Introduction

According to the Global Nutrition Report (2017), over 2 billion people suffer from deficiencies in key micronutrients such as iron (Fe), zinc (Zn), and vitamin A (Vit-A). This widespread deficiency poses significant challenges to achieving Sustainable Development Goal (SDG) target 2.2, which aims to end all forms of malnutrition by 2030. Globally, climate change is expected to reduce the protein and Zn content in major food crops. In the South Asian region, about 95.4% of people suffer from Zn deficiency1,2. In India, the dietary inadequacy of Zn increased from 28% in the early 90’s to 31% in the last decades leading to serious malnutrition issues like underweight, stunting, wasting, etc3,4. Nevertheless, the North-Eastern Himalayas of India, particularly Meghalaya, Mizoram, Nagaland, and Tripura, have more distressing scenarios than the national average5. Zn is an important trace element required for higher plants, as it acts as a cofactor in several enzymes and bio-synthesis of plant hormones, carbohydrates, and protein metabolism6. Whereas, in animals, it plays a substantial role in developing cognitive abilities and immunological functions7. Nearly 50% of cereal-growing regions are geographically identified as having low soil-available zinc (Zn), and about 48.5% of soils in India are considered deficient in Zn8,9. As a result, Zn is ranked as the fourth most yield-limiting nutrient after nitrogen (N), phosphorus (P), and potassium (K)10. The prior study has demonstrated that there is a positive correlation between the soil available Zn, plant uptake, and dietary intake by humans11,12.

Biofortification, either through genetic or agronomic approaches is an innovative agricultural strategy being increasingly adopted to combat micronutrient deficiencies13,14. Being a legume crop, soybean emerges as a suitable crop for Zn biofortification, offering multiple health benefits to humans and animals. In South Asia, soybean (Glycine max L.) plays a crucial role in providing food and nutritional security to billions15. Soybean is used as a valuable animal feed and raw material for different industries, viz., pharmaceutical, textile fibers, and paper coatings, etc16. It contains a decent amount of high-quality protein (42–45%), essential amino acids, vitamins, and micronutrients, along with high polyunsaturated fatty acids17,18,19. Moreover, it can be grown under diverse agro-ecologies and also improves soil fertility through nitrogen (N) fixation, addition organic matter and release of organic acid in the rhizosphere20,21,22.

The proper execution of the 4Rs approach and combined use of Zn through soil and foliar spray increases phyto-availability of nutrients, better translocation in the edible part, and enhance yield & quality23,24,25. The foliar spray of micro-nutrients (Zn and Fe) improved nutrient use efficiency, reduced cost of production, and had a less negative effect on the environment26,27. The combination of soil + foliar application lowers phytic acid & phytic acid to zinc (PA/Zn) molar ratio, besides increasing Zn content in the wheat grain (47%) and straw (37%)28. As per29,30,31the increases in nutrient doses of Zn positively enhanced the Zn content and uptake in seed and stover of soybean. Moreover, several studies have documented Zn biofortification efforts globally and in India across crops such as rice, wheat, maize, soybean, and chickpeas32,33,34,35,36,37,38,39,40,41,42,43. However, in North-Eastern India, limited research has been conducted to comprehensively evaluate the impact of Zn biofortification in soybean within real field conditions24,44. Thus, we have designed the present experiment to study soybean, as the majority of the ethnic population relies on soybean for cheap dietary protein.

Henceforth, we have hypothesized that, the combined application of different Zn sources through soil and foliar spray would improve (i) physiological attributes and crop yield, (ii) Zn biofortification and bioavailability, protein and oil content, and (iii) Zn use efficiency, DTPA extractable-Zn, and soil microbial activities. Additionally, this study will provide insight into doses, timings, and methods of Zn fertilization for enhancing the Zn biofortification.

Materials and methods

Site description

The current field experiment on biofortification of Zn in Soybean was conducted for two consecutive years i.e. 2018 and 2019 at the School of Agricultural Sciences and Rural Development, Nagaland University, located at a coordinate of 25045′ N; 95053′ E, at 310 m above mean sea level India. The site falls under the ’North-Eastern Himalayan’ zone and experiences a sub-tropical moderate climate with an average annual precipitation of 1069 mm, wherein 70–80% of the total rainfall received in the month of July-September. The average annual maximum and minimum temperature ranges from 30.6 to 34.2 °C and 11.2 to 16.0 °C, respectively whereas, the relative humidity (RH) varies from 50.2 to 96.0% (Fig. 1). The pre-experimental soil samples were collected from the upper 0–15 cm soil depth in triplicates using an auger of 5.0 cm diameter. Then the soil samples were shade dried, ground gently, and passed through a 0.2 mm sieve and then stored for laboratory analysis. The initial soil properties were are presented in the Table 1.

Experimental details

A uniformity trial on Mung bean (Vigna radiata L.) was undertaken before the initiation of the experiment in the summer season to find out the fertility gradient. The block was oriented perpendicular to the fertility gradient to ensure uniform environmental conditions across blocks. The field experiment was laid out in a Factorial Randomized Block Design (FRBD) with three replications. The factor ‘A’ consists of 03 popular soybean genotypes namely JS-97−52, JS-335 and local landrace. While, Factor ‘B’ comprised different Zn fertilization sources and application methods, which are presented in Table 2. Randomization was carried out independently within each replication to ensure the unbiased allocation of treatments. All possible genotypes × Zn treatments combinations were randomly assigned to individual plot measuring 13.5 m² (4.5 m × 3.0 m) using a random number table.

Cultural practices and management

Each year, the experimental field was deep ploughed (30–45 cm) using a tractor drawn disc plough in the 2nd fortnight of April followed by harrowing using a cultivator, and then levelled properly. The weeds and previous crop stubbles were removed manually prior to layout and imposition of the treatments. The seeds were dibbled manually @ 50 kg ha−1 in the 1st fortnight of June every year at a spacing of 45 cm x 10 cm and 3–5 cm soil depth. To maintain an optimum plant population the intercultural operations viz. gap filling, thinning, and earthing up was carried out at 15, and 30 days after sowing (DAS). The recommended fertilizer application i.e. 20:60:40 NPK kg ha−1 along with farmyard manure @ 10 t ha−1 on dry weight basis was applied as a basal dose through urea, single super phosphate (SSP), and muriate of potash (MOP). In addition, the Zn fertilizers viz. zinc sulphate (ZnSO4.7H2O) containing 21% and zinc oxide (ZnO) having 80% Zn were applied uniformly as per the plan of experiment. Weeds were managed through manual weeding at 30 and 60 DAS. In North-Eastern region of India, the soybean crops are heavily infested with shoot borer hence, Furadan @ 2–3 granules were applied on the top leaf whorl at 20–30 DAS supplemented by chlorpyriphos spray at 40 and 50 DAS.

Yield measurements

The soybean crops were harvested manually in the 2nd fortnight of October as the pods turned golden brown and leaves dried completely, from a net sampling area of 9.0 m22 (3.0 m × 3.0 m) leaving the border rows from all the sides as described by32. The harvested samples were sun dried in the field and then weighed for biological (grain + stover) yields. The stover yield was calculated by subtracting the grain yield from total plant biomass21.

Growth and physiological parameters

Leaf area index (LAI)

The leaf area of soybean was assessed at 60 DAS using a leaf area meter (Model LICOR-3100 Biosciences, Lincoln, NE, USA), and the LAI was worked out using the following formula given by51:

$$\:{\text{LAI}} = \frac{{{\text{Total}}\:{\text{leaf}}\:{\text{area}}\:({\text{cm}}^{{\text{2}}} )}}{{{\text{Ground}}\:{\text{area}}\:({\text{cm}}^{{\text{2}}} )}}$$
(1)

Total chlorophyll content

The fully matured soybean leaves were collected in a sunny day at 60 days after sowing (DAS), and transported immediately to the laboratory for analysis. A representative fresh leaf sample weighing 01 gram (g) was crushed using mortar and pestle with 20 ml of 80% acetone and then centrifuged at 5000 rpm for 5 min. The supernatant obtained was transported to the volumetric flask of 100 ml using Whatman No. 1 filter paper. Similarly, the process of crushing the samples with 20 ml of 80% acetone was repeated till the filtrate became colourless. The final volume was prepared in the 100 ml volumetric flask along with 80% acetone; and the absorbance of supernatant was measured in the UV-Vis spectrophotometers at 645 nm and 663 nm wavelength to determine the total chlorophyll content with 80% acetone as blank. The total chlorophyll content (chl. A, & chl. B) was calculated using the formula described by52:

$$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{c}\text{h}\text{l}.\:\left(\text{m}\text{g}\:{\text{g}}^{-1}\text{t}\text{i}\text{s}\text{s}\text{u}\text{e}\right)=20.2\:\left(\text{O}.\text{D}.\:\text{a}\text{t}\:645\:\text{n}\text{m}\right)+\:8.02\:\left(\text{O}.\text{D}.\:\text{a}\text{t}\:663\:\text{n}\text{m}\right)\frac{\text{V}}{1000\:\text{X}\:\text{W}}$$
(2)

Where, O.D. = optical density; V = Final volume of chlorophyll extract in 80% acetone; and W = Fresh weight of tissue extracted (g).

Nodulation studies

The plant samples for nodulation studies were collected from the central row through destructive sampling method using spade and khurpi at 60 DAS. The collected samples were thoroughly cleaned in the running tap water followed by dipping in a container for separation of nodules from soil. The samples were gently cleaned using sieve of 0.2 mm mesh to avoid washing over of nodules; and then nodule related observations such as nodule number, and nodule dry weight at 65 °C were recorded as per the procedure given by53.

Protein content and yield

The total crude protein content in soybean seeds was computed using the grain nitrogen (N) concentration multiply with a species-specific conversion factor as given by54. The protein yield (PY) of the soybean was calculated using the formula as described by55:

$$\text{PY (kg ha}^{-1}\text{)} = \frac{\text{Protein (\%)} \text{*} \text{Seed yield (kg ha}^{-1}\text{)}}{100}$$
(3)

Oil content and yield

The seed samples weigh 5 g collected from all the treatments were crushed, placed in a thimble, and extracted with light petroleum ether for 6 h in a soxhlet extraction unit as per the method described by56]. The extract was transferred to weight flask, distilled, and the last traces of the solvent, as well as moisture in the flask was removed by treating it at 100–150˚C. Then, the flask was cooled and reweighed and oil content was calculated as per the following formula56:

$$\:\text{P}\text{e}\text{r}\text{c}\text{e}\text{n}\text{t}\:\text{o}\text{i}\text{l}=\frac{\left(\text{W}2-\text{W}1\right)\text{*}100}{\text{X}}$$
(4)

Where, W2 = Weight of the empty flask (g); W1 = Weight of empty flask + weight of oil (g); and X = Weight of sample taken for extraction (g). Similarly, the oil yield (OY) of the soybean was calculated using the formula:

$$\text{OY (kg ha}^{-1}\text{)} = \frac{\text{Oil content (\%)} \text{*} \text{Seed yield (kg ha}^{-1}\text{)}}{100}$$
(5)

Phytic acid content

The phytic acid or phytate of the finely powdered soybean seed samples was evaluated using phytate/total P assay enzymatic kit, developed by Megazyme International Ltd., Ireland. Similarly, the phytate content was computed using the procedure suggested by57. The determination of phytic acid content using assay kit was based on the amount of P released as “available P” from phytic acid, myo-inositol phosphate n, and monophosphate esters of samples resulting from the action of phytase and alkaline phosphatase. While, the phytic: Zn molar ratio which indicates the bio-availability of Zn in the harvested grains was computed using the following formula:

$$\:\text{P}\text{h}\text{y}\text{t}\text{i}\text{c}\:\text{a}\text{c}\text{i}\text{d}:\:\text{Z}\text{n}\:\text{m}\text{o}\text{l}\text{a}\text{r}\:\text{r}\text{a}\text{t}\text{i}\text{o}=\frac{\text{g}\:\text{k}{\text{g}}^{-1}\text{p}\text{h}\text{y}\text{t}\text{a}\text{t}\text{e}}{660\:}\:\::\:\:\frac{\text{g}\:\text{k}{\text{g}}^{-1}\text{p}\text{h}\text{y}\text{t}\text{a}\text{t}\text{e}}{\:65.4}$$
(6)

Zinc concentration, and uptake

The seed and stover of soybean were collected at the time of harvest from 1 m linear length of crop row. The harvested plant samples were air-dried followed by oven dried at 60 ± 2 °C for 48 h for the estimation of Zn concentration. The grounded samples were digested in a di-acid mixture (HClO4 + HNO3 3:10) and Zn conc. was estimated using an atomic absorption spectrophotometer (Elements AS AAS4141; ECIL, Hyderabad, India), and expressed as mg kg–1 as described by58. The Zn uptake was calculated by multiplying the seed and stover yields with their respective Zn concentration and expressed in g ha−1.

Zinc use efficiencies and economics of soybean cultivation

The different Zn use efficiency indices viz. partial factor productivity (PFP) (kg grain kg−1 Zn), agronomic use efficiency (AE) (kg grain increased kg−1 Zn), crop recovery efficiency of Zn (CREZn) (%), zinc harvest index (ZnHI), and Zn mobilization efficiency index (ZnMEI) were computed based on Zn input applied, and seed yield of soybean using expressions as suggested by59,60,61.

$$\:{\text{PFP}}\:({\text{kg}}\:{\text{grain}}\:{\text{kg}}^{{ - {\text{1}}}} \:{\text{Zn}}) = \frac{{{\text{YT}}}}{{\text{F}}}$$
(7)
$$\:\text{A}\text{E}\:\left(\text{k}\text{g}\:\text{g}\text{r}\text{a}\text{i}\text{n}\:{\text{k}\text{g}}^{-1}\text{Z}\text{n}\right)=\frac{\text{Y}\text{T}-\text{Y}\text{C}}{\text{F}}$$
(8)
$$\text{CREZn (\%)} = \frac{\text{ZnUT} - \text{ZnUC}}{F} \times 100$$
(9)
$$\:\text{Z}\text{n}\text{H}\text{I}=\frac{{\text{Z}\text{n}}_{\text{s}}}{{\text{Z}\text{n}}_{\text{t}}}\:\text{x}\:100$$
(10)
$$\:\text{Z}\text{M}\text{E}\text{I}=\frac{\text{Z}\text{n}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{i}\text{n}\:\text{g}\text{r}\text{a}\text{i}\text{n}\:\left(\text{m}\text{g}\:\:{\text{k}\text{g}}^{-1\:}\text{g}\text{r}\text{a}\text{i}\text{n}\right)}{\text{Z}\text{n}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{i}\text{n}\:\text{s}\text{t}\text{r}\text{a}\text{w}\:\left(\text{m}\text{g}\:\:{\text{k}\text{g}}^{-1\:}\text{s}\text{t}\text{r}\text{a}\text{w}\right)}$$
(11)

Where, YT = Soybean yield under treated plots (kg ha−1); YC = Soybean yield under control plot (kg ha−1); ZnUT = Zn uptake in treated plots (kg ha−1), ZnUC = Zn uptake under control plot (kg ha−1); and F = Amount of Zn applied (kg ha−1), Zns = Zn uptake by grain at harvest, Znt = Zn uptake by whole crop (grain + straw) at harvest.

The cost of cultivation for various soybean cultivars, as well as for different Zn fertilization sources and application methods, was determined based on prevailing market prices of relevant agricultural inputs, expressed in US Dollars ($). Gross returns were computed from the grain and straw yields, using the respective seasonal market prices for grain and straw. Net returns were obtained by subtracting the total cost of cultivation from the corresponding gross returns. The benefit–cost ratio (BCR) was calculated as the ratio of net returns to the cost of cultivation, i.e., BCR = Net returns/Cost of cultivation.

Soil sampling and analysis

The post-experimental soil samples from the upper soil surface (0–15 cm) were collected in triplicate using an auger of 5 cm diameter after the harvesting. The collected soil samples were shade dried, ground, sieved through 2 mm sieve and kept in a sealed polythene bag. To estimate the soil available Zn, finely grounded soil sample (10 g) was taken in the conical flask followed by addition of 20 ml DTPA-extractant (0.005 M DTPA + 0.1 M tri-ethanolamine + 0.01 M CaCl2), and shaken for ~ 2 h at 120 cycles min−1 as suggested by62. Then, the samples were filtrated, and estimated using atomic absorption spectrophotometry (Elements AS AAS4141; ECIL, Hyderabad, India), and was expressed as mg kg–1 of soil. Similarly, for soil microbial parameters viz. soil microbial biomass carbon (SMBC), dehydrogenase activity (DHA), and Fluorescein diacetate (FDA), the samples were collected from the rhizosphere from 0–15 cm soil depth at the flowering stage. Then, the samples were processed, and then stored in a refrigerator at 5 °C for 18–24 h. The method of chloroform fumigation extraction was employed to estimate the soil microbial biomass carbon (SMBC)63. The colorimetric method was used to measure the dehydrogenase activity (DHA)64, and the fluorescein diacetate (FDA) was estimated using procedure described by65.

Statistical analysis

The data generated from the experimental units were subjected to the analysis of variance for the factorial randomized block design to determine the statistical significance of the different treatments66. The Tukey’s honest significant difference test was employed to differentiate the mean effect of the treatments at a 5% level of significance using the SAS 9.4 i.e. SAS Institute, Cary, NC. The correlation coefficient (R2 between different parameters were prepared using MS Excel 2013, TM.

Results

Growth parameters

The crop growth parameters of soybean at flowering stage were significantly (p < 0.05) influenced by the varieties and zinc fertilization strategies including different sources and application methods, over both the years of experimentation (Table 3). Among the soybean varieties studied, the local landrace had significantly higher LAI (4.03 and 4.07), nodules plant−1 (33.3 and 35.5), and nodules dry weight (0.75 and 0.54 g plant−1), at flowering stage during both the years. However, JS-97-52 recorded higher total chlorophyll (chl a + b) content (1.66 and 1.70 mg g−1), although this was statistically similar to that of variety JS-335 during the study period. Irrespective of the source or application method, Zn fertilization significantly improved the growth parameters viz. LAI, total chlorophyll, nodule numbers, and nodules dry weight, compared to the control (no Zn application). Foliar application of 0.5% ZnSO4.7H2O (FA-ZS) recorded an increased LAI (3.33 and 3.49), although it was statistically similar with soil + foliar application of ZnSO4.7H2O (SA + FA-ZS), soil application of ZnO at 5 kg ha−1 (SA-ZO), and soil application of ZnSO4.7H2O at 5 kg ha−1 (SA-ZS) during the first year of the study. However, higher increment in total chlorophyll content, nodules plant−1 and nodules dry weight was observed with SA + FA-ZS, which was, on average 17, 32 and 32% higher than the control, respectively. Overall, ZnSO4.7H2O proved to be more efficient Zn source than ZnO in improving the growth attributes, whereas soil + foliar was more efficient application method than sole application through soil or foliage except for LAI parameter. A significant interaction effect between genotypes and Zn fertilization was observed for LAI in 2018, as well as for nodule number and nodule dry weight in both 2018 and 2019. The local landrace of soybean exhibited a positive interaction with FA-ZS, resulting in a higher LAI. However, this genotype showed increased nodule number and dry weight in response to the SA + FA-ZS treatment.

Crop yield

The yield of soybean, as influenced (p < 0.05) by the varieties and Zn fertilization strategies during the experiment, are presented in Fig. 2. The soybean variety JS-97-52 produced higher seed (1.87 and 1.69 Mg ha−1) and stover (1.90 and 1.77 Mg ha−1) yields in 2018 and 2019, though the stover yield was statistically similar to that of local landrace (1.78 and 1.96 Mg ha−1). Zn fertilization to soybean, regardless of the sources and application method, increased 15% seed yield and 13% stover yield compared to the control. The treatment FA + SA-ZS resulted in a significantly higher seed yield (20–21% increase over control in both the years), though this was statistically comparable to FA-ZS, and SA + FA-ZO in the first year. Conversely, higher stover yield was observed with FA-ZS (20% increase over the control in both the years), which was statistically similar to SA + FA-ZO, SA + FA-ZS, FA-ZO and SA-ZS. Among the application methods, foliar application of ZnSO4.H2O or ZnO was superior to the soil application in increasing seed and stover yield. The interaction effect between main-plot and sub-plot factors was not significant for seed and stover yield throughout the study period.

Zinc biofortification

The Zn biofortification trait, such as Zn content in soybean grain, was significantly (p < 0.05) affected by varieties and Zn fertilization strategies in both the years (Table 4). In 2018, the variety JS-335 recorded significantly higher Zn content in grain (31.5 mg kg−1) and stover (22.8 mg kg−1) following the Zn fertilization, which was statistically similar to that of local landrace. However, in 2019, the local landrace, being at par with the variety JS-335, noted significantly higher Zn content in grain (31.4 mg kg−1) and stover (22.4 mg kg−1). In contract, the variety JS-97-52 and landrace recorded higher Zn uptakes by seed (51.5 g ha−1) and stover (41.5 g ha−1), respectively in both the years. However, the variety JS-97-52 noted a total Zn uptake of 94.6 g ha−1 during 2018 and 87.2 g ha−1 during 2019. Regardless of the sources or methods of application, Zn fertilization improved 19% and 13% of Zn content in seed and stover, respectively. The foliar spray of 0.5% ZnSO4.7H2O resulted in significantly higher Zn content in grain to the tune of 34% and 24% than the control in the first and second years, respectively. Similarly, both the FA-ZS and SA + FA-ZS recorded higher stover Zn content of 23.7 mg kg−1 (17% increase) and 23.2 mg kg−1 (23% increase), during first and second year, respectively, which was at par with SA-ZO and SA-ZS. On average, higher Zn uptake by the seed (52%) and stover (42%) was observed with FA-ZS, which was statistically comparable to SA + FA-ZS. The significantly higher total Zn uptake was observed with FA-ZS (99.4 and 94.3 g ha−1) in both the years of study, which was statistically similar to SA + FA-ZS (95.9 and 92.4 g ha−1). Among the sources of Zn, the ZnSO4.7H2O was more effective than ZnO in enhancing the total Zn uptake. However, there observed no significant interaction between genotypes and Zn fertilization strategies for Zn content and uptake over the course of the study. Regardless of varieties and Zn fertilization strategies, the Zn content in soybean seed demonstrated a poor correlation (R2 = 0.17) with DTPA-Zn content in post-harvest soil (Fig. 4.A).

Zinc bio-availability

The varieties and Zn fertilization strategies significantly (p < 0.05) influenced bioavailability of Zn in soybean grain, as indicated by the phytic acid (PA) content and phytic acid: zinc (PA: Zn) molar ratio in our study (Fig. 3). Upon Zn fertilization, the variety JS-335 demonstrated a significantly lower PA (569 and 585 mg 100 g−1) content and PA: Zn ratio (18.2 and 18.9) in grain during both the years of study, thereby resulting in higher Zn bioavailability. However, the local landrace showed lower Zn bioavailability, as it recorded higher PA content and higher PA: Zn molar ratio in grain during the trial. The Zn fertilization increased Zn bioavailability in the grain Zn by reducing the PA content in grain by 9% and 8% and PA: Zn ratio by 20% and 24% during first and second year, respectively. The treatment SA + FA-ZS significantly lowered the PA content in grain by 15% and 13% and PA: Zn ratio by 33% and 29% over both years compared to control. This treatment was statistically similar to FA-ZS, which showed, on average, a 10 and 30% decrease in PA content and PA: Zn ratio in grain, respectively. Further, these two treatments were followed by SA + FA-ZO, SA-ZS, FA-ZO, SA-ZO and control with increasing order of PA content and PA: Zn molar ratio. Moreover, it was observed that the application of ZnO, either through soil and/or foliage was less effective than ZnSO4.7H2O in enhancing the Zn bioavailability in grain. However, the PA and PA: Zn molar ratio of grain recorded a strong negative correlation (R2 = 0.57 and 0.75) with the Zn content of the grain during the course of experimentation (Fig. 4.B & 4.C).

Protein and oil content

The grain protein content remains similar among the soybean genotypes (Table 5). However, the variety JS-97-52 resulted in significantly higher oil content (17.9 and 17.9%), protein yield (714 and 647 kg ha−1) and oil yield (338 and 305 kg ha−1) in response to the Zn fertilization during both the years. The Zn fertilization strategies significantly influenced the protein and oil content and their yield (Table 5). The FA-ZS recorded higher increment in protein (11 and 10%) and oil content (9 and 8%) in soybean grain compared to the control during both years of the study. This treatment was statistically on par with SA + FA-ZS recording 10% and 10% increase in protein content and 7% and 8% in oil content, during first and second year, respectively and SA + FA-ZO recording 6% and 8% increase in protein content and 4% in oil content, during first and second, respectively. Similarly, higher protein (648 and 624 kg ha−1) and oil yield (292 and 288 kg ha−1) was observed with SA + FA-ZS during both the years, which was similar to that of FA-ZS. However, the interaction effect between the variety and Zn fertilization wasn’t significant, except for oil content in grain. The variety JS-97-52 demonstrated a positive interaction with FA-ZS and recorded a higher oil content in grain during both years of the trial.

Zn use efficiency and economics

Genotypes and Zn fertilization strategies significantly influenced the Zn use efficiency and economics of soybean during the course of study (Table 6; Supplementary Table 1). Significantly higher PFP (443 and 403 kg kg−1), AE (59 and 61 kg kg−1), gross returns (1844 and 1662 US $ ha−1), net returns (1844 and 1662 US $ ha−1) and B: C ratio (1.76 and 1.49) were observed with JS-97-52 during both the years. However, the variety JS-335 recorded higher ZnHI (58 and 61%) and ZnMEI (1.40 and 1.42) during both years. Among the Zn fertilization strategies, FA-ZS, which involves the foliar application of 0.5% ZnSO4.7H2O, resulted in significantly higher PFP (2.8-fold increase over SA-ZS in both the years), AE (4.1- and 4.2- fold), and CREZn (5.4- and 5- fold) during the both years of the study. Further, this treatment was followed by SA + FA-ZS, SA + FA-ZO, FA-ZO, SA-ZS and SA-ZO for AE and CREZn and by SA-ZS, SA-ZO, SA + FA-ZS, FA-ZO, SA + FA-ZO for PFP throughout the study. Likewise, the FA–ZS treatment exhibited higher net returns (929 and 858 US$ ha⁻¹) and benefit–cost ratios (1.39 and 1.29) in both years, although these values were statistically comparable to those obtained under the SA + FA–ZS and SA + FA–ZO treatments. On average, FA–ZS and SA + FA–ZS achieved 20% and 18% greater gross returns, respectively, and 21% and 27% greater net returns, respectively, relative to the control (no zinc application). The SA + FA-ZS, being at par with SA + FA-ZO and FA-ZS recorded significantly higher ZnHI (56 and 57%) and ZnMEI (1.47 and 1.51) during both years. Overall, it was observed that the foliar application of Zn was more efficient than soil application and ZnSO4.7H2O was more efficient than ZnO in soybean. Moreover, the interaction effect of variety and Zn fertilization strategies was significant only for PFP and AE. The variety JS-97-52 showed a significantly higher PFP and AE in response to FA-ZS treatment during both years of the trial.

DTPA-Zn and microbial activity in soil

The DTPA extractable Zn and microbial activity parameters of post-harvest soil in soybean were significantly (p < 0.05) influenced by the genotypes and Zn fertilization strategies (Table 7). Higher DTPA-Zn level (2.84 and 2.90 mg kg−1) in post-harvest soil was observed with the variety JS-97-52, while lower DTPA-Zn level (2.47 and 2.54 mg kg−1) were found with local landrace during both years of the study. Likewise, the variety JS-97-52 recorded significantly higher DHA (31 and 30 µg TPF g−1soil hr−1), FDA (5.3 and 5.4 µg g−1 soil 0.5 h−1), and SMBC (274 and 280 µg g−1) during both years of study, but were statistically similar to the local landrace. Regardless of source and method of application, Zn fertilization increased the DTPA-Zn by 14%, DHA by 12%, FDA by 10% and SMBC by 7%, in the post-harvest soil compared to control. Further, the SA + FA-ZS led to higher DTPA-Zn (21 and 25%) in the post-harvest soil during both the years, which was statistically similar to the treatments SA-ZS, SA + FA-ZO and SA-ZO. the treatment SA + FA-ZS, being at par with SA + FA-ZO, and FA-ZS, recorded significantly higher DHA (32 µg TPF g−1soil hr−1), FDA (5.5 µg g−1 soil 0.5 h−1), and SMBC (275 µg g−1) on average during the experiment. A positive correlation (R2 = 0.42–0.66) was recorded with DTPA-Zn and microbial activities in post-harvest soil (Fig. 4.D-F). However, the interaction effect between varieties and Zn fertilization strategies wasn’t significant during the study (Table 7).

Discussion

Soybean, which is cultivated extensively in the hill and valley regions of North-eastern Himalayan region (NEHR) of India, plays a central role in the diets of indigenous tribes, as it is the primary ingredient in popular ethnic foods such as kinema, hawaijar, tungrymbai, bekang, aakhone, and peruyaan44,67. Thus, enriching soybean grain with Zn through agronomic biofortification could significantly help meet the dietary Zn needs of the local population. However, severe soil acidity and Zn deficiency are major constraints to soybean cultivation in the NEHR of India68,69,70highlighting the need for suitable Zn fertilization strategies to enhance productivity and Zn biofortification. Therefore, the current field experiment was designed with these objectives, the results of which are discussed under the following sections.

Growth and yield

Varietal variation in growth and yield in response to exogenous Zn application has been widely reported in major food grain crops, including rice34wheat28maize71chickpea72and mungbean73. This indicates genetic variation in Zn utilization across crop varieties, which might be attributed to different root-based and shoot-based physiological and biochemical mechanisms58,74. In the present field experiment, the local landrace exhibited a higher LAI and healthier nodules following the Zn application to soil and foliage. However, the variety JS-97-52 demonstrated higher total chlorophyll (chl a + b) content and greater seed and stover yield (Table 2; Fig. 2). These differences might be due to the genetic makeup of the varieties and their differential response to exogenous Zn application methods and sources75,76,77.

Zinc fertilization enhanced LAI and photosynthetic pigment (chlorophyll a and b) content (Table 3). Zn plays a major role in the chlorophyll biosynthesis pathway6,78 and leaf area development, as it is actively involved in the synthesis of growth hormones (tryptophan and indole-3-acetic acid) and the plant defense system against ROS6,79. Therefore, Zn fertilization enhances the photosynthetic efficiency of crops. Similar results have been observed soybean, rice, maize, and beans80,81,82,83. Further, exogenous Zn application enhanced both nodule number and dry weight, potentially due to Zn’s direct involvement in the activity of transcription factors, such as the FUN protein, and signaling molecules like isoflavones in legumes84. These components regulate the expression of genes associated with nodule development and nitrogen fixation53,85,120. The adequate Zn supply promotes root hair deformation and supports the proper expression of early nodulation genes (e.g., GmNFR1A, GmNIN) and nodulin synthesis genes, which are essential for nodule organogenesis and maintenance53,85. Similarly86,87, also observed improvements in nodule health in soybeans due to exogenous Zn application through soil and foliage, respectively. Consequently, the improved growth attributes and symbiotic characteristics resulting from Zn fertilization led to higher grain and stover yields in soybean. The initial Zn status of the experimental soil was high (2.45 mg kg−1); yet, the soybean crop responded significantly to the different methods of Zn fertilization. This was possible because the critical concentrations of Zn in soil are site- and crop-specific10. The response might also be due to the poor availability of the native Zn owing to the severely acidic condition of the soil.

Among the methods and sources of Zn fertilization, both the combined soil and foliar application, as well as the foliar application of ZnSO₄·7 H₂O, had improved soybean yield (Table 3; Fig. 2). Basal application of Zn at 5 kg ha⁻¹ through ZnSO₄·7 H₂O, followed by foliar spray of ZnSO₄·7 H₂O at 0.25% during critical stages such as pre-flowering and pod formation, likely ensured a consistent Zn supply for plant growth and development28,34. Zn applied via foliar spray was found to be easily absorbed and translocated by soybean plants, allowing for rapid correction of deficiencies88. While soil application enriches the soil Zn pool and satisfies the initial Zn requirements of the crop, its availability progressively declines due to unfavorable soil conditions, such as extreme soil pH, high organic matter content, calcareous soil and Zn-fixing clays78,89. In contrast, foliar application bypasses these soil-related constraints by delivering Zn directly to the plant in a readily available form, thereby supporting yield formation more effectively. The response of soybean to foliar Zn application was comparable to that observed with the combined soil + foliar application method and superior to soil application alone, likely due to the crop’s limited responsiveness to soil-applied Zn and poor availability of soil Zn. Zn plays an active role in pollen germination, fertilization, and consequently seed set82,90. Therefore, foliar application of Zn, especially at the anthesis stage, proved effective in promoting healthy grain production in soybean.

The agronomic effectiveness of Zn fertilizers largely depends on the solubility of the Zn source. However, multiple soil factors-such as pH, phosphate, and carbonate content-as well as management practices, including fertilizer placement, influence Zn availability to plants91,92. In our study, the application of ZnSO₄, whether through soil, foliage, or a combination of both, enhanced soybean growth and yield compared to ZnO application (Table 3; Fig. 2). This was likely due to the higher water solubility and dissolution rates of ZnSO₄ in soil compared to ZnO91,93,94. Therefore, two foliar applications of ZnSO4, particularly at pre-flowering and pod formation stages, along with basal Zn application, should be recommended for higher soybean yields in the North-eastern Hill region of India.

Zn biofortification and bioavailability

There is significant variation in grain Zn content across soybean genotypes95, which can be exploited for the development of Zn-rich soybean varieties. However, the performance of these traits is often complicated by the interaction of various factors, especially under field conditions96,97. Therefore, it is essential to adopt appropriate Zn fertilization practices in the field. In our trial, the variety JS-335 and the local landrace showed higher Zn content in grain (31–32 mg kg⁻¹) and stover (22–23 mg kg⁻¹) following Zn fertilization (Table 4). However, the JS-335 variety demonstrated higher bioavailable Zn in grain, as it recorded lower phytic acid (PA) content and a reduced PA: Zn molar ratio (Fig. 3). Thus, JS-335 emerged as a promising variety for higher Zn biofortification and bioavailability in the NEHR and similar agro-climatic conditions. The differential responses among cultivars in grain Zn content may be attributed to variations in Zn uptake, translocation, internal mobilization, and genotype-environment interactions34. This finding is also supported by the higher ZnHI (58–61%) and ZnMEI (1.40–1.42) values of JS-335 and the negative correlation between PA and grain Zn content (Fig. 4.B). The variety JS-97-52, with higher grain yield and Zn uptake, demonstrated lower grain Zn content, indicating an inverse relationship between yield and grain Zn content in soybean. This might be due to mineral dilution effect resulting from increased grain yield or lower Zn translocation efficiency of the variety28,34. However, a larger genotypic study is needed to fully assess and confirm this relationship between yield, Zn uptake and Zn biofortification traits in soybean.

Zn fertilization through various methods improved the Zn content in the seeds and stover of soybean, in line with previous studies by43,88,98. Moreover43,98, observed superior Zn biofortification in soybean with soil application of Zn. In contrast, our experiment revealed that foliar spray of 0.5% ZnSO₄·7 H₂O resulted in higher Zn content (33–34 mg kg⁻¹) and accumulation (51–56 g ha⁻¹) in the grain (Table 4). The high initial DTPA-extractable Zn content in soil might have led to the poor response to soil-applied Zn and the better response to foliage applied Zn. This was also supported by a modest correlation (R2 = 0.17) between DTPA-Zn levels in the soil and grain Zn content (Fig. 4.A). Foliar application of Zn is readily absorbed through the leaf epidermis and translocated throughout the plant before being loaded into the grain99,100as evidenced by the higher ZnHI and ZnMEI values. The foliar spray method proved more effective in enriching grain Zn, as Zn was applied twice during reproductive stages-pre-flowering and pod formation. Similarly, the grain-filling stage is more responsive to Zn biofortification through foliar Zn spray in wheat, soybean, and rice as reported by96,101,102. While both ZnO and ZnSO₄ were equally effective in soil applications, ZnSO₄ proved to be more effective for Zn biofortification when applied as a foliar spray (Table 4)91. in an extensive review, found that ZnSO₄ was a better source for increasing yield and Zn uptake compared to Zn-EDTA and ZnO across various crops and application methods, as ZnSO₄ is more soluble in soil and bioavailable to plants. However, recent developments in nano-ZnO fertilizers suggest they may be more efficient and responsive than conventional ZnO and ZnSO₄ fertilizers in field crops, including soybean103,104.

Zinc bioavailability in food grains is a crucial aspect of Zn biofortification, as it indicates the amount of Zn absorbed by the human body. The presence of phytic acid (PA) in grains reduces Zn bioavailability by forming Zn-phytate complexes, which limit Zn absorption in the human intestine105,106. In our experiment, Zn fertilization increased Zn bioavailability in soybean grain by decreasing both PA content and the PA: Zn molar ratio. Higher bioavailability was recorded with both foliar application and soil + foliar applications of ZnSO₄ (Fig. 3). The increased Zn uptake in soybean grain may have contributed to a reduction in PA and the PA: Zn molar ratio, which is supported by the inverse relationship between grain Zn content and PA and PA: Zn (Fig. 4.B & 4.C). Such inverse relationship between PA/PA: Zn molar ratio and Zn content in soybean grain is fundamentally due to the strong chemical binding of Zn by PA during seed formation, with molecular (gene regulation) and physiological (storage/partitioning) factors reinforcing this outcome105. However, Zn application through soil and/or foliar methods has been reported to interfere with the uptake, translocation, and metabolism of phosphorus28,107potentially explaining the lower PA content in soybean grain, as phosphorus is a key component of PA in grains105. Beyond Zn bioavailability, the protein and oil content of soybean grain also improved significantly with both foliar ZnSO4 and soil + foliar ZnSO₄ applications (Table 5). The role of Zn in enhancing amino acid and protein synthesis is well established in major food crops such as rice, wheat, chickpea, beans and soybean28,108,109,110,111. As an integral part of ribosomes and RNA polymerase enzymes, Zn is essential for protein synthesis and nitrogen metabolism in plants112. Zn activates key enzymes involved in nitrogen metabolism, including nitrate reductase, thereby improving nitrogen uptake and assimilation and supporting increased synthesis of amino acids and proteins in seeds. Zn stimulates nodulation and biological nitrogen fixation, as evidenced in our experiment, which boosts nitrogen supply and enhances protein content. Moreover, oil and protein content in seeds are governed by several genes and are influenced by external factors, as these are quantitative traits113. Zn has been shown to enhance oil content and yield in soybean, as Zn is a cofactor for enzymes involved in lipid metabolism and fatty acid biosynthesis, facilitating the accumulation of oils in soybean seeds. Adequate Zn supports membrane integrity and reduces lipid peroxidation, improving oil synthesis and stability114. Similarly115, also reported increased oil and protein content in soybean seeds with higher ZnSO₄ application.

Therefore, in Zn-sufficient soils, a foliar application of 0.5% ZnSO₄ at the maximum vegetative stage, pre-flowering, and pod formation stages should be recommended. In contrast, for Zn-deficient soils, soil application of Zn @ 5 kg ha−1 through ZnSO4.7H2O followed by foliar spray of ZnSO4.7H2O @ 0.25% at pre-flowering and pod formation stages should be recommended to achieve enhanced biofortification, bioavailability, and quality in soybean in NEHR of India. Our study suggests future research on revealing the effects of agronomic Zn biofortification on Zn content and bioavailability in processed or fermented food products, value added products, and oil content of soybean.

Zn use efficiency, economic and soil properties

The Zn use efficiency (ZUE) of field crops is generally low (< 1), which may be due to factors such as Zn adsorption in soils, poor Zn absorption and utilization by crops, and other limiting factors like pH, EC, organic carbon, and nutrient interactions12,58,73,88. Variation in ZUE was observed across soybean varieties, with JS-97-52 recording significantly PFP and AE in response to foliar Zn application (Table 6). This might be attributed to the variety’s higher yield with lower Zn fertilizer application rates. The higher content of available Zn and microbial activity in soil was recorded under variety JS-97-52 (Table 6), which could have enhanced the ZUE of the variety. Cultivation of the JS-97-52 genotype yielded significantly higher gross and net returns (Supplementary Table 1), primarily due to its superior grain yield. The economic viability of agronomic Zn biofortification could be further enhanced if Zn-biofortified grain were marketed at a premium price. Among Zn fertilization strategies, foliar application of 0.5% ZnSO₄·7 H₂O during the maximum vegetative stage, pre-flowering, and pod formation stages resulted in significantly higher PFP (3-fold), AE (4-fold), and Zn recovery efficiency (CREZn) (5-fold) compared to soil application of ZnSO₄·7 H₂O (Table 6). Foliar-applied Zn is more readily accessible to the plant, allowing for maximum absorption and utilization87, whereas soil-applied Zn undergoes fixation and precipitation within the soil matrix, reducing its bioavailability12,78,88. The higher yields were achieved using lower amounts of Zn fertilizer through foliar application, resulting in enhanced ZUE (PFP and AE). Among the Zn sources, foliar application of ZnSO₄·7 H₂O proved more effective than ZnO in soybean, as ZnSO₄ is more soluble and mobile within the plant35,91. Both soil + foliar and sole foliar application of 0.5% ZnSO₄·7 H₂O resulted in significantly higher gross and net returns (Supplementary Table 1), as the combined fertilizer and application costs were minimal relative to the economic gains. Foliar application incurred lower cultivation costs, thereby yielding the highest net returns. Consequently, foliar Zn fertilization represents a remunerative and highly Zn-use-efficient strategy for soybean production on Zn-deficient soils. Moreover, its cost can be minimized by integrating ZnSO₄ application with routine pesticide sprays, as application expenses account for over 90% of total foliar Zn fertilization costs34.

Among the soybean varieties, JS-97-52 exhibited significantly higher DTPA-Zn and microbial activities in the post-harvest soil (Table 7). This might be due to release organic and low-molecular-weight compounds by roots of soybean into the soil, which enhances Zn availability and promote microbial diversity and activity116,117. Therefore, soybean cultivation should be promoted, especially in the uncultivated foothills and upland areas of NEHR to enhance soil health and cropping intensity. Soil application of 5 kg ha⁻¹ Zn, with or without additional foliar Zn application, led to significantly higher DTPA-Zn accumulation in post-harvest soil, indicating a strong residual effect on labile Zn pools and improved soil Zn fertility (Table 7). While, the soil + foliar and foliar applications of both ZnSO₄ and ZnO enhanced soil microbial activity, as evidenced by elevated DHA, FDA, and SMBC values. A synergistic correlation between soil Zn content and microbial activity was observed (Fig. 4.D-F), which might be due to Zn’s role as a cofactor or activator in various enzymatic processes81,118. Since only a small amount of Zn is removed by a single crop compared to the amount applied, the residual Zn content will be beneficial for subsequent crops91,119. Therefore, the rate and frequency of Zn applications should be tailored considering the soil type and cropping system, with regular monitoring of soil Zn status to prevent Zn toxicity, which could have harmful environmental effects on agro-ecosystems81. Based on our findings, we recommend soil (5 kg Zn ha−1) + foliar Zn (0.25% ZnSO4.7H2O at pre-flowering and pod formation stages) in Zn-deficient soils, while foliar application of 0.5% ZnSO₄·7 H₂O at maximum vegetative stage, pre-flowering, and pod formation stages is advised for Zn-sufficient soils to improve ZUE, as well as the Zn status and microbial properties of the soil.

Conclusion

A micro-nutrient deficiency under the ever-changing climate and dietary habits poses a constant challenge in combating malnutrition. Agronomic biofortification of Zn with a suitable Zn fertilization strategy can enhance Zn content in the edible parts of widely grown crops. The present study clearly demonstrated that JS-97-52 showed superior growth parameters, zinc use efficiency (PFP, AE, and CREZn), crop, protein and oil yield, whereas JS-335 showed lower phytic acid and PA: Zn molar ratio, higher Zn harvest index and Zn mobilization efficiency index. Further, the application of ZnSO₄·7 H₂O, through combined soil and foliar application (SA + FA-ZS) or foliar spray alone (FA-ZS), enhanced growth, chlorophyll content, nodulation, yield, grain Zn content, Zn bioavailability, net returns and benefit: cost ratio. Further, it also significantly (p < 0.05) reduced grain phytic acid and PA: Zn ratio, thereby improving nutritional quality. Notably, FA-ZS had the highest ZUE and Zn uptake by seed, while SA + FA-ZS improves soil Zn availability (DTPA-Zn) and microbial activities (DHA, FDA, SMBC). In a nutshell, the integration of JS-97-52 and JS-335 with foliar or combined soil and foliar application of ZnSO₄·7 H₂O could be a plausible option for achieving higher productivity, improving crop physiological attributes, nutritional quality, Zn use efficiency and soil health under rainfed conditions of the North Eastern Himalayas.

Limitations of the study

The study delivers valuable insights into the role of Zn fertilization and genotype selection for enhancing soybean productivity and nutritional quality. Still, there are certain limitations of the study, which are as follows:

  1. (i)

    The present study assessed three soybean genotypes, which may not fully represent the genetic diversity of the NEH region; thus, the wider screening of local and high-yielding varieties is essential to recognise genotypes with consistent bio-fortification potential.

  2. (ii)

    Since the soils of the NEH region are also deficient in iron (Fe), a multi-nutrient bio-fortification strategy may offer a more holistic approach for improving the nutritional quality of the food products.

  3. (iii)

    Further, the NEH region has a greater spatial variability in soils, rainfall, and altitude, which limits the extrapolation of findings to other agro-climatic regions in India.

  4. (iv)

    Lastly, the impact of bio-fortification on physiology, nutritional quality, soil Zn dynamics and microbial interactions needs long-term assessment across years under variable climatic conditions.

Fig. 1
figure 1

Agro-meteorological conditions at the experimental site during the crop growth periods.

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Fig. 2
figure 2

Effect of different sources, and method of Zn fertilization on grain and stover yield of soybean during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using Tukey’s HSD test. Where, T1− Control; T2-SA-ZS; T3-SA-ZS; T4-SA + FA- ZS; T5- SA + FA-ZO; T6- FA-ZS; T7- FA- ZO.

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Fig. 3
figure 3

Effect of different sources and method of Zn fertilization on grain phytic acid and PA/Zn molar ratios soybean during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using Tukey’s HSD test. Where, T1- Control; T2-SA-ZS; T3-SA-ZS; T4- SA + FA-ZS; T5- SA + FA-ZO; T6- FA-ZS; T7- FA-ZO.

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Fig. 4
figure 4

Correlation between Zn content in grain and soil-DTPA Zn content (a), Zn content in grain and phytic acid content (b), Zn content in grain and PA: Zn molar ratio (c), and soil DTPA-Zn and soil microbial properties (d, e and f) as influenced by the varieties and Zn fertilization strategies.

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Table 1 Initial soil properties of the experimental field (0–15 cm layer).
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Table 2 Description of the treatments employed during the two years of experimentation.
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Table 3 Influence of different sources and method of Zn fertilization on growth parameters at flowering stage of soybean during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using tukey’s HSD test.
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Table 4 Effect of different sources and method of Zn fertilization on Zn concentration and uptake at harvest of soybean during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using tukey’s HSD test.
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Table 5 Effect of different sources and method of Zn fertilization on protein content and oil content of soybean during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using tukey’s HSD test.
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Table 6 Effect of different sources and method of Zn fertilization on Zn use efficiency of soybean during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using tukey’s HSD test.
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Table 7 Effect of different sources and method of Zn fertilization on DTPA-Zn and soil microbial activity of post-harvest soil during the experimentation. Means followed by a similar uppercase letter within a column are not significantly different at p < 0.05 using tukey’s HSD test.
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