Influence of biostimulants on growth, physiological parameters, and yield of soybean in northern transition zone of Karnataka

Introduction

Soybean [Glycine maz (L.) Merrill] is an agronomically and economically significant leguminous crop globally, classified under the family Fabaceae. Often termed the 'cow crop' due to its extensive industrial usage and soy milk production, it is highly valued for its rich protein content (40-42%) and cholesterol-free oil (20%). Furthermore, it is a primary dietary source of all eight essential amino acids, fiber, iron, calcium, and B vitamins. In India, soybean cultivation occupies a prominent position, particularly in states like Madhya Pradesh, Maharashtra, Rajasthan, and Karnataka. Despite its genetic yield potential ranging from 3 to 4 t/ha, the average productivity frequently stagnates below this threshold. In the Northern Transition Zone of Karnataka, yield gaps are commonly attributed to erratic rainfall distribution, periodic moisture stress, and nutritional deficiencies occurring precisely during the critical flower initiation and pod formation stages. Modern agronomic practices have increasingly turned toward the application of plant biostimulants as a viable strategy to enhance resource use efficiency and narrow the yield gap. Biostimulants are defined not as fertilizers intended to correct severe nutrient deficits, but rather as formulations containing organic compounds, microbial inoculants, seaweed extracts, enzymes, and trace elements that stimulate natural physiological processes. They facilitate enhanced nutrient uptake, up-regulate plant genes involved in metabolism, stimulate root proliferation, and fortify antioxidant defenses against abiotic stress (Calvo et al., 2014) [2] . For example, humic and fulvic acids interact with soil nutrients to elicit physiological responses, while amino acid-based biostimulants enhance stress tolerance distinct from standard nitrogen fertilization (Kocira, 2018) [3] . Among the diverse range of biostimulants, products derived from marine algae (seaweed extracts) have demonstrated considerable efficacy. Seaweed extracts contain naturally occurring plant growth regulators, particularly auxin precursors, cytokinins, betaines, and polyamines, alongside complexed micronutrients. When applied as foliar sprays, they have been shown to accelerate vegetative growth, improve chlorophyll retention, and optimize assimilate partitioning (Rathore et al., 2009; Zodape et al., 2010) [7, 10] . 'Crop Max' is a commercial biostimulant formulation containing 24% organic compounds (fermentation metabolites, marine algae extracts, and protein hydrolysates) and 18% mineral components, including essential micronutrients (Zn, Fe, Mn, Cu) necessary for enzyme activation. 'Biozyme' is another growthregulating formulation extracted from the marine alga Ascophyllum nodosum, widely documented for its positive impact on legume productivity (Biradar, 1998; Tandon and Dubey, 2015) [1,8] . While individual applications of these formulations have been studied, there is a distinct need to evaluate their sequential application at specific crop growth stages to maximize synergistic effects. Therefore, the present field investigation was formulated to assess the influence of these biostimulants, applied as foliar sprays at varying concentrations and specific phenological stages, on the sequential growth dynamics, physiological water status, and final yield attributes of soybean.

Materials and Methods

Description of the Experimental Site

The field experiment was conducted during the kharif season of 2021 at the Main Agricultural Research Station (MARS), University of Agricultural Sciences, Dharwad, Karnataka. The site is geographically situated at 15° 29' N latitude and 74° 59' E longitude, at an altitude of 678 meters above mean sea level. The climate of the region is classified under the Northern Transition Zone. Soil samples were collected from a depth of 0-30 cm prior to sowing to determine the baseline physicochemical properties. The experimental soil was a black clay Vertisol with a neutral pH of 7.1 and a normal electrical conductivity of 0.32 dS/m. Organic carbon content was assessed as low (0.44%) using the Walkley and Black wet oxidation method. Available nitrogen was low (263 kg/ha, alkaline permanganate method), while available phosphorus (30.5 kg/ha, Olsen's method) and available potassium (366 kg/ha, flame photometry) were in the high range. The soil also contained 5.9 kg/ha of available sulfur.

Meteorological Conditions

Weather parameters were recorded at the MARS meteorological observatory. During the crop growth cycle, the site received a total rainfall of 402 mm distributed across 34 rainy days, aligning well with the soybean water requirement of 600-650 mm when combined with residual soil moisture. The mean maximum temperature ranged from 27.2°C to 28.3°C, and the mean minimum temperature ranged from 20.5°C to 20.8°C, which fell within the optimal thermal range (15-32°C) for soybean phenology. Relative humidity remained high, oscillating between 82.3% and 87.0%, fostering a conducive microclimate for optimal leaf expansion.

Experimental Design and Treatment Structure

The experiment was structured in a Randomized Complete Block Design (RCBD) comprising 11 distinct treatments, each replicated three times. The gross plot size was 3.6 m x 6.0 m, and the net plot size was 2.4 m x 4.8 m. The treatment details were as follows: T 1: Crop Max @ 375 ml/ha at flower initiation + Crop Max @ 375 ml/ha at pod formation T 2: Crop Max @ 500 ml/ha at flower initiation + Crop Max @ 500 ml/ha at pod formation T 3: Crop Max @ 625 ml/ha at flower initiation + Crop Max @ 625 ml/ha at pod formation T 4: Crop Max @ 750 ml/ha at flower initiation + Crop Max @ 750 ml/ha at pod formation T 5: Crop Max @ 375 ml/ha at flower initiation + Biozyme @ 625 ml/ha at pod formation T 6: Crop Max @ 500 ml/ha at flower initiation + Biozyme @ 625 ml/ha at pod formation T 7: Crop Max @ 625 ml/ha at flower initiation + Biozyme @ 625 ml/ha at pod formation T 8: Crop Max @ 750 ml/ha at flower initiation + Biozyme @ 625 ml/ha at pod formation T 9: Biozyme @ 625 ml/ha at flower initiation + Biozyme @ 625 ml/ha at pod formation T 10: Control-1 (Urea @ 2% and KNO3 @ 1% foliar spray) T 11: Control-2 (Water spray) Foliar applications were executed systematically. The first application coincided with flower initiation (25-35 Days After Sowing), and the second application was performed during the pod formation stage (45-50 DAS).

Crop Husbandry Practices

The land was prepared using a tractor-drawn mould board plough followed by harrowing to achieve a fine seedbed. Soybean seeds (variety MACS-1188, recognized for shattering resistance and high oil content) were treated with Rhizobium and Phosphorus Solubilizing Bacteria (PSB) at 1250 g/ha each. Sowing was carried out via dibbling at a depth of 2.5-3 cm with a spacing of 30 cm x 10 cm, utilizing a seed rate of 62.5 kg/ha. A uniform basal dose of Farm Yard Manure (6.25 t/ha) and chemical fertilizers (40:80:25 kg N:P2O5:K2O/ha + 12.5 kg ZnSO4/ha) was incorporated prior to sowing. Weeds were managed using a preemergence application of Pendimethalin 30 EC (3.25 l/ha) followed by manual weeding at 45 and 60 DAS. Plant protection measures, including a spray of Propiconazol 25 EC (1.5 l/ha) to control rust, were undertaken as required.

Data Collection and Analytical Methods

Five plants were selected randomly in each net plot and tagged for sequential biometric observations. Plant height (from ground level to the tip of the main shoot) and the number of branches were recorded at 30 DAS, 60 DAS, and at harvest. Leaf area per plant was measured, and physiological parameters were assessed. Relative Leaf Water Content (RLWC) was calculated using the formula: RLWC = [(Fresh Weight -Dry Weight) / (Turgid Weight -Dry Weight)] * 100. Leaf chlorophyll content was measured using a SPAD meter on five fully opened leaves from the top of the tagged plants. Yield parameters, including the number of pods per plant, seed yield, and haulm yield, were recorded at physiological maturity following manual harvesting and threshing. The recorded data were compiled and subjected to statistical analysis utilizing Fisher's method of analysis of variance (ANOVA). Where the F-test was significant, mean separation was achieved using Duncan's Multiple Range Test (DMRT) at a 5% level of significance (P=0.05). Standard Error of Mean (S.Em. ±) was also computed for data interpretation.

Results and Discussion

Influence on Morphological Growth Parameters

The application of foliar biostimulants demonstrated a progressive influence on the morphological growth of soybean, with the effects becoming highly pronounced at 60 DAS and at harvest (Table 1). At 30 DAS, prior to the full absorption and physiological integration of the first foliar spray, parameters such as plant height and branch count did not exhibit statistically significant variance among the treatments. However, by 60 DAS and subsequently at harvest, Treatment T 8 (Crop Max @ 750 ml/ha at flower initiation followed by Biozyme @ 625 ml/ha at pod formation) recorded the maximum plant height of 81.5 cm and 82.1 cm, respectively. This was statistically comparable to T 7 (77.7 cm and 79.3 cm) but significantly superior to the water spray control (T 11 ), which recorded 70.4 cm at harvest. The number of branches per plant did not yield statistically significant differences across treatments; however, a numerical maximum of 4.7 branches per plant was observed in T 8 at harvest. These findings corroborate the results of Biradar (1998) [1] and Noli et al. (2021) [5] , who reported significant increases in plant height and vegetative metrics following seaweed extract application. The enhanced cellular elongation and division can be attributed to the bioactive phytohormonesspecifically auxin and cytokinin precursors-present in marine algae extracts. When applied externally, these compounds stimulate meristematic activity and internodal elongation (Vishwanatha et al., 2022) [9] . @ 375 ml ha -1 followed by Crop Max @ 375 ml ha -1 T2-Crop Max @ 500 ml ha -1 followed by Crop Max @ 500 ml ha -1 T3-Crop Max @ 625 ml ha -1 followed by Crop Max @ 625 ml ha -1 T4-Crop Max @ 750 ml ha -1 followed by Crop Max @ 750 ml ha -1 T5-Crop Max @ 375 ml ha -1 followed by Biozyme @ 625 ml ha -1 T6-Crop Max @ 500 ml ha -1 followed by Biozyme @ 625 ml ha -1 T7-Crop Max @ 625 ml ha -1 followed by Biozyme @ 625 ml ha -1 T8-Crop Max @ 750 ml ha -1 followed by Biozyme @ 625 ml ha -1 T9-Biozyme @ 625 ml ha -1 followed by Biozyme @ 625 ml ha -1 T10-Control-1 (Urea @ 2% and KNO3 @ 1%) T11-Control-2 (water spray) Means followed by the same alphabet (s) within a column are not significantly differed by DMRT (P=0.05) DAS-Days after sowing Note 1. RDF: FYM @ 6.25 t + 40:80:25 kg N: P2O5 and K2O + ZnSO4 @ 12.5 kg + Rhizobium and PSB @ 1250 g each per hectare common to all treatments Note 2. For T1 to T10, foliar application at flower initiation (25-35DAS) and at pod formation stage (45 -50 DAS)

Leaf Area Expansion and Physiological Metrics

Leaf area, a fundamental component determining light interception and photosynthetic capability, responded positively to biostimulant application at 60 DAS (Table 2). Treatment T 8 recorded a significantly higher leaf area (11.47 dm2/plant) compared to the control (9.70 dm2/plant). This expansion is likely facilitated by the complexed micronutrients (such as Zn and Mn) in Crop Max, which are vital co-factors for enzymes involved in chlorophyll synthesis and carbon assimilation (Tandon and Dubey, 2015) [8] . Relative Leaf Water Content (RLWC) serves as an effective indicator of the plant's internal water balance and cellular turgidity under field conditions. At 60 DAS, T 8 maintained a significantly higher RLWC (84.77%) compared to the water spray control (79.91%). Seaweed extracts are recognized sources of naturally complexed potassium and organic osmolytes like betaines. These compounds aid in osmotic adjustment, regulating stomatal conductance, and minimizing transpirational water loss, allowing the plant to maintain turgor during transient moisture fluctuations (Pramanick et al., 2013) [6] . Chlorophyll content, measured as SPAD values, displayed numerical enhancements post-spray. Although the statistical differences among the treatments were non-significant at both 30 and 60 DAS, T 7 and T 11 maintained robust SPAD readings (49.0) at 60 DAS. The lack of statistical variance suggests that the baseline chlorophyll biosynthesis was adequate across the field; however, the biostimulants actively delayed leaf senescence, thereby maintaining a functional green leaf area for a prolonged duration (Zodape et al., 2010) [10] . 0.62 0.9 0.6 T1-Crop Max @ 375 ml ha -1 followed by Crop Max @ 375 ml ha -1 T2-Crop Max @ 500 ml ha -1 followed by Crop Max @ 500 ml ha -1 T3-Crop Max @ 625 ml ha -1 followed by Crop Max @ 625 ml ha -1 T4-Crop Max @ 750 ml ha -1 followed by Crop Max @ 750 ml ha -1 T5-Crop Max @ 375 ml ha -1 followed by Biozyme @ 625 ml ha -1 T6-Crop Max @ 500 ml ha -1 followed by Biozyme @ 625 ml ha -1 T7-Crop Max @ 625 ml ha -1 followed by Biozyme @ 625 ml ha -1 T8-Crop Max @ 750 ml ha -1 followed by Biozyme @ 625 ml ha -1 T9-Biozyme @ 625 ml ha -1 followed by Biozyme @ 625 ml ha -1 T10-Control-1 (Urea @ 2% and KNO3 @ 1%) T11-Control-2 (water spray) Means followed by the same alphabet (s) within a column are not significantly differed by DMRT (P=0.05) DAS-Days after sowing Note 1. RDF: FYM @ 6.25 t + 40:80:25 kg N: P2O5 and K2O + ZnSO4 @ 12.5 kg + Rhizobium and PSB @ 1250 g each per hectare common to all treatments Note 2. For T1 to T10, foliar application at flower initiation (25-35DAS) and at pod formation stage (45 -50 DAS)

Yield Attributes and Final Yield Outcomes

The ultimate objective of biostimulant application is the realization of enhanced economic yields. The integration of Crop Max and Biozyme drastically improved the source-to-sink translocation of photoassimilates. Treatment T 8 significantly maximized the number of pods per plant, recording an average of 58.7 pods compared to a mere 41.7 pods in the control (Table 3). This aligns with the observations of Rathore et al. (2009) [7] and Meshram (2013) [4] , who noted that foliar nutrition during early reproductive phases minimizes floral abscission and maximizes pod setting. Consequently, this structural advantage translated into exceptional final yields. Treatment T 8 recorded the highest seed yield of 2693 kg/ha and haulm yield of 3977 kg/ha, which equates to a 9.16% and 4.6% increase over the control, respectively. Treatment T 7 closely followed with a seed yield of 2655 kg/ha. The targeted application of Crop Max (supplying metabolic catalysts) at flower initiation, followed by Biozyme (supplying growth regulators) at pod formation, ensured an uninterrupted supply of metabolic energy precisely when the crop required maximum nutritional support for grain filling (Kocira, 2018) [3] . @ 375 ml ha -1 followed by Crop Max @ 375 ml ha -1 T2-Crop Max @ 500 ml ha -1 followed by Crop Max @ 500 ml ha -1 T3-Crop Max @ 625 ml ha -1 followed by Crop Max @ 625 ml ha -1 T4-Crop Max @ 750 ml ha -1 followed by Crop Max @ 750 ml ha -1 T5-Crop Max @ 375 ml ha -1 followed by Biozyme @ 625 ml ha -1 T6-Crop Max @ 500 ml ha -1 followed by Biozyme @ 625 ml ha -1 T7-Crop Max @ 625 ml ha -1 followed by Biozyme @ 625 ml ha -1 T8-Crop Max @ 750 ml ha -1 followed by Biozyme @ 625 ml ha -1 T9-Biozyme @ 625 ml ha -1 followed by Biozyme @ 625 ml ha -1 T10-Control-1 (Urea @ 2% and KNO3 @ 1%) T11-Control-2 (water spray) Means followed by the same alphabet (s) within a column are not significantly differed by DMRT (P=0.05) DAS-Days after sowing Note 1. RDF: FYM @ 6.25 t + 40:80:25 kg N: P2O5 and K2O + ZnSO4 @ 12.5 kg + Rhizobium and PSB @ 1250 g each per hectare common to all treatments Note 2. For T1 to T10, foliar application at flower initiation (25-35DAS) and at pod formation stage (45 -50 DAS).

Conclusion

The field investigation establishes that the strategic foliar application of biostimulants effectively mitigates physiological constraints and enhances the yield potential of soybean. Applying Crop Max at 750 ml/ha during flower initiation, followed by Biozyme at 625 ml/ha during pod formation (Treatment T 8 ), proved to be the most optimal regimen. This specific combination significantly improved vegetative parameters (plant height, leaf area), maintained superior cellular turgidity (RLWC), and decisively augmented the final seed and haulm yields by optimizing source-to-sink assimilate partitioning. The adoption of this biostimulant schedule presents a highly productive and sustainable strategy for soybean farmers operating in the Northern Transition Zone of Karnataka.

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