Physiological Indicators in Two Lettuce Cultivars Under Saline Stress

Lettuce (Lactuca sativa L.) is considered as the main leafy vegetable in Brazil. In the last decades, there had been many changes in the predominant varietal types in the country, however, issues regarding the use of saline water inhibit the growth by the osmotic effect. The aim of this study is avaliate the effect of water salinity on physiological in lettuce cultivars. The experiment was carried out at the Alagoas Federal University, Arapiraca Campus, in a completely randomized design and with a 5 × 2 factorial scheme, with six replications. Five treatments of water salinity levels were analyzed (ECw: 0.14, 1.54, 2.94, 4.34, and 5.74 dS m at 25 °C) in two types of lettuce crops (Saia Véia and Vitoria Verdinha). Stomatal conductance, net photosynthesis, transpiration rate, water use efficiency, leaf temperature, and green index were assessed at 10, 20, and 30 days after the application of the treatments. The saline stress caused by the increase in saline concentrations decreased the photosynthesis and transpiration rates, which were associated with the reduction of stomatal conductance in both cultivars. Nevertheless, Saia Véia cultivar was higher tolerance in all tested saline levels compared to Vitória Verdinha. The green index for Vitoria Verdinha was seven times higher when compared to Saia Véia from the lowest to the highest saline levels. The cultivars differ in salt sensitivity, which could be useful for producers to choose the cultivar that is most adapted to the region and breeders regarding improvement prospects for adaptation of the lettuce under saline stress. In addition to osmotic stress, which is the first to happen, there are others.


Introduction
Soil salinity and irrigation water have been arousing interest since they are regarded as worldwide issues in agricultural production. It is estimated that soils affected by salt occupy about 10% of land surface and 50% of irrigated land in the world (Ruan et al., 2010). The effects of salinisation can be observed in numerous vital ecological and non-ecological soil functions. Drivers of salinisation can be detected both in the natural and man-made environment, with climate and the foreseen climate change also playing an important role. Global annual losses in agricultural production due to salt-affected soils can be as high as US$ 12 billion (Qadir et al., 2008;Flowers et al., 2010). The salinization process is due to environmental characteristics and anthropic actions (Daliakopoulos et al., 2016). Natural characteristics include the transport of salt sediments from salinized areas to unsalted sites; actions of ascent by capillarity of the soils to surface; high rates of evapotranspiration, among other factors (Pedrotti, 2015;Walter et al., 2018).
The Brazilian Semi-arid (BSA) region known as the Sertão is located in the Northeast part of the country and is one of the two areas most affected by climate change in Brazil (Seddon et al., 2016). The Sertão is characterized by the unique Caatinga biome -mostly consisting in deciduous forests, with uneven rainfall patterns and land distribution, climatic variation and social disparities. Several public policies have been adopted since 1877 to address the issues of the region, predominantly related to water scarcity; yet, the Sertão remains marginalized. Family farmers are the area's most vulnerable social group, in particular diffuse farmers (Machado & Revere, 2018). In these regions, agricultural activity has been a great challenge, especially for family farmers who do not have technical assistance and, plant empirically, leafy vegetables that are highly sensitive to soil salination Medeiros et al., 2016).
Among the leafy vegetables sensitive to soil salinity, lettuce stands out (Lactuca sativa L.), consumed worldwide and is an important crop of agrosystems in semi-arid regions (Ribeiro et al., 2003). Saline environments are mainly located in coastal zones, but they can also be found inland. Natural causes for inland salinization are the presence of salt-rich rainwater or saline groundwater, high evaporation and low precipitation. In such regions, upward-directed soil water movement by capillary rise is a dominant process and rates of precipitation are too low to leach out salts. This leads to an accumulation of salts within the topsoil or at the soil surface. Typically, such conditions are found in arid regions (Colombani, Mastrocicco, & Giambastiani, 2015;Walter et al., 2018).
Accumulation of salts in the soil can alter the water uptake by plants, reducing assimilation and nutrient utilization (Parida & Das, 2005;Tavakkoli et al., 2012). The accumulation of Na + and/or Clin chloroplasts, affects important biochemical and photochemical processes involved in photosynthesis and gas exchange (Xu & Mou, 2015;Amorim et al., 2010). Plants on the basis of adaptive evolution can be classified roughly into two major types: the halophytes (that can withstand salinity) and the glycophytes (that cannot withstand salinity and eventually die). Majority of major crop species belong to this second category. Thus salinity is one of the most brutal environmental stresses that hamper crop productivity worldwide (Flowers, 2004;Munns & Tester, 2008). In addition, the tolerance a crop has to salinity can vary between genotypes of the same species as well as the plant's development stage (Gheyi et al., 1997).
According to the researchers Gupta & Huang (2014) a comprehensive understanding on how plants respond to salinity stress at different levels and an integrated approach of combining physiological tools with and biochemical techniques are imperative for the development of salt-tolerant varieties of plants in salt-affected areas. Recent research has identified various adaptive responses to salinity stress at physiological levels, although mechanisms underlying salinity tolerance are far from being completely understood. Lettuce is considered 'moderately sensitive' to salinity and its potential yield is reached when the electrical conductivity of the saturated extract reaches the threshold value of 1.3 dS m -1 , with a 13% reduction in yield per unit increase of salinity above the value (Ayers & Westcot 1991). In this context, this paper aims evaluate the effect of water salinity on physiological in lettuce cultivars.

Environment and Meteorological Parameters
The experiment was carried out in a greenhouse with 50% shading and anti-UV treatment, located at the Alagoas Federal University (UFAL), Arapiraca Campus (latitude 09°41′53.6″S, longitude 36°41′26.3″W and 244 m altitude) Northeast of Brazil. The outside meteorological parameters in the greenhouse obtained from the university's automatic weather station (maximum, minimum temperature, precipitation and global radiation). The inside meteorological parameters in the greenhouse: photosynthetically active radiation obtained from spectroradiometer (Apogee, model SS-110) which measures received by pots were performed in two moments: 7:00 a.m. to 11:00 a.m. and 12:00 p.m. to 4:00 p.m. The temperature and humidity were measured with the aid of a thermo-hygrometer (model HT-208) and it was installed at the center of the protected environment, 1.0 m above from the ground level.
The climate of the region is of the AS tropical type (rainy with a dry summer), according to the classification of Köppen (1948) and Dubreuil et al. (2018). It features two well established climatic seasons: a warm and dry summer, with occasional rainfall (September to March), and wet and rainy winter (April to August). The mean annual precipitation ranges from 750 to 1000 mm (Xavier & Dornellas, 2005).

Lettuce Varieties
Sáia véia: The cycle of 35 to 40 days after transplantation. Optimum germination range: 10-27 °C. Plant with moderately wrinkled, large, and elliptical smooth leaves, with light green color. It does not form a head and has white seeds.
Vitória verdinha: The cycle of 35 to 40 days after transplantation. Optimum germination range: 10-27 °C. It has smooth leaves, slightly wrinkled and soft texture, dark green in color. It does not form a head and has white seeds.
These two lettuce cultivars used in this experiment are the most planted in the APL region (Aranjo Produtivo Local) in the municipality of Arapiraca in Northeast of the Brazil. The APL is reference in the production of fruits and vegetables and it distributes to consumer markets in Brazil.

Soil Analyses
A soil sample of 1.0 kg was collected at the UFAL area in a 0-0.20 m layer that represents the effective depth of the lettuce root system. Samples were sent for chemical and physical analyses, respectively, to the Soil Fertility Laboratory and Soil Physics Laboratory (Department of Environmental Resources, School of Agronomic Sciences, UNESP, Botucatu, São Paulo).

Water Analyses
The water (control) used in the experiment had the following elements: 3 mg/L of N; 0.3 mg/L of P; 12 mg/L of K; 5 mg/L of Ca; 3 mg/L of Mg; 4 mg/L of S; 4.6 mg/L of Na; 0.12 mg/L of B; 0 mg/L of Cu; 0 mg/L of Fe; 0 mg/L Mn and 0 mg/L of Zn.
The electrical conductivity curves were obtained as a function of sodium chloride (NaCl) concentration, in which the desired electrical conductivity (dS m -1 ) was multiplied by 640 mg L -1 , according to Richards (1954). The different saline levels were obtained by dissolving sodium chloride (NaCl) in water, that was obtained from the supply system of UFAL, in buckets of 14 L. Saline water that was used for treatments irrigation was verified before each irrigation with the aid of a Portable Digital Conductivity Meter (Mod. Cd-4301).

Experimental Design
The experiment was assessed in three periods at interval of 10 days. Saline treatments were applied three days after the seedlings were established, thus starting the first evaluation period. The experimental design was completely randomized, arranged in a 5 × 2 factorial scheme (five salt levels and two lettuce cultivars), with six replicates. The experimental unit was represented by a pot with a plant. The treatments resulted from the combination of five irrigation water salinity levels, as follows: 0.14 (control); 1.54; 2.94; 4.34; and 5.74 dS m -1 . The effect of irrigation water salinity levels on the analyzed variables was measured through the analysis of variance, whose results of the treatments were studied through polynomial regression analysis. The effect of the cultivars was compared by the Tukey's test at 0.05 probability. Statistical tests were performed using the SISVAR software, version 5.3Build 77 (Ferreira, 2014).

Cultivations Conditions
Initially, seedlings of lettuce (Lactuca sativa L.) of the Saia Véia and Vitória Verdinha varieties were produced in Styrofoam trays with 200 cells, filled with the Bioplant substrate. Seedlings were irrigated daily with water (electrical of conductivity of 0.14 dS m -1 ) and every 7 days were fertigated (0.5% of urea in the trays). The transplanting was performed manually 30 days after sowing (DAS), where seedlings that showed more vigor were selected and transplanted into plastic containers and one plant was conditioned per pot, with seedlings with four to six leaves. As a form of irrigation control, plastic vats with a capacity of five liters were drilled and connected to collecting hoses to conduct the fraction of drained water. Each pot had in the bottom filled with 500 g of gravel (3 cm high) and the remaining volume was completed with soil, totaling 5.5 kg. Pots were placed on a bench at 1.0 m of distance from the ground level.
The beginning of the experiment was characterized by raising the soil to the field capacity; for this, recipients were saturated with water and wrapped individually in plastic, to force the loss of water only by drainage. When the drainage ceased, the plastic covers were removed and the recipients were weighed on a digital scale (0.1 g precision), thus obtaining the control weight, corresponding to the field capacity, which was approximately 6.8 kg for each recipient; finally, seedlings were transplanted.
After the beginning of treatments, irrigation was performed twice a day (at 8:00 am and 4:00 p.m.), based on the water consumption of the plants of the previous day, keeping the soil moisture close to the field capacity.
The estimated water volume for daily irrigation was divided by the factor 0.9, to restore soil moisture to field capacity, also, to obtain a leaching fraction (FL) of 10%, using the following formula: (VI-VD)/(1-FL), where VI: is the volume of water to be applied on the irrigation; VA: is the volume of water applied in the previous irrigation, and VD: is the volume of drained water in the previous irrigation (VI, VA, and VD were described in mL).

Physiological Analysis
The physiological analysis was performed with an infrared gas analyzer IRGA (LI 6400, LICOR, Lincoln, USA), with a photosynthetic photon flux density (PPFD) according to the environment of 800 μmol photons m -2 s -1 . Analyses were performed between 8:00 a.m. and 10:00 a.m., at 10, 20, and 30 days after the application of treatments (DAAT). In fully expanded and non-senescent leaves, the following measures were performed: net photosynthesis (A-μmol CO 2 m -2 s -1 ) and transpiration (E-mmol H 2 O m -2 s -1 ) rates, stomatal conductance (gs-H 2 O m -2 s -1 ), leaf temperature (T °C), and water use efficiency (WUE-μmol CO 2 mmol -1 H 2 O) through the A/E ratio. The green index (GI) was measured in 10 fully expanded and non-senescent leaves. For each leaf, 10 readings were collected, and a mean of the green index was obtained. The readings were obtained with a Portable Chlorophyll Meter (Model SPAD-502 ® , Soil Plant Analyzer Development, Minolta, Japan).

Data From the Automatic Meteorological Station
The recorded, for the greenhouse proximal area, where the pots were allocated with the seeds, from germination to the end of the plants' biocycle, temperatures that ranged from 24 to 36 °C ( Figure 1A) and global radiations that ranged from 5 to 28 MJ m -2 day -1 (Figure 1B). Inside the greenhouse, the photosynthetically active radiation was measured, ranging from 558 mol m -2 s -1 from 7:00 a.m. to 11:00 a.m. and 678 mol m -2 s -1 from 12:00 p.m. to 4:00 p.m. The temperature inside the greenhouse ranged from 40 °C (May) to 28 (June) and relative humidity ranged from 30 to 80%.
The precipitation average for the year of this experiment is in line with the averages recorded for the region, as can be seen in the author's paper Xavier and Dornella (2005), in which municipality of Arapiraca has a rainfall regime concentrated in the autumn-winter period, confirming the regional dynamics. Most of your rainfall precipitates in just 3 months (usually, May, June and July).
In the month in which the experiment started (May), the highest temperature of the year was recorded, a fact that repeated itself at the end of November ( Figure 1A), which increased the internal temperature of the greenhouse, as well as the relative humidity. Temperature and relative humidity influence the metabolic functions, transpiration, and mechanisms of thermal control of plants (Teruel, 2010  On the other hand, when the isolated effect of salinity levels was analyzed, there was an effect (p < 0.01) for the three variables (A, E, and gs) in the different periods assessed, which highlights the direct influence of saline water on growth and development of the two lettuce cultivars (Table 1).
The presence of NaCl in the irrigation water influenced the photosynthesis of the assessed cultivars only at 20 DAAT, where the cultivar C1 showed an increase of approximately 3% in this variable compared to C2. Compared to C2, cultivar C1 showed higher values for E at 20 (7%) and 30 (2%) DAAT and gs at 10 (5.6%) and 20 (21.6%) DAAT (Table 1).
Saline stress caused linear reductions in gs in all assessed periods; however, at 20 DAAT, the decrease was more expressive with approximately 5% per unit increase of ECw. The decrease of the lowest (0.14 dS m -1 ) to the highest saline level (5.74 dS m -1 ) was 24.7% ( Figure 2A).   Note. (*) Significant at 0.05 and (**) at 0.01 probability; (NS) Not Significant; (MSD) Minimum Significant Difference. Means followed by the same letter do not differ from each other, in columns, by the Tukey test at p < 0.05 level.
When analyzing the isolated effect of salinity levels (p < 0.01) for the three variables (GI, WUE, and T), the different evaluation periods were observed, and interaction was observed between salt levels and cultivars exclusively for GI at 30 DAAT. The cultivar factor was significant (p < 0.01) at 20 and 30 DAAT. Regarding cultivars C1 and C2, higher values were observed for GI 13.9 and 10.5% at 20 and 30 DAAT, respectively ( Table  2).
The variable WUE was influenced by the presence of NaCl at 20 and 30 DAAT, where cultivar C2 increased by 4.5 and 2.3%, respectively, when compared to C1 (Table 2). At 10 DAAT, LT was higher for C2, which presented an increase of 1.9% about C1 (Table 2).
At 10 DAAT, each increased unit of ECw showed a linear decrease of 1% for the outcome GI; this decrease reached 5.3% when the lowest and highest saline levels were compared ( Figure 3A). At 20 DAAT, plants presented a linear increase of 1.5% at each unit increase of ECw; this increase reached 14.7% when the lowest and highest saline levels were compared ( Figure 3A).

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