Nitrogen (N), one of the essential primary macronutrients, is needed for the proper functioning of chlorophyll biosynthesis, photosynthesis, carbon assimilation, and protein synthesis processes in terrestrial plants (Leghari et al., 2016). Considering the high N concentration (46%), water solubility, and ease of use, urea is the widely used source of N accounting for almost 50% of the total N consumption worldwide (Motasim et al., 2021; Tanan et al., 2021). However, urea is hydrolyzed to ammonia and carbon dioxide in soil, whereas the presence of urease enzyme escalates this process 8 × 10⁷ times (Rana et al., 2021). Most of the soils have high urease activity that results in rapid production and release of ammonia from the applied urea fertilizer (Dejarmakeerthi and Thenabadw, 1996). According to an estimate, 20 to 70% of urea is lost from soils through ammonia volatilization (Suherman and Anggoro, 2011; Naz and Sulaiman, 2016). The crop plants recover as low as 30-40% N from fertilizer sources, like urea, and the value becomes even lower upon the losses through continuous urea hydrolysis (Shi et al., 2012; Soares et al., 2012). Hence, the use of urease inhibitor coated urea fertilizer becomes even more vital for the reduction of N losses through urea hydrolysis (Artola et al., 2011; Li et al., 2017). The inhibition of soil ureases results in a decrease in ammonia volatilization losses from soil-applied urea (Bishop and Manning, 2010).

The compounds with urease inhibition potential were largely developed to inhibit urease in the human stomach by disinfecting Helicobacter pylori. However, their use might not be suitable or economical for soil urease inhibition (Kiss and Simihăian, 2002). Among soil urease inhibitors, N-(n-butyl) thiophosphoric triamide (NBPT) is known for its high urease inhibition potential and non-toxicity to plants (Silva et al., 2017) with few limitations. Firstly, NBPT degrades over time and the addition of organic amendments in the field reduces its shelf life (Watson et al., 2008). Secondly, NBPT-induced decrease in NH₃ loss is dependent on soil type and temperature (Trenkel, 2010). Similarly, hydroquinone (Hq) is another commercially available synthetic urease inhibitor that may prove phytotoxic as reported in cowpea (Mensah et al., 2012) and rice (Pandey et al., 2005). Being a benzene metabolite, Hq may also prove haematotoxic and carcinogenic (Enguita and Leitão, 2013).

Plant materials that have urease inhibition potentials are an alternative to synthetic urease inhibitors. The list of plant materials that have been tested for soil urease inhibition is rather short, with Azadirachta indica, Acacia caven, Quillaja saponaria, Bacharis linearis, Pinus radiata, and Mentha spicata the most promising candidates (Patra et al., 2006; Suescun et al., 2012; Mathialagan et al., 2017). In our previous study, we tested the extracts of 35 plant materials for their potential to inhibit jack bean and soil ureases. In this regard, the extract of Vachellia nilotica leaves was found to be the best at inhibiting jack bean as well as soil ureases and prolonging the urea-N stay in the soil (Rana et al., 2021). In this study, various concentrations of Vachellia nilotica extract coatings on urea, at the half as well as a full dose of recommended urea-N fertilization, were tested in terms of their impact on growth, yield and nitrogen use efficiency (NUE) of wheat cultivated under pot and field conditions.

Nitrogen (N) losses, driven by the activities of soil ureases in urea applied agricultural soils, result in poor crop growth and yield along with excessive N deposition, both in freshwater bodies and atmospheric air, causing various environmental pollution risks (Sigurdarson et al., 2018). Previous studies have reported soil urease inhibition using chemical compounds, like N-(n-butyl) thiophosphoric triamide (NBPT), agrotain, and hydroquinone (Hq), etc. as urease inhibitors (Khan et al., 2013; Silva et al., 2017). However, studies reporting the use of plant extracts, like Vachellia nilotica extract, in various coating rates in comparison to chemical urease inhibitors (Hq) have rarely been reported to date.

Our previous study reported that Vachellia nilotica is a natural inhibitor of urea hydrolysis (Rana et al., 2021). In this study, we further dissected the urease inhibition potential of Vachellia nilotica extract by determining its effective dose for urea coatings and subsequent verifications through the investigations of wheat growth, yield, and N status, in field trials in comparison to Hq, a commercially available chemical urease inhibitor. In the initial pot experiment, Vn.Fl-20 and Vn.Fl-50 proved to be the best treatment, which induced significant increases in the total number of tillers, number of productive tillers, number of spikelets per spike, number of grains per spike, and 1000-grain weight ( Table 2 ). These enhancements in agronomic parameters by Vn.Fl-20 and Vn.Fl-50 applications were further replicated in the significant gains in biological, straw, and grain yields of wheat ( Table 2 ). The aforementioned improvements in growth and yield parameters of wheat, by two of the three, applied Vachellia nillotica extract coated urea treatments (i.e. Vn.Fl-20 and Vn.Fl-50), was the result of the inhibition of urea hydrolysis by Vachellia nillotica extract coating, with involved prolonged use of fertilizer-N in the soil to enable plant uptake, which might otherwise have been lost through ammonia volatilization (Rana et al., 2021). This argument was also supported by the significantly higher grain N concentration, straw N concentration, grain N uptake, and straw N uptake in wheat under the application of Vn.Fl-20 as well as Vn.Fl-50 ( Table 2 ). These increases in N concentrations and uptake could have strengthened the photosynthetic framework of wheat and ensured better carbon-to-nitrogen metabolism for efficient protein and carbon skeleton accumulation (Zhou et al., 2016; Belete et al., 2018; Iqbal et al., 2020), as indicated by the increased biomass and grain yield production ( Table 2 ).

Considering the results of the initial screening experiment, the two best performing treatments of Vachellia nillotica extract coatings on urea, viz. Vn.Fl-20 and Vn-Fl-50, were applied to field grown wheat crops for the determination of their urease inhibition potential under field conditions. Consistent with the findings of the initial screening pot experiment, both Vn.Fl-20 and Vn-Fl-50 significantly enhanced plant height, the total number of tillers, number of productive tillers, spike length, number of spikelets per spike, 1000-grain weight, biological yield, straw yield, grain yield, grain N concentration, straw N concentration, grain N uptake and straw N uptake of wheat in comparison to uncoated urea application under field conditions ( Table 3 ). Although the urease inhibition responsive growth and yield benefits to the wheat crop are not reported in the literature (Li et al., 2015), this could have been influenced by the differences in soil N status and application rates of fertilizer N. A crop cultivated on N fertilized soil or nourished well with an excess amount of N would, obviously, show either no or less response to prolonged urea N stay due to urease inhibition (Zhang et al., 2012). Besides the urea hydrolysis inhibition, the extract coatings could also act as a minor physical barrier in the release of N from coated urea granules. However, it might not be true in the case of Vachellia nilotica extract coatings due to the hydrophilic and water-soluble nature of the extract. Furthermore, the preparation of slow-release fertilizers mostly requires hydrophobic coating materials (Naz and Sulaiman, 2016). The role of coated fertilizers in the slow release of nutrients is a proven fact and their impact on crop growth and yield parameters is already reported in existing studies (Johnson and Raun, 2003). Although the maximum values of growth and yield parameters of wheat were noted at the recommended rate of N ( Supplementary Table 2 ), Vachellia nilotica extract coated urea applied at full dose as well as a half dose of the recommended N rate significantly increased biological, straw, and grain yield along with grain N uptake compared to the respective full and half doses of uncoated urea ( Table 4 ). The Vachellia nilotica extract (Vn.Fl-20 or Vn.Fl-50) coated urea applied at 60 kg N ha^-1 (half of the recommended dose of urea) achieved an almost similar straw yield compared to uncoated urea applied at 120 kg N ha^-1 (recommended dose of urea) ( Table 4 ). Understandably, the urease inhibitory role of Vachellia nilotica extract could have reduced urea losses and increased the availability of N to wheat plants by slowing down the urea hydrolysis in the soil, thus reducing the pool of exchangeable NH₄ ⁺, lowering NH₃ losses (>50%) and enhancing nitrogen use efficiency (Sanz-Cobena et al., 2011; Soares et al., 2012; Khan et al., 2013; Silva et al., 2017). Furthermore, at a half dose of urea application, the wheat plants would have faced N deficiency and, thus, the N saved through urease inhibition proved to be of more utility to them as compared to those wheat plants, which nourished well under a full dose of recommended N supply (Ghafoor et al., 2021).

In the third field experiment, the Vachellia nilotica extract coated urea (Vn.Fl-20) proved to be significantly more effective than Hq coated urea in terms of enhancements in growth, yield, and N status of wheat plants ( Table 5 ). These enhancements further ensured increments in the NPFP, NAE, PNB, and NRE of wheat as compared to Hq coated urea as well as uncoated urea applications ( Table 6 ). As NUE depends on the tradeoff between N demand and the N released from applied fertilizer. The gains in the NPFT, NRE, and PNB (NUE components) of wheat under Vn.Fl-20 coated urea were the result of improved N availability (Fixen et al., 2015), somehow, through slow urea hydrolysis leading to higher biomass and yield production per unit of applied urea-N as compared to that of uncoated urea (Zhang et al., 2012). On the other hand, the internal utilization efficiency (IUE) is defined as the grain yield in relation to the amount of N taken up. A better N uptake requires the controlled release of N from the fertilizer source. A high release of N does not need to ensure higher N uptake by plants. In the case of low N uptake, the IUE remains high (Shewry, 2007; Hawkesford, 2014), as was the case in our study under uncoated urea treatment ( Table 6 ). Physiological efficiency (PE) expresses the yield increase in relation to the increase in crop N uptake. Accumulation and distribution of N in the vegetative and reproductive organs of crop plants are important processes for determining PE. In this study, PE remained non-significant in either Vn.Fl-20 coated or uncoated urea treatments ( Table 6 ), perhaps, due to the dynamic characteristics of N uptake by wheat plants (Zhang et al., 2009; Obi et al., 2022), which should be further investigated.

In our previous study, Hq was used as the reference urease inhibitor for urea hydrolysis in soil up to 50% (Rana et al., 2021). However, in this study, the effect of Hq on growth, yield, N status, and NUE of wheat was either statistically at par or lower than that of Vn-Fl-20 ( Tables 5 , 6 , and Supplementary Table 4 ). We argued that the inhibitory effect of Hq on urea hydrolysis could be of short-term nature as compared to Vn.Fl-20. This argument is further supported by the fact that the application of Hq with urea significantly reduces N₂O emission in the wheat-rice system but does not increase the grain yield of wheat in comparison to the application of urea alone (Malla et al., 2005). Moreover, the phytotoxic effects of Hq reported in the case of cowpea (Mensah et al., 2012) and rice (Pandey et al., 2005), could have also reduced its effectiveness in comparison to Vn.Fl-20. Furthermore, Hq, being a benzene metabolite, possesses haematotoxic and carcinogenic characteristics (Enguita and Leitão, 2013). N-(n-butyl) thiophosphoric triamide (NBPT) is reported to interfere heavily with urea-applied N availability in maize plants, which limits the N influx and assimilation (Zanin et al., 2015). Moreover, their effective inhibition potential is a short period with limited shelf life, which calls for the need for further efforts in their research and development (Cantarella et al., 2018). On the other hand, as compared to Hq, in particular, Vachellia nilotica leaf extract is a natural product that has minimal environmental and hazardous consequences. It may also prove cost-effective and easy to access, especially, in regions abundantly inhabited by Vachellia nilotica trees.

Given its urea hydrolysis inhibition potential, Vachellia nilotica extract coating on urea prills (Vn.Fl.20) enhanced grain N concentration, straw N concentration, grain N uptake, and the straw N uptake of wheat. These conditionings of N, subsequently, resulted in the increased nitrogen partial factor productivity (NPFP), nitrogen agronomic efficiency (NAE), partial nitrogen balance (PNB), and nitrogen recovery efficiency (NRE) of wheat. The increments in the nitrogen use efficiency components by Vn.Fl.20 coated urea, in comparison to both uncoated and hydroquinone (Hq) coated urea under pot as well as field conditions, resulting in the enhancement of the growth and yield attributes of wheat. Hence, Vachellia nilotica leaf extract (Vn.Fl-20) can be used as natural urease inhibitor with minimal phytotoxic consequences and maximum soil N retention benefits.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

XL, RM, and MAR conceived the idea and designed the research. MAR, RM, and FN conducted the research. RM and MAR analyzed the data and wrote the paper. FN, YW, CJ, and XL revised the manuscript. All authors approved the final manuscript.

The study was supported by the China Postdoctoral Science Foundation (2022T150571), Healthy Soil Projects in DongYang (JCZFCG2022-Z37-C303), and an indigenous Ph.D. Fellowship grant (213-64972-2AV2-162-50025766) by the Higher Education Commission (HEC), Pakistan.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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