Nitrogen

This study introduces a novel methodology for predicting cotton yield by integrating machine learning (ML) with mechanistic soil modeling. This hybrid approach enhances yield prediction by combining data-driven ML techniques with soil process modeling. Using the developed yield model, yield curves for various nitrogen (N) levels can be constructed to identify the optimal N dose that maximizes yield. Estimating cotton N requirements is crucial, as growers often apply excessive N, exceeding the amount needed for maximum yield. By comparing the Mean Absolute Error (MAE) between predicted and observed cotton yield values across three ML algorithms, i.e., Random Forest (RF), XGBoost, and LightGBM, the RF model achieved the lowest error (422.6 kg/ha), outperforming XGBoost (446 kg/ha) and LightGBM (449 kg/ha). Additionally, the RF model exhibited high sensitivity to N fertilization, ranking N as the most influential variable in feature importance analysis. Furthermore, phosphorus (P) availability in the soil model was found to be a significant factor influencing the RF yield model, highlighting P’s crucial role in cotton growth and productivity. Full article

Beef-cattle manure (BCM) can be an effective source of nitrogen (N) for crop production. However, N availability from manure can be difficult to quantify across varying environments. The Magruder Plots are a continuous winter wheat (Triticum aestivum L.) fertility study established in 1892 in Stillwater, OK, USA. In the study, wheat grain yield responses to BCM that is applied every four years are compared to those following annual applications of inorganic nitrogen (N), phosphorus (P), and potassium (K) fertilizers. This long-term, comprehensive dataset facilitates the evaluation of manure N availability and uptake across a wide range of growing environments; thus, the objective of this paper was to use 56 years of data from the Magruder Plots to benchmark current N-based manure application guidelines for Oklahoma. The results from this analysis revealed some discrepancies compared to regionally accepted guidelines for manure N availability. Existing guidelines for Oklahoma suggest that 50 to 70% of total N in BCM will become plant-available in the first year after application; however, the Magruder Plots have only averaged 23% total N availability in year one. The three seasons after manure application averaged total N availability of 20, 16, and 14%, respectively, which much more closely align with the existing estimates for Oklahoma of 10 to 20% N availability after year one. This study suggests that N availability of BCM in the first year after application in Oklahoma has a more accurate estimate of 10 to 30% of total N. Full article

Increasing soil salinity is threatening agricultural productivity which implies the development of new sustainable strategies to deal with this challenge. The main objective here is to assess the potential for improving the tolerance of alfalfa to salinity by combining inoculations with rhizobia and AMF. However, the distinguishing feature of this study is the comparison of two alfalfa varieties’ microbial response to salinity. The greenhouse trial was conducted on an Australian variety Siriver and an indigenous Demnate population, which were inoculated with Rhizoglomus irregulare and/or native AMF, and/or a RhOL1 rhizobial strain. The RhOL1 strain was selected from nine rhizobia tested for their plant growth promoting rhizobacteria (PGPR) activities. In addition to its ability to tolerate high salinity levels (769 mM) and solubilize insoluble phosphate as well as potassium, it can also synthesize auxins such as IAA. The application of these biofertilizers was carried out in the absence and the presence of the saline stress (0 and 120 mM NaCl). The double inoculations of native AMF and RhOL1 significantly improve the shoot and root dry biomass, plant elongation, number of formed leaves, and mineral nutrition, as well as the number of nodules and the rate of mycorrhizal root colonization. The synergistic effects between the native AMF and RhOL1 strain have been demonstrated in this study. However, the behavior of alfalfa genotypes towards microbial inoculation was significantly different. The ability to react to the double indigenous RhOL1 + AMF inoculation is more important in the Siriver than in the Demnate population. Thus, the possibility of formulating biofertilizers is based on the AMF–rhizobia–hote tripartite combination for alfalfa production in saline areas. Full article

This study investigated the effects of Bradyrhizobium elkanii inoculation and nitrogen (N) fertilization on the growth of Pseudalbizzia niopoides seedlings in a nursery and their subsequent performance in soil. P. niopoides is a legume tree native to Latin American tropical forests, known to nodulate but with no previously identified rhizobial partner. Seedlings were grown in a nursery under varying N fertilization rates (0, 250, 500, 1000, and 2000 mg L⁻¹) with and without B. elkanii inoculation. Morphological traits, nodulation, and post-planting growth were assessed. Both inoculation and N fertilization significantly enhanced seedling growth in the nursery. However, high N rates suppressed nodulation and caused root toxicity. Inoculated seedlings exhibited improved growth after planting, particularly at lower N rates. Notably, inoculated seedlings without added N demonstrated vigorous new root proliferation after three months, highlighting the beneficial effects of the symbiosis. In terms of nitrogen fertilization in nurseries, a N rate up to 500 mg L⁻¹ produced satisfactory plant growth and no prejudicial effects on the symbiosis establishment. However, it is possible to raise seedlings even in the 0 mg L⁻¹ N rate, with a vigorous root emission during the post-planting growth. This study provides valuable insights into the interaction between a specific rhizobia strain and P. niopoides, with implications for nursery practices and sustainable agroforestry systems. Full article

Alley cropping, an agroforestry system that integrates trees and arable crops, holds the potential to improve both crop yields and soil health. It has been found to be effective for upland crops in many countries of the world. However, the utilization of alley cropping to improve soil health in the terrace ecosystem of Bangladesh is poorly understood. Therefore, this study was undertaken to assess the changes in soil biochemical properties and quantify the cabbage yield under three alley widths of Leucaena leucocephala (3.0, 4.5, and 6.0 m size) and five nitrogen (N) levels [0, 40, 80, 120, and 160 kg N ha⁻¹ (0, 25, 50, 75, and 100% of recommended N rates, respectively) with the addition of pruned materials of L. leucocephala (Ipil-ipil)]. The field experiment was conducted following a split-plot design, where alley width was considered as the main-plot factor and N rate as the sub-plot factor. Within each main plot, the five N rates were replicated thrice. Control plots with similar N doses were applied accordingly without addition of pruned materials to compare the results with alley cropping. Data were collected on the biochemical properties of the soil [soil pH, organic carbon (C), total N, available phosphorus (P), exchangeable potassium (K), microbial biomass C, and biomass N] and the yield of cabbage quantified [edible head weight (kg plant⁻¹) and head yield (t ha⁻¹)] under different alley widths and control. Findings revealed that organic C, total N, available P, exchangeable K, microbial biomass C, and biomass N in the topsoil exhibited maximum values in the L. leucocephala-based alley plot, which is proved to be a possible solution of restoration of degradable land. Additionally, L. leucocephala-based alley cropping improved the soil pH, indicating a potential avenue for more-sustainable land management practices. Results also showed that alley widths and N rates have significant effects on cabbage (Brassica oleracea L. var. capitata) yield. Alley width of 6.0 m along with 100% N provided the highest cabbage yield followed by 75% N in 6.0 m alley, and the control with 100%. The wider alley minimizes tree–crop competition, allowing for optimal cabbage production. These aforementioned results suggest that alley cropping with L. leucocephala is a promising approach to enhance soil fertility and crop productivity in the terrace ecosystem of Bangladesh. Full article

This study explores the economic benefits and challenges associated with biodegradable waste composting, highlighting its role in promoting sustainability through a circular economy framework. We reviewed the relevant literature and found 160 articles for this study, including the keywords “Biodegradable waste”, “Compost production”, and “Economic Potential”. Considering quality studies, we employed the PRISMA technique to conduct a comprehensive data synthesis and evaluate 89 articles for the final review. Our findings highlight that composting offers significant advantages, including waste reduction, cost savings in waste management, carbon credit, a source of nitrogen, job creation, and reduced reliance on synthetic fertilizers. Further, it supports environmental sustainability by improving soil health, mitigating greenhouse gas emissions, and reducing landfill use. However, challenges such as high upfront costs, quality control, and market competition with chemical fertilizers remain barriers to widespread adoption. The study extends the literature by emphasizing that the integration of composting into a circular economy can foster innovation, enhance local economies, and contribute to climate change mitigation. Furthermore, it offers a promising pathway for advancing sustainability. Future research should focus on improving composting technologies, optimizing their applications, and developing stronger policy frameworks to ensure the successful implementation of biodegradable waste composting practices. Full article

Soil nitrogen (N) is a crucial nutrient for agricultural productivity and ecosystem health. The accurate and timely assessment of total soil N is essential for evaluating soil health. This study aimed to determine the impact of bootstrapping techniques on improving the predictive accuracy of indirect total soil N in conventional wheat fields in Al-Muthanna, Iraq. We integrated a novel methodological framework that integrated bootstrapped and non-bootstrapped total soil N data from 110 soil samples along with Landsat 9 imagery on the Google Earth Engine (GEE) platform. The performance of the proposed bootstrapping-enhanced random forest (RF) model was compared to standard RF models for soil N prediction, and outlier samples were analyzed to assess the impact of soil conditions on model performance. Principal components analysis (PCA) identified the key spectral reflectance properties that contribute to the variation in soil N. The PCA results highlighted NIR (band 5) and SWIR2 (band 7) as the primary contributors, explaining over 91.3% of the variation in soil N within the study area. Among the developed models, the log (B5/B7) model performed best in capturing soil N (R² = 0.773), followed by the ratio (B5/B7) model (R² = 0.489), while the inverse log transformation (1/log (B5/B7), R² = 0.191) exhibited the lowest performance. Bootstrapped RF models surpassed non-bootstrapped random forest models, demonstrating enhanced predictive capability for soil N. This study established an efficient framework for improving predictive capacity in areas characterized by limited, low-quality, and incomplete spatial data, offering valuable insights for sustainable nitrogen management in arid regions dominated by monoculture systems. Full article

Nitrate pollution in aquatic environments poses significant environmental and public health issues, mostly due to industrial activities and agricultural runoff. Biological denitrification, the favored method for removing nitrates, typically needs an external carbon source to support microbial processes. Traditional electron donors like methanol, ethanol, and acetate are effective but introduce economic, environmental, and operational challenges such as cost variability, flammability hazards, and excessive residual organic material. Recently, solid-phase carbon sources—like biodegradable polymers and organic agricultural waste—have shown promise as alternatives because they allow for controlled carbon release, improved safety, and enhanced long-term sustainability. This review systematically examines the performance of solid-phase carbon in wastewater denitrification by analyzing peer-reviewed studies and experimental data. The findings suggest that solid-phase carbon sources, including polycaprolactone (PCL) and polyhydroxyalkanoates (PHA), offer stable and extended carbon release, ensuring consistent denitrification effectiveness. Nonetheless, challenges remain, including optimizing biofilm development, balancing carbon availability, and reducing operational costs. Furthermore, the review emphasizes the potential for integrating machine learning in process optimization and highlights the need for more research to enhance the economic viability of these materials. The findings confirm the practicality of solid-phase carbon sources for extensive wastewater treatment and their capability to sustainably address nitrate contamination. Full article

Addressing the challenge of reducing environmental pollution from agricultural practices by improving nitrogen use efficiency (NUE) and water use efficiency (WUE) while ensuring high crop yields is essential for sustainable agriculture. Using a controlled glasshouse experiment, we evaluated the combined effects of biochar and bokashi under different irrigation regimes on NUE, WUE, and yield-related parameters in a wheat cropping system. The experiment followed a completely randomized design with three replications with four treatments: (1) control (C), (2) bokashi only (B₀), (3) bokashi +1% biochar (B₁), and (4) bokashi +2% biochar (B₂). These treatments were evaluated at three irrigation levels—30% (IR₃₀), 50% (IR₅₀), and 60% (IR₆₀) of field capacity (FC), resulting in a total of twelve treatments. Co-application of bokashi–biochar significantly (p < 0.050) improved grain yield (GY), straw yield (SY), total biomass (TB), total nitrogen uptake (TNU), grain protein content (GPC), NUE, and WUE, with the most notable benefits observed at 1% biochar application compared to C and B₀ treatments. In addition, both types of treatment (bokashi and bokashi with biochar) and the level of irrigation had a significant impact on GY, SY, TB, TNU, GPC, NUE, and WUE. The B₁ and B₂ treatments further improved yield and efficiencies compared to bokashi alone. The positive correlation between grain yield and WUE underscores the importance of optimizing irrigation strategies alongside soil amendments for improved crop productivity. These enhancements in yield and efficiency are likely attributed to the increased soil fertility, nutrient availability, and water retention resulting from the combination of biochar and bokashi. Full article

Sweet corn (Zea mays convar. saccharata var. rugosa) is an important crop in the United States (US), particularly in the southeastern region. While effective nitrogen (N) management is essential for optimizing yields, the sandy soils and variable precipitation in this region impact N uptake. This study evaluates the effects of several N rates (ranging from 224 to 336 kg ha⁻¹) and N fertilizer application timing (N fertilizer applied at emergence and side-dress stages) on sweet corn growth and yield under the subtropical environmental conditions of the southeastern US. Field experiments were conducted over three years in the states of Georgia (2020) and Alabama (2021 and 2022). In general, the weather conditions of each season had a direct impact on sweet corn growth, development, and yield parameters. Among all locations, the season in Alabama (2022) allowed for the highest yields (17,380 kg ha⁻¹), which could be attributed to favorable weather conditions that required moderate nitrogen application rates (224–280 kg ha⁻¹). Contrarily, the weather conditions of Alabama in 2021 and Georgia in 2020 impacted soil N availability, consequently leading to negative effects on sweet corn growth. Overall, N fertilizer management strategies are indicated to be region- and season-specific in order to enhance sweet corn production while protecting the environment from excessive N losses. Further research is still required to refine these strategies and improve predictive models for diverse climatic conditions. Full article

The development of critical levels for sap nitrate and chlorophyll meter reading (SPAD test) in the case of various crops is of great importance for growers in characterizing a plant’s N status. A field experiment with spring oat (Avena sativa L.) was carried out on loamy soil in Debrecen, Hungary, using a small-plot design. Ammonium nitrate was broadcast at rates of 0, 30, 60, and 90 kg N/ha in three replicates. The total N content of the plant, sap nitrate content, and SPAD values were measured at jointing when the first node appeared above the soil surface (Feekes 6) and at boot stage (Feekes 10). Regression analysis between total N content and sap nitrate showed cubic and linear relationships with r² = 0.7982 (Feekes 6, whole plant) and 0.9625 (Feekes 10, upper developed leaves), respectively. Optimal grain yield was obtained when sap nitrate exceeded 650 mg/L and 540 mg/L at Feekes 6 and Feekes 10, respectively. There were linear and logarithmic relationships between total N content and SPAD values with r² = 0.8058 and 0.6258 at Feekes 6 and 10. Optimal grain yield occurred over SPAD values of 43 and 48 at Feekes 6 and 10, respectively. Optimal N rate was 60 kg N/ha on the experimental site. Full article

Excessive irrigation in protected vegetable production often results in soil nutrient loss and groundwater contamination. Cucumber (Cucumis sativus L.) is a widely cultivated and important vegetable in the world and a sensitive plant to irrigation water supply. In order to obtain higher water use efficiency (WUE) and to assess the leaching loss of mineral elements under the current strategies of irrigation and fertilization in the production of protected crops, we conducted experiments with three irrigation levels, namely, normal (NI), optimized (OI), and deficit irrigation (DI), on cucumber in a solar greenhouse. The results indicated that the contents of nitrate–nitrogen (NO₃⁻–N) in the top soil layer increased significantly under the reduced irrigation condition (OI and DI) after two cultivation seasons compared with normal irrigation (NI). However, there were no significant differences in the contents of available phosphorus (A–P) and available potassium (A–K) between the three treatments in each soil layer during a single irrigation cycle and for the whole growth cycle. In addition, compared to the NI condition, reducing the amount of irrigation (OI and DI) decreased the amount of leaching of the soil mineral elements by more than half without jeopardizing the fruit yield of cucumber, particularly for DI. Under the three irrigation treatments, the economic yield of cucumber varied from 64,513 to 72,604 kg·ha⁻¹ in the autumn–winter season and from 89,699 to 106,367 kg·ha⁻¹ in the winter–spring season, but the differences among the treatments were not significant. Moreover, the reduced irrigation treatments (OI and DI) substantially improved WUE by 43.9% and 135.3% in the autumn–winter season, and by 82.2% and 173.7%, respectively, in the winter–spring season, compared to the NI condition. Therefore, deficit or optimized irrigation was a potential and suitable irrigation strategy in the solar greenhouse for increasing the water use efficiency, reducing the amount of leached soil mineral elements, and maintaining the economic yield of cucumber crop. Overall, our results provided some insight into the future applications of water-saving irrigation techniques in sustainable greenhouse vegetable production. Full article

An experiment was conducted in spring 2024 to investigate the effects of biochar, biogas slurry, and dicyandiamide (DCD) on N₂O emissions from soil in protected tomato cultivation. Five treatments were applied: conventional fertilization (CK1), biogas slurry alone (CK2), biochar combined with biogas slurry (T1), DCD combined with biogas slurry (T2), and the combination of biochar, biogas slurry, and DCD (T3). The study aimed to assess the response of the soil physicochemical properties and nitrifying ammonia-oxidizing microorganisms in the tomato root zone to these treatments and to determine their impact on soil N₂O emissions. The results showed that adding biochar and biogas slurry increased the soil pH, organic matter content, and levels of nitrate-N and ammonium-N, without affecting ammonia-oxidizing archaea (AOA) but inhibiting ammonia-oxidizing bacteria (AOB). The inclusion of DCD raised the soil pH and ammonium-N levels, enhanced AOA growth, did not alter organic matter content, and significantly reduced nitrate-N levels and AOB activity. Compared to CK1, treatments CK2, T1, T2, and T3 decreased the average N₂O emission flux by 5.83%, 8.24%, 15.27%, and 16.16%, respectively. The application of biochar, biogas slurry, and DCD enhanced the physicochemical properties of the root zone soil and notably reduced N₂O emissions in protected tomato cultivation, with T3 showing the most effective results. The biochar and biogas slurry used in this study, both derived from agricultural waste, promote sustainable agricultural development and enhance economic benefits. However, this study only considered the short-term effects of biochar, biogas slurry, and DCD, necessitating further research to explore their long-term impacts and mechanisms. Full article

Nitrogen, an essential element for plant growth and food production, presents significant challenges in agriculture due to the environmental consequences of synthetic nitrogen fertilizers. This review explores the potential of nitrogen-fixing cyanobacteria as a sustainable alternative for agricultural nitrogen fertilization. The molecular mechanisms underlying nitrogen fixation in cyanobacteria, including key genes such as nif and related biochemical pathways, are examined in detail. Biotechnological approaches for utilizing nitrogen-fixing cyanobacteria as biofertilizers are discussed, alongside strategies for genetic engineering to improve nitrogen fixation efficiency. The review further evaluates the impact of cyanobacteria on soil health and environmental sustainability, emphasizing their role in mitigating the detrimental effects of synthetic fertilizers. While promising, challenges such as oxygen sensitivity during nitrogen fixation and competition with native microorganisms are critically analyzed. Finally, future directions are proposed, including advancements in synthetic biology, integration with conventional agricultural practices, and scalable implementation strategies. This review underscores the transformative potential of nitrogen-fixing cyanobacteria in promoting sustainable agriculture and enhancing global food security. Full article

The use of nitrification inhibitors (NIs) with nitrogen fertilizers represents an effective strategy to reduce nitrogen loss. In addition, nitrification inhibitors are widely applied to improve agricultural yield. However, it is necessary to continue investigating the crop-specific agricultural practice. In this study, a nitrapyrin-based nitrification inhibitor was used to assess its effects on Brassica oleracea L. var. botrytis growth and on the environment. In a pot experiment, cauliflower plants were grown in fertilized soils based on calcium nitrate (SF) and SF + nitrapyrin. At the end of the experiment, the content of nitrogen compounds in soil and percolation water and the cauliflower yield were determined, and the plant tissues were characterized by Fourier-transform infrared spectroscopy. The application of the NI significantly reduced nitrogen losses, increasing nutrient availability in the soil and the element’s absorption in the plant. Co-application of fertilizers and NIs reduced ${NO}_3^{-}$ leaching from 925 to 294 mg/L. Plant tissue characterization by FTIR spectroscopy highlighted variations in the functional groups in response to the application of NIs. These results suggest that applying nitrogen fertilizer in combination with nitrapyrin can mitigate nitrate pollution and improve element absorption and plant growth. Our research has shown that application methods and cropping systems need to be studied to maximize the effectiveness of nitrapyrin-based NIs. Full article

Nitrous oxide (N₂O) is a potent greenhouse gas, and agriculture represents more than fifty percent of total anthropogenic emissions. The production of N₂O in soil is biogenic through nitrification, denitrification, chemonitrification, nitrifier denitrification, etc., which are processes influenced by the soil pH, temperature, moisture, oxygen concentration, organic carbon, and soil nitrogen. Higher N₂O emissions from the soil result in lower nitrogen use efficiency and higher environmental pollution in terms of global warming. Therefore, an understanding of different pathways for N₂O production in soil and the affecting factors is essential to mitigate N₂O emissions from soil to the atmosphere. Nitrification inhibitor application has been reported in many studies, but the impact of nitrification inhibitors in different perennials (orchards) and biennials (rice, wheat, maize, etc.) is not lacking. In this study, we develop an understanding of different N₂O production pathways and different influencing factors. The role of the different nitrification inhibitors was also developed to achieve low N₂O emissions from soils to the atmosphere. Full article

Bark beetle-associated bacteria from the sub-boreal and boreal forests of northern Canada represent a largely unexplored source of bioactive natural products. This study aims to investigate the chemical potential of bacteria isolated from Dendroctonus ponderosae, Dendroctonus rufipennis, Dendroctonus pseudotsugae, and Ips perturbatus by focusing on nitrogen-containing secondary metabolites. Genomic analyses of the bacterial isolates identified diverse biosynthetic gene clusters (BGCs), including nonribosomal peptides (NRPs), NRPS-PKS hybrids, and ribosomally synthesized and post-translationally modified peptides (RiPPs), many of which exhibit low sequence homology, suggesting potential for novel bioactive compounds. Nitrogen-15 NMR spectroscopy was employed to detect nitrogen-containing functional groups in crude extracts, revealing distinct signals for amides, amines, and nitrogen heterocycles. The combination of BGC predictions and NMR data highlighted the genetic and chemical diversity of these bacteria and underscored the potential for discovering novel nitrogen-rich metabolites. These findings provide a foundation for further exploration of bioactive natural products with pharmaceutical and agrochemical applications and potential to contribute to the understanding of the chemical ecology of bark beetle–microbe interactions in northern ecosystems. Full article

Nitrogen (N) deficiency in soil limits the development of forage grasses, while its application can significantly increase productivity. This study aimed to evaluate the effects of increasing N doses and cutting intervals on structural and productive traits, biomass allocation, leaf chlorophyll index, and specific leaf area in Megathyrsus maximus cv. MG12 Paredão. The experiment was conducted with a randomized block design in the field, using a 5 × 2 factorial scheme, with five N fertilization levels (0, 100, 200, 300, and 400 kg N ha⁻¹ year⁻¹) and two cutting intervals (either 28 or 56 days). Our measurements included plant height, number of tillers, dry mass production, fresh shoot weight, root dry mass, leaf and stem biomass, SPAD readings, and specific leaf area. The results indicated a significant increase in SPAD values associated with higher N fertilization levels, so that the 300 kg N ha⁻¹ year⁻¹ dose resulted in the most significant changes compared to the control, with SPAD values increasing from 38.2 in the control group to 54.7. Dry mass production (DMP) was higher at the 28-day cutting interval compared to 56 days, particularly with 400 kg N ha⁻¹. The 400 kg N ha⁻¹ year⁻¹ dose resulted in a 68% increase in DMP compared to the control at 28-day intervals. Additionally, fertilization enhanced the number of tillers, leading to greater biomass accumulation. Significant differences in plant height were observed between cutting intervals, with taller plants recorded at 56 days. N fertilization promoted increased plant height, particularly at doses of 200, 300, and 400 kg ha⁻¹year⁻¹. Therefore, our study suggests the use of 400 kg N ha⁻¹ year⁻¹ dose at 28-day intervals. Thus, cutting frequency directly influenced plant growth. Full article

The hybrid progeny (1-1) resulting from the cross between Caucasian clover and white clover initially demonstrated an inability to fix nitrogen naturally via spontaneous nodulation. However, following inoculation with specific rhizobia strains derived from the Trifolium genus, successful nodulation and nitrogen fixation were observed in the 1-1 progeny, resulting in enhanced biomass production and adaptability. To explore in greater depth the mechanisms driving nitrogen fixation in these hybrid progeny, the inoculation was carried out using the dominant rhizobia strain (No. 5), isolated from Mengnong Clover No. 1. Root samples were collected at 3, 6, and 9 days post inoculation for RNA sequencing. A total of 1755 differentially expressed unigenes were identified between the control and treatment groups. KEGG pathway analysis highlighted key pathways associated with nodule nitrogen fixation. In combination with Weighted Gene Co-expression Network Analysis (WGCNA) and Gene Set Enrichment Analysis (GSEA), several differentially expressed genes were identified, suggesting their potential contribution to nitrogen fixation. Noteworthy among these, the gene TRINITY_DN7551_c0_g1 in the Phenylpropanoid biosynthesis pathway (MAP00940) emerged as a key candidate. This study offers valuable RNA-seq data, contributing significantly to the understanding of the molecular regulatory mechanisms underpinning nodule nitrogen fixation in legumes, thereby laying a solid foundation for future investigations into the hybrid progeny of Caucasian and white clover crosses. Full article