Field scale responses of soil enzymatic activities and microbial indicators to combined bioinoculant-fertilizer management in mustard (Brassica juncea L.)

The global demand for food is projected to rise significantly in the coming decades, driven by population growth and increasing per capita consumption¹. Estimates suggest that feeding a world population of 9.1 billion people by 2050 will require raising overall food production by approximately 70% from 2005/07 levels^2,3. This surge necessitates the expansion and diversification of crop production to ensure food security. Moreover, urbanization and income growth are driving changes in food preferences, with a greater reliance on processed and energy-dense foods, further amplifying the pressure on agricultural systems^4,5. Climate change and the depletion of natural resources add additional layers of complexity to this challenge, emphasizing the need for resilient and resource-efficient crops that can adapt to diverse and changing environments⁶.

In this context, Indian mustard (Brassica juncea L.) emerges as a crop of strategic importance in addressing global food security. Mustard seeds are a rich source of oil and protein, catering to the dual demand for edible oils and nutritional supplements. Its adaptability to various agro-climatic zones, coupled with its ability to thrive under water-limited⁷ and low-nutrient conditions⁸, positions it as a vital crop for regions prone to environmental stress. Furthermore, mustard’s short growing season and its role as a rotational crop in cereal-based systems enhance land-use efficiency and productivity⁹. Incorporating mustard into cropping systems can also aid in diversifying food supplies, countering the trend of increasing homogeneity in global food production. Such diversification is crucial for enhancing food security and nutritional quality¹⁰. Moreover, mustard cultivation can contribute to soil health and act as a biofumigant, suppressing soil-borne pathogens and reducing the need for chemical inputs¹¹.

For mustard to achieve its full productive potential, proper nutrient management is critical¹². Mustard is a nutrient-demanding crop that requires an adequate and balanced supply of essential macronutrients like nitrogen (N), phosphorus (P), potassium (K), and sulphur (S)¹³. These nutrients play pivotal roles in physiological and metabolic processes. However, excessive application of chemical fertilizers, while effective in boosting crop yields, often results in the accumulation of salts and other residues in the root zone, negatively impacting soil structure, fertility, and microbial diversity¹⁴. Naturally derived microbial inoculants are emerging as a potential strategy to address these challenges. Healthy soils are characterized by microbial ecosystems that include diverse populations of bacteria, fungi, protozoa, and other organisms. These microbial communities are critical for maintaining soil functions such as organic matter decomposition, enzymatic activity, and the suppression of soil-borne pathogens¹⁵. Continuous use of microbial inoculants has been shown to support microbial diversity and biomass, creating a dynamic soil environment that sustains nutrient availability over the long term¹⁶.

The combined use of chemical and microbial inoculants in mustard cultivation has shown promising results in enhancing crop yield and soil health. Studies have demonstrated that combining reduced doses of chemical fertilizers with microbial inoculants can significantly boost mustard growth and yield while minimizing environmental impact^17,18. Interestingly, the complementary effects extend beyond yield enhancement. The combined use of organic amendments and reduced chemical fertilizers has shown to improve soil microbial activities, organic carbon content, and nutrient availability in saline soils under mustard cultivation¹⁹.

Considering the current limitations in nutrient-use efficiency associated with conventional fertilization practices, there remains a critical research gap in understanding how biofertilization could enhance soil health indicators and crop productivity. This study aims to comprehensively investigate the combined effects of integrating microbial inoculants with chemical fertilizers to optimize nutrient dynamics, enhance soil enzymatic activities, and stimulate beneficial microbial populations. Specifically, the research intends to elucidate how these integrated fertilization strategies influence soil biological processes and enzymatic functionality.

The novelty of the present study lies in its integrative evaluation of soil enzymatic activities, microbial population dynamics, and nutrient availability as interrelated functional indicators of crop productivity under field condition. By assessing key soil enzymes such as dehydrogenase, urease, and alkaline phosphatase together with microbial biomass, soil nutrient pool and yield attributes, the study adopts a soil function-oriented perspective to examine how combined microbial inoculant along with chemical fertilization can influence nutrient cycling in mustard-based system. Field-scale evidence linking enzyme-mediated soil processes with agronomic performance remains scarce, particularly in subtropical alluvial soils, and such integrated assessments are important for translating biological soil indicators into practical and sustainable nutrient management strategies.

Study area

The experiment was conducted at the Agricultural Research Farm, Institute of Agricultural Sciences, Banaras Hindu University (BHU), during the cropping seasons of 2021–22 and 2022–23. The experimental site is geographically located at 25°15′50.63″ N 82°59′36.38″ E, with an elevation of 103 m above mean sea level (AMSL). It is situated in the Northern Gangetic Alluvial Plains of Uttar Pradesh (Fig. 1). The soil of the experimental field exhibited homogeneity in fertility status and had a sandy clay loam texture. The alluvial soils of the Indo-Gangetic plains are characteristically deep, flat, and well-drained, though they are generally low in available nitrogen and medium in available phosphorus and potassium.

Initial chemical analysis of the soil revealed a pH of 7.21, electrical conductivity (EC) of 0.27 dS m⁻¹, and organic carbon content of 0.39%. The available nutrient concentrations were 207.51 kg ha⁻¹ for nitrogen, 19.21 kg ha⁻¹ for phosphorus (P₂O₅), 208.167 kg ha⁻¹ for potassium (K₂O), and 17.50 kg ha⁻¹ for sulphur (S). Physical properties of the soil included a bulk density of 1.39 g cm⁻³, particle density of 2.01 g cm⁻³, and soil moisture content at field capacity of 14.17%. Initial soil microbial population of the study soil revealed bacteria 25 × 10⁷ CFU g⁻¹, fungi 21 × 10⁵ CFU g⁻¹ and actinomycetes 15 × 10⁶ CFU g⁻¹. All the methods employed for the analyses is given in Table 1. The seasonal meteorological data during the experimental period is shown in Fig. 2.

Experimental design

The experiment was conducted in a split-plot design with three replications arranged as blocks. Nutrient regime was assigned to the main plots and microbial inoculant to the sub-plots. The three main-plot treatments (nutrient regimes) were: 100% recommended dose of fertilizer (RDF) (F₁), 75% RDF (F₂) and an unfertilized control (F₃). Within each main plot, six sub-plot treatments (microbial inoculants) were randomly allocated: Azotobacter (B₁), phosphorus-solubilizing bacteria (PSB) (B₂), potassium-mobilizing biofertilizer (KMB) (B₃), zinc-solubilizing bacteria (ZSB) (B₄), NPK consortia + ZSB (B₅) and an uninoculated control (B₆). Thus, each combination of nutrient regime × microbial inoculant occurred once in each block, giving a total of 3 (nutrient regimes) × 6 (microbial treatments) × 3 (replications) = 54 experimental plots. Randomization of main-plot treatments within each block and of sub-plot treatments within each main plot was done using a random number table. Individual plots measured 6.0 m × 5.0 m, with buffer spaces of 1.0 m between blocks and 0.5 m between adjacent sub-plots reduce interference and edge effects. In the split-plot design, the unfertilized main plot (F₃) combined with microbial inoculant sub-plots (B₁–B₅) effectively represented bioinoculant-only treatments, allowing assessment of microbial effects in the absence of mineral fertilizer inputs. However, these treatments were nested within the split-plot structure and were not treated as an independent experimental factor.

All biofertilizers were obtained from Indian Farmers Fertilizer Cooperative Limited (IFFCO), the details can be availed from the link: https://www.iffco.in/en/organic-and-bio-fertilisers. As per the manufacturer’s specifications, all inoculants contained viable microbial populations ≥ 10⁷ CFU g⁻¹ at the time of application. To ensure good tilth, one deep ploughing was carried out with the help of disc plough and subsequently harrowing was done using a harrow drawn by the tractor to pulverize the soil. After each ploughing, planking was done with the purpose of preserving the soil moisture and to level the field in both seasons. Mustard variety, Giriraj was used for the experimentation and sowing was done on 27.10.2021 for the first year and 19.10.2022 in the second year. RDF of 120 kg N ha⁻¹, 60 kg P₂O₅ ha⁻¹, 60 kg K₂O ha⁻¹, 40 kg ha⁻¹ S was applied as per treatment through Urea, DAP, MOP and elemental S. A full dose of PKS together with half dose of N were used as basal and the remaining 50 per cent of nitrogen will be top dressed at 35 DAS. All microbial treatments were incorporated through seed inoculation as per the standard procedure.

Sampling of soil and plant

A composite surface soil sample (0–15 cm) was prepared from multiple sampling points across the field to assess baseline soil properties. While the composite approach ensured representative characterization, individual subsample values were not analyzed separately; therefore, spatial heterogeneity could not be statistically quantified. The field was selected based on uniform topography and land-use history to minimize variability.

Post-harvest of Indian mustard, five soil samples were randomly collected from each plot at a depth of 0–15 cm. The samples were air-dried under shade, ground, and then passed through a 2 mm sieve for further analysis. Plant samples were thoroughly washed with tap water, followed by rinsing in double-distilled water. They were then dried in the shade for 3 days, and subsequently dried in an air oven at 60 °C until a constant weight was achieved. After drying, the straw and seed dry matter were separately ground using a Willey Mill and stored in butter paper envelopes for subsequent nutrient analysis.

Statistical analysis

All the data collected were subjected to Analysis of Variance (ANOVA) for split plot at p ≤ 0.05 as described by Gomez and Gomez³⁰ and Duncan’s multiple range test (DMRT)post-hoc analysis was performed thereafter³¹. ANOVA, DMRT, principal component analysis (PCA) was performed using the software “statistical tool for agricultural research” (STAR v.2.0.1). For all graphical illustration, OriginPro, Version 2025 (OriginLab Corporation, Northampton, MA, USA) was used. Prior to PCA analysis, all dataset were subjected to Z-score transformation to remove scale effect. Sampling adequacy was evaluated using the Kaisser-Meyer-Olkin (KMO) measure, which gave an overall KMO value of 0.60, indicating acceptable adequacy for multivariate analysis. Bartlett’s test of sphericity was highly significant (P < 0,001), conforming that the correlation matrix was suitable for PCA.

Yield attributes

Data on siliqua number plant⁻¹, seeds siliqua⁻¹, seed yield, and stover yield during the experimental period is presented in Table 2. A significant influence of nutrient regime and microbial inoculant was observed on these parameters. In terms of nutrient regime, significantly higher siliqua plant⁻¹, seed siliqua⁻¹, seed and stover yield were recorded under 100% RDF (F₁), followed by 75% RDF (F₂) and the least of mustard’s yield attributes were observed under control plot (F₃). There was an increment of 11.76% and 38.97% in terms of seed yield under F₁ treatment as compared to F₂ and F₃ treatments and 4.69% and 24.53% respectively, in terms of stover yield.

Varied response was observed under growth and yield characters of Indian mustard due to microbial inoculants. During the study period, attributes including siliqua no. plant⁻¹ and seeds siliqua⁻¹, application of Azotobacter (B₁), PSB (B₂) and KMB (B₃) showed no statistical difference and were at par with each other. While the significantly highest and lowest siliqua plant⁻¹ and seed siliqua⁻¹ were recorded under NPK consortia + ZSB (B₅) and control treatment (B₆) respectively. Interestingly, in terms of seed yield, application of NPK consortia + ZSB (B₅) and Azotobacter (B₁) show no distinct significance, although the former being the superior and recording the highest seed yield of 1894.36 kg ha⁻¹. Similarly, in stover yield, NPK consortia + ZSB treatment resulted in significantly higher stover yield over all the other treatments. Treatment B₆ recorded 34.05% and 22.38% higher values over control treatment for seed and stover yield, respectively.

Microbial population

Data on soil microbial population as influenced by nutrient regime level and microbial inoculant is presented in Table 3. It is evident from the data that application of 100% RDF (F₁) resulted in the highest population of bacteria, fungi and actinomycetes at 30, 90 DAS and harvest, respectively. This was followed by 75% RDF (F₂) treatment, and the lowest microbial count was recorded under control treatment. Interestingly, treatments F₁ and F₂ showed no statistical difference under bacterial population at 30 and 90 DAS, and actinomycetes population at 30, 90 DAS and harvest.

Microbial inoculants also had a significant influence on the microbial population during the experimental period. The application of NPK consortia + ZSB (B₅) consistently resulted in the highest population of bacteria, fungi and actinomycetes at 30, 90 DAS and harvest, respectively. This was followed by the application of Azotobacter (B₁), PSB (B₂), KMB (B₃), ZSB (B₄) and lastly control (B₆). Comparing the percent increase in microbial population with the best treatment over control, application of NPK consortia + ZSB resulted in an increment of bacteria (35, 53.42 and 52.37% at 30, 90 DAS and harvest, respectively), fungi (67, 35.9 and 39.21%, respectively) and actinomycetes (51.68, 56.42 and 61.78%, respectively). Interestingly, treatment B₁ and B₅ showed no statistical difference in terms of bacterial population at 30 DAS and fungi population at 30, 90 DAS and harvest.

Enzyme activity

The data from the graph indicates a significant response of nutrient regime to dehydrogenase (Fig. 3a), urease (Fig. 3b) and alkaline phosphatase (Fig. 3c) with the highest activity recorded under 100% RDF, followed by 75% RDF and the least under control treatment.

Under microbial inoculant treatment, NPK consortia + ZSB (B₅) was found to significantly influenced the dehydrogenase, urease and phosphatase activity in soil and recorded the highest values. Compared to the second highest performing treatment (Azotobacter), treatment B₅ recorded an increase of 11.01, 4.88 and 3.03% respectively, in terms of dehydrogenase, urease and alkaline phosphatase activity.

Soil nutrient status

The data on available soil nutrients, organic carbon (OC), and soil microbial biomass carbon (SMBC) is presented in Table 4. A significant influence of nutrient regime was observed across all parameters. The highest available soil Nitrogen (N), phosphorus (P), potassium (K), sulphur (S), organic carbon (OC), and soil microbial biomass carbon (SMBC) were recorded under 100% RDF (F₁). A slight but significant reduction was observed under 75% RDF (F₂), with values of N-P-K corresponding to 187.69, 18.40, 235.92 kg ha⁻¹, respectively and S-OC-SMBC of 9.47 mg kg⁻¹ soil, 0.473%, and 195.98 mg kg⁻¹ soil, respectively. The lowest nutrient availability and microbial biomass were observed in the control treatment (F₃).

Among microbial inoculants, NPK consortia + ZSB (B₅) displayed the highest available N, P, K, S, OC, and SMBC, and the difference was statistically significant compared to the rest of the microbial inoculant treatments. Azotobacter (B₁) and PSB (B₂) showed statistical similarities in terms of soil available N-P-K-S and SMBC. The lowest values across all parameters were recorded under B₆ (control).

Plant nutrient status

A significant decline in N, P, K, and S content was observed as the level of fertilizer application was reduced from 100% (Table 5). Treatment F₁ (100% RDF) recorded the highest N, P, K, and S content in seed, followed by 75% RDF (F₂). The lowest seed nutrient content was observed in control (F₃). A similar trend was reflected in the nutrient content of stover, where treatment F₁ displayed significantly higher N (0.748%), P (0.406%), K (1.37%), and S (0.178%) content. However, in terms of K content in stover, no significant difference was observed among the main plot treatments.

Among microbial inoculant treatments, NPK consortia + ZSB (B₅) exhibited the highest nutrient accumulation in both seed and straw, surpassing other treatments. Additionally, Azotobacter (B₁) and PSB (B₂) remained at par for K-S content in seed and stover. The lowest nutrient in seed as well as straw was recorded in control treatment (B₆).

Principal component analysis (PCA)

Principal Component Analysis (PCA) was performed on the standardized dataset to identify patterns of covariation among yield attributes, soil nutrient availability, microbial biomass, and enzymatic activities. The scree plot showed a sharp decline after the first principal component, which had an eigenvalue well above the Kaiser threshold of 1 (Fig. 4). PC1 alone accounted for most of the variance in the dataset, while subsequent components contributed only marginally.

Biplot illustrates the clustering of different treatments and their associations with measured parameters (Fig. 5). PC1 explained 83.48% of the total variance, while PC2 accounted for 6.79%, cumulatively explaining over 90.27% (PC1 + PC2) of dataset variability. Most yield attributes, soil nutrient pools, microbial biomass, and enzymatic activities clustered closely along the positive direction of PC1.

Plant growth and yield in Indian mustard showed a clear response to nutrient management in the present study. The higher number of siliqua plant⁻¹ and seeds siliqua⁻¹ recorded under 100% RDF indicates that improved nutrient availability was closely associated with better development of reproductive structures. This suggests that plants grown under adequate nutrient supply experienced fewer nutritional constraints during critical growth stages, allowing greater allocation towards yield components. Treatments receiving 100% RDF consistently produced higher seed and stover yield, which coincided with improved yield attributes. This indicates that enhanced crop performance under adequate fertilization likely reflects the combined effects of improved nutrient availability and overall plant growth conditions, rather than any single physiological process. The observed yield improvement aligns with earlier findings reported by Niu et al.³², who documented a substantial increase in mustard seed yield following application of a multi-nutrient fertilizer compared with unfertilized control plots. In addition, higher stover yield under 100% RDF in the present study suggests improved plant vigour, which may be linked to adequate phosphorus and potassium supply supporting structural development and nutrient transport.

In terms of microbial inoculants, the application of NPK consortia + ZSB resulted in significantly higher seed and stover yield compared to other microbial treatments. This yield advantage was accompanied by higher yield attributes, soil nutrient availability, microbial biomass carbon, and enzyme activities, indicating a strong combined treatment response rather than the effect of any single microbial function. The improved crop performance under NPK consortia + ZSB is likely due to improved soil functional conditions and nutrient availability under this treatment, as observed in the soil biological and chemical indicators. Additionally, plant growth promoting rhizobacteria (PGPR) are known to exhibit traits such as phosphate solubilization, nitrogen fixation, and siderophore production³³, although the study did not quantify these mechanisms, these beneficial effects may have also influenced the outcome. The ability of microbial consortia to enhance yields under combined effect of RDF + microbial inoculants has also been reported by Tinna et al.³⁴, where yield increases of 3.7–8.6% in onion was observed when 75% NPK was combined with microbial inoculants compared with full NPK application alone. In addition, higher soil microbial biomass carbon and enzyme activities recorded under NPK consortia + ZSB in this study suggest improved soil biological functioning, which is often linked with more efficient nutrient cycling³⁵. These biological improvements coincided with higher seed yield, supporting that enhanced soil functional status under combined chemical and biological inputs contributes to improved mustard productivity.

Changes in soil microbial populations observed in this study appear to be closely associated with differences in fertilization intensity rather than direct microbial stimulation. Across all sampling stages, higher bacterial, fungal, and actinomycetes populations were consistently recorded under 100% RDF, followed by 75% RDF, with the lowest counts observed in the unfertilized control. This consistent pattern suggests that improved nutrient availability under fertilized treatments created more favourable soil conditions for microbial proliferation. The observed increases in microbial populations are likely linked to indirect changes in soil properties, such as nutrient status and organic carbon availability, which were also higher under fertilized treatments in the present study. Similar associations between fertilization, soil nutrients, and microbial abundance have been reported by Wang et al.³⁶, who observed that increases in soil nitrogen and phosphorus were key factors associated with shifts in microbial communities under organic fertilization. Additionally, Gondwe et al.³⁷ also reported that partial substitution of chemical fertilizers with organic inputs altered soil organic matter and nutrient levels, which in turn coincided with changes in microbial abundance.

The application of 100% RDF (F₁) has shown to significantly affect soil enzyme activities, particularly dehydrogenase, urease, and alkaline phosphatase. The increase in dehydrogenase activity is likely due to the enhanced microbial activity resulting from the availability of nutrients provided by the fertilizers. Dehydrogenase is an intracellular enzyme that reflects on the overall microbial activity in the soil. Urease, which is involved in the nitrogen cycle, catalyzing the hydrolysis of urea to ammonia and carbon dioxide. The increased urease activity may be attributed to the higher availability of nitrogen substrates from the applied fertilizers, stimulating the production of this enzyme by soil microorganisms. Alkaline phosphatase is an enzyme involved in the mineralization of organic phosphorus compounds. The increase in alkaline phosphatase activity may be due to the enhanced microbial growth and activity stimulated by the balanced nutrient supply from the fertilizers. These findings have been supported by the work of Meena et al.¹⁹, where 100% RDF resulted in 39% higher dehydrogenase, 23% higher urease and 26% higher alkaline phosphatase over control treatment.

Significantly higher soil available N, P, K, OC and SMBC were observed with the application of 100% RDF. Chemical fertilizers directly add readily available forms of nitrogen, phosphorus, and potassium and sulphur to the soil solution, increasing their concentrations³⁸. This immediate increase in nutrient availability may have allowed for rapid uptake by the crop. Long-term chemical fertilization can alter soil properties in ways that affect nutrient availability. For instance, decrease in soil pH over time, which can influence the solubility and availability of certain nutrients³⁹. The acidification effect of chemical fertilizers can increase the solubility of phosphorus compounds, potentially enhancing P availability in the short term^40,41. The availability of nutrients from fertilizers stimulates microbial growth and activity in the soil. This increased microbial population likely contributed to the higher SMBC levels as observed in our study. Higher nutrient availability in the soil likely resulted in enhanced uptake and accumulation of nutrients in plant tissues. The application of 100% RDF ensures an adequate supply of essential nutrients (N, P, K, and S) throughout the growing season, enabling the plant to absorb and utilize these nutrients efficiently. This may have resulted in the higher nutrient content in both seed and stover and supports the finding of our study. The application of microbial inoculants along with reduced mineral fertilizer doses has been shown to improve plant growth parameters and yield attributes in crops like onion⁴². The same mechanisms apply to Indian mustard as well. Microbial consortia containing nitrogen-fixing bacteria such as Azotobacter, phosphate-solubilizing microorganism such as Bacillus, and other plant growth-promoting rhizobacteria (PGPR) can enhance nutrient availability and uptake by the plants⁴³. This explains the higher yield attributes observed in our study under microbial inoculant treatments as compared to control. Similar observation is also reported by Liu et al.⁴⁴ where microbial inoculants allowed for a 25–50% reduction in chemical fertilizer application without compromising yield.

Microbial inoculation resulted in clear and consistent increases in culturable bacterial, fungal, and actinomycetes populations across stages, with the strongest response observed under the NPK consortia + ZSB treatment. These increases were evident at 30 DAS, 90 DAS, and at harvest, indicating that microbial populations responded positively to inoculant application under field conditions. These results represent relative shifts in dominant, culturable microbial groups associated with different inoculation treatments. Studies have reported that microbial inoculation can influence soil microbial abundance under different management systems. For example, Angelina et al.⁴⁵ observed changes in soil microbial interactions following inoculation with Bacillus strains, while Rawat et al.⁴⁶ reported higher bacterial, fungal, and actinomycetes biomass in inoculated soils compared with uninoculated controls. The trends observed in this study are consistent with these reports. Alongside changes in microbial populations, higher soil enzyme activities were recorded under microbial inoculant treatments, particularly NPK consortia + ZSB. The increase in dehydrogenase, urease, and alkaline phosphatase activity coincided with higher microbial counts and soil nutrient availability, suggesting an overall improvement in soil biological functioning under combined treatments. Similar associations between microbial inoculation and increased soil enzyme activity have been reported in studies involving Azotobacter and phosphate-solubilizing bacteria under integrated nutrient management^47,48. In the present study, the concurrent increase in microbial population, enzyme activity, and nutrient availability under inoculated treatments supports the interpretation that microbial inoculants contribute to a more biologically active soil environment, which was positively associated with mustard crop performance.

Beyond individual enzyme responses, the findings highlight a coordinated pattern of soil enzymatic activity under combined NPK consortia + ZSB application. Higher dehydrogenase activity observed under this treatment reflects enhanced overall microbial metabolic activity, which coincided with increased urease and alkaline phosphatase activities. This increase suggests a functional enzymatic cascade associated with nitrogen and phosphorus transformation potential in soil, rather than isolated enzyme responses. This coordinated enzymatic response was accompanied by higher soil nutrient availability and improved yield attributes, which likely indicate that enzyme activities acted as functional indicators of improved soil nutrient cycling efficiency. These findings suggest that combined microbial inoculation supported a more functionally active soil system, which was positively associated with mustard productivity under the agro-ecological conditions of the Indo-Gangetic plains. The PCA further supported this, as enzyme activities, soil nutrients, microbial biomass, and yield attributes clustered along PC1, indicating strong co-variation among these soil functional indicators. To summarize the observed relationship among soil enzymes, nutrient availability, microbial populations and crop yield, a conceptual relationship has been included in Fig. 6.

PCA analysis indicated clear patterns of association among soil enzymatic activities, microbial indicators, nutrient availability, and mustard yield attributes. The inflection observed in the scree plot after the first principal component indicates that PC1 captured most of the meaningful variation in the dataset, whereas subsequent components accounted for comparatively minor variation. The close clustering of yield attributes, soil nutrient pools, microbial biomass, and enzymatic activities along the positive axis of PC1 suggests strong positive associations among these variables. This pattern reflects a common fertility-productivity gradient, where enhanced soil biochemical indicators were consistently associated with higher nutrient availability and improved crop performance, explaining the high variance contribution of PC1. In contrast, PC2 accounted for smaller contrasts, primarily related to differences between seed and straw nutrient concentrations.

This study has some limitations. Initial soil samples were composited to establish baseline soil properties, which precluded statistical estimation of spatial variability within the experimental field. Although the site was selected for uniform topography and prior land-use history, some degree of microscale heterogeneity cannot be ruled out. Consequently, baseline soil variability could not be incorporated into the statistical model, which represents a limitation of the experimental design. The experiment was conducted at a single location using one Indian mustard variety, which may restrict the applicability of the results to other environments and genotypes. Although bioinoculant-only treatments were included under the unfertilized control (F₃), microbial inoculants were evaluated as a sub-plot factor within a split-plot design. Therefore, interpretations focus on microbial responses across fertilizer regimes rather than treating bioinoculants as an independent management system. Microbial responses were assessed using culture-based and enzymatic approaches, without molecular-level characterization of microbial community structure and diversity. The present study did not directly measure enzyme production pathways. Therefore, the observed increases in enzyme activity should be interpreted as indicators of enhanced soil biological activity rather than evidence of specific microbial mechanisms. In addition, the findings are based on two cropping seasons, so longer-term studies across multiple locations and years are needed to assess the stability and wider relevance of these results.

The application of 100% Recommended Dose of Fertilizer (RDF) demonstrated significant improvements in overall productivity of Indian mustard. Additionally, the incorporation of microbial inoculants, particularly the NPK consortia + Zinc Solubilizing Bacteria (ZSB), resulted in the highest soil microbial populations, enzyme activities, and nutrient availability. Although 100% RDF resulted in the highest productivity, reducing it to 75% RDF combined with NPK consortia + ZSB is advocated. However, this recommendation is specific to the study conditions and requires validation in other environments and seasons. Further multi-location and long-term trials are necessary to confirm these findings.