Introduction

Strawberry cultivation in the semi-hydroponic system, popularly known as soilless cultivation, is a is a practice applied in several regions of the planet (^{Recamales et al., 2007}; ^{Neri et al., 2012}; ^{Rampazzo, 2016}). Instead of soil, it uses a wide range of raw materials to produce the substrates, such as perlite, vermiculite, peat, pine bark, charred rice husk, and coconut fiber (^{Lieten et al., 2004}; ^{Jarosz and Konopinska, 2010}; ^{Marques, 2016}). The substrats used in semi-hydroponic system usually have good physical and chemical characteristics and have good nutrient bioavailability for plant development (^{Abad et al., 2005}; ^{Ilha, 2013}). They also, are free of pest and diseases, provide more rational water and nutrients use, and are more ergonomics for labors (^{Rampazzo, 2016}).

One of the most critical points in strawberry semi-hydroponic system is the nitrogen management by fertigation (^{Othman et al., 2019}). Nitrogen availability affects strawberries productivity and fruits quality (^{Nestby et al., 2004}; ^{Jarosz and Konopiska, 2010}). Both nitrogen forms, nitrate and ammonium salts, are absorbed and metabolized by plants (^{Roosta et al., 2009}), and play important metabolic functions, such as growth, development, production and affect fruits appearance, durability and flavor (^{Tabatabaei et al., 2008}; ^{Krüger et al., 2012}; ^{Samec et al., 2016}).

Despite this, there is little information about nitrate and ammonium concentration in nutrient solutions for growing strawberries. In the existing ones, the variation is quite expressive, between 4 and 12 mmol L^-1 for NO₃ ^- and 0.2 and 2.5 mmol L^-1 for NH₄ ⁺ (^{Hennion and Veschambre, 1997}; ^{Paranjpe et al., 2003}; ^{Furlani and Fernandez, 2004}). This research aimed to determine the best NO₃ ^-:NH₄ ⁺ ratio for strawberry production by using the semi-hydroponic fertigation system.

Material and methods

This trial was performed in Curitiba, Paraná State, Brazil (25° 24’ 38.5” S; 49° 14’57.5” W). Each ‘San Andreas’ strawberry runners, a neutral cultivar to the photoperiod, was planted in 8-liter black polypropylene pots on 09/07/18, filled with organic substrate (Table 1) composed by rice husk and pine bark.

The experimental design applied was completely randomized with five repetitions per treatment, and each repetition with three pots. The treatments consisted of proportions of nitrate and ammonium (NO₃ ^-:NH₄ ⁺) in the nutrient solution of 100:0; 75:25; 50:50; 25:75; and 0:100%, with 150 mg L^-1 nitrogen concentration applied in all pots, based and modified by ^{Furlani and Fernandes Júnior (2004)} (Table 2).

Other nutrients were applied in the same concentrations in all pots: 170 mg L^-1 of Ca, 200 mg L^-1 of K, 40 mg L^-1 of HPO₄ ^-, 40 mg L^-1 of Mg, 32 μmol L^-1 of Fe, 10 μmol L^-1 of Mn, 30 μmol L^-1 of B, 3.1 μmol L^-1 of Zn, 1.3 μmol L^-1 of Cu, and 0.17 μmol L^-1 of Mo. The following salts were used to formulate the nutrient solution: Ca(NO₃)₂, KNO₃, KH₂PO₄, MgSO₄, NH₄H₂PO₄, CaCl₂, KCl, (NH₄)₂SO₄, H₃BO₃, CuSO₄5H₂O, MnSO₄H₂O, ZnSO₄7H₂O, Na₂MoO₄2 H₂O, Fe-EDDHA (6%).

Fertigation was applied in each pot once a day, to maintain the substrate water retention capacity at 60%, in such way approximately 500 mL were daily applied per pot. Electrical conductivity (EC) on the nutritional solution was maintained between 1 300 and 1 800 mS cm^-1 and the pH of 6 ±0.2.

Leaf evaluations were performed at 60 and 120 days after planting (Dap), corresponding to the vegetative and productive stages, respectively (^{Nepar, 2019}). The leaf area was obtained by measuring the length and width of the leaflets of the newly developed 4th leaf of the plant with a digital caliper and using the equation La= (π / 4) [(L + W) / 2]² (^{Pires, 1999}). Were La= leaf area, L= length and W= width. From the same leaflets, a tissue sample of approximately 0.17 g was collected to evaluate chlorophyll a, b and total leaves (^{Porra, 2002}).

The material was macerated with 10 ml of 80% acetone and centrifuged at 12 000 rpm for 10 minutes and a spectrophotometer reading at 645 and 663 nm wavelengths was performed. The remainder of the 4th collected leaf was used for nutritional analysis (S, Ca, K, Mg, P, Fe and Zn) by wet digestion with nitric acid (HNO₃) and hydrogen peroxide (H₂O₂), and reading were done with the inductively coupled plasma optical emission spectroscopy (ICP-OES) (Varian 720-ES™). Total N was analyzed by the Dumas combustion method in Elemental Analyzer (^{Carmo et al., 2000}).

All senescent leaves were collected through the plants’ growth and dried at 65 ºC until reaching constant mass. At the end of trial, all remained leaves were also dried at 65 ºC until reaching constant mass. Dry weigh per plant were obtained by adding the senescence dry weight to the final leaves dry weight.

Physical and chemical fruits features were analyzed on ten fruits harvested per replicate, in three harvest dates (20/12/2017; 20/01/2018; 20/02/2018). Those with at least 3/4 reddish epidermis, minimum weight of 6 g and without injuries, diseases or deformations were chosen. First of all, pulp firmness was determined by using a texture analyzer (Brookfield CT3^™), with 2 mm tip, 5 mm penetration and 5 mm s^-1 penetration speed. Afterwards, strawberry juice was extracted, soluble solids content was measured on the by using a refractometer. Juice solution was diluted in water to 10% and titration was performed with 0.1 M sodium hydroxide solution (NaOH) under constant agitation, to reach the pH of 8.2.

The Bartlett test was used to verify the homogeneity of variances (p> 0.05) of the data, followed by Anova. In case of significant variation, regression analysis was used to verify the effects of the NO₃ ^-:NH₄ ⁺ ratios. For the statistical analysis, the software Assistat 7.7 (^{Silva, 2002}) was used.

Results and discussion

The NO₃ ^-:NH₄ ⁺ proportions did not affect the most leaves’ nutrient content (S=1.19 g kg^-1; Ca =10.64 g kg^-1; K=18.13 g kg^-1; Mg= 3.75 g kg^-1; P= 2.89 g kg^-1; Fe= 69.42 mg kg^-1; Zn= 23.38 mg kg^-1). These nutrients levels are the similar foliar level reported in strawberry leaves by other authors (^{CQFS RS/SC, 2004}; ^{Nepar, 2019}). It was expected did affect the Ca, K and Mg levels under the highest NH₄ ⁺ level in the nutritive solution, through competition between ions of the same valence, either by a protein channel or for binding to a transport protein in cell membrane (^{White, 2012}), but it not happened in this research.

We also didn’t see a change in pH in the rhizosphere due to NH₄ ⁺ absorption (^{Hawkesford et al., 2012}), which has a direct effect on nutrient bioavailability (^{Waller and Wilson, 1984}). The substrate pH changed very few during this trial (5.65 before planting- 5.27 in 0-100% of NO₃ ^-:NH₄ ⁺), probably due to its organic composition. The substrate composition plays an important role since ^{Choi et al. (2011)} found a decrease in the substrate pH, from pH 7.0 with the use of nutrient solution without NH₄ ⁺ to pH 5.8 with NH₄ ⁺, and also a decrease in the levels of K⁺, Ca²⁺ and Mg²⁺ in strawberry leaves using a mineral substrate.

Among all nutrients evaluated in the leaf, only the total nitrogen (N) was affected by the different proportions of NO₃ ^-:NH₄ ⁺ from the nutrient solutions. The content found was 20.64 g kg^-1, when 100% of the nitrogen applied was in the form of NH₄ ⁺, representing an increase of approximately 14% of N in relation to the absence of NH₄ ⁺ (Figure 1). A similar result was obtained by ^{Tabatabaei et al. (2008)}, where the application of the maximum dose of 75% of NH₄ ⁺ in the ‘Selva’ strawberry variety found an increase in the leaf content of N around 16.8%, and by ^{Choi et al. (2011)} with an increase of 14.2% of N when using 100% NH₄ ⁺ in nutrient solution.

Figure 1 Content of total nitrogen in leaves of the ‘San Andreas’ strawberry cultivar according to NO₃ ^-:NH₄ ⁺ proportions in nutritive solution, in vegetative period (60 days after planting) and reproductive period (120 days after planting), in semi-hydroponic cultivation. 

Most plants absorb the two nitrogen forms which are mediated by specific transport proteins (^{Roosta et al., 2009}). Nitrogen forms can be metabolized in the same root cell where it was absorbed or translocated unchanged to the aerial part of the plant. NH₄ ⁺ is assimilated into root cells and its reduced form is carried to the leaves, while most of NO₃ ^- is carried to the leaves and then reduced to be assimilated to amino acids. The energy requirement for NH₄ ⁺ assimilation is lower than for NO₃ ^- assimilation, since the first does not need to be reduced for its incorporation into amino acids (^{Bloom et al., 1992}; ^{Hawkesford et al., 2012}; ^{Taiz and Zeiger, 2017}). Possibly, the NH₄ ⁺ assimilated in the roots and transported in the form of amino acids, ended up accumulating in the leaves and increasing the levels of N in these tissues (^{Majerowicz et al., 2000}).

High concentrations of NH₄ ⁺ in the nutrient solution caused necrosis in the leaves (Figure 2), consequently increasing the dry mass of the aerial part (Figure 3), indicating a toxic effect on the plant. ^{Choi et al. (2011)} also observed that in high concentrations of NH₄ ⁺ in the nutrient solution, the young strawberry leaves had a matte green color, with withered and curled edges, while the older leaves were dry and tanned. The higher N content in the leaf (Figure 1) favored the production of NH₄ ⁺ through photorespiration, considered the main metabolic pathway for the production of this compound in plants (^{Bittsánszky et al., 2015}), leading to toxicity.

Figure 2 Symptoms related to NH₄ ⁺ toxic effect in strawberry plants under 0:100% treatment of NO₃ ^-:NH₄ ⁺ in nutrient solution, where: a) symptom onset; b) aggravated symptom; c) aerial view of the plant; and d) leaf death. 

Figure 3 Dry mass of ‘San Andreas’ cultivar after the period of 120 days after planting the seedlings, according to NO₃ ^-:NH₄ ⁺ proportions in nutrient solution in semi-hydroponic cultivation. 

Toxicity occurs when the rate of NH₄ ⁺ assimilation into amino acids and amides becomes lower than the rate of absorption. In the leaves, the accumulation of NH₄ ⁺ causes damage to the structures of the chloroplast, lowering the pH of the cells to intolerable levels. This fact dissipates transmembrane proton gradients necessary for electron transport in photosynthesis, which results in a decrease in the photosynthetic rate (^{Bittsánszky et al., 2015}).

Chlorophyll levels varied similarly on 60 DAP and 120 DAP periods, under the proportions of NO₃ ^-:NH₄ ⁺ (Figure 4). The maximum chlorophyll contents were 0.25 mg g^-1, in the proportions of 36.2% NH₄ ⁺. ^{Tabatabaei et al. (2008)} also observed chlorophyll content increase using the proportion of 50% NH₄ ⁺ in nutrient solution. In our study, increasing NH₄ ⁺ concentration above 36% decreased leaf chlorophyll content, probably due the NH₄ ⁺ phytotoxicity discussed earlier. Maximum levels of chlorophyll were obtained with NO₃ ^- and NH₄ ⁺ in the fertigation solution, in which the proton generated by NH₄ ⁺ assimilation can be used to reduce NO₃ ^-, thus making it easier for plants to regulate intracellular pH when both forms of nitrogen are provided (^{Hawkesford et al., 2012}).

Figure 4 Chlorophyll content and leaf area on ‘San Andreas’ strawberry leaves according to the proportions of NO₃ ^-:NH₄ ⁺ in nutrient solution in vegetative period (60 days after planting) and reproductive period (120 days after planting) in semi-hydroponic cultivation. 

The adequate combination of NO₃ ^-:NH₄ ⁺ has a stimulating effect on plants because reduce the spent of energy in nitrogen uptake process, done by root cells, and in assimilation into amino acids. Therefore, the cell with your metabolism regulated and with greater energy efficiency can assimilate the nitrogen into glutamine and glutamate, as well as incorporate other amino acids that play an important role in protein and molecule formation, such as chlorophyll (^{Taiz and Zeiger, 2017}).

The ratio 71:29 to NO₃ ^-:NH₄ ⁺ was the one that provided the highest leaf area, both in 60 DAP and 120 DAP, reaching 93 cm² (Figure 4). These values approximate those obtained in the studies conducted by ^{Tabatabaei et al. (2008)} and ^{Choi et al. (2011)}, in which the application of 25% of the nitrogen in the form of NH₄ ⁺ resulted in larger leaf areas. A greater leaf expansion was possible, since the osmotic and energetic regulation provided by the two forms of N (NO₃ ^- and NH₄ ⁺) benefited the cell elongation and plant growth (^{Cao and Li, 2003}; ^{Li et al., 2013}).

Leaf area results (Figure 4) show the same tendency as chlorophyll contents (Figure 4), since chlorophyll is the basis for the formation of various components of the plant photosynthetic apparatus, providing greater leaf development. However, there was a lower leaf growth on plants supplemented with high NH₄ ⁺ concentration (75% and 100%), probably due to higher demand of carbohydrates that are channeled for assimilation and detoxification of the large amount of this cation.

The pulp firmness was the only physical-chemical characteristic that guides the quality of the fruit affected by the proportions of NO₃ ^-:NH₄ ⁺ and decreased up to 25% with increasing NH₄ ⁺ levels in the nutrient solution (Figure 5). Its similar to the results found by ^{Sokri et al. (2015)} in apples, which the highest concentration of NH₄ ⁺ tested in the solution decreases the firmness of the pulp.

Figure 5 Pulp fruit firmness of ‘San Andreas’ strawberry cultivar according to NO₃ ^-:NH₄ ⁺ proportions in nutrient solution in semi-hydroponic cultivation. 

The firmness of the pulp is closely linked to Ca since it is linked to polygalacturonic acids such as pectins. They form the Ca-pectates in the middle lamella, whose function is essential for the formation of cell walls and plant tissues. Pectate degradation is mediated by polygalacturonase, an enzyme strongly inhibited by high Ca concentrations (^{Wehr et al., 2004}). Thus, when Ca deficiency occurs, polygalacturonase activity is increased (^{Konno et al., 1984}). A typical symptom of Ca deficiency is the disintegration of cell walls and the breakdown of affected tissues, which causes loosening and loss of fruit firmness (^{Ho and White, 2005}). The reduction in pulp firmness may be related to the lower Ca concentration in fruits (^{Alan, 1989}). In this experiment, although leaf concentrations of Ca were not affected by NH₄ ⁺, it is likely that Ca concentrations in the fruit may have been influenced.

Fruits had mean values of total soluble solids (TSS) of 8.5, in °Brix, total titratable acidity (TTA) in % of citric acid of 0.97; TSS/TTA (ratio) of 8.9 and pH of 3.5. According to ^{Kader (1999)} for the strawberry flavor to be acceptable, the minimum amount of TSS recommended is around 7 °Brix. The maximum values of acidity recommended are between 0.8%, and the SS/TA ratio of at least 8.75. It was expected that SST levels would decrease and TTA levels would increase with higher concentrations of NH₄ ⁺ in the nutrient solution.

This is probably because, according to ^{Sokri et al. (2015)} and ^{Zhang et al. (2019)}, the application of NH₄ ⁺ decreases the influx of K by the plant, due to the antagonism of these cations. Cation concentration is reduced with increasing ammonium concentration, which can lead to an increase in the acidity of the fruits. On the other hand, in soybean seeds and apple fruit (Malus domestica Borkh. cv. Fuji) fertilization with K increased TSS (^{Tu et al., 2017}; ^{Zhang et al., 2017}). Although leaf tissues show symptoms of NH₄ ⁺ toxicity, in this trial, leaf K contents did not vary with the increase in NH₄ ⁺, maintaining the chemical characteristics of the fruits.