Salt balance in substrate cultivated with ‘Sunki’ mandarin x ‘Swingle’ citrumelo hybrids

Balanço de sais em substrato de cultivo de híbridos de tangerineira ‘Sunki’ com citrumelo ‘Swingle’ R E S U M O Em fase inicial de desenvolvimento da planta, objetivou-se avaliar o balanço de sais no substrato de cultivo de 10 híbridos do cruzamento tangerineira ‘Sunki’ comum (TSKC) x citrumelo ‘Swingle’ (CTSW), todos com potencial de uso como porta-enxertos. Como testemunhas incluiu-se o limoeiro ‘Cravo Santa Cruz’, a tangerineira ‘Sunki Tropical’ e o híbrido LVK (limoeiro ‘Volkameriano’) x LCR (limoeiro ‘Cravo’) 038, somando um total de 13 genótipos avaliados. Foram coletadas amostras do substrato em experimento desenvolvido em ambiente protegido da Universidade Federal de Campina Grande, Pombal, PB, de dezembro de 2015 a junho de 2016. Foram testados dois níveis de salinidade da água de irrigação (0,3 e 3 dS m-1), em esquema fatorial 2 x 13, com 4 repetições, usando-se, como substrato, a casca de pinus, o húmus e a vermiculita na proporção 1:1:1. Considerando água de irrigação com nível de salinidade 3 dS m-1, o substrato mostra-se menos salinizado em relação aos híbridos TSKC x CTSW 044, TSKC x CTSW 045, TSKC x CTSW 048 e TSKC x CTSW 049, assim como para o limoeiro ‘Cravo Santa Cruz’. Por outro lado, a maior concentração de sais foi obtida no substrato em que são cultivados TSKC x CTSW 042, TSKC x CTSW 047, TSKC x CTSW 041, TSKC x CTSW 053, TSKC x CTSW 055 e TSKC x CTSW 057.


Introduction
Salinity of soil and water is among the main problems in agriculture leading to reduction in crop yield (Gheyi et al., 2016).Despite being a global problem, salinity is more evident in arid and semi-arid regions, such as Northeast Brazil, for being characterized by low and irregular rainfall levels (Medeiros et al., 2003).
In addition, the predominance of waters with high levels of electrical conductivity in these regions should also be considered, reflecting in increased risk of salinization, if adequate management practices of plant, soil and water are not adopted (Araújo Neto et al., 2014;Dalastra et al., 2014).
The effects of salinity on agricultural production encompass osmotic effects, reducing water absorption by plants (Willadino & Câmara, 2010), and ionic effects, which can cause phytotoxicity and nutritional imbalance.These effects culminate in reduction of growth and potential of plants considered as sensitive (Taiz et al., 2015), such as citrus, which have mean salinity threshold of 1.4 dS m -1 (Mass, 1993).
Nonetheless, according to the literature, the effects of salts on crops can vary depending on species, cultivar, phenological stage, and intensity and duration of the saline stress (Silva et al., 2012;Sousa et al., 2017).Thus, using salt-tolerant rootstocks can be an alternative to guarantee successful citrus production in Northeast Brazil.
Based on studies conducted with citrus in recent years (Fernandes et al., 2011;Hussain et al., 2012;Silva et al., 2012;Hussain et al., 2015;Sá et al., 2015;Brito et al., 2016;Barbosa et al., 2017;Sá et al., 2017) to obtain genetic materials with potential for tolerance to salinity, it becomes necessary to evaluate new crosses and hybrids, which can be done by studying parameters that can help interpret tolerance mechanisms.
In this context, this study aimed to evaluate the balance of salts in the substrate used for cultivation, under saline water application, of citrus rootstocks considered as tolerant and belonging to the progeny resulting from the cross between 'Sunki' mandarin and 'Swingle' citrumelo.

Material and Methods
The experiment was carried out from December 2015 to June 2016 in a protected environment (greenhouse) at the Center of Sciences and Agri-food Technology (CCTA) of the Federal University of Campina Grande (UFCG), Brazil (6º 47' 20" S,37º 48' 01" W,altitude of 194 m).The local climate is classified as BSh (hot and dry semi-arid), with mean annual rainfall of 750 mm and mean annual evapotranspiration of 2000 mm.
All factors combined led to 26 treatments (2 salinity levels x 13 genotypes), repeated in 4 blocks, and each plot consisted of 1 plant, totaling 104 plots.
Seedlings were initially prepared in a protected environment at Embrapa Cassava and Fruits, considering all criteria for the initial production of rootstocks, such as the use of seeds from reputable companies, pest control and selection of nucellar plants.
At 75 days after sowing (DAS), the seedlings were transferred to 2000-mL black polyethylene bags and taken to the protected environment of the CCTA/UFCG, where the experiment was conducted.During growth period in the protected environment at Embrapa until the 90 DAS, the seedlings received public-supply water with low electrical conductivity (ECw = 0.3 dS m -1 ).
At 90 DAS, solutions with different salinity levels began to be applied and irrigation depths were daily determined based on the water balance, obtained by drainage lysimetry, adding a leaching fraction (LF) of 0.20.In this process, the volume applied per bag (Va) was obtained by difference between the total volume applied in the previous night (Vta) and the volume drained (Vd) in the next morning, applying the leaching fraction, as indicated in Eq. 1 for each treatment.

Va
Vta Vd LF mL = − − ( ) ( ) Drained water was collected through a hose attached to the bottom of each bag and connected to a container, to measure the drained volume.
Nutritional management and all cautions with respect to weed control, and prevention and control of pests followed the recommendations for citrus seedling production proposed by Mattos Júnior et al. (2005).
Irrigation water of 3.0 dS m -1 was prepared in such a way to obtain an equivalent proportion of 7:2:1, of Na:Ca:Mg, respectively, using NaCl, CaCl 2 .2H 2 O and MgCl 2 .6H 2 O salts.This ratio corresponds to the ions present in most water sources used for irrigation in small properties of Northeast Brazil (Audry & Suassuna, 1995;Medeiros et al., 2003).
To prepare the solution with the desired electrical conductivity (ECw), the salts were added to the public-supply water, which had ECw of 0.3 dS m -1 , corresponding to the first salinity level studied.After preparation, the solutions were stored in 60 L plastic containers, one for each ECw level, properly protected to avoid evaporation and contamination with materials that could compromise their quality.Every two days, electrical conductivity was measured in the solutions using a portable conductivity meter, with value automatically corrected to 25 ºC, and its value was adjusted when necessary.
When the rootstocks had adequate diameter for grafting, about 0.5 to 0.7 cm, which occurred at 210 DAS, plants were (1) cut close to the soil and roots were collected.The material (shoots and roots) was packed and dried in a forced-air oven for 72 h to obtain the dry matter or total dry phytomass (TDP), measured with an analytical scale, and the data were expressed in grams per plant.
Then, the substrates filling the bags were collected, dried, sieved, stored and labeled in plastic bags for analyses at the Laboratory of Soil and Plant Nutrition of the CCTA/UFCG, where the ions Ca +2 , Mg +2 , Na + and K + , soluble Cl -and ECw were determined using the methodologies described in EMBRAPA (1997).
The obtained data were subjected to analysis of variance (ANOVA) by F test.In the cases of significance for the factor genotypes, means grouping test (Scott-Knott, p < 0.05) was applied for each water salinity level.To verify the differences between salinity levels in each genotype, the F test was conclusive (Ferreira, 2011).

Results and Discussion
Based on the balance of salts (Table 1), the use of saline water caused alterations in soil chemical characteristics, increasing the electrical conductivity of the saturation extract (ECse) and Ca +2 and Na + concentrations as the salinity levels increased, which caused the substrate to be classified as saline when irrigated with 3 dS m -1 water, resulting in ECse higher than 4 dS m -1 in all containers.Lowest ECse values occurred in the substrate cultivated with the genotypes x CTSW -044, TSKC x CTSW -045, TSKC x CTSW -048, TSKC x CTSW -049 and LCRSTC, irrigated with high-salinity water.
The lowest ECse values also coincided with the lowest Na contents in the substrate cultivated with first two hybrids.
Such increase in the concentration of ions was due to the use of Na, Ca and Mg salts to prepare the solution with the desired ionic concentration corresponding to the second salinity level (3 dS m -1 ).These results corroborate those obtained by Brito et al. (2015), studying salt balance in the substrate and growth of 'Tahiti' acid lime grafted with 'Sunki' mandarin hybrids, under saline stress.These authors observed that the increase of salt concentration in the irrigation water led to linear increase in the concentration of ions in the substrate.
Variation of ionic concentration in the substrates, due to the hybrids cultivated in it, can be interpreted as a quantitative difference in the nutritional demand between the genetic materials, i.e., the absorbed contents of nutrients usually vary between genotypes, as mentioned by Epstein & Bloom (2006).In the literature on plant physiology, there are references to the selective permeability of the plasma membrane, adjusting the cell to the incorporation of ions according to plant needs, which vary depending on genetic constitution, development stage and conditions of soil and climate (Meer et al., 2008;Taiz et al., 2015).In addition, plants showed different growths, as can be observed in Table 2, based on the total dry phytomass.
For Mg +2 concentrations in the substrate solution (Table 3), there was no statistical difference between salinity levels for TSKC x CTSW-041, TSKC x CTSW -042, TSKC x CTSW -044, TSKC x CTSW -045 and LVX x LCR -038, although  Table 2. Test of means between citrus genotypes and between water salinity levels for total dry phytomass of citrus hybrids under water salinity MgCl 2 6H 2 O was added to the water, in the treatment with highest salinity (3 dS m -1 ), which can be related to greater fixation to soil colloids and/or absorption by the genotypes, which had higher demand for the nutrient.
Regarding K contents in the extract, the increase in salinity raised the concentration in the substrate cultivated with the hybrids TSKC x CTSW-042, TSKC x CTSW -047, TSKC x CTSW -048, TSKC x CTSW -053, TSKC x CTSW-055, TSKC x CTSW -057, as well as 'Sunki Tropical' mandarin, which can be attributed to the reserves (stock) of this nutrient adsorbed to the colloids, besides the fertilizations.
Additionally, greater presence of Ca +2 , Mg +2 and Na +2 ions, applied through irrigation water, may have consequently increased the competition for the adsorption sites; Na +2 , Ca +2 and Mg +2 are attracted and bound to the colloids, releasing K + to the solution.
Garcia-Sánchez et al. ( 2006), cultivating 7-year-old plants of 'Clemenules' mandarin (C.Clementina hort.ex.Tanaka) grafted onto two rootstocks ['Cleopatra' mandarin (C.reshni hort.ex Tanaka) and 'Carrizo' citrange (C.sinensis x P. trifoliata)], irrigated with water containing NaCl at the concentrations of 3, 15 and 30 mM, for three years, observed increase of toxic ions (Cl -and Na + ) and reduction of N, P and K contents in the leaves.Reduction of K + concentration in the plant due to increasing salinity is among the most studied effects, and the selective K + adsorption capacity associated with Na +2 exclusion is known as the tolerance mechanism of some plants to the saline stress (Willadino & Camara, 2010).In addition, elevated concentrations of Ca +2 and Mg +2 reduce K absorption by competitive inhibition, although low Ca +2 concentrations have synergetic effect on the nutrition of a few species (Faquin, 2005).
Chlorine is considered as an essential element to plants, but at the concentration of a micronutrient; at high concentrations, it is toxic.According to the data in Table 3, there was Cl accumulation in the substrate under irrigation with EC = 3.0 dS m -1 .Nonetheless, even under such conditions and although chlorine is the most harmful element to citrus species, when at high contents (Hussain et al., 2012;Syvertsen & Garcia-Sanchez, 2014;Brito et al., 2015), plants maintained their growth.
As already mentioned previously, besides reducing ECse and Na + content in the substrate cultivated with some genotypes, more evident in TSKC x CTSW -044 and TSKC x CTSW -045, in these hybrids and also in TSKC x CTSW -048, chlorine contents were lower when plants were irrigated with high-salinity water.
To survive under these conditions, plants may have used mechanisms of tolerance to the stressful condition because, according to Gheyi et al. (2016), Cl --sensitive plants may exhibit symptoms of toxicity, which consist of burn on tip of the leaves, reaching the edges in advanced stages; in general, premature leaf abscission occurs, and these symptoms appear when chloride concentration reaches 0.3 to 1.0%, based on leaf dry matter.Also according to these authors, the maximum chloride level (in mmol c dm -3 ) in the saturation extract for Cleopatra mandarin, bitter orange and sweet orange cannot exceed 25, 15 and 10 mmol c dm -3 , respectively, which are lower than the values observed in the present study, as the contents of Na + ions.
In the same context, Ayers & Westcot (1999) cite 10 mmol c dm -3 as maximum limit allowed of chloride for citrumelo, which would make the salinity observed in the present study even more harmful, considering the values found in the substrate cultivated with the 'Sunki' x 'Swingle' hybrids (TSKC x CTSW), denoting the tolerance capacity of these genotypes.
Complementing this discussion, phytomass data of the genotypes are presented in Table 2, showing differences between rootstocks at each level of water salinity.Even with the use of water containing low salt concentration (0.3 dS m -1 ), it is noticeable the difference of vigor between the genotypes, particularly TSKC x CTSW -047, LVK x LCR -038, TSKC x CTSW -055 and TSKC x CTSW -057.These latter two were affected by salinity when plants were irrigated with saline water (3 dS m -1 ), but even in this condition they remained in the group of higher phytomass.