Introduction

The cowpea bean (Vigna unguiculata L. Walp) is an important source of food in the tropics and subtropics, provides adequate contents of protein, dietary fiber, carbohydrates, vitamins and phytochemicals (^{Devi et al., 2015}). Dry beans, green pods, growing tips are consumed from cowpea beans and the whole plant is used as fodder (^{Márquez-Quiroz et al., 2015}). For what is an important source of food for the rural population of the states of Campeche, Chiapas, Guerrero, Jalisco, Oaxaca, Tabasco, Veracruz, Yucatan and Tamaulipas (^{Lagunes-Espinoza et al., 2008}; ^{Apaez-Barrios et al., 2011}).

Zinc is an essential micronutrient in biological systems, which is required in small quantities. It is involved in the formation and activation of enzymes that impact on the growth, development and production of plants (^{Poblaciones and Rengel, 2016}). Its deficiency impacts on growth, pollen viability, flowering and grain production (^{Pandey et al., 2006}). In humans, its deficiency is associated with problems of growth and learning capacity in children, and increases the risk of infections, cancer and DNA damage (^{Ahmed et al., 2014}). It is present in around one third of the world population, which represents the fifth risk factor for diseases in developing countries (^{Shahzad et al., 2014}).

Biofortification is the agronomic process by which the content of elements and microelements in the edible parts of the cultivation plants is increased, which are applied in a foliar or edaphic form (^{Guillén-Molina et al., 2016}). In this regard, ^{Prasad et al. (2015)} mention that the success of biofortification depends on the mobility of the element in the soil and the plant, zinc, is one of the most suitable microelements for biofortification, presenting its greatest absorption when applied as a foliar ZnSO₄. While ^{Cakmak et al. (2010)} report that the most effective method to increase the zinc content in the wheat grain is the edaphic and foliar application at the same time.

In cabbage (Brassica oleracea cv. Bronco) it is reported that the application of doses between 80 and 100 μM of Zn increase the concentration of zinc in the edible part of the plant (^{Barrameda-Medina et al., 2017}). While ^{Sida-Arreola et al. (2015b)} report that with 50 μM dose of zinc sulfate has the highest growth and yield of Phaseolus vulgaris.

Regarding cowpea beans, it is reported that the zinc content in the grain is low, especially when planted in soils with deficiencies of elements (^{Morales-Morales et al., 2016}). While ^{Guillén-Molina et al. (2016)} report that the zinc content in the cowpea bean grain is increased when it is biofortified with Zn chelate in an edaphic way. Therefore, the objective of this work was to know the effect of foliar application of different doses of zinc chelate (Zn-EDTA) and zinc sulfate (ZnSO₄) on the yield and mineral content of cowpea beans (V. unguiculata L.).

Materials and methods

Results and discussion

Flowering in biofortified plants with different Zn-EDTA doses occurred between 46.0 and 51.0 dds, while plants biofortified with doses of ZnSO₄ flowered between 46.3 and 51.7 dds, while analysis of orthogonal contrasts detected statistical differences (p≤ 0.01) between the dose of 7 mM L^-1 of ZnSO₄ and the doses with 14 and 28 mM L^-1 (Table 1). Because flowering at all doses (treatments) occurred after 45 days, it is considered late (^{Manggoel and Uguru 2012}). The different doses of Zn-EDTA and ZnSO₄, decreased the days to flowering, with a higher precocity of the plants treated with 7 mM L^-1 of Zn-EDTA or ZnSO₄. For grain weight per plant values ​​were found between 22.20 and 34.03 g per plant for Zn-EDTA doses, while ZnSO₄ doses had grain weights between 20.97 and 24.50 g, with statistical differences (p≤ 0.01) in the contrast of the control treatment (0 mM L^-1) and the doses of 7, 14 and 28 mM L^-1 of ZnSO₄.

In general, these yields are greater than the 7 g reported by ^{Apaez-Barrios et al. (2011)} and the 13 g of grain per plant obtained by local producers of cowpea beans (^{SIAP, 2013}); but they are equal to the average grain weights reported by ^{Márquez-Quiroz et al. (2015)} for biofortified cowpea beans. The highest performance of the treatments with the doses of 7, 14 and 28 mM L^-1 of Zn-EDTA or ZnSO₄, coincides with that reported by ^{Quddus et al. (2011)} who indicate that the yield increases with increasing zinc dose.

The number of pods per plant ranged between 14.33 and 25.67 for the different doses of Zn-EDTA, and from 15.33 to 19.67 for the doses of ZnSO₄. These values are higher than the 8.75 pods obtained by ^{Guillén-Molina et al. (2016)} for cowpea beans biofortified with zinc. The differences may be due to the time of year in which the experiments were planted, since the present work was planted in the autumn winter cycle, while the work of ^{Guillén-Molina et al. (2016)} was sown in the spring-summer cycle, with average temperatures higher than those presented in the present work. The effect of temperature on the flowering of cowpea beans is known to be sensitive to high temperatures in the reproductive stage, which causes flower abortion and decreased yield (^{Hanumantha et al., 2016}).

The number of grains per plant ranged between 163.67 and 181.33 grains for the doses of Zn-EDTA, and from 164.67 to 177.33 grains for the doses of ZnSO₄. For the weight of 100 grains values between 12.92 and 16.67 g were found for the different doses of Zn-EDTA, while the doses of ZnSO₄ had weights of 100 grains between 12.50 and 14.39 g. Observing statistical differences (p≤ 0.01) between the control doses and the different doses of Zn-EDTA and ZnSO₄ (table 1). On average, the doses of 7, 14 and 28 mM L^-1 of Zn-EDTA had a 28.25, 6.97 and 19.20% greater weight of 100 grains than the control (0 mM L^-1), while the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ had weight increases of 100 grains of 15.12, 3.20 and 4.80% with respect to the control.

In the treatments with Zn-EDTA and ZnSO₄, the 7 mM L^-1 dose with the highest average weight of 100 grains stands out. In general, the weights of 100 grains of all the treatments are higher than the 12.0 and 12.4 g reported by ^{Lagunes-Espinoza et al. (2008)} and ^{Apaez-Barrios et al. (2011)}, but according to ^{Márquez-Quiroz et al. (2015)} only the doses with 7 and 28 mM L^-1 of Zn-EDTA had the higher weights of 100 grains of biofortified cowpea beans.

The doses of 7, 14 and 28 mM L^-1 of Zn-EDTA and ZnSO₄ decreased the days to flowering, and increased the weight of grain, number of pods per plant, number of grains per plant and the weight of 100 grains, which indicates that the different doses Zn-EDTA and ZnSO₄ increase the production of biomass and grain in the plants of cowpea beans, which coincides with ^{Ratto and Miguez (2005)} and ^{Quddus et al. (2011)} who indicate that total dry matter and yield increase in plants biofortified with zinc, while ^{Sida-Arreola et al. (2015a)} and ^{Ibrahim and Ramadan (2015)} indicate that the application of the adequate dose of Zn increases biomass and yield.

The total nitrogen (N) content ranged between 4.14 and 4.38% for the doses of Zn-EDTA, while for the doses of ZnSO₄ it was 4.15 to 4.32% (Figure 1B). These values are higher than those reported by ^{Márquez-Quiroz et al. (2015)} and ^{Guillén-Molina et al. (2016)}. According to the factor of 5.45 to convert the nitrogen content to crude protein of V. unguiculata L. (^{Muranaka et al., 2016}), the protein content ranged between 22.56 and 23.87% for the different doses of Zn-EDTA and from 22.62 to 23.54% for the doses of ZnSO₄ (Figure 1A ); presenting the highest total crude protein contents at 14 mM L^-1 doses of ZnSO₄ (23.89%) and Zn-EDTA (23.54%). These crude protein contents are higher than the 20.3% reported by ^{Guillén-Molina et al. (2016)} and 22% of protein reported by ^{Márquez-Quiroz et al. (2015)} for biofortified cowpea beans.

Figure 1 Content of total crude protein (A), nitrogen (B), phosphorus (C), potassium (D), calcium (E) and magnesium (F) in the bean seed of cowpea (V. unguiculata L.) biofortified with different doses of chelate and zinc sulfate. 

But they are low with respect to the maximum value of the total crude protein range of 21 to 30.70% reported for V. unguiculata (^{Timko and Singh, 2008}). The highest contents of total crude protein were obtained with the doses of 7 and 14 mM L^-1 of Zn-EDTA (23.82 and 23.87%) and ZnSO₄ (23.05 and 23.54%), decreasing the content of crude protein in 4.28% in the dose of 28 mM L^-1 of Zn-EDTA and 4.06% in the dose of 28 mM L^-1 of ZnSO₄. What can be due to higher doses of Zn cause oxidative stress to the plant, which increases the production of active oxygen species, which damages the cellular components and the production of proteins (^{Muhammad et al., 2016}).

It can also be due to the problem of toxicity that occurred with the application of 28 mM L^-1 doses of Zn-EDTA and ZnSO₄ at 21 dds, manifesting with problems of leaf necrosis and reduced plant growth. With the doses of 14 mM L^-1 of Zn-EDTA and of ZnSO₄, the highest content of nitrogen and total crude protein (Figure 1A) and the lowest grain yields per plant were observed (Table 1), which may be due to the negative correlation between nitrogen content and grain yield (^{Olunike, 2014}).

When comparing the phosphorus content (P) of the control with the doses of 7, 14 and 28 mM L^-1 of Zn-EDTA, increments of 18.46, 18.46 and 1.85% are observed, respectively; while the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ had increases of 20.75, 20.75 and 18.86%, respectively (Figure 1C). There was a slight decrease in phosphorus with the doses of 28 mM L^-1 of Zn-EDTA and ZnSO₄, which indicates that with the zinc doses used, the maximum limit was not reached to observe the pronounced decrease in phosphorus reported by ^{Cakmak et al. (2010)}.

For the content of potassium (K) it was found that, with the doses of 7, 14 and 28 mM L^-1 of Zn-EDTA was increased by 6.87, 7.63 and 7.63%, respectively, the potassium content; while the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ the potassium content increased by 0, 3.82 and 7.63%, respectively (Figure 1D).

The contents of potassium found in all doses are within the contents reported for cowpea beans (^{Frota et al., 2008}; ^{Márquez-Quiroz et al., 2015}). With regard to the content of calcium (Ca), values between 0.08 and 0.9% were found in the different doses of Zn-EDTA and ZnSO₄ (Figure 1E), with increases in the calcium content with the doses of 7 and 14 mM L^-1 of Zn-EDTA of 12.5%, while with ZnSO₄ the decrease in calcium content was observed with the dose of 28 mM L^-1. For the content of magnesium (Mg) values between 0.22 and 0.25% were found with the doses of Zn-EDTA and 0.24 and 0.25% with the doses of ZnSO₄ (Figure 1F), observing that with the dose of 14 mM L^-1 of Zn-EDTA and ZnSO₄ have the highest Mg contents, while with the dose of 28 mM L^-1 of ZnSO₄ there is a decrease of 8.33% with respect to the control.

The increase in mineral contents with the doses of 7 and 14 mM L^-1 of Zn-EDTA and ZnSO₄, indicate the influence of biofortification with zinc on the mineral content of cowpea beans (^{Guillén-Molina et al., 2016}). For the content of crude protein and macroelements, it was found that the highest contents were obtained with doses of 7 and 14 mM L^-1 of Zn-EDTA and of ZnSO₄, but with a tendency to present the highest values at doses of 7 and 14 mM L^-1 of Zn-EDTA, which may be due to the greater assimilation of the chelated fertilizers and that some elements are characterized by being inside the plant in a chelated form (^{Sánchez, 1984}).

For the content of iron (Fe) it was observed that with the doses of 7 and 14 mM L^-1 of Zn-EDTA there were increases of 11.14 and 9.19% of the iron, respectively; while with doses of ZnSO₄, only with 7 mM L^-1 there was an increase of 0.71% of iron. Meanwhile, with the doses of 28 Mm L^-1 of Zn-EDTA and ZnSO₄, the iron content decreases 4.37 and 14.61% (Figure 2A). Similar effects have been observed in V. unguiculata, with reports indicating that doses greater than 15 mM L^-1 of ZnSO₄ decreases iron content by 31.10% (^{Morales-Morales et al., 2016}). In general, iron content is within the range of 48 to 79 ppm reported for V. unguiculata (^{Timko and Singh, 2008}). For the zinc content there were increases of 5.81, 8.98 and 2.11% with the doses of 7, 14 and 28 mM L^-1 of Zn-EDTA, while the doses of ZnSO₄ had increases of 11.28, 19.99 and 23.61%, respectively (Figure 2B).

Figure 2 Content of iron (A), zinc (B), boron (C), manganese (D), copper (E) and molybdenum (F) in seeds of cowpea beans (V. unguiculata L.) biofortified with different doses of chelate and zinc sulfate. 

The higher zinc contents found in the present work with respect to what was reported by ^{Timko and Singh (2008)} and ^{Guillen-Molina et al. (2016)}, it can be because the foliar application of zinc is more effective than the edaphic application, to increase its content in the grain (^{Yue-Qiang et al., 2012}). The higher zinc content in the biofortified grains with the ZnSO₄ doses, may be due to the biofortification with ZnSO₄ in foliar form is more effective to increase the zinc content in the grain, than that made with Zn-EDTA (^{Prasad et al., 2015}). The increase in Zn and the decrease in Fe, with the increase in the dose of Zn-EDTA and ZnSO₄, is due to the antagonistic effect between Zn and Fe (^{Fasaei and Ronaghi, 2015}).

Regarding ^{Shekari et al. (2015)} report that the increase in the dose of Zn, decreased the Fe content in the wheat grain. With the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ and with 14 mM L^-1 of Zn-EDTA there were 64.10, 68.00, 71.20 and 61.90 ppm, values that are higher than the 61 ppm established as critical level of zinc in plants, to have a zinc sufficiency status in human nutrition (^{Huett et al., 1997}).

The boron content was 16.90 to 21.10 ppm for the doses of Zn-EDTA, while for the ZnSO₄ doses the boron content was 20.60 to 23 ppm (Figure 2C). For the different doses of Zn-EDTA it is observed that the control (0 mM L^-1) had the highest boron content, while the doses of 7, 14 and 28 mM L^-1 decrease 15.17, 3.32 and 19.91% on the boron content. Whereas the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ increased the boron content by 1.94, 3.99 and 11.65%. For the manganese content values between 20.30 and 26.70 ppm were found for the doses of Zn-EDTA, while in the doses of ZnSO₄ the content was 18.70 to 19.40 (Figure 2D).

The copper content ranged between 10.90 and 17.60 ppm for the different doses of Zn-EDTA, and from 9.84 to 15.20 ppm for the doses of ZnSO₄ (Figure 2E), with increases of 61.47, 53.21 and 4.59% in the doses of 7, 14 and 28 mM L^-1 of Zn-EDTA, while, in the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ the increase was of 54.47, 49.39 and 16.87%. In general, it is observed that the copper content increases with the doses of 7 and 14 mM L^-1 of Zn-EDTA and of ZnSO₄, but decreases with the doses of 28 mM L^-1. For the molybdenum content values between 10 and 15.10 ppm were found in the doses of Zn-EDTA, while the ZnSO₄ doses had contents of 13.40 to 17.70 ppm (Figure 2F).

Conclusions

The biofortification of cowpea beans with different doses of Zn-EDTA and ZnSO₄ have effects on grain yield, total crude protein content and mineral content. The doses of 7, 14 and 28 mM L^-1 of Zn-EDTA had the greatest increases in grain yield per plant with respect to the control. The doses of Zn-EDTA and ZnSO₄, decreased the days to flowering, and increased the number of pods, number of grains and the weight of 100 grains with respect to the control. The highest contents of nitrogen, crude protein, phosphorus, potassium, calcium, iron, manganese and copper were obtained with the doses of 7 and 14 mM L^-1 of Zn-EDTA.

The zinc content increased proportionally with the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ in 11.28, 19.99 and 23.61% presenting the doses of 7, 14 and 28 mM L^-1 of ZnSO₄ and the dose of 14 mM L^-1 of Zn-EDTA values higher than 61 ppm, which is considered as critical value to have a zinc sufficiency status in the plants cultivated for human consumption.