Morphophysiology of Eggplant Irrigated With Wastewater and Nitrogen and Phosphorus Doses in the Semi-arid Region of Brazil

Water is a scarce resource in semi-arid regions, therefore, the correct water management is an essential practice. In this research we evaluated the use of nitrogen, phosphorus and treated wastewater on the growth and morphophysiology of eggplants (Solanum melongena L.) in the semi-arid region of Brazil. The experiment was conducted in Pombal, Paraíba, Brazil, using a randomized block design, in a 4 × 4 + 1 factorial scheme: wastewater with four nitrogen doses (N1 = 0.22; N2 = 0.39; N3 = 0.56; and N4 = 0.73 g N dm) and four doses of phosphorus (P1 = 0.96; P2 = 1.68; P3 = 2.40; and P4 = 3.12 g P dm), and the controls – distilled water fertilized with 0.56 g of N dm and distilled water fertilized with 2.40 g of P dm. Each treatment was replicated 4 times. The nitrogen and phosphorous interaction did not influence the growth and physiological aspects of eggplant plants. Excess growing media nitrogen significantly decreased gaseous exchanges of eggplant plants, being found decreased of 4.4 μmol m s the CO2 assimilation.


Introduction
Eggplant (Solanum melongena L.) is a vegetable of the Solanaceae family, easily adapted tropical climatic conditions (Lima et al., 2015).The Brazilian semi-arid areas have favorable edaphoclimatic conditions for eggplant cultivation.The semi-arid region of the northeast of Brazil is characterized by an irregular spatial and temporal rainfall distribution (Medeiros et al., 2017) and irrigation is needed to ensure agricultural production in the region (Medeiros et al., 2018a).
According to Gonçalves et al. (2013), the planned use of treated wastewater as an alternative water source saves surface water and allows agricultural production in regions with water restrictions.In addition, wastewater provides macro, such as nitrogen and phosphorus, and micronutrients to plants (Medeiros et al., 2017).The reuse of nutrients and reduction of the application of synthetic fertilizers are examples of the benefits of reusing water (Gonçalves et al., 2013;Medeiros et al., 2018b).Nitrogen and phosphorus are essential macronutrients for plant growth and development, and play important roles in the respiration and photosynthesis (Weber et al., 2016).
Despite the benefits of nitrogen and phosphorus for growth, physiological processes and plant production, high concentrations can be harmful during growth and development, causing, among other factors, nutritional imbalance, toxicity and soil salinization (Santos et al., 2016;Weber et al., 2016;Souza et al., 2017).Nitrogen and phosphorus provide higher growth and production in eggplant crops (Souza et al., 2017), but little is known about the optimal concentration levels of these nutrients when using wastewater irrigation.In this research we evaluated the use of nitrogen, phosphorus and treated wastewater on the growth and morphophysiology of eggplants (Solanum melongena L.) in the semi-arid region of Brazil.

Localization, Experimental Design, Treatments and Plant Material
The greenhouse experiment was conducted at the Centro de Ciências e Tecnologia Agroalimentar (CCTA) of the Federal University of Campina Grande-UFCG, Pombal, PB.The site is situated at 6º48′16″ S, 37º49′15″ W, with an average altitude of 144 m.
The experimental design was randomized blocks, with treatments arranged in a 4 × 4 + 1 factorial scheme, with four replications each, totalizing 68 experimental units.The factors consisted of four nitrogen doses (N 1 = 0.22, N 2 = 0.39, N 3 = 0.56 and N 4 = 0.73 g dm -3 of soil) and four doses of phosphorus (P 1 = 0.96, P 2 = 1.68,P 3 = 2.40 and, P 4 = 3.12 g dm -3 of soil), corresponding respectively to 40, 70, 100 and 130% of the recommended fertilization for eggplant pot production (Malavolta, 2006).In addition to these treatments, the control treatment consisted of 100% of the recommended nitrogen and phosphorus does irrigated with drinking water.The control was compared with the treatments that received the minimum (40%) and recommended (100%) doses of nitrogen and phosphorus fertilization irrigated with wastewater.
We produced the eggplant seedlings in expanded polystyrene trays of 128 cells, using a commercial substrate based on pine bark, hummus, and vermiculite.After twenty-five days of sowing (DAS), two seedlings were transplanted per pot, and at 17 days after transplanting (DAT), we performed the thinning leaving the more vigorous seedlings.During the experiment, the soil of the pots were maintained near the field capacity approach described by Medeiros et al. (2017) using wastewater and water supply, according to the treatments.
We used urea as nitrogen source (45% N).To avoid possible losses due to volatilization and/or leaching, nitrogen fertilization was divided in seven applications, every 7 DAT.
The source of phosphorus was superphosphate (18% of P 2 O 5 ), added as a basal fertilization five days before transplanting.Potassium fertilization was also carried out with potassium chloride in the amount of 0.31 g dm -3 , fertilized in the same period of nitrogen fertilizations.
The wastewater used in the experiment came from showers, sinks and urinals of the bathrooms of the UFCG, Campus de Pombal, collected by pipes and deposited in a septic tank.The tank was connected by a tube inserted at the lower end to a plastic container with 500 L capacity, functioning as effluent distribution tank.The distribution occurred connecting three different intermittent aerobic filters (each filter receiving 50 L of wastewater every 6, 8 and 12 hours).The effluent produced was stored in a plastic container with a 500 L capacity.
The filters were constructed adapting plastic containers with a capacity of 250 L each.The containers have three layers: a bottom layer of 10 cm of gravel, a layer in the middle composed by 50 cm of sand and in the upper part another layer of 5 cm of gravel, to standardize the flow.
The physical-chemical characterization of the wastewater (mean values) before and after the treatment with sand filter (Sousa, 2015) is shown in Table 2.   Note.pH: Hydrogenionic potential; EC: Eletric Conductivity; DO: Dissolved Oxygen; Ca: Calcium; Mg: Magnesium, Cl -: Cloretum; P: Fosforum; N: Nitrogen; Na: Sodium; K: Potassium; COD: Chemical Oxygen Demand; BOD: Biochemical Oxygen Demand; SAR: Sodium Adsorption Ratio. 1 These values were compatible with those recommended for agricultural use, according to CONAMA resolution (Brazil, 2005).

Data Sampling
Physiological and growth assessments were performed at the beginning of flowering, 40 days after transplanting (DAT).Eggplant plants growth was evaluated by plant height (PH), stem diameter (SC), leaf number (LN) and leaf area (LA).Plant height (mm) was the distance between the plant collar and the apex of the main stem.Stem diameter (mm) was determined at 3 cm of the plant collar using a digital caliper.For number of leaves, were only the leaves with at least 50% of photosynthetic active area and 1cm minimum width.Leaf area (cm²) was obtained according to the methodology provided by Maldaner et al. (2009).
We used an analysis of variance by the F test (p < 0.05) to determine the effects of phosphorous and nitrogen doses, and their interaction on the growth and physiological aspects of eggplant.When signicant, a polynomial regression analysis was performed.A Tukey test was performed to compare the averages of the control treatment (drinking water + 100% of nitrogen and phosphorus dose) with the treatments that used wastewater for irrigation with 40% (N 1 P 1 ) and 100% (N 3 P 3 ) of nitrogen and phosphorus.The analyses were performed at the statistical software SISVAR (Ferreira, 2014).

Results
The results of the analysis of variance for the growth of eggplant plants show that nitrogen doses significantly influenced plant height (PH) and leaf area (LA) (p < 0.01).Phosphorus doses and interaction between nitrogen and phosphorus did not affect the plant growth variables.The different types of water used for irrigating the plants (wastewater and drinking water) did not significantly influence the studied variables (Table 3).

Discuss
The For the photosynthesis process, the plant needs to open the stomata and absorb the atmospheric CO 2 necessary to perform the biochemical processes, since the greater stomatal opening favors the entry of CO 2 into the leaf mesophyll, thus increasing the intracellular concentration, and consequently photosynthesis (Silva et al., 2015).
In our study, the net CO 2 assimilation rate was reduced when the plants received doses above the estimated level of 0.45 g of N dm -3 .Excess growing media nitrogen decreased significantly by 4.4 μmol m -2 s -1 the CO 2 assimilation.
The positive effects on the net photosynthetic rate observed up to the optimal dose of 0.45 g of N dm -3 may be a result of the nitrogen function during plant growth and development, required for the synthesis of several cellular compounds, such as for chlorophyll and ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco), that act in the photosynthetic process during the photochemical and biochemical phases, respectively (Lima et al., 2014;Souza et al., 2016Souza et al., , 2017)).However, plants submitted to excessive doses of nitrogen have a reduction in stomatal opening due to the negative effect of the high doses on the mesophyll conductance (Souza et al., 2016).Abrantes (2014) found the highest value of net photosynthetic rate of 17.2 μmol m -2 s -1 at the N dose rate of 0.55 g of dm -3 , results similar to those found here.
The greater production of foliar biomass raised the transpiration rate as the nitrogen dosed increased to 0.44 g of N dm -3 , resulting in increments of the net photosynthetic rate and demand (Silva et al., 2015).Freitas et al. (2012) reported that the stomatal behavior controls the transpiratory water demand determining the loss of water to the environment.The reduction in stomatal conductance probably caused the negative effect on transpiration rate, reducing the water loss by the plants, but also the net photosynthetic rate (Lima et al., 2014;Weber et al., 2016;Souza et al., 2017).The reduction in stomatal conductance can also be a result of the possible reduction of soil water potential due to the increase of solute concentration in the soil solution (Vieira et al., 2016).
The increase in nitrogen fertilization also decreased the intracellular concentration of CO due to two factors.(i) the reduction of CO 2 uptake by foliar tissues, by decrease in the enzymatic activity of Rubisco, a fact evidenced by the decline in photosynthetic rate under high nitrogen concentration.And, (ii) by the increase in oxygenize activity of Rubisco enzyme, instead of the carboxylase (Ramos et al., 2016, Santos et al., 2016).The nitrogen excess probably promoted a phytotoxic effect on plants which possibly caused the partial closure of the stomata due to osmotic effects (Soares et al., 2013, Silva et al., 2015;Vieira et al., 2016).According to Ramos et al. (2016), the reduction in stomatal conductance results in the decline of photosynthesis caused by the decrease in CO 2 pressure within intracellular spaces.
Under optimal nutritional and water availability, plants, in general, have high transpiration rates (Silva et al., 2015, Souza et al., 2016, Ramos et al., 2016).However, under some stress, plants decrease perspiration to minimize water loss (Soares et al., 2013).Therefore, reductions in the photosynthetic and transpiration rates, intracellular CO 2 concentration and stomatal conductance in eggplants submitted to greater nitrogen doses were probably by nutritional excess.Lorenzoni et al. (2018) found similar results of nitrogen on sweet pepper (Capsicum annuum L.) plants for the above variables.According to Lorenzoni et al. (2018), an excess of nitrogen or an imbalance with other nutrients trigger detrimental effects on plant performance, which may be a result of the physiological imbalance.

Conclusion
Nitrogen and phosphorus doses interaction did not affect the growth and physiological aspects of eggplant.
The increase of the nitrogen doses reduced the leaf area of the eggplant when irrigated with wastewater.
The estimated nitrogen dose of 0.47 g dm -3 and irrigation with wastewater resulted in the greatest plant height.
Excess growing media nitrogen significantly decreased gaseous exchanges of eggplant plants, being found decreased of 4.4 μmol m -2 s -1 the CO 2 assimilation. 22

Table 1 .
Chemical and physical attributes of the soil used during the experiment

Table 2 .
Physical-chemical characteristics of the wastewater (WW) of the septic tank and after the sand filters