Manganese Supply Improves Bread Wheat Productivity, Economic Returns and Grain Biofortiﬁcation under Conventional and No Tillage Systems

: Manganese is an important essential micronutrient, and its deﬁciency causes latent health issues in humans. Agronomic biofortiﬁcation can promisingly improve the plant nutrient concentration without changing the genetic makeup of plants. This study was designed to assess the best method of Mn application to enhance productivity and grain Mn contents under conventional tillage (CT) and no tillage (NT) systems. Manganese was delivered through seed coating (250-mg kg − 1 seed), osmopriming (0.1- M Mn solution), soil application (1 kg ha − 1 ), and foliar application (0.25- M Mn solution). A general control with no seed Mn application was included, whereas hydropriming and water spray were used as positive control treatments for Mn seed priming and Mn foliar spray, respectively. No tillage had a higher total soil porosity (9%), soil organic carbon (16%), soil microbial biomass carbon (4%), nitrogen (2%), and soil nutrients in the CT system. Manganese nutrition through various methods signiﬁcantly enhanced the yield, grain biofortiﬁcation, and net beneﬁts for CT and NT systems. Averaged across two years, the maximum improvement in grain productivity was recorded with osmopriming (28%) followed by foliar application (26%). The highest grain Mn concentration (29% over no application) was recorded with Mn foliar applications under both tillage systems. Moreover, the highest economic returns and marginal net beneﬁts were recorded with osmopriming. To improve the wheat production, proﬁtability, and grain Mn concentration, Mn application through priming and foliar application may be opted.


Introduction
Hidden hunger is an emerging issue that is adversely affecting the global population and has become a major challenge [1,2]. Globally, there are around 0.8 billion people who are persistently hungry and malnourished [3]. Above two billion people suffer from hidden hunger, particularly in developing countries [4]. The global population is escalating with rapid growth, and food demand is also rising at the same rate. After the green revolution, the major focus of the research was to increase the food quantity, without any focus on the quality of food [5][6][7]. About 50% of the global population is influenced

Plant Material
Anaaj-2017 cultivar of wheat was procured from Wheat Research Institute, Faisalabad. Germination potential and moisture contents of the seeds were determined according to [39] and were 94% and 12%, respectively.

Experimental Details
The experiment was designed as a randomized complete block design under a splitplot arrangement; where tillage systems were kept in the main plots and Mn application treatments were placed in subplots. The tillage system was comprised of conventional tillage and no tillage systems. There were seven Mn treatments viz. no application, Mn seed coating, hydropriming, osmopriming, basal application, foliar application of Mn, and water spray. For performing a coating of the seeds, an adhesive solution was prepared with Arabic gum, and Mn (250 mg kg −1 seed) was added in sticky solution, and the seed was added in the solution for 30 min and allowed to adhere on seeds. For seed priming, seeds were dipped in aerated distilled water (hydropriming) or 0.1-M aerated Mn solution (osmopriming) for 12 h with 1:5 seed weight to a solution: volume ratio. The aquarium

Plant Material
Anaaj-2017 cultivar of wheat was procured from Wheat Research Institute, Faisalabad. Germination potential and moisture contents of the seeds were determined according to [39] and were 94% and 12%, respectively.

Experimental Details
The experiment was designed as a randomized complete block design under a splitplot arrangement; where tillage systems were kept in the main plots and Mn application treatments were placed in subplots. The tillage system was comprised of conventional tillage and no tillage systems. There were seven Mn treatments viz. no application, Mn seed coating, hydropriming, osmopriming, basal application, foliar application of Mn, and water spray. For performing a coating of the seeds, an adhesive solution was prepared with Arabic gum, and Mn (250 mg kg −1 seed) was added in sticky solution, and the seed was added in the solution for 30 min and allowed to adhere on seeds. For seed priming, seeds were dipped in aerated distilled water (hydropriming) or 0.1-M aerated Mn solution (osmopriming) for 12 h with 1:5 seed weight to a solution: volume ratio. The aquarium pump was used for the provision of artificial aeration to the solution during soaking. From the soaking solution, seeds were removed, washed by using distilled water, and dried for gaining their original weight. Mn soil application was carried out by its broadcasting at the rate of 1 kg ha −1 before seed drilling. For foliar application, 0.25-M Mn solution (Foliar Mn) or water was sprayed using a manual sprayer at the booting stage (BBCH-40) [40]. Hydropriming and water spray were considered as a positive control for osmopriming and Mn foliar application. For the determination of soil bulk density (BD), total soil porosity (TSP), soil organic carbon (SOC), total N, available P, and extractable K, soil samples were taken at final harvest from two sampling depths (0-10 cm and 10-20 cm). Whereas the soil was sampled at the anthesis stage (BBCH-69) [40] for the estimation of soil microbial biomass carbon (SMBC) and nitrogen (SMBN). Data regarding BD [41] and TSP [42], SOC [43], total N [44], available P [45], and extractable K [46] were estimated. Chloroform extraction method was used for the determination of SMBC and SMBN [47,48].

Yield Parameters
The number of productive tillers were determined from a 1 m −2 unit area in each plot from four random points at final harvest. Twenty spikes were randomly selected from each plot and after threshing grains were separated. From the same spikes, grains were counted to record grains per spike. The crop was manually harvested from each plot, tied into bundles, sundried for a week, and weighed to record the biological yield. The crop, from each plot was threshed with the help of a mini-thresher, grains were separated, and grain yield was recorded. For 1000-grain weight, three subsamples of 1000 grains from each plot were taken and weighed using digital weighing beam. The harvest index was recorded as the ratio of dry grain yield to biological yield and expressed in percentage.

Grain and Straw Mn Concentrations
Mature samples of grain and straw were taken and prepared by wet ashing [49]. Samples were oven-dried, crushed, and weighed. Afterward, these samples were digested in a di-acid mixture (HClO 4 :HNO 3 at 3:10 v/v), and Mn concentration in grains and straw was determined on atomic absorption spectrophotometer (Perkin Elmer, San Jose, CA, USA).
where GY Mn is grain yield of Mn fertilized plots, GY C is the yield of unfertilized plots, Mn a is the total amount of Mn applied, Y Mn is the grain and straw yield of Mn-treated plots, Yc is the grain and straw yield of unfertilized plots, U Mn is the Mn uptake in the grain and straw of Mn-fertilized plots, and U C is the uptake of Mn in the grain and straw of untreated plots.

Economic Analysis
For the estimation of net benefits and the benefit:cost ratio (BCR), an economic analysis was performed following [51]. For deriving adjusted grain and straw yield, the actual grain and straw yield was reduced by 10%. Seed, irrigation, fertilizers, plant protection, labor cost, and harvest were included as a fixed cost, whereas tillage operations and Mn nutrition were included as a variable cost. The marginal analysis was performed by the following [52].

Statistical Analysis
Data were statistically analyzed using computer software Statistix 8.1. For mean separation, Tukey's HSD (honest significant difference) test was applied at the 5% probability level [53]. SigmaPlot 10.0 was used for graphical representation of data.
Total N, available P, and extractable K were also affected by tillage systems during 2017-2018 and 2018-2019 ( Figure 2), whereas the results were nonsignificant for total N during the first year of study. Total N was 12% higher in the NT system, compared to CT during 2018 to 2019. The highest available phosphorus was recorded (9% and 8%) with a NT system compared to CT during both study years. Likewise, NT showed the highest values (6% and 7%) for extractable K compared with CT during the 2017 to 18 and 2018 to 19 ( Figure 2).

Yield Parameters
A manganese application considerably influenced the productive tillers during both study years. However, WTs had no considerable impact on the productive tillers. Across different WTs, the highest value for the number of productive tillers i.e., 12% higher during each year, was observed with osmopriming, compared with those in no Mn application treatment. Grains per spike were substantially improved with Mn application, and 23% and 27% higher grains per spike were observed with osmopriming during the first and second years of study, respectively. Results were statistically at par for the first year to foliar-applied Mn in case of grains per spike. The highest 1000-grain weight (20% and 32% over control) was noted with osmopriming during 2017-2018 and 2018-2019, respectively. However, osmopriming was statistically at par with Mn foliar application during both years. The highest biological yield was found by soil-applied Mn in the CT system and NT system for the first and second years, respectively. The grain yield was highest (36% and 26% over control) with osmopriming in the NT system during both study years. Grain yield (26% over control) was found with osmopriming for the second year. The harvest index was highest with a foliar application of Mn for both years (Table 3).

Grain and Straw Mn Concentrations
Manganese nutrition significantly improved the grain and straw Mn accumulation. For both years, the highest grain Mn content was recorded by its foliar application under both WTs (Figure 3). The highest straw Mn concentration was noted with osmopriming during both years in the CT and NT systems (Figure 3).

Manganese Use Efficiency Indices
The application of Mn considerably influenced the efficiency indices (Table 4). However, the effects of WTs were not significant for efficiency indices. Mn seed coating showed the highest AgE during both years. The PE was highest, with soil-applied Mn during both study years. Likewise, the AgPE was highest, with soil-applied Mn for the first year, whereas the highest AgPE was observed with soil-applied Mn in a CT system during the second experimental year; the results were statistically similar to the soil-applied Mn in the NT system. ARE and UE were highest by seed coating with Mn for both experimental years. MnHI was highest with foliar-applied Mn during both study years, and the results were statistically at par to seed coated with Mn during the second year (Table 4).

Economic and Marginal Analysis
The economic analysis showed that manganese application improved the net benefits of wheat through either application method grown in both tillage systems. Nevertheless, the highest net benefits and BCR were noted with osmopriming in the CT and NT systems. Among WTs, the NT system had the highest net benefits compared with the CT system (Table 5). Likewise, the highest marginal rate of return was recorded with osmopriming ( Table 6).

Discussion
The results supported the hypothesis that Mn nutrition in wheat by different application methods increased the productivity, net benefits, grain Mn concentration, and its efficient use in conventional, as well as conservation, tillage systems (Tables 3-6 and Figure 2); nevertheless, a variation was found for various Mn application methods and WTs. The no tillage system enhanced the soil health attributes in comparison to the CT system, as depicted by a decrease in soil BD and improvement in total soil porosity, soil organic carbon, soil microbial biomass nitrogen, soil microbial biomass carbon, total N, available P, and extractable K for both years of experimentation (Table 2 and Figure 2). Soil BD was higher under the CT system owing to intensive tillage and soil compaction with heavy tillage implements [54,55]. Lower BD under the NT system due to minimizing the soil disturbances, which makes its way towards improving the soil pores' continuity [56]. It is well-documented that with a decrease in soil BD, TSP increased [57]. Moreover, residues holding on to soil surface results in firm aggregates formation and improves TSP [58][59][60], as less soil disturbance improves the transmission and storage pores of soil [61]. Furthermore, the reduction in soil compaction under the NT system provided a feasible environment for the microbial population in the soil, thus improving the SMBC, SMBN, and SOC [62]. Higher residues retention on the soil cover amended the soil health owing to a greater availability of carbon for decomposition; as reduced soil disturbance in the NT system provide organic C continuously for soil microfauna, thus enhancing the microbial activity and biomass [63][64][65]. No tillage (NT) decreases the decomposition of SOM and reduces the soil C losses and improves the SMBC, SMBN, and SOC [66]. Soil microbial biomass carbon and SOC were improved under the NT system, which may be due to the conservation of mineralizable C from reserved residues that improve the results of biological activities of soil by increasing the concentration of soil enzymes, including phosphatase and urease [67]. Contrarily, the CT exposed stored soil carbon due to an intensive disturbance of soil that may lead to the depletion of SOC, lessening the biological activity, and active microbial biomass [68,69]. In addition, under the CT system, the dispersal of soil particles owing to intensive soil disturbances intensifies carbon-rich macropores and free the SOM particles having higher degradability and poor stability results in SOC loss [70]. Concentrations of total N and available P were improved under the NT system, because N and P are directly associated with the presence of crop residues, as it enhances the storage of N and N in the top layer of the soil [71]. Likewise, no tillage leads to more contents of P stratification near the soil surface [72]. In a CT system, the soil is highly exposed to the aerial environment that leads to N volatilization. Moreover, higher nitrate leaching was observed in a CT system compared with the NT system [73]. In CT, during plowing, soil inversion shifts fertile subsoil to the surface, leading to the possibility of leaching [74].
Both WTs and methods of Mn applications significantly affected the yield and associated traits (Table 3). Better results regarding the productive tillers, grain weight, and grain yield with the NT system were due to better soil properties and nutrient dynamics [75]. Under both tillage systems, Mn nutrition through either method significantly enhanced the grain yield of wheat (Table 3). Manganese has a significant role in photosynthesis and assimilates translocation toward grains during grain development [76]. A deficiency of Mn leads to the poor development of anthers, pollens infertility, reduction in assimilates translocation, poor seed setting, and declined grain yield [77].
Among the application methods, osmopriming and foliar applications effectively improved the yield and yield contributing components. Manganese osmopriming produced the highest number of productive tillers owing to uniform and vigorous stand establishment. Primed seeds have excessive metabolites that are readily available during planting [78]. It plays a significant role to readily start and completes the process of germination, which can lead to uniform crop standing even under adverse conditions [79] and leads to improved seed setting and grain weight [33]. Manganese nutrition improves the number of tillers and seeds set due to better pollen germination and fertilization [80]. After osmopriming, foliar-applied Mn enhanced the wheat yield for both WTs, because it is an efficient method of application owing to various reasons, including lower application rates, uniform application and distribution, and rapid plant response [81]. The lower response of Mn basal application was might due to lower SOM and alkaline calcareous soils that reduced the Mn availability to plants [82]. Grain weight with Mn application on foliage might be due to an improved source-sink relationship that ensures maximum assimilates supply during grain development [83]. Such findings could be helpful for model and non-model plants [84,85].
The manganese foliar application might improve the grain yield; as Mn foliar fertilization at the anthesis stage efficiently translocates the Mn towards reproductive parts, and then accumulates in grains [86,87]. In addition, the foliar application of Mn resulted in absorption in the leaf epidermis, and after remobilization, it was transported into developing grains via xylem [88]. Furthermore, foliar-applied Mn improved the grain Mn contents that may be due to the accumulation of Mn on flag leaf and better translocation toward grains, with the appropriate balance of photosynthates among vegetative, as well as reproductive parts.
Wheat tillage systems, different Mn application methods, and their interactions significantly affect their net benefits and BCR (Table 5). Maximum net income and BCR were achieved by osmopriming in the NT system. The lowest income and BCR were recorded where Mn was not applied in the CT system (Table 5). Nonetheless, the marginal study showed that osmopriming is the better profitable approach of Mn application with the highest marginal rate of return (Table 6).

Conclusions
The application of Mn through either method enhanced the productivity, economic returns, and grain biofortification under both tillage permutations. The highest wheat yield and economic returns were achieved with osmopriming followed by foliar-applied Mn, particularly under the no tillage system. The variations in the efficacy of Mn application methods in relation to the grain Mn contents was also found among different tillage systems, because the Mn foliar approach gave the maximum grain of Mn accumulation under both tillage systems. Among the tillage systems, the no till system improved the soil health attributes, as shown by higher TSP, SMBC, SMBN, and SOC due to the improved microbial activity and nutrient concentration compared with a conventional till system. In crux, wheat cultivation under the no tillage system with Mn application as osmopriming and foliar application is helpful in attaining better yield and grain biofortification.