Assessing the Effectiveness of Zn Acetate and Oxide as Alternatives for Corn and Soybean Seed Treatment in Sandy and Clay Soil

Zinc (Zn) is the micronutrient with the lowest availability in agricultural soils, and consequently 50 % of the world’s soils present Zn deficient. To test the viability of alternative Zn sources (Zn acetate and Zn oxide) to corn and soybean seed treatments, we ran an experiment using these two alternatives at contrasting application rates (0; 0.25; 0.50; 0.76 and 1.01 g kg) applied to soybean and corn seeds that were subsequently sowed in sandy and clay soils. We measured: Zn accumulation, dry matter and germination, and analyzed this data using uni (LSD-test) and multivariate analysis (Principal Component Analysis, PCA). Results of the PCA showed that the sandy soil yielded higher dry matter and Zn accumulation than the clay soil. The corn provided higher dry matter while the soybean showed enhanced Zn accumulation and germination. The LSD test showed that corn presented positive Zn accumulation in response to Zn rates in both sandy and clay soil. For soybeans, this effect was only observed in sandy soil, while the clay soil presented decreases in dry matter and germination due to Zn rates. Overall, our findings reveal that both Zn acetate and Zn oxide are viable alternatives for supplying Zn to corn seed treatment in sandy and clay soil, and to soybean seed treatment in sandy soil. We suggest that more research should be undertaken to understand the response of soybean seed treatments to Zn supply, especially in clay soil.


Introduction
Zinc (Zn) is widely considered to be the micronutrient with the lowest availability in agricultural soils (Alloway, 2011), and consequently, 50 % of the world's agricultural soils have been classified as Zn low level (Fageria et al., 2002).Under normal conditions, Zn is found within the soil superficies due to its low mobility and strong relationship with organic matter (Dechen & Nachtigall, 2006) and clay present in the soil (Alloway, 2011).
With respect to plant metabolism, Zn plays an essential role in nitrogen metabolism (Faquin, 2005), and enzymatic activation (Dechen & Nachtigall, 2006;Nonogaki et al., 2010), which are in turn strongly related to crop development and the resulting productivity of grain and cereal crops (Fageria et al., 2002), and the Zn causes decrease in radicular development.
Zn fertilization of plants can be performed through direct addition to soils, or application to seeds and leaves.Soil Zn fertilization has been shown to enhance corn, Zea mays L. (Pereira et al., 1973;Galrão, 1996) and soybean (Glycine max L. Merril) productivity (Sousa et al., 1993).On the other hand, Zn fertilization of seeds has been shown to improve crop productivity, seed germination and plant growth (Nonogaki et al., 2010), which presents advantages in terms of application uniformity and small rates with precision (Lopes & Souza, 2001), and can be considered the best alternative to Zn fertilization (Boneccarrére et al., 2004;Ribeiro et al., 1994).However, the seed treatment efficiency should be tested according to the seed characteristics, Zn application rates and sources.

Experiment Installation
In the greenhouse facility, the sandy and clay soils were prepared using the rate of 1.99 and 6.26 g pot -1 calcium carbonate, and 0.85 and 2.68 g pot -1 magnesium carbonate, respectively, of according of soil tests to achieve 60% of base saturation.Prior to being incubated, samples were maintained at 70% of the field capacity for two weeks, aiming the time reaction.After this period, fertilizers (5.1 mg kg -1 of urea, 100 mg kg -1 of Mono-Ammonium-Phosphate and 50 mg kg -1 of Potassium sulfate) were broadcast evenly across the soils and mixed thoroughly.Micronutrients were also provided during planting in the form of 0.2; 5.0; 3.0; 0.1; and 1.5 mg kg -1 of Boric acid, EDTA Iron, Manganese oxide, Ammonium molybdate and Copper sulfate, respectively, according to plant needs and soil test (Table 1).
Posteriorly, pots were filled with soil (4.5 kg soil pot -1 , total volume of 4.3 L).Zn acetate and oxide application rates were prepared by dissolving the appropriate volumes of material in deionized water and applied at soybean (Cultivar TMG 1168 RR) and corn seeds (Hybrid 2B810PW).Six seeds were planted in every pot at a depth of 3 centimeters.
Fifteen days after sowing, seedlings were thinned two plants per pot and another application of fertilizer was performed via nutrient solution containing 12.5; 12.5; 18.7 and 0.1 mg kg -1 of Urea, MAP, Potassium chlorate and boric acid, respectively.Throughout the experiment, plants were irrigated every day with deionized water and humidity was maintained at about 60 % of the soil field capacity.

Parameters Measured and Data Analysis
On the 8 th and 13 th day after sowing, the number of emerging plants were counted to check the seed germination (GD) per pot, using the physiological criterion of protrusion (≥ 5 mm), of the first visible structure.The dry biomass production of the aerial part was collected on the 30 th day after sowing (V6-V8 corn stage and R6 soybean stage), dried to a constant weight at 75±2 °C and weighed.Dry biomass samples were used to determine the plant zinc accumulation as described in Malavolta et al. (1997), with the value (Zn concentration) multiplied by the aerial dry biomass to obtain the total zinc accumulated.
The descriptive statistics assessed included: the mean, standard deviation, minimum and maximum values.The assumptions of normality were evaluated using the Shapiro-Wilk test and outliers were identified by the Grubbs' test (Grubbs, 1969) and homogeneity of variance was assessed using the Bartlett test.
We used Principal Component Analysis (PCA) to identify variation between soil textures and plant species using the variables: dry matter, zinc accumulation, 8 th and the 13 th germination day after sowing.Therefore, initially, the date group was standardized to obtain a zero mean and constant variance (Sneath & Sokal, 1973), and a Euclidean matrix was calculated (Manly, 2008) using the Ward algorithm to group similar data points (Hair et al., 2010).The Principal Component (CP) with eigenvalues greater than 1, was used because it provides relevant information about the original variables (Kaiser, 2002).The results of the PCA were presented separately as Bi-plots for each soil texture and plant species to identify the groupings of information. jas.ccsenet.

Soil Te
PCA was a with the m significant influence s for the 8 th

Zinc Rates
For sandy soil, the effect of Zn application rates on Zn accumulation by soybean plants fitted a linear response curve (R 2 = 88.5 %; P < 0.01), as did corn plants (R2 = 81.7 %; P < 0.01), Table 3 and Figure 3. Table 3. Mean estimates of dry matter (DM), Zn accumulation (Zn Accu.), 8 th and 13 th germination day (GD) for corn and soybean seeds in sandy soil
Furthermore, in sandy soil the control treatments provided the lowest Zn accumulation by plants with the decrease of 43 % and 9 % compared to the smallest Zn rate tested, respectively for soybean and corn plants.The effect of Zn application rate was not significant for estimates of dry matter weight or germination counts.For all tested variables, there was no significant interaction between Zn rates and sources (Table 3).
Figure 3.The effect of Zn rate on Zn accumulation (µg pot -1 ) for corn and soybean seeds in sandy soil In clay soil, the effect of varying Zn application rate on soybean seeds fitted a negative quadratic response curve for dry matter (R 2 = 95.1 %; P < 0.01), and for the 8 th (R 2 = 95.7 %; P < 0.01) and 13 th (R 2 = 40.3%; P < 0.01) germination day (Table 4 and Figure 4).
Additionally, in clay soil corn seeds presented a positive interaction between Zn sources and application rates with respect to Zn accumulation and dry matter content (Table 4 and Figure 4).Zn acetate provided an average increase of 7.6 % and 8.9 % in Zn accumulation and Dry matter when compared to Zn oxide.
Figure 4.The effect of Zn application rates on dry matter content (g pot -1 ), 8 th and 13 th germination day for soybean seeds in Clay soil For Zn oxide, a positive curve described the relationship between application rate and both Zn accumulation (Linear, R 2 = 76.3 %; P < 0.01) and dry matter (Quadratic, R 2 = 66.3 %; P < 0.01).While, for Zn acetate it was possible to fit a quadratic response curve to describe Zn accumulation (R 2 = 92.7 %; P < 0.01), and dry matter (R 2 = 83.9%; P < 0.01), with increment by the rate of 0.6 and 0.5 g kg -1 seeds (Table 4 and Figure 5).According to these trends, it was possible to recommend a Zn oxide application rate of 1.0 g kg -1 , and a Zn acetate application rate of 0.5-0.6 g kg -1 to obtain the highest Zn accumulation and dry matter for corn seed treatments.

Zinc So
When app showed no difference accumulat cultivated worth poin among all

Clay Soil
dry matter (g po atments, Zn ac (Figure 6).Th with the exc xide for corn p µg pot -1 for so nificant differ pot -1 ), 8 th and y soil

Soil Texture and Plant
PCA results showed that the variance concentration along the axis CP1 and CP2, accounted for 74.2 % of the total variance, and attending the criteria of at least 70 %, described by Senath and Sokal (1973).Therefore, PCA appears to be an appropriate statistical approach to examine the relationship between soils and plant.
The differentiation of soil type into two distinct groups (sand vs. clay) can be attributed to the increases of 50 % and 70 % dry matter and Zn accumulation in clay soils, which may be governed by the soil Zn dynamics, and the fact that this element is intrinsically associated with soil minerals, mainly clay (Malavolta, 2006;Inocêncio et al., 2012) and organic matter (Malavolta, 2006).Both, were higher in clay soil, and are able to adsorb Zn from the soil over relatively short periods to trigger Zn decrement for plants and seeds.This is backed up by the observation that prior to experiments, the clay soil presented higher concentrations of Ca 2+ , Cu 2+ and P, which are known to inhibit Zn absorption.Interestingly, there was no effect on germination, probably because of adequate water availability during the experiment for both soils.
PCA also highlighted the clear disparity between corn and soybean, through the observation that the dry matter vector was closer and higher with respect corn, representing an increase of 35 % when compared to soybean.This may be partially explained by plant physiology, as corn plants given adequate temperature and soil moisture achieve the V3-V4 stage at 30 days and present greater development, three developed leaves (Magalhães & Durães, 2002).Over a similar period of time, the soybean plants also presented the V3-V4 stage, but showed a dramatic increase in visible root nodules (Fehr & Caviness, 1977) and greater root development.In this way, our results reflect the differential development of plant crops, with soybean showing rapid initial developed of roots while corn showed greater increases in above ground part.
Interestingly, we observed that soybean presented higher germination on the 8 th and 13 th day in sandy and clay soil, which represented an increase of almost 47 % compared to corn.While the positive effect of Zn application have been shown for both soybean germination (Santos et al., 1986), and corn seeds (Ribeiro, 1993), these benefits are probably more pronounced for corn seeds than soybean.The Zn applied to seeds triggers the translocation of Zn from the seed to the plant during and after germination (Muraoka, 1981).

Zinc Rates
Corn and soybean usually present high responsiveness to Zn addition in soils that have pronounced Zn deficiency.Our results concur with this observation, showing that in sandy soil, corn and soybean fitted a linear response curve for the relationship between Zn accumulation and Zn application rates, as has been affirmed by Prado et al. (2007) testing Zn rates using corn seed treatments.The addition of 1 g kg -1 of Zn, regardless of source, appears to have a positive effect on Zn accumulation and it is associated lower Zn level and clay concentration, before the experiment.This is apparent when we compare the control treatments, which yielded the lowest Zn accumulation by plants with the lowest Zn application rate (i.e., decreases of 43 % and 9 %, respectively for soybean and corn plants).It is also likely that plants found adequate soil chemical conditions which facilitated root growth that allowed development through the soil and absorb the Zn due to the essentiality and the Zn absorb by seeds (Malavolta et al., 1987), which was manifest by higher Zn accumulation in plants (Prado et al., 2007).
Interestingly for clay soil, Zn rates presented a negative effect on soybean seed germination following a decrease and a small increase.Yagi et al. (2006) have previously shown a negative effect of Zn on the germination of Sorghum bicolor sp. that was related to the amount of Zn applied to the seeds.It should be kept in mind that any negative responses may be because soybean seeds have a higher phytic concentration, as well as high protein and lipid concentration and low carbohydrates in the endosperm when compared to corn.The high level of phytate, protein bodies, in soybean seed has be described by Gupta et al. (2015), located in endosperm and representing 10.7% of phytic acid on soy seeds (Lehrfeld, 1994).Therefore, we hypothesized that phytate may have blocked zinc absorption during the germination process, in a way similar to that described by Couzy et al. (1998) who showed phytate to be a potent inhibitor of Zn absorption in humans.
On the other hand, corn seeds responded positively to Zn oxide in terms of Zn accumulation and dry matter, leading us to recommend an application rate of 1.0 g kg -1 for corn seed.With respect Zn acetate, we recommend a lower rate between 0.5 and 0.6 g kg -1 seeds.The positive outcomes of using Zn acetate on soybean seed treatment have been observed by Milléo and Cristófoli (2015) and Gomes et al. (2016), who reported increases in soybean plant height, plant number and root dry mass (Milléo & Cristófoli, 2015), while for corn seed there have been reports of reduction in the time of emergency (Gomes et al., 2016).

Zinc Sources
The absence of major differences in the effect of Zn acetate and Zn oxide as a fertilizer for corn and soybean seed treatment is a promising result.Although there was a small disparity of 0.1 g pot -1 for dry matter among treatments in clay and sandy soil, for practical purposes this can be considered insignificant.Even though Zn acetate presented low Zn (78 % lower) and N content (4 % higher) it appeared sufficient to supply additional Zn to plants.Our results contrast those done on other Zn sources such as Zn sulfate described by Malavolta et al. (1987), andPrado et al. (2007) which have been reported as less viable when compared to Zn oxide.One explanation for our findings is that both fertilizers tested here are soluble and the amount of Zn necessary to elicit a response may be lower in seed treatments.
Our findings are important because they show that Zn supply can be effective at the seed treatment stage, which has the advantages of permitting uniform application and the need for comparatively lower application rates because of the homogeneous distribution.Soil having Zn deficiency may be more responsive to corn and soybean seed treatment, which can be supplied as either Zn acetate or Zn oxide because both seem to be viable alternative sources with little difference between them.We do not recommend using the soybean seed treatment in clay soil because of the germination decrease, but we do draw attention to the need for further research aimed at understanding the relationship between soybean treatments and Zn supply.

Table 1 .
Soil chemical and physical characterization and seed characterization

Table 4 .
Summary of dry matter (DM), Zn accumulation (Zn Acc.), 8 th and 13 th GD for corn and soybean seeds treated with Zn Acetate (Zn Ac) and Zn oxide (Zn Ox.) in Clay soil