Physiological Responses of Cowpea ( Vigna unguiculata ) Under Irrigation With Saline Water and Biostimulant Treatment

Cowpea (Vigna unguiculata) is one of the world’s main crops, and it is a fundamental source of protein for semiarid regions population. In these regions, the use of high salts concentration water in irrigation systems is one of the major factors that contributes to reduced cowpea yield. One way to alleviate the negative effects of salinity is through the biostimulants application, which is a product that has beneficial substances to the plants metabolism. The aim of this study was to evaluate the application of biostimulant in cowpea cultivars under irrigation with saline water. The study was carried out in the Agrarian Sciences Center, of the Department of Agronomic and Forest Sciences of the Federal Rural University of the Semi-Arid, in the city of Mossoró, RN. The experimental design was completely randomized, with four replications. The treatments were arranged in 5 × 2 × 2 factorial scheme, with five doses of biostimulant (0, 15, 30, 45 and 60 mL L), two electrical conductivities of the irrigation water (0.5 and 5.0 dS m), and two cowpea cultivars (IPA-206 and BRS Guariba). The evaluated characteristics were: chlorophyll content index, stomatal conductance, net photosynthesis, internal CO2 concentration, transpiration rate, shoot height, stem diameter and shoot dry mass. The biostimulant application was not efficient in attenuating the salinity stress effect on the development of cowpea cultivars. The higher biostimulant concentrations along with the use of saline water increased the negative effects of salinity on the cowpea plants physiology. There was no difference between the cultivars regarding the tolerance to saline stress and the application of biostimulant.


Introduction
The cowpea (Vigna unguiculata (L.) Walp.) is one of the main sources of vegetable protein in tropical and subtropical regions of the world (Santos et al., 2014).In this scenario, Brazil is classified as the world's third largest producer, with estimated production of 749.4 thousand tons for the 2017/2018 harvest (CONAB, 2018).In the Brazilian national ranking, the Northeast region is the largest producer, with approximately 51% of the national production, produced in an area of 1,134.3hectares.Despite the high productive potential, the average yield of cowpea in the Northeast is still low, with only 336 kg ha -1 (CONAB, 2018).
The development of irrigation management strategies, especially those aimed at mitigating the effects of saline stress on plants (Silva et al., 2013;Oliveira et al., 2015), is one way to increase the cowpea productivity in regions with salinity problems.Among these strategies, the application of natural or synthetic substances that alleviate the salinity stress on plants may be an important alternative (Oliveira et al., 2017).Several studies have been conducted aiming the development of techniques that might alleviate the negative effects of irrigation with saline water, the use of biostimulants is among the techniques studied (Oliveira et al., 2013(Oliveira et al., , 2017)).Biostimulants are products that can assist plants in overcoming abiotic stresses, mainly due to their roles as hormonal and nutritional stimulants (Oliveira et al., 2016).
Many commercial products based on the seaweed extract Ascophyllum nodosum have been used as a biostimulant, which is an alternative source of nutrients to plants that leaves no residues or pollutants in the environment.The Acadian ® (Acadian Seaplants liquid, Canada) stands out as one of the most used biostimulants in agriculture (Hurtado & Critchley, 2018).Acadian is the trade name for the seaweed A. nudosum L. This is commonly used in agriculture as a growth stimulant, which contributes to the quality of different crops, in addition to increasing the photosynthetic rate.This is rich in many growth regulators such as auxins, gibberellins, cytokinins, macro and micronutrients such as Ca, K and Mo.This substances are beneficial to the plant metabolism, which gives it the biostimulating effect (Acadian Agritech, 2009).
Several studies have already proven the beneficial effects of biostimulants applications (Oliveira et al., 2015(Oliveira et al., , 2017)).However, in most of these researches the biostimulant used was the Stimulate ® (plant growth regulator of the chemical group composed of the hormones cytokinin, gibberellin and indolalkanoic acid).There is little information in the literature about the biostimulant Acadian ® being used in cowpea, especially under salinity stress conditions.Due to the above mentioned considerations, the hypothesis was raised that the biostimulant application can reduce the effects of salinity stress on cowpea.The objective of this work was to evaluate the effects of biostimulant application on the physiological responses of cowpea cultivars (Vigna unguiculata (L.) Walp.) irrigated with saline water.

Material and Methods
The study was carried out from April to June 2016 in a greenhouse in the Didactic Vegetable Garden of the Agrarian Sciences Center of the Department of Agronomic and Forest Sciences of the Federal Rural University of the Semi-Arid (UFERSA), in Mossoró, RN, Brazil (5°11′31″ S; 37°20′40″ W; altitude 18 m).
A mixture of soil and commercial substrate (Plantmax ® ) in a 3:6 ratio, was the material used as substrate.The soil used is classified as Eutrophic Haplic Planosol (EMBRAPA, 2013), collected in the 0-20 cm depth, and its physico-chemical characteristics are presented in Table 1.

Physical characteristics
Granulometric fraction (g kg Note: OM = Organic matter; CEC = Cation exchange capacity. The experimental design was completely randomized, with the treatments arranged in a 5 × 2 × 2 factorial scheme.The first factor was two electrical conductivities of the irrigation water (ECw) (0.5 and 5.0 dS m -1 ); the second two cowpea cultivars (IPA-206 and BRS Guariba); and the third five biostimulant doses applied via leaves (0, 15, 30, 45, 60 mL of Acadian ® per liter of water) with four replicates.Each experimental unit was represented by a 1.6 dm -3 volumetric capacity plastic bag containing one plant.Five cowpea seeds were sown in each plastic bag, at a depth of two centimeters.Seven days after sowing, the thinning was performed, leaving in each bag the most vigorous plant.After that, the treatments with saline water irrigation started.
For the lowest ECw (0.5 dS m -1 ), water from a deep well located at the UFERSA Central Campus was used.To prepare the water with the highest ECw (5.0 dS m -1 ), a mixture of the following salts: NaCl, CaCl 2 •2H 2 O and MgCl 2 •6H 2 O was added to the 0.5 dS m -1 water, in a 7:2:1 ratio (Rhoades et al., 2000).The biostimulant used was the seaweed extract of the species Ascophyllum nodosum (Acadian ® ), composed of: N-8.12; P-6.82; K-12.00;Ca-1.60;Mg-2.03;S-8.16 g kg -1 ; B-5.74; Cu-13.60;Fe-11.5;Mn-0.04;Zn-24.40 and Na-20000 mg kg -1 ; potassium hydroxide, with 61.48 g L -1 of water-soluble K 2 O; 69.60 g L -1 of total organic carbon; and a density of 1.16 g dm -3 (Silva et al., 2016).Two applications of biostimulant were carried out at 7 and 25 days after sowing.The applications were performed in the morning, starting at 8:00 am.The whole aerial part of the plant was sprayed until runoff, using a 5 L capacity hand sprayer, applying a water volume equivalent to 300 L ha -1 (Abrantes et al., 2011).For the treatments that did not receive the biostimulant dose (0 mL of Acadian ® ), the plants were sprayed only with water, applying the same volume of the other treatments.
The following variables were evaluated at 40 days after sowing: chlorophyll content index (CCI), stomatal conductance (gs; mol H 2 O m -2 s -1 ), net photosynthesis (A; μmol CO 2 m -2 s -1 ), internal CO 2 concentration (Ci; μmol CO 2 m -2 s -1 ) and transpiration rate (E; mmol H 2 O m -2 s -1 ).The gs, A, Ci and E measurements were performed using an infrared gas analyzer (IRGA, portable model LI-6400, li-color, Lincoln, Nebraska, USA), and the readings were performed between 08:00 and 10:00 am.The CO 2 contents were set at 400 μmol m -2 s -1 and the luminous intensity at 1500 μmol photons m -2 s -1 .Young newly expanded, undamaged and well lit leaves (when the light intensity was greater than 1000 μmol of photons m -2 s -1 ) were evaluated.The CCI was determined using a portable chlorophyll-meter (model CCM-200, Opti-Science), and two readings were performed per plant, always in the third and fourth leaf of each plant, counting from the apex.
At 45 days after sowing, the plants were harvested.The following variables were evaluated: shoot height (SH), measured with a ruler graduated in cm; stem diameter (SD), determined at 3 cm from the ground, using a digital caliper; and shoot dry mass (SDM), where the aerial plant part (leaves + stem) was packed in paper bags and placed in a forced air oven at a temperature of 65±1 °C until reaching a constant mass.After that, the dried shoot was weighed in analytical balance to obtain its dry mass.
The data obtained were submitted to analysis of variance by the F test (p ≤ 0.05), and the results were analyzed using the SISVAR software (Ferreira, 2011).The effect of the salinity and cultivars factors were analyzed using the Tukey's test (p ≤ 0.05), while the biostimulant effect was evaluated by regression analysis.

Results and Discussions
There were triple interactions (cultivar × salinity × biostimulant) for the internal CO 2 concentration, stomatal conductance, net photosynthesis, chlorophyll content index and shoot dry mass.For the shoot height, there was a double interaction between cultivar and biostimulant and between salinity and biostimulant.For the transpiration rate, there was an isolated effect of the cultivar and interaction between salinity and biostimulant.For the stem diameter, there was an isolated effect of the factors (Table 2).Note.SV = Source of variation; DF = Degree of freedom; CV = Coefficient of variation; (**) Significant at 1% probability by F-test; ( ns ) not significant.
In general, cowpea cultivars irrigated with the highest ECw level (5.0 dS m -1 ) presented lower values of internal CO 2 concentration (Ci), stomatal conductance (gs) and net photosynthesis (A), in relation to those that were irrigated with water of lower ECw (0.5 dS m -1 ).The reduction of these variables is possibly a reflection of the ionic and osmotic effects caused under salinity stress conditions.This first effect (ionic) results from reduced soil water potential, while the second one (osmotic) is caused by the ions accumulation in plant tissues (Munns & Tester, 2008).Salinity stress can lead to stomatal closure and, as a result, reduce stomatal conductance.This results in lower intercellular CO 2 availability in the leaves and carbon fixation inhibition, while reducing transpirational water loss, resulting in lower photosynthetic rates (Praxedes et al., 2010;Huang et al., 2015).In the literature, several researchers have already reported the negative effects of salinity on cowpea gas exchanges (Neves et al., 2009;Souza et al., 2011;Silva et al., 2013;Prazeres et al., 2015).
In relation to Ci, in the plants of cultivar IPA-206 (Figure 1A) submitted to ECw of 0.5 dS m -1 , there was a positive linear effect as the biostimulant concentrations increased and, according to the regression equation, plants treated with 60 mL L -1 of Acadian ® provided the highest values, with 263.17 μmol CO 2 m -2 s -1 , representing a 15.9% increase compared to plants that did not receive biostimulant doses.In the ECw of 5.0 dS m -1 , increased biostimulant concentrations provided a quadratic effect, with a lower value (142.74 μmol CO 2 m -2 s -1 ) recorded in the absence of the biostimulant, and a higher (236.55 μmol CO 2 m -2 s -1 ) at the concentration of 33.4 mL L -1 .
The increase of the biostimulant doses at the two levels of salinity promoted a quadratic effect on the Ci values in the cultivar BRS Guariba (Figure 1B) and, according to regression equations, the lowest values were verified in the absence of biostimulant (0 mL of Acadian ® ).At the ECw of 0.5 dS m -1 , the highest value was 251.67 μmol CO 2 m -2 s -1 , obtained in the biostimulant dose of 33.7 mL L -1 , whereas, at the ECw of 5.0 dS m -1 , the highest value was 271.59 μmol CO 2 m -2 s -1 , recorded at the dose of 51.22 mL L -1 .
For the cultivar IPA-206 (Figure 1C), at the ECw of 0.5 dS m -1 , there was a quadratic response in relation to the biostimulant concentrations to variable gs, with maximum value (0.3586 mol H 2 O m -2 s -1 ) in the treatment with 8.9 mL L -1 , decreasing (0.1144 mol H 2 O m -2 s -1 ) in concentration with lower value in 60 mL L -1 .On the other hand, at the ECw of 5.0 dS m -1 , a higher value (0.0699 mol H 2 O m -2 s -1 ) was observed when 45.7 mL L -1 was applied, and the lowest value (0.0276 mol H 2 O m -2 s -1 ) was obtained in the absence of the biostimulant.The obtained results differ from those found by Anjos et al. (2015), who assessed different doses of Stimulate ® , Booster ® and Biozyme TF ® on common bean (Phaseolus vulgaris L.) plants and did not observe any significant results for gs.
Regarding the cultivar BRS Guariba (Figure 1D), there was small oscillations in the gs values at the two ECw levels with the increase of biostimulant concentrations.The highest gs values were obtained in the biostimulant concentrations of 34.3 and 22.3 mL L -1 , at both ECw levels, whereas the lowest gs values were obtained in the concentrations of 60 and 0.2 mL L -1 , at the salinity levels of 0.5 and 5.0 dS m -1 , respectively.This may have happened due to the fact that in high concentrations of salts (which hinders or reduces the absorption of water) the plants tend to close their stomata and, consequently, reduce their stomatal opening.Similarly to gs, at the ECw of 0.5 dS m -1 , A in the cultivar IPA-206 had a quadratic response to the effect of the biostimulant concentrations, presenting the higher value (22.48 μmol CO 2 m -2 s -1 ) at the dose of 9.4 mL L -1 (Figure 1E).From this concentration on, a reduction was observed, reaching the lowest value (10.94 μmol CO 2 m -2 s -1 ) in the dose of 60 mL L -1 .At the ECw of 5.0 dS m -1 , increasing biostimulant concentrations resulted in a linear decrease response in A, and according to the regression equation, the lowest A value (4.69 μmol CO 2 m -2 s -1 ) was found in the highest biostimulant concentration (60 mL L -1 ).
Regarding the A in BRS Guariba cultivar (Figure 1F), there was a linear decreasing response to the biostimulant concentrations at the two ECw levels.The highest A values were found in the absence of biostimulant and the lowest values in the dose of 60 mL L -1 .The results found in the present study are similar to those of Prazeres et al. (2015), who verified a linear reduction in the cowpea net photosynthesis as the salinity of the irrigation water increased.

Table 1 .
Physical and chemical characteristics of the substrate used in the study