respectively, hints at slightly increased malic acid levels early during nodule primordia formation and reduced sugar levels at later stages, possibly the consequence of an increased carbon usage of the enlarged nodules, contributing to the enhanced senescence. Taken together, these results suggest that strigolactones regulate the development of nodules, which is probably mediated through NIN, and available plant sugars.

Rising vapor-pressure deficit increases nitrogen fixation in a legume crop.

Atmospheric vapor-pressure deficit (VPD) is increasing in many regions and has a large impact on plant productivity. A VPD increase leads to raising transpiration rate (TR) and soil-water demand, risking productivity penalties. Like water, nitrogen is critical to productivity, but the effect of VPD on legume nitrogen fixation is undocumented. To address this, we developed a portable system for quantifying nitrogen fixation noninvasively and at a high temporal resolution by tracking the rate of hydrogen gas evolution by root nodules. Combining field and controlled-environment experiments where we measured leaf gas exchange and H2 production by nodules, we confirmed the ability of the system to track nitrogen fixation dynamics. Raising VPD from 0.5 to 3 kPa within c. 2.5 h under well-watered conditions increased nitrogen fixation by up to 25% in addition to TR, consistent with the hypothesis that raising VPD in that range might have alleviated nitrogenase feedback inhibition. Genotypic differences were found in this response, indicating a potential for breeding. Our study provides evidence for an important environmental effect on nitrogen fixation that is not taken into account in current crop and vegetation models, pointing to untapped avenues for better understanding climate change effects on legumes and nitrogen cycling.

North-south differences in hypoxia and nitrogen cycle of the East China Sea over the last century indicated by sedimentary bacteriohopanepolyols

Coastal hypoxic zones are expanding due to anthropogenic activities and climate changes. Increased hypoxia has greatly affected the marine nitrogen cycle. There are two typical seasonal hypoxic core areas in the north and south of the East China Sea (ECS). However, the interannual variations, north-south differences in hypoxia and their effects on the nitrogen cycle are still poorly understood. This study investigated bacteriohopanepolyols (BHPs), especially newly developed marine anammox bacteria and hypoxic biomarker bacteriohopanetetrol stereoisomer (BHT-x) and cyanobacterial nitrogen fixation biomarker 2-methyl BHT in two sediment cores, aiming to reconstruct hypoxia and nitrogen cycle records in the northern and southern hypoxic zones of the ECS over the last century. The results showed that BHT-x ratio in northern core 3100–2 presented higher values since 1960s (>0.2) and increased after 2000 (0.25–0.57), while it decreased in southern core DH6–1 after 2000 (0.03–0.11), indicating the continuously intensified hypoxia in the northern area and significantly weakened hypoxia in the southern area. Hypoxia is mainly associated with organic matter degradation and seawater stratification. The similar temporal increases of BHT-x ratios and marine-derived organic matter (MOM) contents in core 3100–2 suggest that eutrophication-driven MOM degradation is the main reason for hypoxia formation, which is exacerbated by strong water stratification there. Differently, although MOM contents still increased in core DH6–1 after 2000, the hypoxia weakened and even disappeared due to lessened stratification. In addition, the temporal variations of BHT-x and 2-methyl BHT in both cores indicated that anammox and nitrogen fixation simultaneously increased during periods of hypoxia. The co-variation suggests that hypoxia promotes anammox, and nitrogen loss may induce an increase in nitrogen fixation to compensate for nitrogen availability.

Stable isotope tracing reveals compartmentalized nitrogen assimilation in scleractinian corals

Corals form symbiotic relationships with dinoflagellate algae of the family Symbiodiniaceae, bacteria, and other microbes. Central to that relationship is the regulation of nutrition flux between the animal host and the photosynthetic Symbiodiniaceae that it is reliant on for the majority of metabolic needs. Nitrogen availability controls the growth and density of Symbiodiniaceae within coral tissues and has been proposed to play a role in host derived symbiosis regulation. Warming ocean temperatures and subsequent increases in dissolved organic carbon can potentially increase nitrogen fixation and lead to bleaching. We investigated the importance of nitrogen metabolism in vivo with LC-MS based stable isotope tracing using nubbins from three species of Hawaiian coral, the more heat tolerant Montipora capitata and Porites compressa and the more heat sensitive Pocillopora acuta, that were collected from reefs in Kāne’ohe Bay, O’ahu. In addition to 15N incorporation into nucleotides, amino acids, and urea cycle metabolites, we also observed significant isotopic labeling in dipeptides, supporting their previous identification as major heat stress response metabolites. Surprisingly, the dipeptides are highly enriched in 15N compared to free amino acids, which are the biosynthetic precursors for dipeptides. This suggests that there is a high turnover of dipeptide pools and distinct biosynthetic mechanisms that separately mediate amino acid and dipeptide production. These preliminary data show that nitrogen assimilation in the coral holobiont is likely compartmentalized, with rapid assimilation and quick dipeptide turnover occurring in one region of the holobiont and slow turnover of other nitrogen containing metabolites in other region(s).

Characterization of diazotrophic root endophytes in Chinese silvergrass (Miscanthus sinensis)

BackgroundPhytoremediation is a potentially cost-effective way to remediate highly contaminated mine tailing sites. However, nutrient limitations, especially the deficiency of nitrogen (N), can hinder the growth of plants and impair the phytoremediation of mine tailings. Nevertheless, pioneer plants can successfully colonize mine tailings and exhibit potential for tailing phytoremediation. Diazotrophs, especially diazotrophic endophytes, can promote the growth of their host plants. This was tested in a mine-tailing habitat by a combination of field sampling, DNA-stable isotope probing (SIP) analysis, and pot experiments.ResultsBacteria belonging to the genera Herbaspirillum, Rhizobium, Devosia, Pseudomonas, Microbacterium, and Delftia are crucial endophytes for Chinese silvergrass (Miscanthus sinensis) grown in the tailing, the model pioneer plant selected in this study. Further, DNA-SIP using 15N2 identified Pseudomonas, Rhizobium, and Exiguobacterium as putative diazotrophic endophytes of M. sinensis. Metagenomic-binning suggested that these bacteria contained essential genes for nitrogen fixation and plant growth promotion. Finally, two diazotrophic endophytes Rhizobium sp. G-14 and Pseudomonas sp. Y-5 were isolated from M. sinensis. Inoculation of another pioneer plant in mine tailings, Bidens pilosa, with diazotrophic endophytes resulted in successful plant colonization, significantly increased nitrogen fixation activity, and promotion of plant growth.ConclusionsThis study indicated that diazotrophic endophytes have the potential to promote the growth of pioneer plant B. pilosa in mine tailings.EAsdjSo3ZCTEVznKh83dpJVideo

Expression and Variation of the Genes Involved in Rhizobium Nodulation in Red Clover.

Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency, in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency.

Nitrogen content and C/N ratio in straw are the key to affect biological nitrogen fixation in a paddy field

Straw amendment can increase nitrogen fixation in paddy field, however, the efficiency of carbon sources with different biochemical properties to enhance N2 fixation and nitrogen fixation activity is still unclear. A 15N2-labelling system was used in the field environment to determine biological nitrogen fixation (BNF) under the addition of three kinds of straw. The nifH gene (DNA) and nifH RNA gene (cDNA) of soil were amplified by real-time fluorescent quantitative PCR. The diversity and community composition of nitrogen fixing microorganisms were studied by high-throughput sequencing. The study is expected to reveal how different carbon sources impact biological nitrogen fixation and its mechanism in the paddy field system. Results showed that the absolute abundance diazotrophs in the treatment of mature stage wheat straw (MWS) was 4.88 times as high as that in the CK treatment, but jointing stage wheat straw (JWS) and poplar branch (PB) did not induce any significant changes. Straw amendment had no impact on cyanobacteria abundance. The proportion of N2 fixation increased by MWS was 2.07 times, but which was much lower than the increase proportion of the heterotrophic diazotrophs, leading to a decrease of diazotrophic nitrogen fixation activity. Mature wheat straw addition increased biologically fixed nitrogen in paddy field by increasing the number of heterotrophic nitrogen fixing bacteria. The results indicated that to increase biological N2 fixation in paddy system, straws with low nitrogen content and high C/N ratio were recommended.

Feedstock-dependent abundance of functional genes related to nitrogen transformation controlled nitrogen loss in composting

The objective of this work was to explore how selection of feedstock affects nitrogen cycle genes during composting, which eventually determines the nitrogen loss. Four composting mixes (CM: chicken manure; SM: sheep manure; MM1/3: mixed manure with CM: SM = 1:3 w/w, MM3/1: CM: SM = 3:1 w/w) were investigated. Results showed that adding 25 % and 75 % SM to CM reduced 26.5 % and 57.9 % nitrogen loss, respectively. CM contained more ammonification genes and nrfA gene, while SM had more denitrification genes. Nitrogen fixation genes in CM were slightly higher than that in SM at the initial stage, but they sharply dropped off as the composting entered the high temperature stage. MM1/3 showed significantly reduced ammonification genes than CM, and increased nitrogen fixation and NH4+ assimilation genes. Therefore, adding SM to CM could change the abundance of genes and enzymes related to nitrogen cycle to reduce nitrogen loss.

High-throughput screening of soybean di-nitrogen fixation and seed nitrogen content using spectral sensing

Symbiotic di-nitrogen fixation of grain legumes has a substantial impact on crop performance, harvest product quality, and nitrogen (N) balance of crop rotations, particularly under organic management regimes. In soybean breeding, selection for increased nitrogen fixation is desirable for improving seed protein content and N balance of cropping systems. However, the lack of high-throughput screening methods for direct measurement of N2 fixation rates prohibits practical breeding efforts. Therefore, hyperspectral canopy reflectance measurement as a field-based phenotyping method was evaluated in three environments for indirect estimation of N fixation and uptake of soil nitrogen in a set of early maturity soybean genotypes exhibiting a wide range in seed protein content. Reflectance spectra were collected in repeated measurements during flowering and early seed filling stages. Subsequently, various spectral reflectance indices (SRIs) were calculated for characterizing nitrogen accumulation of individual genotypes. Moreover, prediction models for seed protein content as an end-of-season target trait were developed utilizing full spectral information in partial-least-square regression (PLSR) models. A number of N-related SRIs calculated from spectral reflectance data recorded at the beginning of the seed filling stage were significantly correlated to seed protein content. The best prediction of seed protein content, however, was achieved in PLSR models (validation R2 = 0.805 across all three environments). Environments lower in initial soil mineral N content appeared as more favorable selection sites in terms of prediction accuracy, because N fixation is not masked by soil N uptake in such environments. Hyperspectral reflectance data proved to be a valuable method for determining genetic variation in crop N accumulation, which might be implemented in high-throughput screening protocols for N fixation in plant breeding programs.

Evaluation of maize/peanut intercropping effects on microbial assembly, root exudates and peanut nitrogen uptake

Legume/cereal intercropping has been widely studied within ecosystem function, owing to its overyield potential, predominantly by dinitrogen (N2) fixation. In our 2-year peanut/maize intercropping field experiment, land equivalent ratio (LER) showed an average yield-increase by 41.48%. Performance index of intercropped peanut (IP) functional leaves exhibited significant improvement (2.02-fold). Moreover, IP increased dry nodule weight by 58.82% as compared to mono-cropped. Also, the ratio of nodules to aboveground biomass in IP reduced by 65.8%. In pot experiment, higher urease activity was found in rhizosphere (22.73%). The abundance of Rhizobium and niƒH gene in the rhizosphere of IP were significantly enhanced by 71.91% and 208%, respectively. To analyze root exudates, we performed hydroponic coculture, the proportion of total isoflavonoids in peanut root exudates were increased distinctly by 22.4%. Our findings certainly helped in filling one the information gaps, that how intercropping increases nitrogen fixation in rhizosphere. Lastly, it can further facilitate to understand functional significance of intercropping system for agricultural ecological sustainability and efficient resource utilization.

Soybean (Glycine max (L.) Merr.) Growth, Yield, and Nodulation in the Early Transition Period from Conventional Tillage to Conservation and No-Tillage Systems

Soybean (Glycine max (L.) Merr.) is the most important protein crop globally, with its cultivation area in Europe on the increase. To investigate how alternative tillage systems affect soybean growth, yield performance, and nitrogen fixation capacity in the early conversion period from conventional tillage to conservation and no-tillage practices, a field study was conducted in 2020 under the humid central European climatic conditions of Slovenia. A complete randomized block design with four repetitions was used for the three different tillage systems (conventional, conservation, and no-tillage). The results show that the majority of the studied soybean growth parameters (e.g., plant density, nodes per plant, and shoot and root dry matter) and the yield components (e.g., pods per plant, and 100-seed mass) were greatest for the reference conventional tillage system. The conventional system also showed significantly greater dry nodule mass (p < 0.01) and proportion of large-sized nodules (>4 mm) on both the tap root (p < 0.05) and the lateral roots (p < 0.001). A positive linear correlation between nitrogen content and nodule production in the roots also suggested increased nitrogen fixation for the conventional system. The less intensive conservation and no-tillage systems resulted in significantly greater soil compaction, which negatively affected early plant establishment and resulted in significantly decreased plant densities. Despite the large differences in plant stands and individual plant performances, no significant differences were seen for dry seed yields between these tillage systems. Dry seed yields for the conventional and conservation systems were 4.54 and 4.48 t ha−1, respectively, with only minor (non-significant) yield reduction for the no-tillage system, at 4.0 t ha−1. These data show that soybean cultivation in the early transition period to less intensive tillage systems have no major yield losses under these less suitable agro-climatic conditions if correct crop and weed management measures are implemented.

Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields.

Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 ("Kv137-1036") retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Effect of Predation by Colpoda sp. in Nitrogen Fixation Rate of Two Free-Living Bacteria.

Biological nitrogen fixation is limited to several groups of prokaryotes, some of them reduce nitrogen as free-living nitrogen-fixing bacteria. Protozoa predation on these latter releases sequestered nitrogen that may enhance the formation of new bacterial biomass and possibly increase nitrogen fixation within soil microbial communities. We aim to evaluate the predation effect of Colpoda sp. on two nitrogen fixers: Azospirillum lipoferum and Stenotrophomonas sp. during their lag, early exponential, and exponential phases. The kinetics of bacterial population growth was determined in the predators' presence or absence and the effect of predation on the rate of N fixation was evaluated through the reduction of acetylene to ethylene technique. Colpoda sp. showed a non-significant difference in preferences between the two species offered as prey. Consequently, the abundance of A. lipoferum and Stenotrophomonas sp. decreased significantly due to predator's pressure and both species responded by increasing their specific growth rate. Likewise, predation promoted greater nitrogen fixation rate by CFU during the lag phase in A. lipoferum (0.20nM/CFU with predation vs 0.09nM/CFU without predation) and Stenotrophomonas sp. (0.22nM/CFU vs 0.09nM/CFU respectively). During early exponential phase (29h), the rate diminished to 0.13 and 0.05nM/CFU in A. lipoferum and to 0.09nM/CFU and 0.05nM/CFU in Stenotrophomonas sp. Finally, during the exponential phase (52h), only A. lipoferum without predation produced 0.003nM/CFU of ethylene. Thus, the nitrogenase activity was higher in the lag and the early exponential phases when predator activity was involved.

Experimental evolution can enhance benefits of rhizobia to novel legume hosts.

Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host–symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host–symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts.