PEER-REVIEW ARTICLE PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8136 Soil and Leaf Nutrient Responses in Strawberry to Nano- urea and Azotobacter Applications Shaifali,a Manish Bakshi,a Shailesh K. Singh,b Rajeev Kumar Gupta,c,d,* S. Sreethu,c Saud Alamri,e and Manzer H. Siddiqui e Nanofertilizers release nutrients slowly and in a controlled manner, matching the plants’ growth needs. By reducing the need for chemical inputs such as fertilizers and pesticides, nanotechnology contributes to more sustainable agricultural practices. The present study used nano-urea with Azotobacter on strawberry cv. ‘Winter Dawn’ under protected cultivation to evaluate its impact on soil fertility status and leaf nutrient content. Formulations as per the treatment requirements were sprayed onto the strawberry plants. The plants provided with nano-urea formulations exhibited enhanced levels of nitrogen (2.49%), phosphorus (0.37%), and potassium (2.60%) compared to the plants treated with conventional urea. Soil nutrient analysis showed enhanced levels of NPK in soil samples from traditional application treatments. A higher concentration of microorganisms in the soil was observed when urea was applied at the nano level. Nano-urea combined with Azotobacter can significantly impact the soil’s NPK levels. This innovative blend enhances nutrient availability in the soil and promotes sustainable agricultural practices. By leveraging the nanotechnology-infused urea alongside the nitrogen-fixing process of Azotobacter bacteria, the soil experiences a synergistic boost in nitrogen, phosphorus, and potassium content. This dynamic duo fosters a conducive environment for plant growth while minimizing nutrient leaching and environmental degradation. DOI: 10.15376/biores.19.4.8136-8153 Keywords: Microbial load; Nano-urea; Plant nutrients; Soil fertility; Strawberry Contact information: a: Department of Horticulture, School of Agriculture, Lovely Professional University, Punjab, India, 144411; b: School of Agriculture, Lovely Professional University, Punjab, 144411; c: Department of Agronomy, Lovely Professional University, Punjab, India, 144411; d: Department of Soil Science, Punjab Agricultural University, Ludhiana, Punjab, 141004; e: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia; * Corresponding author: rajeev.30662@lpu.co.in INTRODUCTION Strawberry (Fragaria × ananassa) is a high-value fruit crop renowned for its flavour, nutritional benefits, and economic importance (Barbey et al. 2021). Achieving optimal growth and yield in strawberry cultivation is contingent upon a complex interplay of factors, including soil fertility, and nutrient availability (Mukherjee 2022). Traditional fertilization practices, while effective, often face challenges related to nutrient use efficiency and environmental sustainability (Swify et al. 2023). Introducing innovative agronomic technologies, such as nano-fertilizers and beneficial microorganisms, presents a promising alternative to enhance plant nutrition and growth (Goyal et al. 2023). Nano-urea, a novel form of nitrogen fertilizer, has emerged as a potential game- changer in nutrient management due to its enhanced delivery mechanisms and improved PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8137 nutrient use efficiency (Salama et al. 2024). The nanoscale formulation of urea allows for controlled release and targeted delivery, which could potentially mitigate nutrient losses and reduce the frequency of application (Sales et al. 2024). This technology aligns with the growing emphasis on precision agriculture and sustainable farming practices (Singh et al. 2024; Pratama and Rahayu 2024). Simultaneously, Azotobacter, a free-living nitrogen-fixing bacterium (Yilihamu et al. 2020), has been recognized for its capacity to improve soil fertility and promote plant growth (Ibrahim et al. 2022). Azotobacter not only contributes to the nitrogen economy of the soil, but it also enhances the bioavailability of essential nutrients and supports plant health through the production of growth-promoting substances (Basu et al. 2021; Ibrahim et al. 2022). Its integration into crop management strategies could complement the effects of nano-urea, offering a synergistic approach to optimizing soil fertility and plant nutrition (Jayara et al. 2023; Abd-Elsalam 2024). Additionally, increased dependence on conventional chemical fertilizers threatens the soil microbial community (Iqbal et al. 2022). Chemical fertilizers are reported to be expensive (Abebe et al. 2022) and have adverse effects on soil microbial population. In such aspects, biofertilizers can stand as the best alternative for enhancing the fertility of the soil as these are environment-friendly (Kour et al. 2020), economical, and can provide a better yield to the crop (Nagananda et al. 2010; Nosheen et al. 2021). These produce growth regulators auxin, cytokinins, and gibberellins (Mourya and Singh 2022) and can fix between 2 to 15 mg N/g carbon sources (Gurikar et al. 2022). Exopolysaccharide production by Azotobacter has been proven (Ali et al. 2023) to play an important role in improving soil porosity and transport of heavy metal pollutants (Hindersah et al. 2018). These bacterial cells (Azotobacter spp.) are highly sensitive to acidic pH, temperature, and high salt conditions in soil (Mikić 2023). The beneficial effect of Azotobacter has been reported on crop growth and yield (Zhou et al. 2023), stimulation of rhizospheric microbes (Azzawi and Kamal 2023), biosynthesis of biologically active substances (Kaur and Sharma 2024) and producing phytopathogenic inhibitors (Pattaeva et al. 2023). After the death of the Azotobacter cell, the protein of the cell is mineralized in the soil (Wang et al. 2023). It also helps modify nutrient uptake, ultimately boosting the biological nitrogen fixation in the soil and crop (Jehan et al. 2023). Despite the promising potential of these interventions, there has been limited research on their combined effects on soil fertility and leaf nutritional composition in strawberry cultivation. Understanding how nano-urea and Azotobacter interact to influence soil nutrient dynamics, plant growth parameters, and leaf nutrient profiles is critical for advancing sustainable agricultural practices and improving strawberry production systems. This study aims to evaluate the effects of nano-urea and Azotobacter interventions on soil fertility and leaf nutritional composition in strawberry plants. This research seeks to provide valuable insights for optimizing strawberry cultivation practices and contributing to the broader goal of sustainable agriculture. MATERIALS AND METHODS The research study was performed at Horticulture farm, Lovely Professional University, Punjab, India, from 2022 to 23 and 2023 to 24. Planting material for strawberries was sourced from the ICAR regional centre, Shimla. All intercultural operations and fertility management practices were carried out as per the standard in the PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8138 Package of Practices of Cultivation of Fruit (Thind and Mahal 2021). The experiment was laid out in Randomized Block Design (RBD) under protected conditions, having three replications per treatment and sixteen treatments in total (as given under the treatment details section). Each replication consisted of ten plant units spaced at 45 cm x 30 cm. Only the dosage of applied traditional urea was varied (25%, 50%, and 75% urea) in RDF (Recommended Dose of Fertilizer); the rest of the dosages of phosphorus and potassium were applied as recommended without any variation. Nano-urea was used as a foliar application to the strawberry plants as per the treatment specifications along with Azotobacter inoculation (@ 2mL per litre soil drenching) and the results were compared to the conventional (basal) application of the chemical fertilizers. Initial soil sampling was done at depths of 15 and 30 cm using a composite sample of the experimental soil. Specimens were analyzed for soil organic carbon and soil N, P, and K. Post-harvest data regarding soil fertility status at 120 DAP was collected from 15 and 30 cm depth within each replicated plot. Soil organic carbon (SOC) was determined by the method as described by Walkley and Black (1934). Soil NPK was estimated by the method described by Tel (1984). Azotobacter colonies were cultured using Jenson’s media (Allen 1953). For leaf sampling, fully matured leaves were selected to determine the leaf nutrient analysis for NPK. Total nitrogen (N) was analyzed using a nitrogen analyzer (Kjello Plus, Pelican, Chennai). The phosphorus (P) was estimated by using the phosphovanadomolybdate method (Jackson 1973), and potassium (K) by using a flame photometer (Labtronics, Panchkula, India). Treatment Details A total of 16 treatments were used, designated by symbol T as per the details: T1: RDF (PAU recommendation), T2: 25% RDF + N1, T3: 25% RDF + N2, T4: 50% RDF + N1, T5: 50% RDF + N2, T6: 75% RDF + N1, T7: 75% RDF + N2, T8: 25% RDF + N1+ Azotobacter, T9: 25% RDF + N2+ Azotobacter, T10: 50% RDF + N1+ Azotobacter, T11: 50% RDF + N2+ Azotobacter, T12: 75% RDF + N1+ Azotobacter, T13: 75% RDF + N2+ Azotobacter, T14: 25% RDF + Azotobacter, T15: 50% RDF + Azotobacter, T16: 75% RDF + Azotobacter. Note: N1: 300 ppm nano-urea and N2: 400 ppm nano-urea Statistical Analysis The data generated were statistically analyzed using IBM SPSS Statistics (SPSS Inc., IBM Corp., Armonk, NY, USA) software at a 5% significance level to arrive at the homogenous subsets. Variables having non-significant differences are superscripted with the same alphabet. RESULTS AND DISCUSSION Plant Nutrient Analysis Underapplication of nano-urea resulted in significant variations in the leaf nitrogen content; however, the variable treatments failed to produce any considerable change in the phosphorus and potassium levels in the strawberry plants (Table 1 and Fig. 1 a-c). A thorough examination of the data indicated a noteworthy impact of nano-urea on the nitrogen levels of the strawberry cultivar ‘Winter Dawn’ throughout the two years of the research experiment. The nitrogen percentage in the leaves ranged from 1.42% to 2.51% during the two years. Application of 25% of the recommended dose of fertilizers along PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8139 with 400 ppm nano-urea and azotobacter inoculation resulted in a maximum nitrogen content of 2.51% in the strawberry leaves. (a) Plant nutrient status 2022 (b) Plant nutrient status 2023 PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8140 (c) Plant nutrient status (Pooled) Fig. 1 (a-c). Impact of nano-urea and Azotobacter application on plant nutrient content However, the application of varying doses of nitrogen failed to make any considerable impact on the leaf phosphorus and potassium content of strawberry plants. Phosphorus in leaves ranged from 0.33 to 0.37%, whereas potassium levels in leaves ranged from 2.66 to 2.78%. Since nitrogen dosage and source were varied among the treatments, a significant impact was observed in the leaf nitrogen content. In contrast, there was statistically non- significant variation among other treatments for phosphorus and potassium levels. Treatments having higher doses of nano-urea applications outperformed conventional nitrogen applications to the strawberry plants. Nano-urea, characterized by its nano-scale particles, provides a distinct advantage over conventional urea through more efficient and rapid absorption by the leaf surface (Iqbal et al. 2019; Dimkpa et al. 2022). This method bypasses the soil-plant interface, reducing nitrogen losses that are commonly associated with leaching, volatilization, or immobilization in the soil (Yadav et al. 2023). The direct availability of nitrogen to the photosynthetic tissues ensures immediate utilization for amino acid synthesis and other nitrogen-demanding metabolic processes, which are crucial for plant growth and productivity (Ji et al. 2023). The results of the current study align with the findings of Abdel-Aziz and Abdel-Hakim (2021), who observed that the levels of primary nutrients, as well as micronutrients like Fe and Mn in Capsicum annuum and lettuce, were much more remarkable when treated with nano fertilizer compared to plants fertilized with conventional fertilizers. Furthermore, Sharaf- Eldin (2022) was of the view that the utilization of nano fertilizers (N) enhanced the effectiveness of fertilizer usage at a dosage lower than the amount that is recommended. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8141 Table 1. Synergistic Impact of Nano-urea and Azotobacter in Enhancing Plant NPK Uptake Treatments Plant Nutrient Analysis (NPK) Nitrogen Phosphorus Potassium 2022 2023 Pooled 2022 2023 Pooled 2022 2023 Pooled T1 1.56d 1.42d 1.49d 0.37a 0.36a 0.37a 2.66a 2.66bc 2.66ab T2 2.00g 2.06g 2.03g 0.37a 0.37a 0.37a 2.73a 2.78c 2.75b T3 2.14h 2.16h 2.15h 0.36a 0.34a 0.35a 2.67a 2.64b 2.65ab T4 1.89e 1.93e 1.91e 0.36a 0.37a 0.37a 2.66a 2.58ab 2.62ab T5 2.00g 2.02fg 2.01fg 0.35a 0.33a 0.34a 2.64a 2.60ab 2.62ab T6 1.88e 1.91e 1.90e 0.37a 0.37a 0.37a 2.62a 2.62ab 2.62ab T7 1.93f 1.97ef 1.95f 0.37a 0.33a 0.35a 2.69a 2.49a 2.59a T8 2.42j 2.36j 2.39j 0.37a 0.36a 0.37a 2.69a 2.52ab 2.60a T9 2.51l 2.47k 2.49k 0.38a 0.36a 0.37a 2.63a 2.57ab 2.60ab T10 2.33i 2.28i 2.30i 0.38a 0.35a 0.37a 2.70a 2.60ab 2.65ab T11 2.46k 2.37j 2.42j 0.38a 0.35a 0.37a 2.62a 2.58ab 2.60a T12 2.31i 2.28i 2.29i 0.35a 0.34a 0.35a 2.66a 2.63b 2.65ab T13 2.17h 2.14h 2.16h 0.37a 0.35a 0.36a 2.69a 2.61ab 2.65ab T14 0.99a 0.96a 0.98a 0.37a 0.36a 0.36a 2.68a 2.61ab 2.65ab T15 1.11b 1.05b 1.08b 0.38a 0.34a 0.36a 2.70a 2.65b 2.67ab T16 1.21c 1.16c 1.19c 0.37a 0.37a 0.37a 2.67a 2.63b 2.65ab T1: RDF (PAU recommendation), T2: 25% RDF + N1, T3: 25% RDF + N2, T4: 50% RDF + N1, T5: 50% RDF + N2, T6: 75% RDF + N1, T7: 75% RDF + N2, T8: 25% RDF + N1+ Azotobacter, T9: 25% RDF + N2+ Azotobacter, T10: 50% RDF + N1+ Azotobacter, T11: 50% RDF + N2+ Azotobacter, T12: 75% RDF + N1+ Azotobacter, T13: 75% RDF + N2+ Azotobacter, T14: 25% RDF + Azotobacter, T15: 50% RDF + Azotobacter, T16: 75% RDF + Azotobacter. Note: N1: 300 ppm Nano-urea, N2: 400 ppm Nano-urea Application of acetobacter in the treatments where reduced dosage of conventional nitrogen fertilizer was applied were compensated with sufficient availability, efficient absorption and uptake in strawberry plants. Multiple studies suggest that applying biofertilizers such as Azotobacter can impact the mineral composition of plants by enhancing the nutrient content in plants (Kolega et al. 2020). Azotobacter applied to the soil enhances nitrogen levels through biological fixation while promoting root health and nutrient uptake (Aasfar et al. 2021). The present result aligns with other research showing a positive correlation between nitrogen supply in the nutrient solution and the nitrogen content in leaves when Azotobacter inoculation is used (Razmjooei et al. 2022). This dual approach ensures sustained nitrogen availability in leaves, which is vital for photosynthesis and growth, potentially explaining increased nitrogen levels without significant variation in phosphorus and potassium, given the consistent dosage. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8142 Soil Nutrient Analysis A significant impact of nano-urea was observed on the final soil nutrient status (NPK) in the Winter Dawn strawberry cultivar, especially for soil nitrogen, across two experimental years. Soil nitrogen levels ranged from 151 to 235 kg per ha. Treatments where the recommended dosage of nitrogen through the soil was applied recorded maximum soil nitrogen content. Phosphorus levels in the soil ranged from 20.8 kg per ha to 28.5 kg per ha. Similarly soil potassium levels ranged from 200 to 216 kg per ha. Table 2. Synergistic Impact of Nano-urea and Azotobacter in Enhancing Soil NPK Uptake Treat- ments Soil Nutrient Analysis (NPK) Nitrogen Phosphorus Potassium 2022 2023 Pooled 2022 2023 Pooled 2022 2023 Pooled T1 234.65j 222.10h 228.38k 20.76a 22.61a 21.69a 204.45a 205.56ab 205.01a T2 178.39ab 162.32ab 170.36ab 22.89bc 22.52a 22.70abcd 207.96ab 203.61ab 205.79a T3 173.87a 151.28a 162.58a 21.90ab 22.89a 22.40ef 209.54ab 208.01b 208.77a T4 195.65de 180.16cd 187.90cd 22.28b 22.15a 22.22ab 208.32ab 200.97a 204.65a T5 209.56fg 199.61efg 204.59fg 25.87ef 21.91a 23.89de 210.98ab 200.34a 205.66a T6 221.34hi 217.04gh 219.19ij 24.87de 22.93a 23.90de 213.87ab 201.95ab 207.91a T7 227.86ij 218.18h 223.02jk 23.87cd 22.54a 23.21bcd 212.76ab 206.67ab 209.72a T8 183.98bc 178.00bcd 180.99c 22.67bc 22.58a 22.62abc 210.65ab 204.85ab 207.75a T9 169.00a 173.08bcd 171.04b 23.67cd 22.63a 23.15bcd 209.45ab 203.42ab 206.44a T10 190.45cd 199.48efg 194.97de 24.87de 22.53a 23.70cde 213.75ab 202.09ab 207.92a T11 187.76bcd 197.91ef 192.84de 26.90fg 22.53a 24.72ef 210.91ab 204.00ab 207.46a T12 213.67gh 213.52fgh 213.60hi 26.45fg 23.25a 24.85ef 214.30ab 201.91ab 208.11a T13 202.75ef 213.87fgh 208.31gh 28.53h 22.88a 25.71f 215.71b 205.26ab 210.49a T14 172.21a 165.26abc 168.74ab 24.87de 22.48a 23.68cde 212.76ab 202.28ab 207.52a T15 211.78fgh 184.00de 197.89ef 23.98cd 22.55a 23.27bcd 214.72ab 202.79ab 208.75a T16 218.45ghi 222.43h 220.44ijk 27.39gh 22.09a 24.74bcd 213.36ab 202.55ab 207.96a T1: RDF (PAU recommendation), T2: 25% RDF + N1, T3: 25% RDF + N2, T4: 50% RDF + N1, T5: 50% RDF + N2, T6: 75% RDF + N1, T7: 75% RDF + N2, T8: 25% RDF + N1+ Azotobacter, T9: 25% RDF + N2+ Azotobacter, T10: 50% RDF + N1+ Azotobacter, T11: 50% RDF + N2+ Azotobacter, T12: 75% RDF + N1+ Azotobacter, T13: 75% RDF + N2+ Azotobacter, T14: 25% RDF + Azotobacter, T15: 50% RDF + Azotobacter, T16: 75% RDF + Azotobacter. Note: N1: 300 ppm nano-urea, N2: 400 ppm nano-urea PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8143 (a) Soil nutrient status (2022) (b) Soil nutrient status (2023) PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8144 (c) Soil nutrient status (pooled) Fig. 2 (a-c). The impact of nano-urea and Azotobacter in enhancing soil NPK levels The basal application of nitrogen (N) is critical for enhancing the availability of nitrogen in the soil (Yadav et al. 2017), thereby optimizing crop growth and yield (Hammad et al. 2018). Nitrogen, being volatile and prone to leaching, may sometimes be unavailable to the plant for more prolonged durations after application. Foliar application of nano-urea targets the leaf tissues and proves to be more economical than soil application of the chemical nitrogen. Overall, the basal application of nitrogen is crucial in enhancing nitrogen availability in soil. Soil Organic Carbon As indicated by Table 3 and Fig. 3 (a, b, c), nano-urea significantly influenced soil nutrient analysis (available organic carbon) of the Winter Dawn strawberry cultivar over the two experimental years (2022-23 and 2023-24). Soil organic carbon ranged from 3.90 kg per ha to 3.38 kg per ha. As per the observations, a higher level of soil organic carbon was recorded in the treatments where reduced soil application of chemical nitrogen fertilizer was done. Treatments involving nano-urea and azotobacter application had a higher level of soil organic carbon. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8145 Table 3. Synergistic Impact of Nano-urea and Azotobacter in Enhancing Soil Organic Carbon and Azotobacter Count Treatments Soil (Organic Carbon) Azotobacter Count 2022-23 2023-24 Pooled 2022-23 2023-24 Pooled T1 3.50a 3.38a 3.44a 2.63a 2.67a 2.65a T2 3.80cd 3.69ghi 3.74ef 4.35b 4.83c 4.59c T3 3.90d 3.75i 3.82f 4.33b 4.67bc 4.50c T4 3.70bc 3.62efg 3.66cde 3.34ab 4.00bc 3.67b T5 3.60sb 3.53cd 3.56bc 3.66ab 4.00bc 3.83bc T6 3.60ab 3.47bc 3.53ab 3.00a 3.67b 3.33ab T7 3.50a 3.39ab 3.45a 3.00a 3.67b 3.33ab T8 3.80cd 3.76i 3.78f 11.33ef 11.83f 11.58g T9 3.90d 3.74hi 3.82f 11.00ef 11.17f 11.08g T10 3.80cd 3.66fgh 3.73def 8.67d 9.83e 9.25e T11 3.80cd 3.66gh 3.73def 8.33cd 9.17e 8.75e T12 3.70bc 3.58def 3.64cd 7.67cd 8.17d 7.92d T13 3.60ab 3.54cde 3.57bc 7.33c 8.00d 7.67d T14 3.90d 3.69ghi 3.79f 12.00f 11.50f 11.75g T15 3.70bc 3.54cde 3.62bc 10.33e 10.00e 10.17f T16 3.50a 3.40ab 3.45a 7.67cd 8.17d 7.92d T1: RDF (PAU recommendation), T2: 25% RDF + N1, T3: 25% RDF + N2, T4: 50% RDF + N1, T5: 50% RDF + N2, T6: 75% RDF + N1, T7: 75% RDF + N2, T8: 25% RDF + N1+ Azotobacter, T9: 25% RDF + N2+ Azotobacter, T10: 50% RDF + N1+ Azotobacter, T11: 50% RDF + N2+ Azotobacter, T12: 75% RDF + N1+ Azotobacter, T13: 75% RDF + N2+ Azotobacter, T14: 25% RDF + Azotobacter, T15: 50% RDF + Azotobacter, T16: 75% RDF + Azotobacter. Note: N1: 300 ppm nano-urea, N2: 400 ppm nano-urea The basal application of conventional nitrogen fertilizers can decrease available organic carbon in soil (Li et al. 2017). This is attributed to heightened microbial activity stimulated by nitrogen influx, which accelerates microbial metabolic processes (Ge et al. 2010; Hutchins et al. 2022). Consequently, microbial communities utilize organic carbon for energy and growth, depleting the available pool (Garcia-Pausas and Paterson 2011). Chemical fertilizers can enhance microbial activity and hasten the breakdown of soil organic materials (Arunrat et al. 2020). Specific microbial taxa, favoured by the elevated nitrogen availability (Fierer et al. 2012), may exhibit enhanced competitiveness over others, leading to shifts in community composition and function (Herren and McMahon 2018). This alteration in microbial diversity and activity can result in the preferential decomposition of organic carbon compounds, further reducing their availability in the soil matrix. The current findings align with the research conducted by Gong et al. (2009), who proposed that using chemical fertilizers has led to significant negative impacts on the PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8146 physical and chemical characteristics of soil, such as the decline in soil organic carbon levels. Additionally, elevated nitrogen levels stimulate plant growth and alter root exudation patterns, affecting organic carbon inputs to the soil (Yin et al. 2013; Murphy et al. 2017; Meng et al. 2024). Changes in plant physiology may decrease below ground carbon allocation, reducing available carbon for microbial use (Savage et al. 2016; Gross and Harrison 2019). Understanding these dynamics is crucial for devising sustainable nutrient management strategies (Lei et al. 2023). (a) OC and azotobacter count (2022) (b) OC and azotobacter count (2023) PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8147 (c) OC and azotobacter count (pooled) Fig. 3(a through c). The impact of nano-urea and Azotobacter in enhancing organic carbon and Azotobacter counts Azotobacter Count Table 3 and Fig. 3 (a, b, c) depict Azotobacter count (CFU x106) variation over the experimental years (2022-23 and 2023-24) for the Winter Dawn strawberry cultivar. Nano- urea significantly impacted Azotobacter count throughout both years, highlighting its influence on soil microbial dynamics. During the initial year of trial (2023-24), T14 exhibited the highest Azotobacter count (11.33 x106 cfu (Fig. 3a). In the second-year trial (2023-24), T8 displayed the highest Azotobacter count (11.83 x106 cfu (Fig. 3b). Pooled data also revealed maximum Azotobacter count (11.75 x106 cfu) under treatment T14. Minimum azotobacter count was recorded in the soil samples under control treatment as the chemical nitrogen dosage was higher compared to other treatments. In the study of soil microbiology and fertility, the application of conventional nitrogenous fertilizers has been noted to impact various microbial populations, including the diazotrophic (nitrogen-fixing) bacteria such as Azotobacter (Aasfar et al. 2021). Notably, basal application of nitrogen fertilizers appears to decrease the population of Azotobacter in soil, a phenomenon that can be attributed to several interconnected factors (Jnawali et al. 2015). Higher concentrations of nitrogen fertilizer suppress the activities of nitrogen-fixing microorganisms. The results are in line with the above findings. Bacterial proliferation is frequently constrained by the scarcity of easily accessible carbon substrate, even in soils with a high carbon-to-nitrogen ratio (Demoling et al. 2007; Singh et al. 2015). When synthetic nitrogen fertilizers are applied to the soil, particularly as a basal dose, they provide plants with readily available inorganic nitrogen. This abundant nitrogen supply reduces the ecological niche and the competitive advantage for Azotobacter, whose nitrogen-fixing capability becomes redundant in the presence of high nitrogen levels. The decrease in Azotobacter count following nitrogen application is also linked to the metabolic burden that nitrogen fixation imposes on bacteria (Han et al. 2024); when nitrogen is readily available, the energy-intensive process of nitrogen fixation is unnecessary, leading to a competitive disadvantage for nitrogen-fixers. Research findings have shown that bio- PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Shaifali et al. (2024). “Soil fertility & leaf nutrients,” BioResources 19(4), 8136-8153. 8148 fertilization has led to a higher microbial population than mineral fertilization (El-Sawah et al. 2018; Gao et al. 2020). CONCLUSIONS A reasonable conclusion came from the current study regarding applying nitrogen to strawberry plants. Foliar application of nano-urea at 400 ppm along with a 25% recommended dose of nitrogen through conventional nitrogenous fertilizers as soil application and augmentation with azotobacter led to enhanced leaf nutrient status of the strawberry plants concerning nitrogen content. Similarly, soil nutrient analysis recorded the highest nitrogen in treatments where the recommended dose of fertilizer (urea) was used. To have better nutrient availability and soil microbial load, it is recommended that nitrogen should be applied in nano form, as soil microfauna is sensitive to higher soil acidity caused by the application of chemical fertilizers. 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