Introduction

Poinsettia is a native species from Mexico used as an ornamental plant for the holidays worldwide (^{Colinas et al., 2006}), being the state of Morelos the main producer in the country. It is possible to obtain poinsettia plants of good quality when grown under proper conditions to obtain healthy, strong and attractive plants, so it is necessary to emphasize in the study of factors that influence their production. Nutrition is one of the most crucial factors in the production of ornamental plants for the impact on growth and quality will have an effect on flower size, as well as leaf area, obtaining stronger colors and in the development of the root system (^{Pineda et al., 2008}).

Plant nutrition should be performed taking into account factors such as: a) water quality; b) substrate properties; c) phenological stage of the plant; d) weather; e) the type of fertilizer; f) culture technique; and g) nutrition goals. Mineral nutrition must be specific based on demand and according to phenological stage, emphasizing between these stages as follows: 1) root growth; 2) vegetative growth and 3) flowering (^{Vázquez and Salome, 2004}). Knowing the amount of nutrients absorbed by the plant in each phenological phase, it provides information that allows establishing appropriate and fractionated fertilization programs that will satisfy the needs during critical periods of development determined by crop phenology (^{Funnell et al., 1998}; ^{Fageria et al., 2006}).

Nitrate (NO₃^-) is the main source of nitrogen (N) for most crops. Nitrogen promotes leaf area and leaf area index (^{McCullough et al., 1994}; ^{Escalante, 1999}), intensifies the green color of the leaves, and is a constituent of essential cellular components such as amino acids, proteins and nucleic acids; also regulates the absorption of phosphorus (P), potassium (K⁺) and other nutrients, improving the degree of succulence of many crops and favors photosynthesis due to the increase in chlorophyll concentration (^{Aroiee and Omidbaigi, 2004}; ^{Taiz and Zeiger, 2006}; ^{Sedano et al., 2011}). Both deficit and excess of N have a negative impact on plants, causing a decrease in production (^{He et al., 1999}; Gonzalez et al., 2005), increasing the susceptibility to pest insects (^{Jauset et al., 2000}; ^{González et al., 2005}) and to pathogens (^{Duffy and Défago 1999}; ^{González-Raya et al., 2005}), thus the incidence of disease (^{Marschner, 2012}).

Calcium (Ca²⁺) is a basic nutrient for proper plant growth and is essential for cell division and expansion (^{White et al., 2000}). On poinsettia, Ca²⁺ deficiency reduces leave growth, cause shortening of internodes near the apical bud, induces weak stems and necrosis in bracts (^{Stromme et al., 1994}; ^{Ayala et al., 2008}). Ca²⁺ is involved in regulatory mechanisms that allow the plant to make adjustments under stress conditions caused by high and low temperatures, and by osmotic stress caused by drought and salinity (^{Liang et al., 2009}).

Ca²⁺ absorption, depending on their relative concentration in the nutrient solution may be substantially reduced due to antagonism with K ⁺, magnesium (Mg^{2 +}) and ammonium (NH₄⁺); however, its absorption is stimulated by NO₃^- (^{Jones et al., 1991}; ^{Villegas et al., 2005}). In Mexico there has been little research regarding poinsettia nutrition in function of phenological stages. In the above context, the present study was established in order to understand how the growth and quality of poinsettia are influenced by the change in the ratio NO₃^-: Ca^{2 +} in nutrient solution, as well as to determine the optimum ratio in phenological stages: 1) root formation; 2) vegetative growth; and 3) flowering.

Materials and methods

The experiment was conducted in a greenhouse with plastic cover on the experimental field of the Faculty of Agricultural Sciences at the Universidad Autonoma del estado de Morelos, located on the Chamilpa campus (18º 58 '52.87 "N and 99º 13' 57.92" W, altitude 1875 m) in Cuernavaca, Morelos. The environmental conditions during the experiment included an average minimum temperature and maximum temperature of 13.7 and 34.7 °C respectively, while relative humidity ranged between 55% and 73%. Photosynthetically active radiation incident daytime averaged 203 μmol m^-2 s^-1.

Prestige poinsettia rooted cuttings were transplanted on July 1, 2012 in black plastic containers 15.2 cm in diameter with a substrate made from a mixture of soil sifted leaf, coconut fiber and red volcanic rock (with particle size between 0.1 to 0.5 cm diameter) in a ratio 60:20:20 (%).

Three concentrations of NO₃^- (10, 12 and 14 mol₍₊₎ m^-3) and three of Ca²⁺ (7, 9 and 11 mol₍₊₎ m^-3) were evaluated, designed from a modification of the ^{Steiner (1984)} universal nutrient solution in the percentage ratio of ions, while maintaining the mutual ratios between cations and anions and the total amount of ions. Such modification was made within certain limits of relative concentration from the ions involved as otherwise the interaction between them can strongly influence the absorption and distribution or function of any other nutrient by the plant, inducing deficiencies or toxicities (^{Schwarz, 1995}; ^{Villegas et al., 2005}). NO₃^- concentration was modified based on the ratio NO₃^- + phosphate (H₂PO₄^-) + sulfate (SO₄^2-) of 12:1:7 (mol₍₊₎ m^-3), and Ca²⁺ based on the ratio K⁺ + Ca²⁺ + Mg²⁺ of 7:9:4 (in mol₍₊₎ m^-3); osmotic potential to -0.072 MPa with an electrical conductivity of 2 dS m^-1 and adjusting pH to 5.5.

Considering the interaction of both factors there were obtained nine nutrient solutions (Table 1) which were administrated in each of the phenological stages of poinsettia: root growth, vegetative development and pigmentation. The stage of root growth was considered from transplant to plant pruning, which was made when the root was visible on the outside of the root ball. The vegetative growth stage was considered from pruning to appearance of bracts transition, while pigmentation stage was considered from the appearance of bracts transition till the presence of visible pollen.

Table 1 Chemical composition of nutrient solutions. 

Before the vegetative and pigmentation stage the plants were irrigated with ^{Steiner (1984)} nutrient solution, but upon reaching the respective phenological stage, plants were watered with nutrient solutions from the treatments. Watering was done manually with a volume of 250 mL per plant every third day or when the plant required it.

Growth variables evaluated included bracts area (LI3100C, LI-COR, Inc., Lincoln, Nebraska, USA) and total dry weight and from each organ (bracts, leaves, roots and stems), which was recorded after drying the tissue in an oven with forced air circulation at a temperature of 70 °C for 72 h. To define the extraction of nutrients and relate it to phenological stage, at the end of each a destructive sampling was performed to remove the plants and separate the organs, to which were analyzed the mineral concentration in aerial vegetative tissues (leaf + stem), root and bracts (when present) in selected treatments (NO₃^-: Ca²⁺ in concentrations of 10:7 and 14:11 mol₍₊₎ m^-3 during the root growth stage, 10: 9 and 14: 11 mol₍₊₎ m^-3 during the vegetative stage of development, and 10:9 and 12:7 mol₍₊₎ m^-3 in the pigmentation stage) being this, the treatments with higher and lower effect on the variables evaluated in each of the stages. The concentration of total N was determined based on the semi-microKjeldahl method (^{Chapman and Pratt, 1973}; ^{Brearen and Mulvaney, 1982}) and P, K, Ca and Mg via wet digestion (^{Alcántar and Sandoval, 1999}); subsequently, the extracts were read in spectrophotometry equipment induction coupled plasma ICP-AES VarianTM, Liberty II. Sulfur (S) was determined by turbidimetry (^{Alcántar and Sandoval, 1999}), with a spectrophotometer (Thermo Spectronic-Genesys10uV).

The data obtained was analyzed through an analysis of variance for the main factors (NO₃^- and Ca²⁺) and the interaction in SAS 8.1 (SAS Inst., Cary, North Carolina, USA); the arrangement of treatments was full factorial 3² with four replicates per treatment. In those cases in which the analysis of variance indicated that at least one treatment was different from the others in terms of results (p≤ 0.05), the means were separated through means comparison test Tukey (p≤ 0.05).

Results

In the root growth stage, NO₃^- had an adverse effect on plant growth by raising the concentration to 14 mol₍₊₎ m^-3 because dry matter of stem and leaves had a significant decrease (Table 2); also, the increase in Ca²⁺ concentration was associated with a decrease in dry matter of root (Table 2).

Table 2 Dry matter production and area poinsettia bracts due to the concentration of nitrate (NO₃^-) and calcium (Ca²⁺) in the nutrient solution in three phenological stages. 

Medias con la misma literal en la columna son iguales estadísticamente de acuerdo con la prueba Tukey (p ≤ 0.05), ns = no significativo.

In the vegetative development stage, the increase in the concentration of NO₃^- caused a decrease in plant growth; whereas, the main effect of Ca²⁺ was not statistically significant in dry matter from the organs evaluated (Table 2). The interaction of factors in study showed that regardless of the concentration of Ca²⁺, the increase in NO₃^- concentration from 10 to 12 mol₍₊₎ m^-3 showed a decrease in dry matter of root, same that increased again with the highest concentration of NO₃^- both with 9 as with 11 mol₍₊₎ m^-3 (Figure 1A). As for stem (Figure 1B) and leaves (Figure 1C), the interaction indicated that by increasing concentration of NO₃^- there was a decrease in the weight of the dry matter when Ca²⁺ concentration was 9 and 11 mol₍₊₎ m^-3, but with 7 mol₍₊₎ m^-3 of Ca²⁺ ,dry matter increased only when the solution with 12 mol₍₊₎ m^-3 of NO₃^- is used.

Figure 1 Effect of concentration of nitrate (NO₃) and calcium (Ca²⁺) in the nutrient solution applied during the vegetative growth stage of the accumulation of dry matter of root (A), stem (B) and sheet (C) good night. 

During pigmentation stage the effect from NO₃^- was not significant on weight of the dry matter of the analyzed organs and bracts area. There was a positive effect on a decreasing dose of Ca²⁺ in dry matter of leaves and bracts, as well as in bracts area (Table 2). The interaction of the factors under study in the bract area shows that a concentration of 7 mol₍₊₎ m^-3 Ca²⁺ had an increase in both the area and dry matter of bracts by increasing the concentration of NO₃^- , response that is detected with Ca²⁺ concentration of 11 mol₍₊₎ m^-3 and 14 mol ₍₊₎ m^-3 of NO₃^- (Figures 2A and 2B). In contrast, with 7 mol₍₊₎ m^-3 of Ca²⁺ showed greater area and dry matter of bracts with NO₃^- levels of 10 and 12 mol₍₊₎ m^-3.

Figure 2 Effect of the concentration of nitrate (NO₃^-) and calcium (Ca²⁺) in the nutrient solution applied during the stage of pigmentation in the area (A) and dry matter of bract (B) in poinsettia plants. 

The concentration of N, Ca, Mg, and S in shoots and roots of poinsettia plants, were not significantly affected by the ratio NO₃^- : Ca²⁺ from the nutrient solution during the stage root growth (Table 3). Similar results are observed in the vegetative growth stage, where there is no effect from the ratios evaluated in the concentrations of N, K, Mg and S in shoot or in the concentrations of N, P, Ca, Mg and S in root (Table 4).

Table 3 Concentration of N, P, K⁺, Ca²⁺, Mg² + and S in shoot and root of poinsettia plants during the period of root growth, the effect of the relationship NO₃^-: Ca²⁺ in the nutrient solution. 

Medias con la misma literal en la columna, son iguales estadísticamente de acuerdo con la prueba Tukey (p≤ 0.05). DMSH, diferencia mínima significativa honesta.

Table 4 Concentration of N, P, K⁺, Ca²⁺, Mg²⁺ and S in shoot and root poinsettia during the vegetative stage of development, the effect of the relationship NO₃^-: Ca²⁺ in the nutrient solution. 

Medias con la misma literal en la columna, son iguales estadísticamente de acuerdo con la prueba Tukey (p≤ 0.05). DMSH, diferencia mínima significativa honesta.

At the stage root growth (Table 3), the concentration of P in shoots of the plant, was statistically higher with with NO₃^-: Ca²⁺ ratio of 10:7 mol₍₊₎ m^-3. Similarly, during the vegetative growth, with the ratio 10:9 mol₍₊₎ m^-3 of NO₃^-: Ca²⁺, obtained the highest concentration of in aerial parts (Table 4).

Similarly, K⁺ concentration in the aerial part of the plants increased by reducing the concentration of NO₃^- and Ca²⁺ in the nutrient solution during the radical growth stage (Table 3); K⁺ concentration in root and vegetative stage, on the contrary, it increases by increasing the concentrations of NO₃^- and Ca²⁺ in the nutrient solution (Table 4).

During the pigmentation, N, P, Ca and Mg concentration in aerial part and bracts were not influenced by the ratios NO₃^-:Ca²⁺ evaluated in the nutrient solution. So in roots, the concentration of P, Ca and Mg were not statistically different (Table 5).

Table 5 Concentration of N, P, K ⁺, Ca²⁺, Mg²⁺ and S in the aerial part, root and bract poinsettia during the stage of pigmentation due to the relationship NO₃^-: Ca²⁺ in the nutrient solution. 

Medias con la misma literal en la columna, son iguales estadísticamente de acuerdo con la prueba Tukey (p≤ 0.05). DMSH, diferencia mínima significativa honesta.

At the pigmentation stage, the concentration of K⁺ was higher in all plant organs when the nutrient solution contained 10 mol₍₊₎ m^-3 of NO₃^- and 9 mol₍₊₎ m^-3 Ca²⁺; on the contrary, with this ratio S concentration decreased in aerial parts and bracts (Table 5). In roots, S concentration increased significantly with this nutrient solution.

Discussion

There have been few studies on the effect of NO₃^-:Ca²⁺ in plant growth; however, the importance of conducting evaluations of the interaction between these nutrients is exposed in this research because the results suggest that there is a response of poinsettia plants to ratios between NO₃^- and Ca²⁺, which was also in function of the phenological stage.

During the initial growth stage, Ca²⁺ concentration had no effect on plant growth, while NO₃^- played an important role in the stage of root development, as greater biomass was accumulated in the aerial part and radical when low to moderate concentrations of NO₃^- (Table 2) were used.

In the vegetative growth stage, the response to NO₃^- concentration was influenced by the concentration of Ca²⁺; overall, there was greater biomass from stem and leaves when low levels of NO₃^- (10 mol₍₊₎ m^-3) were used in combination with high levels of Ca²⁺ (9 to 11 mol₍₊₎ m^-3). It is also possible to obtain the greatest plant growth by increasing NO₃^- to 12 mol₍₊₎ m^-3 if this is combined with a decrease in the concentration of Ca²⁺ to 7 mol₍₊₎ m^-3 (Figure 1). Therefore, it is concluded that in plant development and root growth stage are not recommended high levels of NO₃^- for this species, which may be due to low concentrations NO₃^- is an activator of radical development but has an inhibitory effect when concentrations are high (^{Zhang et al., 1999}; ^{Crawford and Forde, 2002}; ^{Antolinez-Delgado and Rodríguez-López, 2008}). Radical development implies greater allocation of reserves to the root, thereby generating an increase in the surface to obtain water and nutrients (^{Agreen and Franklin, 2003}), which ultimately is reflected in a better plant development.

The results of the present study contrast with those reported by ^{Conley et al. (2002)}, who indicated that poinsettia plants are obtained with larger size and better color and form with high levels of N, from 14.3 to 19.6 mol₍₊₎ m^-3. It has been shown that higher absorption of N on poinsettia occurs in early growth stages before floral induction, but from floral induction to flower formation, the uptake of P, K⁺, and Mg²⁺ increases, while Ca²⁺ uptake remains unchanged between the initial phase and induction (^{Scoggins and Mills, 1998}); the latter it is also in contrast with the results obtained in this study as it was found that in the flowering stage, bracts growth was markedly influenced by the concentration of Ca^2+, although this response depended on NO₃^- concentration.

The results of this study suggest that during the stage of pigmentation, NO₃^- levels between 10 and 12 mol ₍₊₎ m^-3 allow greater bracts growth if combined with 9 mol₍₊₎ m^-3 Ca²⁺ (Figure 2). This is consistent with the information submitted by ^{Rose and White (1994)}, who indicate that during floral induction, an adjustment is made on the accumulation of dry matter toward the bracts and cyathia, causing a decrease in N requirements. ^{Paparozzi et al. (1994)}, mention that in ornamental plants on pots require less fertilizer once it reaches anthesis, which was confirmed by ^{Kuehny et al. (2000)} in several poinsettia cultivars, indicating that it is necessary to adjust the fertilization dose.

P has a great influence on root growth of plant, and specifically on poinsettia where high quality was obtained with the application rate of 1.13 mol₍₊₎ m^-3of P (^{Khandan-Mirkohi et al., 2015}), which could explain the reduction in root growth observed when NO₃^- and Ca²⁺ concentrations increased as to maintain a homogeneous osmotic potential between the nutrient solution under evaluation, it was necessary to decrease the concentration of H₂PO₄^- and SO₄^2- in those solutions with higher concentrations of NO₃^-. Similarly, the highest concentration of P in the aerial part that was present during the radical and vegetative growth (Table 3 and 4) in plants treated with low levels of NO₃^- and Ca²⁺ could be due to increased concentration of H₂PO₄^- in the nutrient solution, so the plant had higher availability of this nutrient. Due to the negative impact of a deficiency of P in the root growth, growth in aerial parts can also be affected (^{Olivera et al., 2007}), since it depends on nutrients transport and other substances from the roots, which could also explain the reduction in dry matter observed in the present study in each of the organs by increasing the concentration of NO₃^-.

A remarkable fact was that despite lowering the concentration of K⁺ in the nutrient solution to adjust osmotic potential, the plants managed to accumulate a higher concentration in different organs during the three phenological stages, suggesting that K⁺ is a nutrient absorbed in higher amounts by poinsettia (^{Oliveira et al., 2004}) and that there was an adjustment by the plant to increase its absorption despite the decrease in the external concentration.

The highest concentration of S observed during pigmentation in the aerial part and root was recorded with 12 mol₍₊₎ NO₃ m^-3 (Table 5), which may be due to the relationship between the absorption and metabolism processes of N and S in superior plants (^{Koprivova et al. 2000}). The highest concentration of N in plants despite of the lowest concentration of NO₃^- in the nutrient solution was observed in the pigmentation stage which can be explained by the fact that during floral induction makes an adjustment in the translocation of dry matter towards the bracts and cyathia, causing a decrease in the requirements of N (^{Rose and White, 1994}); in addition Ca²⁺ is involved in the absorption of NO₃^- (^{Tuna et al., 2007}) and the concentration of Ca²⁺ increases in the bracts of poinsettia flowers by decreasing the concentration of N and K⁺ in the nutrient solution (^{Starkey and Nielsen, 2001}). It has been reported that even concentrations of Ca²⁺ and Mg²⁺ are higher in poinsettia plants when N is provided in the form of NO₃^- instead of NH₄⁺ and that this species prefers N in the form of NO₃^- as it obtains greater biomass (^{Scoggins and Mills, 1998}). From the above data a change in the ratio per phenological stage between NO₃^- and Ca²⁺ in the nutrient solution for an optimal growth of poinsettia plants is suggested.

Conclusions

The optimal ratio between the concentration of NO₃^- and Ca²⁺ was based on the phenological stage in poinsettia flower. During the stage of root growth accumulated greater biomass when low concentrations of NO₃^- (10 to 12 mol₍₊₎ m^-3) were used, with no effect of Ca²⁺ ; while in the vegetative stage these low levels of NO₃^- (10 mol₍₊₎ m^-3) should be combined with high levels of Ca²⁺ (9 to 11 mol₍₊₎ m^-3). In the pigmentation stage, NO₃^- levels between 10 and 12 mol₍₊₎ m^-3 allow greater growth of bracts when combined with Ca²⁺ concentrations of 9 mol₍₊₎ m^-3.