Productivity variables

The productivity variables analyzed in this research are the following:

Yield. In each experimental unit, 10 m² were harvested manually; grain moisture was established in physiological maturity, whose records were used to express the yield at 12% moisture content of the grain.

Weight of 1000 grains (W1000G). In each genotype 100 grains obtained at random were weighed in a Sartorius Basic microbalance with a sensitivity of 0.001 g; subsequently, the result in grams was multiplied by 10.

Number of grains per square meter (G/m²). The number of G/m² was estimated by the quotient between the yield (on dry basis) and the individual weight of the grain. This was established with the average of two samples of 200 grains each, dried in a forced air oven for ten days.

Grain quality variables

The grain quality variables measured in this study are presented below:

Hectoliter weight (hw). Hectoliter weight was estimated according to method 55-10 (^{Approved Methods of the American Association of Cereal Chemists [aacc], 1998}) and expressed in kg/hL.

Flotation index (fi). The FI was obtained from 100 healthy grains placed in an Erlenmeyer of 250 mL, containing 170 mL of a mixture of kerosene-carbon tetrachloride (d = 1.305 at 25 °C). After a brief agitation with a glass rod, the grains that floated were counted. The flotation index was calculated as the percentage of grains that floated (^{Robutti, Borrás, González, Torres, & De Greef, 2002}).

Grain hardness (gh). Grain hardness was determined by increasing it fivefold in a 10-grain sample, carrying out a penetration resistance test measured with a texture analyzer (TAXT2) provided with a conical punch (30° angle), at a speed of 0.5 mm/s, and a penetration distance of 2 mm. The hardness of the grain was reported in kg-f.

Crude protein (cp). Crude protein was established through the Kjeldahl method (^{AACC, 1998}) and expressed in percentage.

Statistical analysis

The data were analyzed with the statistical program Statistix 8.0. An analysis of variance was carried out according to the experimental design used, and the means obtained in the combinations of the different levels of fertilization were compared through Tukey's multiple comparisons test. Correlations were also made between the variables measured using Pearson´s method.

Yield and its components

The hybrid × fertilization interaction was statistically significant (p≤ 0.001) for all the measured variables; this behavior is related to the yield potential of each hybrid and the type of endosperm, which determined the response of the materials evaluated. In table 2 is shown the effect of the fertilization with nitrogen and sulfur on the yield and its components for each corn genotype evaluated. The DK-5277 hybrid with semi-dent (soft) endosperm obtained the highest yields (10,274 kg/ha) when the fertilization level applied was N₂₀₀S₅₀, with an increase of 42.2% compared to the control, and of 39.8% and 38.7% compared to the flint and semi-flint genotypes, respectively.

Commonly, higher yields are observed in corn with soft or semi-dent textures compared to hard or flint textures, which may be determined by a decrease in the protein content in the grain (Ciampitti & Vyn, 2013; Salvagiotti, Castellarín, Ferragutti, Dignani, & Pedrol, 2010). On the other hand, Ferragutti, Castellarín, and Salvagiotti (2016) also obtained a greater response in yield in a hybrid with semi-dent texture compared to one with a flint texture, when they evaluated the nutritional requirements of these hybrids. All the genotypes evaluated reached the highest yields when they were fertilized with N₂₀₀S₅₀, with statistically significant differences between fertilization levels compared to the control (p ≤ 0.05).

Capurro et al. (2002) found that the yield response of corn to the aggregate of S was significant with doses of N higher than 60 kg/ha and higher to increasing doses of N; besides, these showed that there is a significant N*S interaction. Conversely, corn genotypes with high yield potential have very high nutrient requirements, so their response is greater at higher doses of N (^{Salvagiotti et al., 2010}). Similarly, the interaction between nutrients can increase the efficiency of the applied fertilizer and improve the utilization of N by the crop (^{Stewart, 2007}).

Other cereals have also experienced similar responses to the application of S; Hoffman, Fassana, Mazzilli, Berger, and Ernst (2016) found that in wheat, the addition of N in the presence of S increased the absorbed amounts of N. The response of the genetic materials to S of this trial is related to the low mean contents of organic matter and the reduced levels of this nutrient in the soil (table 1). Furthermore, also to the long agricultural history of the area without contributions of sulfur fertilizers, which is the determining cause of S deficiency in soils (^{Pagani, Echeverría, & Sainz, 2009}).

Regarding the variable W1000G, statistically significant differences (p ≤0.05) were observed between the levels of fertilization compared to the control (table 2) for all genotypes, detecting response to fertilization with N and S. All materials evaluated reached the highest W1000G with the application of 200 and 50 kg/ha of N and S, respectively; with this fertilization level the values obtained were significantly different (p ≤ 0.05) compared to the values found in the rest of the fertilization levels applied.

The increase of the W1000G by increasing the dose of N generally depends on the relationship between the weight of the grain and the growth of the plant during the filling of the grains, and this is why in the control treatment, this increase was limited by the smaller amount of assimilates (Amin, 2011). In this case, the addition of S improves both the grain yield and the W1000G with the highest dose of N, even though this variable is not a component that is as directly associated with the yield as the number of G/m² (^{Pérez, 2015}). These results are indicative that the characteristics of each genotype and their ability to remobilize assimilates towards the grain can favor the yield and its components.

Regarding the number of G/m², considering the factor most closely related to variations in yield (^{Capurro et al., 2002}; ^{Muchow, 1998}), the highest values were obtained with the fertilization level of N₂₀₀S₅₀ for all hybrids, with significant statistical differences (p ≤ 0. 05) between fertilization levels compared to the control. These results indicate that the increase in the number of G/m² in response affects the individual weight and quality of the to the addition of S, was a decisive factor in yield grains (^{Pagani et al., 2009}). Ferragutti et al. (2016) increase in the corn genotypes evaluated, and that obtained a similar response when they evaluated fertilization with N and, to a lesser extent with S, fertilization with nitrogen and sulfur in corn modify the supply of assimilates and nutrients, and hybrids with flint and semi-dent textures.

Table 2. Effect of nitrogen-sulfur fertilization on the yield, the weight of a thousand grains and the number of grains per square meter, of three corn hybrids with different endosperm textures 

^Note.Different letters within a column indicate significant differences (p ≤ 0.05) according to Tukey's multiple comparisons test.

Source: Elaborated by the authors

Table 2. (cont.) Effect of nitrogen-sulfur fertilization on the yield, the weight of a thousand grains and the number of grains per square meter, of three corn hybrids with different endosperm textures. 

^Note.Different letters within a column indicate significant differences (p ≤ 0.05) according to Tukey's multiple comparisons test.

Source: Elaborated by the authors

Physical and nutritional quality of the grain

In table 3 is shown that the HW values showed significant differences (p ≤ 0.05) between the levels of fertilization compared to the control for all hybrids evaluated, which shows that the genotype x fertilization interaction significantly influenced the HW of the grains. In this study, the flint and semi-flint genotypes (DK-5632 and DK-440) obtained higher HW values compared to the semi-dent hybrid, because grains with flint endosperm are denser and heavier than those with a soft endosperm. This confirms that when there is higher endosperm hardness, the HW is also higher. Authors as Barrios et al. (2016) reported similar results.

In general, the HW is a quality parameter that responds to nitrogen fertilization and, taking into consideration that the absorption of N is favored when the crop is fertilized with S (Salvagiotti et al., 2009) the HW values reached were expected. On the other hand, even when the quality of the flint genotype was higher compared to the HW, the yield was lower in comparison with the hybrid of soft or semi-dent texture (table 2). This behavior is commonly observed in the field since the dent, and semi-dent corn textures have higher yield potential, a product of the genetic improvement (^{Calderone, Torres, Papucci, Cruciani, & González, 2008}).

The semi-dent hybrid DK-5277 also showed more significant response in the HW compared to the other two hybrids with the application of N₂₀₀S₅₀, if we consider that this genotype experienced an increase of 8.9% compared to the control and in comparison with the increases experienced by the hybrids DK-5632 and DK-440 (6.4 % and 7.7 %, respectively). These results indicate a greater efficiency in the use of nitrogen with the addition of 50 kg/ha of sulfur and represents an improvement in the grain quality of this genotype due to the increase in HW values. Since S is found in proteins as an amino acid component, among others methionine and cystine, the application of sulfur fertilizers could have important effects not only on the increase in protein content, but also on changes in texture and density of the endosperm, increasing the resistance to grain breakage (^{Cruciani, González, Papucci, & Pedrol, 2014}).

Nonetheless, the FI represents one of the most important indicators of hardness for the dry milling process (^{Ferragutti, Salvagiotti, & Castellarín, 2012}). In this trial, significant differences were obtained (p ≤ 0.05) for the FI between the levels of fertilization compared to the control for each of the genotypes evaluated (table 3), where the hybrids of hard and semi-hard texture, i.e., DK-5632 and DK-440, showed lower FI values. In this experiment, the application of the fertilization level N₂₀₀S₅₀ allowed a significant decrease in the FI values, which is an indication of an improvement in grain quality in terms of hardness (^{Zepeda et al., 2009}). This tendency occurred primarily in the case of the semi-dent hybrid DK-5277. Considering studies that have shown that a greater absorption of N occurs in crops fertilized with S (^{Salvagiotti et al., 2009}), these results corroborate that the nitrogensulfur fertilization strategy has an important effect on the texture of the endosperm, increasing the density of the grain through the increase of the proportion of zeins and glutelins in the flint endosperm (^{Barrios et al., 2016}; ^{Tsai, Dwerkat, Huber, & Warren, 1992}).

In relation to the hardness of the endosperm, this is a property of the grain associated with a higher density and vitrosity of the flint endosperm, factors that depend on the union between starch and proteins, especially zeins, a fraction abundant in sulfur amino acids (Cirilo, Actis, Andrade, & Valentinuz, 2011). In this study, significant differences (p ≤ 0.05) between the levels of fertilization compared to the control for the hardness test were obtained for all the evaluated genotypes. Genotype DK-5277 of soft endosperm reached lower values compared to the flint and semi-flint genotypes, as expected, as in the flint fraction of the endosperm the starch and the protein are strongly bound, and in the mealy fraction the link is weaker, although fertilization with N and S allowed a much more evident response in this genotype. This is because the increase concerning the control was more than 100 % compared to the values of 15.99 % and 20.18 % obtained with the flint and semi-flint hybrids, respectively. The improvement of the hardness test values in response to nitrogen fertilization in all hybrids evaluated corroborates that timely nutrition with S and N, which ensures adequate flow of these nutrients to the grains, would favor its hardness by increasing the concentration of both elements (^{Cirilo et al., 2011}).

Regarding the CP content, the means obtained with the fertilization level N₂₀₀S₅₀ for all hybrids differed significantly with those of the rest of the fertilization levels and compared to the control (p ≤ 0.05). These results confirm that the protein content increases by an adequate substitution of N and S, and that this increase positively influences grain density, reducing the susceptibility to cracking as a consequence of the higher zein content and the higher flint/floury endosperm ratio (^{Cirilo et al., 2011}; ^{Cruciani et al., 2014}). On the other hand, gamma and beta zeins also require S for their synthesis, due to the presence of sulfur amino acids in their composition (^{Wu & Messing, 2010}), which would explain the response to this fertilization strategy.

The correlation between the measured variables is presented in table 4. For the three genotypes, CP was positively and significantly associated with yields and GH, and positively and highly significant with HW and W1000G, while the FI showed a negative and highly significant correlation. The fertilization strategies evaluated in this trial allowed an increase in the physical quality of the grain through a decrease in the FI of each hybrid. The HW was associated in a negative and highly significant way with the FI, while the W1000G and the yield were associated in a positive and highly significant way.

Table 3. Effect of nitrogen-sulfur fertilization on the hectoliter weight, the flotation index, the grain hardness and the crude protein content of three corn hybrids with different endosperm textures 

^Note.Different letters within a column indicate significant differences (p≤0.05) according to Tukey's multiple comparisons test

Source: Elaborated by the authors

Table 3. Effect of nitrogen-sulfur fertilization...(Cont) 

^Note.Different letters within a column indicate significant differences (p≤0.05) according to Tukey's multiple comparisons test

Source: Elaborated by the authors

Table 4. Coefficients of linear correlation between grain hardness (GH), grains per square meter (G/m²), flotation index (FI), hectoliter weight (HW), the weight of 1.000 grains (W1000G), percentage of crude protein (CP) and yield, in three corn hybrids with different endosperm textures 

Source: Elaborated by the authors