Influence of integrated bioinoculants and bioenhancers on biochemical quality attributes of strawberry (Fragaria × ananassa Duch.) cv. winter dawn

Introduction

Strawberry (Fragaria × ananassa Duch.) is one of the most important small fruit crops cultivated worldwide and is valued for its pleasant flavour, attractive colour, and high nutritional quality. Strawberry fruits are rich in vitamins (particularly vitamin C and folate), dietary fibre, organic acids, and a wide range of bioactive phenolic compounds such as flavonoids and other polyphenols, which together contribute to their strong antioxidant and anti-inflammatory activities. These bioactive constituents, including anthocyanins and phenolic acids, play a key role in the health-promoting properties of strawberries (Newerli-Guz et al. 2023) [8] . Regular consumption of strawberries has been associated with beneficial effects on human health, including improved antioxidant status and reduced inflammation, with potential protective effects against cardiometabolic disorders (Charoenwoodhipong et al. 2024) [2] . Among the nutritional components of strawberry, ascorbic acid (vitamin C) plays a major role in determining fruit quality and antioxidant potential. In addition to vitamin C, phenolic compounds and anthocyanins are major contributors to the radical-scavenging capacity and overall antioxidant activity of strawberry fruits (Cianciosi et al. 2025) [3] . The concentration of these bioactive compounds is not constant and varies with cultivar, environmental conditions, and stage of fruit maturity, indicating that both genetic and environmental factors strongly influence strawberry fruit quality (Charoenwoodhipong et al. 2024) [2] . From a commercial and consumer point of view, biochemical parameters such as total soluble solids (TSS), titratable acidity, and ascorbic acid content are considered key indicators of strawberry fruit quality.

TSS mainly reflects sugar accumulation, while titratable acidity contributes to flavour balance and overall sensory acceptability. These attributes directly affect consumer preference and market value, and are known to respond to differences in genotype, nutrient availability, and growing conditions (Patel et al. 2023) [9] . In intensive strawberry production systems, chemical fertilizers are commonly used to achieve high yields and acceptable fruit quality. However, prolonged and excessive use of inorganic fertilizers can negatively affect soil health by altering nutrient balance and soil microbial community structure, reducing soil biological activity, and decreasing nutrient use efficiency, raising concerns regarding the longterm sustainability of strawberry cultivation and environmental safety (Bai et al. 2023) [1] . In recent years, plant growth-promoting rhizobacteria (PGPR) and other microbial inoculants have emerged as promising biofertilizers that can complement or reduce the use of synthetic fertilizers in sustainable agriculture. These beneficial microorganisms colonize the rhizosphere and enhance plant growth through multiple mechanisms, including solubilizing phosphorus, fixing atmospheric nitrogen, producing phytohormones, and improving nutrient availability, cycling, and uptake, thereby supporting improved soil health and long-term crop productivity while mitigating environmental impacts associated with conventional chemical inputs (Yang et al. 2024) [17] . Plant growth-promoting bacteria improve plant performance through several mechanisms, such as biological nitrogen fixation, solubilization of insoluble phosphorus compounds, and the production of phytohormones and siderophores that support root development and nutrient acquisition. Their application has been shown to enhance soil fertility and crop productivity while reducing dependence on synthetic fertilizers (Timofeeva et al. 2023) [16] . Among these microorganisms, Azotobacter species are freeliving diazotrophs capable of fixing atmospheric nitrogen under non-symbiotic conditions. In addition to nitrogen fixation, Azotobacter spp. are known to produce growth promoting substances such as indole-3-acetic acid (IAA), which stimulate root growth and enhance nutrient uptake. Some strains have also been reported to solubilize phosphate, thereby contributing to both nitrogen and phosphorus nutrition in plants (Rathod et al. 2025) [11] . Phosphate-solubilizing bacteria (PSB) play an important role in improving phosphorus availability in soils where phosphorus is largely present in insoluble forms. These microorganisms solubilize inorganic phosphates through the secretion of organic acids and phosphatase enzymes, thereby converting unavailable phosphorus into plant-available forms. In strawberry, inoculation with PSB and other beneficial bacteria, such as Azotobacter and Azospirillum, enhances phosphorus uptake, root growth, fruit yield, and overall plant performance, highlighting their potential as sustainable alternatives to chemical phosphorus fertilizers (Singh et al. 2024) [13] . In recent years, traditional organic bioenhancers such as Jeevamrit and Amritpani have received increasing attention in sustainable and natural farming systems. These liquid organic formulations, prepared from cow dung, cow urine, jaggery, pulse flour, and soil, are rich in beneficial microorganisms and nutrients. Their application has been shown to stimulate soil microbial activity, enhance enzymatic processes, and improve nutrient mineralization, thereby supporting plant growth and soil health (Kumar et al. 2025) [5] . Jeevamrit and Amritpani are also believed to act as microbial inoculants that improve nutrient availability and enhance crop productivity under low-input conditions. In addition to microbial inoculants, organic bioenhancers used in organic and low-input farming systems can stimulate soil biological activity and enhance soil enzymatic processes, thereby increasing the potential for nutrient transformation and supporting sustained nutrient cycling and plant growth in strawberry (Maková et al. 2024) [7] . The combined application of microbial inoculants and organic bioenhancers may offer synergistic benefits by improving soil biological health while enhancing nutrient uptake by plants. Integrated nutrient management approaches have been reported to improve fruit biochemical attributes, including TSS, titratable acidity, and vitamin content, in several horticultural crops. Considering the growing emphasis on sustainable horticultural practices and quality-oriented fruit production, the present study was undertaken to evaluate the effects of selected bioinoculants and bioenhancers, applied individually and in combination, on key biochemical quality parameters of strawberry (Fragaria × ananassa Duch.) cv. Winter Dawn. The study focuses on total soluble solids, titratable acidity, and ascorbic acid content, with the aim of generating practical information for sustainable nutrient management under subtropical agro-climatic conditions.

Materials and Methods Experimental Site and Plant Material

The experiment was conducted during the winter season of 2022-23 at the Horticulture Research Farm, Department of Horticulture, Naini Agricultural Institute, Sam Higginbottom University of Agriculture, Technology & Sciences, Prayagraj, under subtropical agro-climatic conditions. Healthy and uniform runners of strawberry (Fragaria × ananassa Duch.) cv. Winter Dawn were used as planting material.

Experimental Design and Treatments

The study was laid out in a Randomized Block Design (RBD) with nine treatments and three replications. The treatments consisted of the recommended dose of fertilizers (RDF), which served as the control (T₀), and different bioinoculant and bioenhancer applications applied individually and in various combinations. Treatments T₁ to T₈ included applications of Azotobacter, phosphatesolubilizing bacteria (PSB), Jeevamrit, and Amritpani. The details of the treatment combinations are presented in Table 1. Azotobacter @ 400 g/100 m² T2 PSB @ 400 g/100 m² T3 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l T4 Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l T5 Azotobacter @ 400 g/100 m² + Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l T6 PSB @ 400 g/100 m² + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l T7 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l + Azotobacter @ 400 g/100 m² T8 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l + PSB @ 400 g/100 m²

Cultural Practices

Standard agronomic and horticultural practices recommended for strawberry cultivation were uniformly followed for all treatments throughout the crop period. Irrigation, weeding and plant protection measures were carried out as and when required to ensure normal crop growth.

Observations Recorded

Fruit samples were collected at commercial harvest maturity for the assessment of biochemical quality parameters. The biochemical attributes analyzed included total soluble solids (TSS), titratable acidity and ascorbic acid content. Total soluble solids were determined using a hand refractometer and expressed as °Brix. Titratable acidity was estimated by titration against a standard sodium hydroxide solution and expressed as percentage citric acid. Ascorbic acid content was determined by the 2,6-dichlorophenol indophenol dye method and expressed as mg per 100 g fresh weight.

Statistical Analysis

The recorded data were subjected to analysis of variance (ANOVA) appropriate for the randomized block design. Treatment means were compared using critical difference (CD) at the 5% level of significance. Statistical analysis was performed using OPSTAT software.

Results

Total Soluble Solids (TSS)

Total soluble solids (TSS) content of strawberry fruits varied significantly among different bioinoculant and bioenhancer treatments (Table 2), and the treatment-wise variation is clearly illustrated in Fig 1 . The highest TSS value was recorded in treatment T₈ (Jeevamrit + Amritpani + PSB), which registered (7.98 °Brix). This was followed by treatment T₆ (PSB + Amritpani), which recorded a TSS value of (7.86 °Brix). Both treatments recorded significantly higher TSS compared to the control (T₀), which showed the lowest value of (4.48 °Brix). Among the individual treatments, Jeevamrit (T₃) recorded a TSS value of (5.87 °Brix), while Amritpani (T₄) recorded (6.01 °Brix). Treatments receiving combined applications of bioinoculants and bioenhancers generally recorded higher TSS values than individual applications. Azotobacter @ 400 g/100 m² 5.25 T2 PSB @ 400 g/100 m² 5.57 T3 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l 5.87 T4 Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l 6.01 T5

Azotobacter @ 400 g/100 m² + Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l 6.34 T6 PSB @ 400 g/100 m² + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l 7.86 T7 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with nonchlorinated water) @ 250 ml/l + Azotobacter @ 400 g/100 m² 6.64 T8 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with nonchlorinated water) @ 250 ml/l + PSB @ 400 g/100 m² 7.98 Values represent mean ± SE (n = 3)

Titratable Acidity

Titratable acidity of strawberry fruits showed significant differences among treatments (Table 3), and the treatmentwise variation is clearly illustrated in Fig 2 . The lowest titratable acidity was recorded in the control treatment (T₀) with a value of (0.57%). Higher acidity values were observed in treatments receiving bioinoculants and bioenhancers.

The highest titratable acidity was recorded in treatment T₈ (1.57%), followed by T₆ (1.33%) and T₅ (1.30%). Among the individual treatments, PSB (T₂) and Amritpani (T₄) recorded acidity values of (0.93%), while Jeevamrit (T₃) recorded (0.90%) acidity. Azotobacter @ 400 g/100 m² 0.60 T2 PSB @ 400 g/100 m² 0.93 T3 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l 0.90 T4 Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l 0.93 T5

Azotobacter @ 400 g/100 m² + Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l 1.30 T6 PSB @ 400 g/100 m² + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l 1.33 T7 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with nonchlorinated water) @ 250 ml/l + Azotobacter @ 400 g/100 m² 1.17 Values represent mean ± SE (n = 3)

Ascorbic Acid Content

Ascorbic acid content of strawberry fruits differed significantly among the treatments (Table 4), and the treatment-wise variation is clearly illustrated in Fig 3 . The maximum ascorbic acid content was recorded in treatment T₈ (58.82 mg 100 g⁻¹ fresh weight), followed closely by T₆ (58.63 mg 100 g⁻¹). The control treatment (T₀) recorded the lowest ascorbic acid content of (51.97 mg 100 g⁻¹). Among individual treatments, Amritpani (T₄) recorded an ascorbic acid content of (56.24 mg 100 g⁻¹), while Jeevamrit (T₃) recorded (55.64 mg 100 g⁻¹). Azotobacter @ 400 g/100 m² 53.08 T2 PSB @ 400 g/100 m² 54.76 T3 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l 55.64 T4 Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l 56.24 T5

Azotobacter @ 400 g/100 m² + Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l 56.77 T6 PSB @ 400 g/100 m² + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l 58.63 T7 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l + Azotobacter @ 400 g/100 m² 54.14 T8 Jeevamrit (3 times diluted with non-chlorinated water) @ 250 ml/l + Amritpani (3 times diluted with non-chlorinated water) @ 250 ml/l + PSB @ 400 g/100 m² 58.82

1.56 Values are mean of three replications. CD = critical difference at 5% level of significance. S = significant.

Discussion

The results of the present study clearly indicate that the application of bioinoculants and organic bioenhancers, either alone or in combination, significantly influenced the biochemical quality attributes of strawberry fruits. Among the treatments, combined applications, particularly Jeevamrit + Amritpani + PSB (T₈), consistently showed superior performance in terms of total soluble solids, titratable acidity, and ascorbic acid content, highlighting the synergistic effects of bioinoculants and organic bioenhancers.

Total Soluble Solids (TSS)

The enhancement of total soluble solids under combined bioinoculant and bioenhancer treatments may be attributed to improved nutrient availability and enhanced microbial activity in the rhizosphere, which can stimulate photosynthetic efficiency and carbohydrate metabolism.

Increased availability of phosphorus through phosphatesolubilizing bacteria plays a crucial role in energy transfer and sugar synthesis, while organic bioenhancers such as Jeevamrit and Amritpani may improve soil microbial dynamics and nutrient mineralization. Similar improvements in TSS due to biofertilizer application have been reported in strawberry and other horticultural crops (Kumar et al. 2019; Singh et al. 2025) [6,15] . Enhanced TSS content is generally associated with improved sweetness and overall fruit quality, which is a desirable trait from a consumer perspective.

Titratable Acidity

Titratable acidity was observed to increase under bioinoculant and bioenhancer treatments compared to the control. Organic acids are important intermediates in plant metabolism, and their accumulation in fruits is influenced by nutrient uptake and physiological activity during fruit development. Improved nutrient availability under integrated bio-based treatments may have enhanced metabolic processes, leading to higher organic acid synthesis. Similar trends of increased acidity under biofertilizer and organic input application have been reported in strawberry and other fruit crops. Importantly, the concurrent increase in TSS and acidity under integrated treatments suggests an improved sugar-acid balance, which is a key determinant of flavour quality in strawberries (Quddus et al. 2025) [10] .

Ascorbic Acid Content

The significant increase in ascorbic acid content observed under combined bioinoculant and bioenhancer treatments may be related to improved micronutrient availability and enhanced enzymatic activity involved in ascorbic acid biosynthesis. Beneficial microorganisms are known to influence the uptake of micronutrients such as iron and zinc, which play important roles in enzymatic pathways associated with vitamin C synthesis. Earlier studies have also reported enhanced ascorbic acid content in strawberry fruits following biofertilizer and organic amendment application (Kaur et al. 2024) [4] . Increased vitamin C content enhances the nutritional value of fruits and contributes to their antioxidant capacity.

Integrated Effect of Bioinoculants and Bioenhancers

The superior performance of combined treatments observed in the present study supports the concept of integrated nutrient management using bioinoculants and organic bioenhancers. Bioinoculants such as Azotobacter and PSB improve nutrient availability through biological nitrogen fixation and phosphorus solubilization, while organic formulations like Jeevamrit and Amritpani stimulate microbial activity and nutrient cycling in the soil. The combined use of these inputs creates a favourable rhizosphere environment that supports sustained plant growth and improved fruit quality. Similar synergistic effects of integrating biofertilizers with organic inputs have been reported in strawberry, where organic manure combined with Azotobacter significantly influenced fruit quality traits, including TSS and sugar content (Reddy et al. 2020) [12] , and where biofertilizer combinations with Azotobacter, PSB, and Azospirillum enhanced quality and economic returns (Singh et al. 2024) [13] . These studies confirm that integrated nutrient management not only improves nutrient availability but also promotes sustainable and low-input horticultural production systems. Overall, the findings of the present study demonstrate that integrated application of bioinoculants and organic bioenhancers can effectively improve the biochemical quality of strawberry fruits while reducing reliance on chemical fertilizers, thereby supporting sustainable strawberry production under subtropical agro-climatic conditions.

Conclusions

The present study demonstrated that integrated application of bioinoculants and bioenhancers significantly influenced the biochemical quality of strawberry fruits cv. Winter Dawn under subtropical conditions. Among the treatments, combined application of Jeevamrit, Amritpani and phosphate-solubilizing bacteria (PSB) resulted in the highest total soluble solids (TSS), indicating improved fruit sweetness and quality. Treatments receiving combined inputs consistently outperformed individual applications, highlighting the synergistic effect of bioinoculants and bioenhancers on fruit biochemical attributes. The findings suggest that integrated use of organic bioinputs can serve as a sustainable and effective strategy for enhancing strawberry fruit quality while reducing dependence on chemical fertilizers.

Acknowledgement

AcknowledgementsThe authors acknowledge the institutional facilities provided for conducting the experiment and sincerely thank Dr. Samir E. Topno for his academic guidance and valuable suggestions.

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