Effect of different levels of nitrogen and bio-fertilizers on growth, yield and economics of Rice (Oryza sativa L.)

Introduction

Rice (Oryza sativa L.), a member of the Poaceae family, is the primary staple food for more than 60% of the global population, occupying over 43.42 million hectares worldwide and contributing approximately 25% of global digestible caloric intake and 20% of total dietary protein [1,2] . India ranks second globally in rice production, accounting for approximately 21% of world output, with Punjab alone contributing 205.24 lakh tonnes of paddy across 31.68 lakh hectares during 2022-23 [3] . The rice-wheat cropping system of the Indo-Gangetic Plains remains central to India's food security, necessitating sustained productivity improvements to meet the demands of a projected global population of 9 billion by 2050 [4] . Nitrogen (N) is the mos t critical macronutrient for rice growth, influencing amino acid and chlorophyll synthesis, tiller production, leaf area index (LAI), photosynthetic rate, and ultimately grain yield [5] . However, low nitrogen use efficiency (NUE) in flooded rice paddies-arising from denitrification, volatilization, leaching and runoff poses significant agronomic and environmental challenges [6] . Excessive N application can increase susceptibility to bacterial leaf blight (Xanthomonas oryzae pv. oryzae) and blast (Pyricularia oryzae) causing up to 50-100% yield losses under severe conditions, and contributes to reactive nitrogen pollution and greenhouse gas emissions [7,8] . Bio-fertilizers incorporating plant growth-promoting rhizobacteria (PGPR) such as Azospirillum offer a sustainable strategy to complement mineral N by fixing atmospheric nitrogen, producing phytohormones (indole-3-acetic acid, gibberellins, cytokinins), solubilizing phosphorus, and enhancing root architecture and nutrient absorption [9,10] . Integrated nutrient management (INM) combining reduced mineral N with bio-fertilizers has been reported to achieve partial N substitution (10-30%) without yield penalty, improve microbial biomass and soil organic carbon, and raise net economic returns [11,12] . Despite growing evidence for INM benefits, optimum combinations of nitrogen rates and Azospirillum inoculation remain site-and cultivar-specific. Limited systematic data exist for the PR-131 variety grown under semi-arid Punjab conditions. The present study was therefore designed to: (i) determine the effect of nitrogen levels on growth and yield of rice; (ii) evaluate the combined effect of nitrogen and Azospirillum on crop performance; and (iii) assess the economic implications of various nutrient management strategies.

Materials and Methods

Experimental site and season

The field experiment was conducted during the kharif season (June-September 2025) at the Agricultural Research Farm, Guru Kashi University, Talwandi Sabo, Bathinda, Punjab, India (29°57'N, 75°7'E; altitude 213 m above sea level). The site has a semi-arid subtropical climate with extreme summers and mild winters, receiving most annual rainfall during June-August. Maximum temperature peaked at 43.2°C in July and minimum temperature at 19.6°C in June; total rainfall during the experiment was 441.6 mm (Table 1).

Experimental design and treatments

A Randomized Block Design (RBD) with nine treatments and three replications was employed. Each plot measured 6 m × 4 m (gross) with a net plot area of 5 m × 3 m. The rice variety PR-131 (Punjab Agricultural University), a high-yielding, shortduration (123-25 days) cultivar suited to water-scarce conditions, was used throughout. Treatments are detailed in Table 3. The recommended dose of nitrogen (RDF) for rice in Punjab is 120 kg N ha⁻¹ [13] . N @ 75% RDF (90 kg N ha⁻¹) T3 N @ 100% RDF (120 kg N ha⁻¹) T4 N @ 125% RDF (150 kg N ha⁻¹) T5 N @ 150% RDF (180 kg N ha⁻¹) T6 N @ 75% RDF + Azospirillum T7 N @ 100% RDF + Azospirillum T8 N @ 125% RDF + Azospirillum T9 N @ 150% RDF + Azospirillum

Agronomic management

Nursery beds (2 m × 2 m) were raised with high-germination seeds; urea was applied in two splits (at seeding and 10 days later). Phosphorus (60 kg P₂O₅ ha⁻¹ as single superphosphate) and potassium (40 kg K₂O ha⁻¹ as muriate of potash) were incorporated basally during final land preparation. Nitrogen was applied as urea in three equal splits: one-third each at basal, tillering and panicle initiation stages. For bio-fertilizer treatments, seedling roots were dipped in an Azospirillum suspension (10 g L⁻¹) for 20-30 minutes prior to transplanting. Twenty-five-day-old seedlings were transplanted at 20 cm × 15 cm spacing (two to three seedlings hill⁻¹). Gap filling was completed within one week. Plots were flood-irrigated, maintaining 2-3 cm water depth up to panicle initiation stage.

Manual weeding was performed twice (21 and 35 days after transplanting, DAT). A minor leaf folder incidence was managed by spraying chlorpyriphos at 2 mL L⁻¹.

Observations recorded

Five randomly tagged plants per plot were used to record growth measurements at 30, 60 and 90 DAT and at harvest. Plant height (cm) was measured from ground level to the tip of the tallest leaf. Tiller count per hill was recorded at each sampling stage and at maturity. Leaf area index (LAI) was calculated as total leaf area per unit ground area. Dry matter accumulation (g hill⁻¹) was determined by oven-drying fresh samples at 70°C for 24 hours. At physiological maturity, panicle length (cm), number of grains panicle⁻¹ and test weight (1000-grain weight, g) were recorded. Grain yield, straw yield and biological yield were expressed in q ha⁻¹. Harvest index (HI) was calculated as: HI (%) = (grain yield / biological yield) × 100.

Economic analysis

Cost of cultivation, gross return, net return and benefit-cost (B:C) ratio were calculated based on prevailing market prices at the time of harvest. Net return = gross return -cost of cultivation; B:C ratio = gross return / cost of cultivation.

Statistical analysis

Data were subjected to analysis of variance (ANOVA) using a Randomized Block Design as described by Cochran and Cox [14] . Treatment means were compared using the least significant difference (LSD) at p = 0.05.

Results and Discussion

Plant height

Plant height increased progressively from 30 DAT through harvest across all treatments (Table 4). At harvest, T 7 (100% N + Azospirillum) recorded the tallest plants (93.13 cm), followed by T 9 (89.50 cm) and T 8 (89.24 cm); the control T 1 recorded the shortest (61.07 cm). The positive effect of combined N and biofertilizer on plant height is attributable to enhanced nitrogen availability, improved root architecture from Azospirillumproduced auxins (IAA), and stimulated vegetative elongation [15,16] . These results align with findings of Wijebandara et al., [15] and Pandey and Kumar [17] , who observed linear increases in plant height with increasing N rates and biofertilizer inoculation.

Number of tillers hill⁻¹

Tiller number per hill increased significantly with nitrogen levels and biofertilizer application throughout the crop season (Table 5). At harvest, T 7 produced the greatest number of tillers (26.30 hill⁻¹), significantly exceeding T 1 (17.73 hill⁻¹). The enhanced tillering under integrated N-Azospirillum treatments is explained by sustained nitrogen supply during the critical tillering phase and Azospirillum-mediated phytohormone production, which stimulates lateral bud development [15,18] . Similar results were reported by Mannan et al., [19] and Bokado et al., [20] , who noted progressive increases in tiller number with ascending nitrogen doses. consistent with enhanced photosynthetic surface area driven by improved nitrogen assimilation and canopy expansion [21] . Shivay et al., [22] similarly observed increased LAI with higher N levels, mediated through enhanced leaf production and expansion.

Dry matter accumulation

Dry matter accumulation increased progressively from 30 DAT to 90 DAT and declined marginally at harvest across all treatments (Table 7). T 7 recorded the highest dry matter at all stages, peaking at 103.53 g hill⁻¹ at 90 DAT and 101.72 g hill⁻¹ at harvest, compared to T 1 values of 69.29 and 68.08 g hill⁻¹, respectively. The Azospirillum-mediated production of IAA, auxins and gibberellins enhances root density and nutrient uptake capacity, thereby supporting greater biomass accumulation [15,23] . Islam et al., [24] reported similar findings, attributing improved dry matter production to balanced nutrient availability at critical growth stages facilitated by bio-fertilizers.

Yield attributes and grain yield

Panicle length, grains panicle⁻¹ and test weight all increased significantly with combined N and Azospirillum application (Table 8). T 8 (125% N + Azospirillum) recorded the highest grains panicle⁻¹ (204.6) and test weight (22.7 g), while T 7 had the longest panicles (27.80 cm). Grain yield differed significantly among treatments, ranging from 31.6 q ha⁻¹ (T 1 ) to 65.6 q ha⁻¹ (T 8 ) (Table 9). Enhanced grain yield under integrated treatments is attributable to higher tiller number, longer panicles with more fertile spikelets, and improved assimilate translocation supported by sustained N supply and biofertilizer activity [25,26] . Sattar et al., [27] and Garai et al., [28] reported analogous yield improvements with Azospirillum inoculation and optimized N management in paddy.

Economic analysis

The cost of cultivation ranged from ₹40,250 ha⁻¹ (T 1 ) to ₹55,250 ha⁻¹ (T 9 ), reflecting higher input costs at elevated N and biofertilizer levels (Table 10). Despite higher cultivation costs, T 8 recorded the maximum gross return (₹1,76,544 ha⁻¹), while T 4 achieved the highest net return (₹1,25,460 ha⁻¹). The highest B:C ratio was obtained under T 2 (2.72), followed closely by T 3 (2.70) and T 4 (2.66), indicating that moderate nitrogen combined with Azospirillum offers superior economic efficiency. These results corroborate findings of Udayj et al., [29] and Patra et al., [30] , who reported improved B:C ratios under Azospirillumintegrated nutrient management compared to sole mineral N application. Based on these findings, the integrated use of nitrogen at 100-125% RDF combined with Azospirillum biofertilizer is recommended for sustainable and profitable rice production in comparable agroclimatic environments. Further multi-location and multi-year trials are needed to confirm these results under variable soil and climate conditions.

Acknowledgement

AcknowledgementsThe authors gratefully acknowledge the Department of Agronomy, Faculty of Agriculture, Guru Kashi University, Talwandi Sabo for providing research infrastructure and field facilities. The support of the advisory committee and department field staff is sincerely appreciated.

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