Bioactive Zn (BAZ) was formulated using the patent method of Niha Corp, Ontario, CA, United States as a fine dry powder of biologically solubilized and organically encapsulated Zn (Tariq et al., 2017). BNFF Urea Z was developed by First Biotech LLC, Lahore, Pakistan an associate of Niha Corp., United States, using the patented process (Tariq et al., 2017). Engro Fertilizers Ltd. (EFERT), having exclusive marketing rights of BNFF Urea Z for Pakistan, launched under the brand name Zabardast Urea (ZU) in 2017. ZU contains 42% nitrogen, 1% BAZ, and a consortium of beneficial microbes 10³ CFU g^–1 of ZU material. The beneficial microbes are a consortium of Zn-mobilizing bacteria with multiple plant benefits (Tariq et al., 2017).

Cross-ecological trials were carried out during the wheat season 2019–2020 to evaluate the ZU application on the yield and quality of 15 bread wheat (Triticum aestivum) varieties. Eight replicated trials were conducted spread over eight different sites (i.e., Shujabad, Multan, Chichawatni, Lahore, Sahiwal, Faisalabad, Kasur, and Chiniot) on farmer fields; in addition, 119 demonstration trials were conducted at 42 different sites. Random soil sampling was done at a depth of 0–20 cm. Samples were pooled, homogenized, air-dried, sieved (2 mm), and characterized for different parameters such as pH, EC, available phosphorous and potassium (US Salinity Laboratory Staff, 1954; Page et al., 1982), and extractable metal (Zn) (Soltanpour, 1985). The soil data are presented in Supplementary Table 1.

The treatment plan at replicated field trials at eight locations is as follows:

The layout of the field experiment was RCBD split-plot design with treatments as main plots and variety as subplots with three replicates at each location. The plot size per replicate was 252.9 m².

For 119 farmer field trials at 42 locations, the treatments were as follows:

The plot size at farmer sites was 0.5 acres (2,023.4 m²) per treatment for each trial.

Sowing was performed from November 15 to December 15, 2019. Uniform application of nitrogen, phosphorus, and potassium was done across all treatments at the rate of 250, 185, and 150 kg ha^–1, respectively. Diammonium phosphate (DAP) was used as a phosphorus source and mutate of potash (MOP) as a potassium source, while the use of nitrogen and Zn varied as explained above in the treatment plan. All P and K fertilizers were applied at the time of sowing, whereas nitrogen was applied in two splits, i.e., half 25 days after sowing and the remaining half 55 days after sowing using the broadcast method followed by flood irrigation. In ZU-treated plants (T4), the ZU was applied during the first split dose, while normal urea was applied in the second dose. The plants were irrigated with canal water as and when required.

Productive tillers (m^–2) were determined 70 days after sowing as an average of three random locations from each replicate plot using a 1 m^–2 steel template. A sample of 15 random plants was selected from each treatment at each location for data readings related to growth and yield at harvest (125 days after sowing). Plant height and spike length were measured using a meter scale. Shoot fresh biomass was recorded for a random sample of 15 plants of each treatment (5 from each replicate) at all locations. The number of grains per spike was determined from 15 spikes collected randomly from the sample of 15 plants of each treatment (5 plants per replicate plot). After recording aforesaid data, plant samples were threshed and 1,000 kernel weight was determined by weighing 100 grains of three sets, then multiplying their average by 10, and recorded for each treatment, accordingly. For final yield data, the wheat crop of each plot was harvested, threshed, weighed, and recorded for grains and straw, separately for statistical analysis. The farmer field trial data were harvested from the whole plot and yield was determined.

This was a random sample, farmer field trial study that precisely detected the differences in the varietal response within and across the environments. Engro field trial data at eight different sites were subjected to analysis of variance using computer software Statistix version 8.1 (Analytical Software, United States). The treatment data were averaged over locations to calculate the mean response of ZU across the cross-ecological trials. The treatment means were compared using the least significant difference test (Steel et al., 1997) at a 5% probability level. Similarly, the farmer field data were averaged over varieties and 125 locations to analyze the varietal response. Correlation analysis, regression, and principal component analyses were performed using IBM SPSS software for Windows (SPSS Version 20, NY, United States).

Statistical analysis of data obtained from field trials at eight locations revealed that the application of ZU produced the highest number of productive tillers as compared to other Zn treatments and non-Zn controls (Figure 1A). The average productive tillers were significantly high in ZU-applied plots (396) with a percentage increase of 6.45 over non-Zn control (372). However, the productive tillers with Zingro (388) showed an increase of 4.30 and with ZnSO₄ (386) a 3.5% increase over non-Zn controls. The ZU-treated plots also produced the longest spikes (9.9 cm) as compared to non-Zn control (9.0 cm) and other sources of ZnSO₄ (Figure 1B). The percentage increase by ZU was 9.9%, followed by Zingro and ZnSO₄ treatments with 5.9 and 3.7% increase, respectively, over non-Zn controls.

The number of grains per spike showed a statistically non-significant effect (Figure 2A) by different Zn application treatments from field trials at eight locations. On average, ZU application showed maximum grains per spike (49.3) followed by Zingro (48.6) and ZnSO₄ (48.0) compared with non-Zn controls (46.9). The 1,000 grain weight, however, showed a significant treatment response where maximum grain weight was observed in ZU-treated plots (34.0 g) followed by Zingro (33.2 g) and ZnSO₄ (32.5 g) compared with non-Zn controls (31.6 g) (Figure 2B). The percentage increase in grain weight was 7.5 with ZU treatment, 3.6 with Zingro, and 2 with ZnSO₄ over non-Zn controls.

The total biomass obtained from eight field trials at eight locations (Figure 3) showed a significant increase by the application of ZU (13.5 t ha^–1) with a percentage increase of 12 over non-Zn controls (12.0 t ha^–1). This was followed by other Zn treatments, i.e., Zingro (13.3 t ha^–1), and ZnSO₄ (12.8 t ha^–1) with an increase of 10 and 6%, respectively, over non-Zn control.

All Zn treatments generally exhibited a positive impact on the grain yield (Figure 3), but comparative analysis showed that ZU treatment produced the maximum grain yield (4.9 t ha^–1), where the percentage increase over control was 11.7% followed by Zingro (4.7 t ha^–1), and ZnSO₄ (4.5 t ha^–1) with an increase of 7.2 and 2.8% over control, respectively. A similar trend was observed for the data obtained for the straw yield (Figure 3) where the maximum yield (8.6 t ha^–1) was obtained in the ZU plots with an increase of 12.6%, followed by Zingro (yield: 8.5 t ha^–1; percentage increase 11.8%) and ZnSO₄ (yield: 8.2 t ha^–1; percentage increase 8.0%).

The data for the impact of different Zn treatments on harvest index (HI) from field trials at eight locations is given in Figure 4. The HI data showed a statistically non-significant response of treatments. However, the HI of ZU plots were relatively higher (0.37) than ZnSO₄ and Zingro (0.35 and 0.36, respectively).

The statistical analysis of grain Zn contents data obtained from field trials at eight locations (Figure 4) shows that the impact of Zn treatments was statistically significant. ZU application showed the maximum increase in grain Zn contents (57.00 μg/g) with a percentage increase of 171.4 over non-Zn controls. This was followed by Zingro (38.17 μg/g) with a percentage increase of 81.8 and ZnSO₄ (31.63 μg/g) with an increase of 56% over non-Zn controls (21 μg/g). The average Zn contents in grains of all wheat varieties were maximum in ZU-treated plots at all sites.

The ZUE of different Zn sources used is mentioned in Supplementary Table 3. The data obtained from field trials at eight locations show that ZUE of ZU plots is highest, i.e., 410 followed by the Zingro, i.e., 21, and then ZnSO₄ with 8.

From the data obtained from farmer field trials (n = 119), total yield and Zn contents were measured, and a comparative analysis was made among the ZU vs. farmer practice/check plots. The range of average yield in different varieties in the check plots was 2.67–4.45 t ha^–1 compared with ZU-treated plots with a yield range of 2.97–4.84 t ha^–1. An increase in yield with ZU application was observed as a general trend in all 15 varieties when data were averaged over 119 locations, but the yield of 11 varieties showed an increase of more than 10% over the check plots (Supplementary Figure 1A). The varieties, such as Akbar 2019, Ujala, Faisalabad 2008, and Abdul Sattar, showed more than a 20% increase in yield when the data were averaged over locations. The percentage yield increase was less than 10% in only four varieties (Sehar, Punjab 11, Gandum 1, and TD1).

Likewise, average Zn contents of different varieties ranged from 13.00 to 57.33 μg/g with farmer practice while 27.50–69.00 μg/g with ZU treatment (Supplementary Figure 1B). In control plots, except for one variety TJ-83 that showed an inherent potential of high grain Zn contents (57.33 μg/g), all varieties showed Zn contents ≤ 35 μg/g, which are significantly lower than the minimum limit recommended for human consumption. In contrast, ZU treatment substantially increased the grain Zn in all the varieties with a 20–214% increase over the check. Maximum Zn accumulation was observed in a variety Shahbaz that showed 69 μg/g grain Zn, followed by TJ 83 (68.67 μg/g) although it has an inherent ability to accumulate high Zn (Supplementary Figure 1B); wheat variety Al-Ghazi followed with 56 μg/g and Galaxy and Abdul Sattar with ∼46 μg/g Zn in grains. The Zn accumulation in TJ-83 grain shows that it has an inherent ability for Zn accumulation, but the varieties, such as Shahbaz, Al-Ghazi, Sehar, and Ujala, showed an increase in Zn mobilization and accumulation only with the application of ZU.

When data were compared concerning soil (Figure 5), it was observed that Zn contents in wheat varieties vary at different locations/farmer sites. As the varieties recommended at each location/region differ, a general comparison is difficult to draw, but the difference in varietal response may be attributed to the soil conditions, farmer practice, and climate conditions.

A positive linear relationship was found among different parameters from the data obtained from farmer fields. Linear regression effectively modeled the positive relationship of grain yield with grain Zn contents, accounting for 65% of the total variance. A positive linear regression (R² = 0.83) was observed for yield and Zn contents in the check plots and ZU-treated plots (R² = 0.89) (Figures 6A,B). Higher regression value for ZU-treated plots shows the key varietal response toward ZU for Zn uptake and grain accumulation. The CAT-PCA (Figures 7A,B) captured more than 89% of the variance and clearly demonstrated the key environmental differences in the treatment response. The effect of soil was more pronounced (Figure 8A) than the treatments that loaded variably on different quadrants (Figure 8B) in different soils.

The ZU exhibited a significantly positive response to facilitate Zn translocation from the rhizosphere to the grains compared with chemical Zn fertilizers in all wheat varieties. Nutrient uptake correlates with grain accumulation and shows a positive impact on plant metabolism and growth (Cakmak and Kutman, 2018; Ahmad et al., 2019). Zn controls several important growth- and yield-regulating processes in plants, e.g., photosynthesis and protein synthesis. It is anticipated that BAZ from ZU made more Zn available for plant uptake. On average, at replicated trials, ZU increased the grain Zn from 21 μg/g (control) to 57 μg/g (171% of the increase), whereas at farmer field demonstration plots ZU increased the grain Zn from 26 μg/g (control) to 44 μg/g (69% of the increase), which is reasonably higher than the desired levels of Zn in wheat grain, i.e., 40 μg/g recommended for human consumption. At farmer fields, the varieties Shahbaz, TJ-83, Al-Ghazi, and Abdul Sattar expressed higher Zn accumulation (>44 μg/g), which can be recommended for the widescale cultivation. Human consumption of this biofortified wheat in a selected population can further validate the implications of ZU technology in terms of reducing malnutrition.

Apart from the beneficial role of BAZ, the ZSB are involved in the increased bioavailability of Zn in plants through the solubilization of insoluble soil Zn fractions present in the rhizosphere. ZSB possibly increased the higher Zn availability during the grain filling stage, which increased the activity of source (flag leaf and stem) and thus more accumulation in grain as previously reported by Cakmak et al. (2010). Enrichment of cereal grains has been reported by ZSB alone and with the combination of organic matter (Hussain et al., 2020). This microbe-mediated grain accumulation of Zn may also cause a reduction in the antinutrient agent, e.g., phytic acid, gluten, tannins, oxalates, lectins, leptins, and saponins, which is helpful to improve the bioavailability of nutrients for human consumption (Vaid et al., 2014; Naz et al., 2016). Phytic acid in grains is not bioavailable and binds to Fe and Zn in grains and makes them unavailable to humans (Thompson, 1989). Reduction in phytic acid accumulation in grains has been related to the increased Zn contents and could be a possible reason for the grain biofortification in this study as reported earlier by Ramesh et al. (2014). The grain phytic acid or the amount of other antinutrients were not determined in this study but may be tested further to see the bioavailability of the accumulated Zn in the grain.

This study also establishes enhanced “ZUE” as indicated by the active uptake, translocation, and accumulation of Zn in grains of all the wheat varieties. It is established that application of Zn in soil, or as foliar treatment, increases the grain Zn concentration in wheat varieties, e.g., Punjab-11, Faisalabad-2008, and Sehar (Kiran et al., 2021). The translocation and mobilization of Fe and Zn in the grains depending on their concentration in the vegetative tissues of the plant, N status of soil, and nature and type of the plant species or cultivars (Cakmak et al., 2010; Rehman et al., 2018; Singh et al., 2018; Kiran et al., 2021). Our study demonstrates that ZUE of wheat can be increased significantly (up to several folds) by using ZU technology despite 12-folds less Zn (1.235 kg) compared with conventional application practice (15 Kg).

The available Zn in soil and other nutrient concentration cause a significant impact on the uptake of Zn in the grain. The treatment loading response was variable depending upon the soil condition as evident from the PCA analysis. In Multan, the available P, K, and Zn were relatively lower compared with Shujaabad, which ultimately was reflected on the differential loading of treatments on the PCA plot, although both of these locations are situated in the similar geoclimatic zone. Similarly, Faisalabad and Chiniot, Kasur and Lahore, and Sahiwal and Chichawatni are not very different from each other from a climatic perspective, but the P, K, Zn, B, and SO₄ available in soil are significantly different, so the treatment response and loading are different.

The data from the farmer fields further show that the ZUE of ZU is higher compared with other Zn recommended forms in all types of soils, i.e., nutrient-sufficient soils or nutrient-deficient soils, and explain the differences in uptake at different locations of the same variety and vice versa. The uptake of micronutrients in plants is affected by soil conditions. During farmer field demonstrations at Lodhran, wheat variety Shahbaz showed a yield increase of only 11% but Zn contents were increased up to 214% with ZU application. At the same site Lodhran, another variety Galaxy grown at two locations, the yield increase was 8.9 and 7.9%, while Zn content increase was 34 and 113%, respectively. It has been reported that at farmer fields in Lodhran and Multan, the plant-available Zn and organic matter in the surface soil are 0.1–1.2 mg/kg and 3–17 g/kg, while they are 0.0–0.9 mg/kg and 0–10 g/kg in the sub-surface soil (Maqsood et al., 2014). This high variation in organic matter affects the Zn enrichment of cereal grains (Hussain et al., 2020). A great variation in the nutritional status of Lodhran soil might have responded to this exceptionally high uptake of Zn in grain in this study.

Widescale field application of ZU demonstrates a significant increase in the growth, yield, and quality of wheat along with a significant increase in the grain Zn contents. ZU outperformed all treatments including chemical Zn fertilizers (farmer practice) at all locations by producing an average yield of 4.06 t ha^–1, which is 13% higher than controls (3.59 t ha^–1). This overall increase in plant health and growth could be the synergistic response of BAZ and the supporting activities of microbes, such as P-solubilization, ACC-deaminase activity, production of siderophores, and indole-3-acetic acid potential (Hussain et al., 2015). These microbial traits enhance not only Zn uptake but also other nutrients, establish an extensive root system, and contribute toward better plant health and growth (Whiting et al., 2001). The beneficial microbes produce a variety of organic acids that reduce the pH of the surrounding environment and shift the dynamic equilibrium of minerals from non-labile to labile form which ultimately improves the nutrient uptake, P, Fe, etc., and accumulation in plants (Wani et al., 2007). The microbe-mediated root development and proliferation enhances the capacity of the plant to uptake more nutrients from the soil and provides stronger anchorage to resist lodging at the later stage (Ditta et al., 2018).

It has been recently reported that the application of foliar Zn in wheat varieties Faisalabad-2008, Punjab-11, Saher, and Lasani-2008 shows a significant increase in crop growth rate, plant height, leaf area, total chlorophyll, spikelet per plant, spike length, grains per spike, number of tillers, and productive tillers (Kiran et al., 2021). The Zn application also increased biological yield, harvest index, and grain yield, but the impact was statistically non-significant (Kiran et al., 2021). Similarly, this study reports a statistically significant increase in different growth parameters by application of BAZ, but the impact on harvest index was statistically non-significant. The results have a wide impactful implication of the product to find out the fate of BAZ and beneficial microbes fortified Zn sources in the plant cell viz. translocation in the cellular system and the possible interplay in food remobilization during plant growth.

Apart from the application form, Zn nutrition significantly enhanced the benefit-cost ratio. The data show a value to cost ratio (VCR) of 16.0 for ZU, 2.8 for Zingro, and 1.1 for ZnSO₄ (Supplementary Table 2). This means that spending one rupee will result in a benefit of Rs. 16 with ZU, while 2.8 for Zingro and 1.1 for ZnSO₄. This cost economics is the most significant factor for the farming community that tends to look for profit maximization. Product profitability is principally associated with the farmer inputs and the yield obtained. Higher VCR for ZU treatment is due to higher yields as well as minimum inputs that subsequently ascertain the monetary benefit to the farmers. As ZU is a urea-coated product, so is more user-friendly as farmers do not need extra application (Zn fertilizer) in the field. It will also contribute significantly in optimizing the cost of Zn to farmers than using Zn separately from the range of chemical products available in the market.

The novel BNFF technology has the inherent potential to transform a range of essential plant nutrients into bioactive form. The technology has shown potential and effectiveness in increasing nutrient use efficiency, optimizing the cost of agricultural production, and improving the economics of farmers. The technology has tremendous potentials for future food security and biofortification program of cereals or other agricultural comodities with necessary micronutrients identified as a potential threat to human and animal health not only in Pakistan but in the world at large. The technology can be replicated for cereals, fodders, fruits, and vegetables without changing the fertilizer-application procedure of farmers. The user-friendliness of BNFF technology will help quicker expansion in its application area and crops. The growing use of this technology will continue to contribute positively to improving farmer economics in terms of better yield and quality.