Introduction

The cultivation of pepper (Capsicum annuum L.) is one of the most important crops in Mexico, for its large consumption in the population (^{Namesny, 2006}) in Mexico, the harvested area is 143 975 hectares with a yield average 16.2 t ha^-1 (^{SIAP, 2015}).

The Ca²⁺ is an alkaline earth (^{Feyerabend et al, 2008}), with ionic radius of 9.9 nm (^{Hu et al., 2004}) element having high correlation with the supply of La (lanthanum), such that with the supply of lanthanum rises the accumulation of Ca²⁺ in leaves per plant (^{Ramírez et al, 2012}). It is abundant in most soils and rarely acts as a limiting factor, except in acidic soils where the contribution of calcium salts (^{Bonilla, 2008}) may be necessary to complement the nutritional requirement from chili peppers, consisting of: N of 2.4-4 kg t^-1 fruits, phosphorus (P₂O₅) of 0.4-1, potassium (K₂O) of 3.4-5.3, calcium (CaO) of0.6-1.8 and magnesium (MgO) of 0.3- 0.5, although this requirement varies in types, organs and systems of production jalapeno pepper, sweet pepper (^{Salazar and Júarez, 2013}). According ^{Charlo et al. (2012)}, the pepper can extract 81.3 kgha^-1 and it can be found 0.84 kg t^-1 of fruit; ^{Azofeifa and Moreira (2004)} report 38 kgha^-1 and 0.82 kgt^-1, respectively; ^{Fontes et al. (2005)} found that the pepper extract 114 kg ha^-1 and containing 2.2 kg t^-1.

The Ca²⁺ ion diffuses into the ground for a concentration gradient and transported out with the aid of dependent pumps of ATP (^{Salisbury and Ross, 2000}), and symptoms of deficiency are always more pronounced in young tissues (^{Kirkby and Pilbeam, 1984}), such that the meristematic zones of the roots, stems and leaves, where there cell divisions, are most susceptible, possibly because calcium is needed to form a new middle lamella in cell plate that appears between the daughter cells (^{Salisbury and Ross, 2000}).

In the cell occurs reaction sequences require Mn²⁺, Ca²⁺ and Cl^- linked to a set of polypeptides, and other experimental data suggest that induced responses of phytochrome require intermediaries such as Ca²⁺ and calmodulin and that these substances play a role in phosphorylation nuclear proteins (^{Lea and Leegood, 1993}). The Ca²⁺ is an essential element for plants, since part of the Photosystem II (PS II) is composed of six integral polypeptides (intrinsic) that are connected together noncovalently, wherein the Ca²⁺ is essential to photolysis of water, and even where it is incorporated using three extrinsic polypeptides (peripheral) that are encoded by nuclear genes (^{Salisbury and Ross, 2000}).

It is now recognized that all agencies remain unexpectedly low concentrations of free Ca²⁺ in the cytosol, usually less than 1 μM (^{Hepler and Wayne, 1985}). This is true even when calcium is so abundant in many plants, especially legumes, such as phosphorus, sulfur and magnesium. Most plants containing calcium found in the central vacuoles, cell walls and is attached to certain polysaccharides called pectates (^{Kinzel, 1989}).

In the vacuoles, usually calcium precipitated as insoluble oxalate crystals. In some species, also in the form of carbonate, phosphate or sulfate insoluble. The low concentrations of calcium, almost micromolar, should be kept in part to prevent the formation of insoluble calcium salts, obtained from ATP and other organic phosphates. Furthermore, Ca²⁺ concentrations in the micromolar range above inhibit cytoplasmic streaming (^{Williamson, 1984}). Although enzymes are activated by Ca²⁺, many others are inhibited, which makes it even more necessary that the cells remain very low concentrations of Ca²⁺ in the cytosol, where many enzymes (^{Salisbury and Ross, 2000}) exist. An important part of Ca²⁺ existing in the cytosol binds directly to several enzymes, such as small protein called calmodulin, with which it binds reversibly (^{Robert et al., 1986}), making said protein is modified in structure and then to activate various enzymes (^{Salisbury and Ross, 2000}).

The Ca²⁺ also acts on the spindle during cell division, which is required for normal functioning of cell membranes, and has been implicated as second messenger in response to environmental conditions and hormonal signals (^{Sanders et al. 1999}). The application of Ca²⁺ in the production of cabbage is essential (^{Amador et al, 2008}), because this element ensures processes such as the synthesis of cell walls, in the middle lamella, which form calcium pectate which provides stability and maintains the integrity of these (^{Gordillo et al. 2004}).

The calcium is very mobile and tends to accumulate in older organs, while higher metabolic activity (growing leaves, flowers, fruits and apical meristems) are the tissues that need a greater contribution; therefore this macronutrient deficiency primarily affects the parties in forming and growing meristems, where it is fixed and practically motionless in their cell walls. Because of this immobility, older leaves may have normal levels of calcium, while young leaves, fruits or other organs, may have levels below normal (^{Chiu and Bould, 1977}).

Transpiration and high water content in the soil favorable mass flow and in turn the mobility of Ca²⁺ (^{Ansorena, 1994}), such that with high doses of Ca²⁺ quantities of this element that fail to enter the cytosol remain in the cell wall situation creates heavier cells (^{Marschner, 2002}).

The objective of this research was to determine the efficacy of formulations (CaNO₃)₂ líq, (CaNO₃)₂ sol-1, (CaNO₃)₂ sol-2 and (CaNO₃)₂ sol-3, greenness index leaf and yield per unit area and the best dose of each solution to induce the production of more dry matter in plants and fruits pimento.

Materials and methods

This research was conducted during the season autumn-winter 2014-2015 in a greenhouse chapel type, installed in the experimental field of the Technological University of Culiacan, located at km 2 of the Culiacan-Imala highway, colony the Angeles, Culiacan, Sinaloa, with coordinates 24° 50' 30" north latitude and 107° 50'30" west longitude, height of 58 masl. According to ^{Garcia (1988)}, the climate is B₁S₁, semi-arid with rains in summer and winter and 670 mm of annual precipitation. The average annual temperature is 24 °C, with highs of 41 °C in summer and minimum of 5 °C in winter, and average relative humidity of 66.6% annually.

The cultivating pimento (Capsicum annuum L.) used was Capia Rojo' Syngenta®; the substrate consisted of a mixture of peat moss, perlite, vermiculite and sand in proportions volume:volume (v:v) of 1:1:1:1; before depositing the substrate in the pots made with plastic bags with a capacity of 12 L, they were treated by soaking in solution with Trichoderma harzianum (2x10¹² ufc g^-1), Bacillus subtilis (2x10¹² ufc mL^-1) and Bacillus thuringiensis (2x10¹² ufc mL^-1) in order to promote root growth and prevent post-transplant stress. The transplant took place on October 19, 2014, with one plant per pot. Tutored plants two stems were handled with plastic sustained raffia strands of wire stretched horizontally along the greenhouse; defoliation and pruning plants were as they required.

The sources of calcium (treatments) were: liquid calcium nitrate [(CaNO₃)₂ líq] formulated 18-0-0-16 (CaO), calcium nitrate soluble [(CaNO₃)₂ sol-1] with formulation 10.1-0-0 17.3 (CaO), calcium nitrate soluble [ (CaNO₃)₂ sol-2] with formula 15.5-0-0-26.5 (CaO) and calcium nitrate soluble ble [ (CaNO₃)₂ sol-3] with formula 15-0-0-26 (CaO), from which the seven doses or treatments were made following: 21, 20, 19, 18, 17, 16 and 15 Lha^-1, the considered base they are 18 L^.ha^-1 (control one) of each formulation which is commercially recommended; each dose was applied three times a week until the last harvest of fruits by 1 200 mL per pot; as witness two ^{Steiner (1961)} solution was applied daily, except when it is watered with calcium solution, throughout the culture period (up to the third cut) with the same amount of mL per pot. The experimental design was randomized complete blocks, with three replicates (three pots with one plant each) per treatment dose and 21 plants for each source of calcium.

Through trays placed below the pots drained water captured to analyze the pH and CE of the same. After 18 days of treatment applied, samples were taken every third day for a total of 25 samples. The pH= 1.8 and pH= 4.5 input output were determined with portable meter HANNA model HI-98128 and CE= 1.2 input and CE= 1.6 d Sm^-1 output were determined with portable meter HANNA model HI-98331.

The study variables were chlorophyll content, plant height, fresh and dry weight of plants, fresh weight and dry fruit and yield per hectare. The greenness index was quantified by a chlorophyll meter Minolta SPAD-502 previously calibrated height was measured with a tape measure, the dry weight was obtained by oven drying at 85 °C to constant weight, so that the fresh plants and fruits dry weight and yield were determined with precision scale Ohaus brand. The data were analyzed statistically with proc glm procedure of SAS Institute (1985) version 6.12, using the multiple comparison test of means Duncan, with α≤ 0.05, and the correlation between variables was done with the procedure proc corr the same statistical program.

Results and discussion

The greenness index (Table 1) where it can be noted that the averages were statistically similar; however, in plants grown with (CaNO₃)₂ sol-3 an increase of 4.1% over the average of the plants that were handled with(CaNO₃)₂ líq and 3.5% compared to the average of the plants had cultured with (CaNO₃)₂ sol-1. Furthermore, with the solution (CaNO₃)₂ líq the respective increases were 2.6 and 3.5% compared to the averages of plants that were applied (CaNO₃)₂ sol-1 and (CaNO₃)₂ sol-2.

Table 1 Contents of chlorophyll, plant height, fresh and dry weight of plants cultivated pimento with four formulations of calcium. 

Medias con la misma letra en la columna son estadísticamente iguales Duncan, α≤ 0.05).

The average height, fresh weight and dry plants were not statistically different, but (CaNO₃)₂ sol-3 and (CaNO₃)₂ líq height increments of 5.1 and 2.4% were obtained in comparison with that of those which were grown with (CaNO₃)₂ sol-1 and (CaNO3)2 sol-2, respectively; the biggest increase (24.8%) of fresh weight was obtained (CaNO₃)₂ líq compared to the average of plants (CaNO3)2 sol-3, but with respect to that cultured with (CaNO₃)₂ sol-1 was 1.8% and 9.1% in relation to expressing those handled with (CaNO₃)₂ sol-2. The dry weight of the plants had its greatest expression with the solution of (CaNO₃)₂ líq, so that compared to what they achieved those cultured with (CaNO₃)₂ sol-1, (CaNO₃)₂ sol-2 and (CaNO₃)₂ sol-3, the respective increases were 7.2, 7.6 and 12.1%.

The correlation between chlorophyll and plant height was r= 0.93* (p= 0.05); between height and fresh and dry weight of plants respective ratios were r= 0.99** (p= 0.001) and r= 0.90* (p= 0.05). The value of the relationship between chlorophyll and fresh weight of the plants was r=0.92* (p= 0.005) and the dry weight reached the value of r= 0.83 ns (p= 0.09); while the ratio between the fresh weight and dry weight of plants was r=0.83 ns (p= 0.16).

With the values in Table 2 could be calculated that with the solution (CaNO₃)₂ sol-1 fresh weight of fruits was 13.1% higher in relation to that obtained with solution (CaNO₃)₂ líq; also, 23.8 and 16.7% over what was achieved with the respective solutions (CaNO₃)₂ sol-2 and (CaNO₃)₂ sol-3; however, dry weight solution (CaNO₃)₂ líq which caused increases was 24.5, 32 and 44.4% compared to what was obtained with solutions (CaNO₃)₂ sol-1, (CaNO₃)₂ sol-2 and (CaNO₃)₂ sol-3, respectively. In turn, the dry weight obtained from fruits harvested from plants grown with (CaNO₃)₂ sol-1 exceeded 6% was achieved with (CaNO₃)₂ sol-2 and 16% compared to that obtained with (CaNO₃)₂ sol-3; whereas with (CaNO3)2 sol-2 weight in question exceeded the estimated 9.4% from the fruits harvested from plants with (CaNO₃)₂ sol-3. So that the performance of plants (CaNO₃)₂ líq exceeded 11.2, 26.8 and 60.8% to the averages were obtained (CaNO₃)₂ sol-1, (CaNO₃)₂ sol-2 and (CaNO₃)₂ sol-3, respectively. The correlation between dry weight of fruits and yield per hectare r= 0.97 (p= 0.0001).

Table 2 Average fresh weight, dry and fruit yield cultivated pimento with four formulations of calcium. 

Medias con la misma letra en la columna son estadísticamente iguales Duncan, α≤ 0.05).

With the values of Table 3 could be calculated with the dose of 17 L^.ha^-1 of (CaNO₃)₂ líq, average fresh fruit weight exceeded the average 43.5% was obtained with the solution Steiner and 26.9 % that was achieved with the commercial dose (18 Lha^-1) of said solution; dose of15 Lha^-1 of (CaNO₃)₂ sol-1 the average exceeded 77.1 and 51.5% of the respective averages were achieved with Steiner solution and the commercial dose of said formulation, while with a dose of 16 L ha^-1 increases were 82.6 and 56.3%; with (CaNO₃)₂ sol-2 the best dose was the commercial (18 L ha^-1), and an increase of52.2% over the average achieved Steiner solution was held; whereas with (CaNO₃)₂ sol-3 the best dose was 16 L ha^-1, since with it the increase was 24.6% compared to the average obtained with Steiner solution, and 23.4% to that obtained with the commercial dose of the same formulation.

Table 3 Fresh weight of pimento cultivated with different doses of calcium solutions. 

Medias con la misma letra en la columna son estadísticamente iguales Duncan, α≤ 0.05).

From the averages listed in Table 4 could be calculated with the dose of 17 L ha^-1 of (CaNO₃)₂ líq, the dry fruit weight 15.8% increase relative to the average was achieved with Steiner solution and 33.2% compared to the commercially recommended dose; the best dose of (CaNO₃)₂ sol-1 was L ha^-1, since the increase was 2.5 times more compared to the average achieved with the Steiner solution and 61% compared to that obtained with the dose commercial. From (CaNO₃)₂ sol-2, the best response was observed where the same commercial dose (18 L ha^-1), which exceeded the average 58.4% dry weight obtained with the Steiner solution was applied; while the (CaNO₃)₂ sol-3,, the dose of19 L ha^-1 was what brought greater response, since the increase was 37.4% compared to that achieved with the Steiner solution, and 41.5% compared produced with the commercial dose.

Table 4 Dry weight of pimento cultivated with different doses of calcium solutions. 

Medias con la misma letra en la columna son estadísticamente iguales Duncan, α≤ 0.05).

The relative increases of greenness index, height, fresh and dry weight of crop plants with the solution of (CaNO₃)₂ liq, indicate that this is a practice which can be applied Ca²⁺ to the ground, and that comes with it more diffusion into pepper plants, since according ^{Salisbury and Ross (2000)} the Ca²⁺diffuses into the ground for a concentration gradient and transported out with the aid of dependent pumps ATP. So that the solution (CaNO₃)₂ líq meristematic zones of the roots, stems and leaves, where cell divisions occurred, were the main beneficiaries, perhaps because calcium is needed to form new middle lamella in the cell plate they must appear between the daughter cells.

Also they related to what was reported by ^{Lea and Leegood (1993)}, who found that in the cell occurs sequences of reactions requiring Mn²⁺, Ca²⁺ and Cl^- linked to a set of polypeptides, which perhaps occurred in pepper plants, all once activated enzymes that favor the chlorophyll synthesis, this increased and therefore, the process of photosynthesis to generate more fresh and dry matter in fruits and plants in general. Matter that perhaps due to increased phosphorylation of proteins in the nucleus, with the consequences in cell division and the yield per unit area.

These results are related to the reporting ^{Salisbury and Ross (2000)}, since when there is more calcium available it can be absorbed and incorporated into the cell parts and influence the division of cells to induce growth and fruit weight, are two components of pepper yield per unit area. They also allow better understand the differences in Ca²⁺ extraction mentioning ^{Charlo et al. (2012)}, as they report that the pepper can extract 81.3 kg ha^-1, while Azofeifa and Moreira (2005) found 38 kg ha^-1 and ^{Fontes et al. (2005}) mention that the pepper extract 114 kg ha^-1. That is, depending on the state of the Ca²⁺ ion and the dose in this case each of the formulations which were prepared doses, will be the ease with which it can be absorbed by plants.

The results were achieved with formulations (CaNO₃)₂ líq and (CaNO₃)₂ sol-1, in terms of dry matter yield per unit area, also have strong relationship with those reported by ^{Gordillo et al. (2004)}, since they found that Ca²⁺ is the element that ensures processes, such as synthesis of cell walls in the middle lamella, which form calcium pectate providing stability and maintaining the integrity thereof. Also, the results of this research are related to ^{Chiu and Bould (1977)}, as they relate to the Ca²⁺ is not very mobile and tends to accumulate in older organs, while the most metabolically active (leaves growing, flowers, fruits and apical meristems) greater contribution, which is fixed and practically motionless in their cell walls is needed. The relationship is also evident with those reported by ^{Marschner (2002)} who mentioned that high doses of Ca²⁺ amounts of this element that fail to enter the cytosol are in the cell wall, a situation that generates heavier cells.

The effects at lower doses of Ca²⁺ (15 and 17 Lha^-1) of the solutions made with the formulations (CaNO₃)₂ sol-1 and (CaNO₃)₂ líq, respectively coincide with those reported by ^{Williamson (1984)}, who mentions that low concentrations of Ca²⁺, almost micromolar, should be kept in part to prevent the formation of insoluble calcium salts, obtained from ATP and other organic phosphates. Furthermore, Ca²⁺ concentrations in the micromolar range above inhibit cytoplasmic streaming. So that the doses contained herein (15 and 17 L ha^-1) perhaps was more limited the formation of insoluble calcium salts and cytoplasmic current is favored, so that in pepper plants occur chemical reactions necessary and such plants produce more dry matter.

Conclusions

With solutions (CaNO₃)₂ líq and (CaNO₃)₂ sol-1 the red pepper was grown in a more sustainable way with solutions (CaNO₃)₂ sol-2 and (CaNO₃)₂ sol-3, since for better response performance needed a dose less than the recommended commercially and, accordingly, the possibility that decreased over time and high concentration calcium become a pollutant on the substrate used.