Introduction

Corn (Zea mays L.) in the High Valleys of Mexico (2 200 to 2 600 masl) is produced by small and medium producers who do not have economic resources and adequate technologies in the conduct of their cultivation, this limit reaching maximum potentials of grain yields. Although there are already improved materials with yield stability (^{Vázquez et al., 2020}), the lack of agricultural innovations in the agronomic management of corn contributes to its low productivity, in addition, adverse edaphoclimatic conditions in contrasting environments significantly reduce production (^{Turrent et al., 2016}).

The above, it will be necessary to consider actions of adjustments in the technological packages in the agronomic management and plant breeding programs that allow the producer to select their seed and adopt the best agricultural practices such as the efficient management of chemical and organic fertilization, finer practices of foliar nutrition to the foliage (^{Fernández et al., 2015}; ^{Zamudio et al., 2018}) and other technological innovations that provide better answers in the agronomic components of the crop.

In recent years, the use of biostimulants has been a strategy within the fertilization program as a complement to fertilization applied to the soil (^{Zamudio et al., 2018}). Studies on the effects of biostimulants have been intensified in different crops (^{Du Jardin, 2015}), although most of the research is reported in horticultural crops and under greenhouse conditions (^{Grabowska et al., 2012}; ^{Mattner et al., 2013}; ^{Petrozza et al., 2013}), with few studies associated with corn (^{Quezada et al., 2015}; ^{Tejada et al., 2018}; ^{Zamudio et al., 2018}).

According to ^{Calvo et al. (2014}), biostimulants can be synthetic or natural and are composed of substances such as plant hormones, macro and micronutrients, amino acids, proteins and microorganisms, so they can be defined by their composition and mode of action, by their impact on the plant (^{Yakhin et al., 2017}), by the functions they exert on plants, or by the final response on crop yield (^{Du Jardin, 2015}). These compounds affect the physiology of the plant when applied in small amounts. ^{Du Jardin (2015)} classified biostimulants into three main groups: algae extracts, hydrolyzed proteins (peptides and free amino acids (FAs)) and humic substances (humic and fulvic acids).

According to ^{Battacharyya et al. (2015}), algae extracts are a mixture of organic and inorganic compounds from seaweed biomass that contain, in the source material, carbohydrates (mannitol), minerals, osmolytes (betaines), secondary metabolites (eg. phenols), amino acids, vitamins and plant growth hormones. Algae extracts have high resistance to osmotic stress, reduction in protein degradation, preventing oxidation of chloroplasts and a delay in foliar senescence that prolongs the photosynthetic activity of the plant (^{Jannin et al., 2013}). In addition, by functioning as metal ion chelates, they improve the absorption of nutrients when applied under suboptimal growing conditions or under environmental stress (^{Crouch and Van Staden, 1994}).

The industrial synthesis of amino acids includes chemical or enzymatic hydrolysis, or both, of agro-industrial by-products of animal or vegetable origin and biomass of specific crops (^{Cavani et al., 2006}). According to ^{Rai (2002}), physiological processes in plants are regulated by short-chain free amino acids. Amino acids can act as anti-stress agents (^{Mladenova et al., 1998}), source of nitrogen and hormone precursors (^{Rai, 2002}; ^{Zhao, 2010}; ^{Maeda and Dudareva, 2012}), as additives with insecticides and fungicides (^{Cavani et al., 2006}) and as chelating agents (^{Ashmead, 1986}). The application of any nutrient together with amino acids is most effective during the process of its assimilation and incorporation into the vegetable tissues of the plant. Nutrients chelated with amino acids form very small, electrically neutral molecules that accelerate their absorption and transport within the plant (^{Ashmead, 1986}).

The absorption of amino acids by plants has an energy advantage by avoiding the processes of chemical transformation of nutrients in their different forms of assimilation. Amino acids are absorbed and assimilated by the plant, quickly incorporated into plant metabolism, as if they were synthesized by them without energy expenditure (^{Jones and Kielland, 2002}). In this sense, it improves crop nutrition, especially in the critical stages of plant development, exhibits higher yield and improves the quality of the grain or fruit harvested (^{Parrado et al., 2008}; ^{Tejada et al., 2016}). In the application of biostimulants, it is expected that the plants show optimal development and present better characteristics and grain yield at the end of the production cycle.

Although in other countries research work is carried out in corn to observe the yield response as a function of the application of biostimulants (Quezada et al., 2005; ^{Gazola et al., 2014}; ^{Galindo et al., 2015}), in Mexico there is no information regarding the foliar fertilization of biostimulants (amino acids and seaweed extracts) for the cultivation of corn that verifies its efficiency in the agronomic characteristics of the crop. In this context, the hypothesis was that the foliar application of biostimulants will differentially impact the genetic response of the corn seed in a higher grain yield and better responses in agronomic characteristics by favoring the synthesis and assimilation of photosynthates. The objective of this research was to determine the effect of foliar fertilization of biostimulants based on amino acids and seaweed extracts on the yield and agronomic characteristics of corn hybrids in the High Valleys of the State of Mexico.

Materials and methods

The experiments were established in three environments of the High Valleys of the State of Mexico in the spring-summer (SS) 2017 agricultural cycle. The first experiment was carried out in Jocotitlán (JOC) (19° 43’ north latitude, 99° 51’ west longitude and 2 700 masl), with average annual rainfall of 669.0 mm and the temperature ranges between 3 and 24 °C and the sowing was carried out on April 10, the second experiment was located in Temascalcingo (TEM) (19° 55’ north latitude, 100° 00’ west longitude, 2350 masl), with average annual rainfall of 874.6 mm and 10.6 and 23 °C. The sowing was done on April 22 and the third experiment corresponded to Jilotepec (JIL) (19° 59’ north latitude, 99° 30’ west longitude and 2 500 masl) with average annual rainfall of 850 mm and the temperature varies from 8 °C to 24 °C, the sowing was carried out on May 20, 2017. In the three experimental areas, the soils are classified as Phaeozem dark color phase (haplic Phaeozem (Jocotitlán), luvic Phaeozem (Temascalcingo) and gleyic Phaeozem (Jilotepec)), of loam-clay texture (30, 38, 32% of clay, silt and sand respectively) (^{Sotelo et al., 2011}). A soil analysis was performed in all three environments to determine its chemical characteristics (Table 1).

Eleven white grain hybrids were evaluated, four released by UNAM (TSIRI PUMA; ATZIRI PUMA; TLAOLI PUMA; IXIM PUMA) and seven released by the National Institute for Forestry, Agricultural and Livestock Research (INIFAP, for its acronym in Spanish) (H-50, #46#48; H-66; H-76; H-77; H-47AE; H-49AE), the latter two and the four of UNAM are trilinear hybrids generated with ‘androsterile’ progenitors. The sowing density was 85 000 plants ha^-1, each experimental plot had four furrows of 0.8 m by 8 m in length and the two central lines (4.8 m²) were considered as useful plot.

Prior to sowing, the seeds were treated with Crusier^® (thiamethoxam 50 ml ha^-1) and Force^® insecticide (10 tefluthrin 15 kg ha^-1) for soil pest control. The fertilization dose was 250-60-60 kg ha^-1 of N-P-K, and the application was carried out in three moments. At the sowing, 60 kg ha^-1 of N was applied, 100% of phosphorus (P₂O₅) and potassium (K₂O) at a rate of 60 kg ha^-1 for both cases, based on urea, diammonium phosphate and potassium chloride, respectively. The second and third applications used urea with 120 kg ha^-1 of N between V₄-V₆ and 70 kg ha^-1 of N between V₁₀-V₁₂, respectively.

The weed was controlled in the initial vegetative stage (V_2-V₄) mechanically with hoeing and subsequently Lumax^® herbicide (S-metolachlor, atrazine and mesotrione 4 L ha^-1) was applied. For the control of foliage insects, Karate Zeon^® (Lambda cyhalothrin 250 ml ha^-1) and Denim^® (emamectin benzoate 100 ml ha^-1) were applied. For the prevention of diseases, Priori^® Xtra (azoxystrobin, cyproconazole 350 ml ha^-1) was applied. The biostimulants were applied foliarly with the use of a backpack sprayer in two phenological stages of the crop, between V₅-V₆ and V₈-V₁₀, respectively. Foliar applications were made in the early hours of the morning (07:00 to 09:00 am). The chemical composition of the biostimulants (amino acids and seaweed extracts) of each foliar treatment are shown in Table 2.

Once physiological maturity was reached (R₆), the harvest was carried out to quantify the yield (YIE) (t ha^-1) with the dry weight of the grain, adjusted to 14% moisture and extrapolated per hectare (^{Tadeo et al., 2015}). In addition, the following agronomic components were recorded: straw production (SP) (t ha^-1), weight of 200 g (W200) (g), rows per cob (RC), grains per row (GR), number grains per cob (NGC), cob diameter (mm) (CD) and volumetric grain weight (VW) (kg hl^-1).

The experimental design was completely randomized blocks with four repetitions, in a 3×5×11 factorial arrangement and environments (E), biostimulants (B), hybrids (H) and their interactions, as sources of variation. To determine the simple effects and interactions of the treatment design in relation to the response variables, a combined analysis of variance was performed with the SAS statistical program version 9.4 (^{SAS, 2002}). The comparison of means was with the Tukey test (p≤ 0.05) and a correlation analysis (Pearson).

Results and discussion

Differences in grain yield and yield components due to the environment (E) were significant (p≤ 0.01), except for RC and CD. Among the hybrids (H), there was a highly significant difference (p≤ 0.01) in all variables. In the biostimulant (B) factor, two groups of significance were identified in YIE, VW, NGC and W200 (p≤ 0.01) and in RC and GR (p≤ 0.05). In the interaction E*H, it had a significant effect on all variables (p≤ 0.01); in the interaction E*B, in YIE, SP, RC, GR, NGC and W200 there was significance (p≤ 0.05); in the interaction H*B in YIE, VW, SP, NGC and W200, and in the interaction E*H*B in YIE, SP, W200 and RC there was high significance (p≤ 0.01) (Table 3).

In the interactions (E*H, E*B, H*B and E*H*B), there were significant differences for grain yield, which means that the hybrids evaluated present differential behavior in yield for both the environment factor and the biostimulants, and at least three biostimulants (B2, B3 and B4) had positive response in the three environments (Table 3). As for the coefficient of variation, it was 6.4% for grain yield, which guarantees the reliability of the statistical results.

The final average yield was 12.3 t ha^-1, higher than the region’s mean of 6 t ha^-1 (^{Tadeo et al., 2015}). The yields on average by environments, the highest production with 13.5 t ha^-1 corresponded to Temascalcingo, in contrast, in Jilotepec, it presented lower yield with 10.2 t ha^-1. The grain yield in Jilotepec decreased 24.5% (3.3 t ha^-1) with respect to Temascalcingo, due to the delay in the sowing, which led to fewer days to complete the cycle and probably affected the grain yield due to the unfavorable weather conditions, such as poor distribution of rains, low temperatures and frosts.

In Jocotitlán and Temascalcingo, a difference of 0.5 t ha^-1 was observed, which may be influenced by the date of sowing close to each other. ^{Tadeo et al. (2015}) evaluated four corn genotypes on different sowing dates (May 17; June 1) in Cuautitlán Izcalli (2 240 masl) and verified that the sowing carried out on May 17 had a higher grain yield in relation to the second sowing. Therefore, as the sowing of corn in the High Valleys is delayed, it is affected by the irregular distribution of rainfall, reduction of solar radiation and low temperatures (frosts) during the cycle, thus reducing the productive potential of the crop.

In relation to hybrids, they presented a highly significant difference in all the agronomic variables evaluated (Table 4), which indicates that the genotypes used present a great phenotypic diversity. The yield of the hybrids ranged from 11.5 (Tsiri Puma) to 13 (H-66) t ha^-1, with an average yield of 12.3 t ha^-1 of grain. The hybrids H-66, H-50 and H-76 stand out with average yield of 13, 12.6 and 12.5 t ha^-1, respectively. The hybrids Tsiri Puma (11.5 t ha^-1) and Atziri Puma (11.7 t ha^-1) had relatively low yields in this study (Table 4).

The results of this study exceed the average yield of the region and those reported in other studies (Martínez et al., 2018; ^{Tadeo et al., 2020}; ^{Vázquez et al., 2020}), in which yields were relatively low for these same hybrids. This was due to foliar fertilization with biostimulants that directly causes a significant increase in grain yield (^{Quezada et al., 2015}; ^{Tejeda et al., 2018}; ^{Zamudio et al., 2018}). However, the continuous study of the foliar fertilization of biostimulants in the cultivation of corn is still pertinent in order to know their mechanisms of action and the morphological and physiological response of the plants in the different hybrids and depending on the contrasting environments.

Regarding biostimulants, in the localities of Temascalcingo and Jilotepec, the best responses were observed with the foliar application of B2 and B3. In both localities, they present slightly alkaline soils with pH ≥ 6.8 (Table 1). In contrast, in Jocotitlán, it is characterized by soils of pH of 5.6, (Table 1), it allowed foliar fertilization with biostimulant (B4) to exceed the other foliar treatments, which contains in its composition 8 and 2% of % of CaO and MgO, respectively (Table 2). In these soils that have characteristics such as low or high pH, presence of scarcely available minerals, plants lack optimal levels of essential nutrients, particularly micronutrients, which could be provided by foliar fertilization.

Absorption by aerial parts is the only practical means of supplying specific nutrients. The availability of micronutrients in the soil is closely related to their solubility, since the leaf and root tissue have the same morphological structure (they originate in the meristem tissue), plants can quickly absorb dissolved minerals (^{Martinka et al., 2014}). Therefore, micronutrient deficiency in soils can be successfully managed by foliar fertilization (^{Barbosa et al., 2013}; ^{Tejada et al., 2018}). The effects of foliar biostimulants were favored during application in all three environments by low temperatures, higher relative humidity, the combination of both factors implies a lower vapor pressure deficit (^{Fernández et al., 2015}) and at the same time is associated with stomatal opening, which allowed greater efficiency in the absorption of foliar biostimulants.

In corn components, biostimulants produced statistically different effects, except for straw production (Table 4). Regarding the control (B1), for each biostimulant, an increase in corn grain yield of 11.4, 9.6, 8.8 and 7.9% was detected for B3, B4, B2, B5, respectively (Table 4). In a study of foliar fertilization with biostimulant based on free amino acids from chicken epithelial tissue, on average of two agricultural cycles, ^{Tejada et al. (2018}) observed a 14% increase in corn grain yield. ^{Ahmad et al. (2007}), in a field study with compost enriched with N and tryptophan to the soil, observed a 21% increase in corn grain yield.

In contrast, ^{Quezada et al. (2015}) did not observe an increased in grain yield in soil fertilization of tryptophan and lysine biosynthesis by-product, but they did detect a significant difference for harvest index (HI) in relation to the control, so they suggest that there is potential in amino acids as sources of N in complement to nitrogen fertilization (nitrate and ammonium sulfate) to increase grain yields.

On average of the eleven hybrids, the best response was observed in B3 with an 11.4% increase in yield, which represents 1.3 t ha^-1 of corn grain (Table 4) compared to B1, followed by B4, B2 and B5. B3, in addition to free amino acids, contains micronutrients (in percentage Zn 1, Mn 0.5, Fe 0.5, B 0.1, Mo 0.01) of easy assimilation for plants due to the chelating effect of amino acids on micronutrients (Cu, Mn, Zn and B) within the plant, which facilitates their absorption and transport when applied together, also positively affect the permeability of the cell membrane (^{Ibrahim et al., 2010}), they are also particularly involved in the reproductive phase of the plant and therefore, in the determination of the yield and quality of the harvested crop (^{Parrado et al., 2008}; ^{Tejada et al., 2018}).

Amino acid-based biostimulants combined with essential micronutrients such as Zn and B represent an alternative for the soils of the High Valleys due to the low concentrations of these micronutrients for these agroecosystems (Table 1). It is observed that, when applying B2, which contains 3% boron, it exceeds the control (B1), with 8.8% grain yield that represents 1.1 t ha^-1 of corn grain production. The presence of these nutrients in biostimulants allows correcting their deficiency in soils, optimizing vegetative and reproductive activities to plants. Micronutrients once assimilated by plants and incorporated into plant tissues are effectively transported by amino acid complexes, and affect grain yield (^{Ashmead, 1986}).

B5, which has in its composition seaweed extracts, presented an increase of 7.9% in grain yield with respect to the control and presented a similar response with B2 and B4 of this study. Although the mechanism of action of seaweed extract to improve agricultural crop productivity is unknown (^{Mohanty et al., 2013}), positive responses in grain yield have been observed with foliar fertilization of biostimulant based on seaweed extracts as a complement to soil fertilization. ^{Galindo et al. (2015}) observed an increase in the yield of corn grain with Egeria densa extract, compared to the control, of 8 and 6.2%, in a single application (VT) and in two applications (VT and R2), respectively. ^{Kumar and Sahoo (2011}) reported higher yield in Triticum aestivum in the foliar application of 20% Sargassum wightii extract, with a 13.69% increase in seed number and a 22.86% increase in the dry weight of the seeds compared to the control.

In this study, B5, in addition to its composition (10% organic carbon and 1% organic nitrogen), contains in the source materials a source rich in minerals, vitamins, carbohydrates and growth promoters, which would be an alternative for use as an organic foliar fertilizer since it impacts on grain yield (Table 4). The agronomic variables evaluated in this study presented significant differences for the biostimulant factor, except for straw production, which did not vary between treatments (Table 5). This is attributed to the high percentage of CV measured for straw production (18.5%), compared to the other variables that ranged from 1.2 to 13.5% (Table 3), acceptable values of experimental variability.

B2 stands out, which resulted in higher density of the corn grain (80.6 kg hl^-1), rows per cob (16.5), weight of 200 grains (66.9 g) and cob diameter (48.9 mm) followed by B3, B4, B5, respectively, compared to the control (B1). This B2 response is probably due to the presence of 3% B, which, among other functions, participates in the development of fruits and seeds, responsible for sugar transport, hormone development and cell division (^{Goldbach, 2001}). In pioneering studies on the effect of boron on cob formation and corn grain yield components, ^{Mozafar (1987}); ^{Vaughan (1977}) conclude that, in the deficiency of B, the stigmas become little receptive to pollen, this is because the B is immobile in the phloem of the plant, which leads to a lower number of rows and grains per row on the cob, so the application of B is recommended for better pollination and grain formation, which is evident in the present study (Table 5).

The significant differences for rows per cob, grains per row and number of grains per cob in the biostimulant factor show consistency of the three variables, which influenced the grain yield both in the treatment with the highest yield (B3) and the control (B1) that presented lower yield. A positive correlation was detected between RC vs NGC (r= 61) and GR vs NGC (r= 77). Rows per cob, grains per row, and number of grains per cob provided values of 16-16.6; 29.7-31.1; 473.3-515.6, respectively (Table 5). This last variable, on average of the four treatments with biostimulants (B2, B3, B4 and B5), represents an increase of 6.9%, compared to the control (B1). ^{Tejada et al. (2018}) verified an increase of 8.9 to 15.8% in the number of grains per cob when they applied increasing doses of amino acid-based biostimulants, in two agricultural cycles (Table 5).

The above data also exceed those reported by Martínez et al. (2018), ^{Zamudio et al. (2018}); ^{Tadeo et al. (2020}); ^{Vázquez et al. (2020}), with hybrids adapted to environmental conditions similar to this study. The positive responses observed in RC, GR and NGC are associated with the genetic quality of the seed of the hybrids, complementary foliar fertilization (Zamudio et al., 2018) and the timely application of nutrients in times of increased demand. The foliar biostimulants affected the volumetric weight, presenting very dense grains that range from 79.9 (B1) to 80.6 (B2) kg hl^-1 (Vázquez et al., 2020), these values exceed what was reported by Zamudio et al. (2018) in a work with foliar fertilization with different products enriched with nitrogenous organic material.

Thus, what is required by the dough and tortilla industry (DTI) and the nixtamalized flour industry (NFI), which demand grains with a VW greater than 74 kg hl^-1 (SE, 2002), is met. In addition, a close positive correlation between VW and YIE (r= 60) was observed through Pearson’s correlation analysis, which allows deducing that the hybrids evaluated with efficient agronomic management such as foliar fertilization, specifically with the use of biostimulants, may be suitable or favorable for the industry.

There was a significant difference in the weight of 200 grains between the biostimulants and it ranged from 64.8 in B1 to 66.9 g in B2. The increase in W200 was probably due to the application of nitrogen products that maintained the photosynthetic activity of the plant for a longer period, allowing greater accumulation of reserves in the grains and finally greater weight of the harvested grains. The result of this study corroborates what was reported by ^{Gazola et al. (2014}), on the positive correlation between grain mass and corn yield (r= 60) (Table 5).

The cob diameter showed significant differences for the biostimulant factor (Table 5) and ranged from 47.7 (B1) to 48.9 (B2) mm, respectively. Like the previous variables, all four treatments exceed the control (B1). The observed values of CD are similar to those reported by ^{Zamudio et al. (2018}). The cob diameter is associated with the genetic factor of the seeds, agronomic management and the environmental conditions that prevail during the cycle. The results of this study coincide with those of ^{Quezada et al. (2015}); ^{Galindo et al. (2015}); ^{Zamudio et al. (2018)}; ^{Tejada et al. (2018}) on the effect of biostimulants on corn plants, which significantly increased grain yield and agronomic characteristics of the crop.

Conclusions

The application of biostimulants in the critical moments of growth and development of the plant favors the productive potential of the hybrids evaluated for the environments of the High Valleys of the State of Mexico, especially in conditions of osmotic stress. The environment with the best response corresponds to Temascalcingo, followed by Jocotitlán and Jilotepec. On average, the hybrids H-66, H-50 and H-76 stand out, however, all genotypes exceeded the regional yield average. In the application of biostimulants, grain yield increased from 0.9 to 1.3 t ha^-1. Technically, the best responses were observed in the biostimulants, B2, B3 and B4. Within B2, the hybrids with the best responses were Tlaoli Puma, H-47AE, H-66 and #46#48, for B3 (Ixim Puma, H-47AE, H-66, #46#48), in B4 (Tlaoli Puma, Ixim Puma, H-47AE, H-50, H-66 and #46#48), and for B5 in H-66. The easy assimilation due to amino acid complexes is responsible for the increase in grain yield. In this sense, foliar biostimulants are an alternative in complementary fertilization to increase production in the cultivation of corn.