SALINE STRESS AND EXOGENOUS APPLICATION OF PROLINE IN CASHEW ROOTSTCK

1 Doctor in Agricultural Engineering from the Federal University of Campina Grande (UFCG). Technology and Natural Resources Center, Campina Grande (PB), Brazil. E-mail: engenheiropadua@hotmail.com 2 Professor of the Department of Science and Technology of the Federal Rural University of the Semi-arid (UFERSA), Caraúbas RN, Brazil 3 Visiting Senior National Professor / CAPES, Federal University of Recôncavo da Bahia, Cruz das Almas, BA, Brazil 4 PhD student in Agricultural Engineering at the Federal University of Campina Grande (UFCG). Technology and Natural Resources Center, Brazil. 5 Agronomist, Federal University of Campina Grande (UFCG). Agri-food Science and Technology Center, Pombal (PB), Brazil


INTRODUCTION
The cashew tree (Anacardium occidentale L.) is one of the most prominent fruit species cultivated in the Brazilian Northeast, being responsible for generating employment, due to the industrialized products derived from its fruit and pseudofruit. The main producing states are Ceará, Piauí and Rio Grande do Norte, which have a planted area of 522,478 hectares, representing 92.60% of the cultivated area in the country (IBGE, 2018).
Despite the importance of this fruit tree, the great genetic variability of the cashew tree found in this region is considered a limiting factor in the expression of the productive potential of the crop. Therefore, in order to try to encourage the expansion of the orchards in these areas, the use of improved plants (early dwarf cashew) has been highlighted to replace the old orchards whose plants were propagated via seeds. These improved genotypes promote precocity and productivity superior to conventional plants, as well as a reduced size, which facilitates harvesting and cultural treatments (SOUZA et al., 2020).
The Northeast region, despite standing out as the largest cashew producer in Brazil, has irregular rainfall, high temperatures and high evapotranspiration rates, which limits water availability for irrigation. Thus, the use of saline water for irrigation appears as an alternative to supply the water demand of the crops, especially in the dry season, when the weirs in the region have reduced volumes of water and higher levels of salts in the water (SILVA et al., 2015). Furthermore, in this region there is a large volume of low quality water.
Despite the possibility of using inferior quality water to irrigate crops, the excess of accumulated salts in the soil can reduce the availability of water for plants, due to the reduction of the osmotic potential in the soil solution (MEDEIROS et al., 2016). Furthermore, it can promote specific toxicity and nutritional imbalance, inducing morphological, structural and metabolic changes in plants, negatively affecting seedling quality, growth and crop production (BEZERRA et al., 2016).
In this context, several studies have a positive correlation between the accumulation of organic solutes or osmolytes (proline, glycine betaine, trehalose, sucrose, among others) and the tolerance of cultures to salt stress (ASHRAF et al., 2011). The increase of these solutes increases the osmotic pressure of the cells, maintaining the water absorption and the cell turgor pressure, which contributes to the continuity of the physiological processes (MARIJUAN; BOSCH, 2013). Ejaz et al. (2012) found an increase in proline in sugarcane plants submitted to 100 mM NaCl and a significant increase in the values of this osmolyte when, together with saline treatment, ascorbic acid was added. Lima et al. (2016) using application of proline via leaf observed that the highest values for fresh mass and number of sweet pepper fruits were obtained when using water with 0.6 dS m -1 and doses of proline of 12.0 and 17.0 mM, respectively. According to Molazem and Azimi (2015), salt stress reduces growth and increases the content of proline in corn plants.
Thus, the objective of this study was to evaluate the influence of foliar applications of proline on growth and phytomass production of BRS 226 Planalto precocious dwarf cashew rootstocks irrigated with increasing salinity levels of irrigation water.

MATERIAL AND METHODS
The work was performed from October to December 2017, in a protected environment (greenhouse), at the Agri-Food Science and Technology Center at the Federal University of Campina Grande (CCTA / UFCG), in the municipality of Pombal -PB, located at 6º47'03 "S, 37º49'15" W and altitude of 193 m.
A randomized block design was used, in a 5 x 4 factorial scheme, corresponding to five levels of water electrical conductivities -CEa (0.3; 1.0; 1.7; 2.4 and 3.1 dS m -1 ) and four concentrations of proline (0; 4; 8 and 12 mM), with three replications and two plants per plot, totaling 120 plants. The sketch of the experimental area is shown in Figure 1.
Information on the application of proline in cashew or other fruit trees is scarce. Then, it was necessary to use the doses determined by Lima et al. (2016) who verified greater growth of pepper plants cv. All Big subjected to salt stress when 12.17 mM proline concentrations were used. The salt levels were based on studies by Sousa et al. (2011), who observed a marked inhibition in the growth of cashew seedlings BRS 274 and BSR 275 from the saline level of 1.58 dS m -1 .
The preparation of saline waters was performed by adding the salts of sodium chloride (NaCl), calcium (CaCl 2 .2H 2 O) and magnesium (MgCl 2 .6H 2 O), in the local water supply (CEa of 0.3 dS m -1 ), maintaining an equivalent ratio of 7: 2: 1. This ratio is predominant in the main sources of water available for irrigation in Northeast Brazil (MEDEIROS, 1992), using the ratio between CEa and the concentration of salts (mg L -1 = 640 x CEa) (RHOADES et al., 2000).
The BRS 226 Planalto cashew clone was used due to the characteristics of rusticity and adaptation to the climatic conditions of the semiarid (FERREIRA-SILVA et al., 2009). The seeds came from a commercial exploration area located in the city of Severiano Melo -RN.
Polyethylene bags with a capacity of 1250 mL were used for the formation of rootstocks, drilled at the base, for free water drainage. The substrate used was composed of eutrophic fluvic neosol (95%) + tanned bovine manure (5%). The soil was collected in the 0-0.20 m layer in Lot 14, Sector I, of the Irrigated Perimeter Várzeas de Sousa-PB. After filling, the bags were placed on a metal bench (angle bracket), at a height of 0.80 m from the ground to facilitate the handling and application of treatments.
The physicochemical characteristics of the substrate used in the experiment (Table 1) were determined according to the methodology proposed by Claessen (1997) at the Irrigation and Salinity Laboratory at UFCG / Campina Grande-PB.
Sowing was performed using one seed per bag. The substrate was in the field capacity using water supply (ECa of 0.3 dS m -1 ). The seed was inserted in the vertical position with the base facing upwards (insertion point of the chestnut into the peduncle), at a depth of approximately 1 cm of soil, according to the recommendations of Embrapa -CNPAT (CAVALCANTI JÚNIOR; CHAVES. 2001).
The application of saline water started 25 days after sowing (DAS), with daily watering shift. The irrigation was performed in the late afternoon (5:00 p.m), manually. The applied blade was estimated by the drainage lysimetry principle: difference between the applied and the drained volume in the previous irrigation ( Figure 2). In order to reduce the accumulation of salts in the soil, a leach fraction of 0.15 was applied every 10 days.
The application of proline concentrations SOUZA, L. P. et al. Weeding was done whenever necessary to control the incidence of invasive plants, harmful to the culture of interest. The effects of the treatments were evaluated by determining the absolute and relative growth rates for plant height (TCAap and TCRap) and stem diameter (TCAdc and TCRdc), in the period between 25 to 65 DAS. Fresh phytomass of stem (FFC) and leaves (FFF) and dry phytomass of stem (FSC), leaves (FSF) and roots (FSR) from cashew at 65 DAS, as well as the ideal time for the production of cashew rootstocks were also determined.
The determination of the absolute growth rate (TCA) was obtained using the methodology proposed by Benincasa (2003), as described in equation 1: (1) Where: A2 = plant growth (stem diameter or height) at time t 2 ; A 1 = plant growth at time t 1 ; and

Figure 2. Collection of drained water
Engenharia na Agricultura, v.28, p. 488-498, 2020 t 2 -t 1 = time difference between samples. The relative growth rates (TCR) were obtained by equation 2. The growth is measured as a function of pre-existing matter, adapting to plant stem height or diameter according to Hunt et al. (2002). (2) Where: A 2 = plant growth (stem diameter or height) at time t 2 ; A 1 = plant growth at time t 1 ; t 2 -t 1 = time difference between the samples; and ln = natural logarithm In order to determine the accumulation of phytomass, the stem of each plant was cut close to the soil. Then, the different parts (stem and leaf) were separated and weighed immediately on a precision scale (0.001 g) to determine the fresh phytomass. After determining fresh phytomass, the different parts of the plant (leaves, stem and root) were packed separately in paper bags properly identified and put to dry in a forced air circulation oven at 65 ºC until dry phytomass of leaf (FSF), stem (FSC) and root (FSR) were obtained.
The variables were assessed using analysis of variance by the F test (0.01 and 0.05 probability) and in cases of significant effect, linear and quadratic polynomial regression analysis was performed using the SISVAR statistical software (FERREIRA, 2011).

RESULTS AND DISCUSSION
Based on the results of the analysis of variance (Table 2) we can verify a significant effect of the interaction between the factors (irrigation water salinity x proline concentration) on the absolute growth rate of the height of the BRS 226 Planalto cashew rootstock. However, for relative growth rate of plant height and absolute and relative growth rate of stem diameter, there is a significant effect only for the irrigation water salinity factor.
Increasing levels of water salinity in interaction with different concentrations of proline inhibited the growth rate of plant height of BSR 226 cashew rootstocks (Figure 3). We can observe that the highest TCAap (0.69 cm d -1 ) was obtained when the plants were irrigated with CEa of 0.3 dS m -1 in the absence of proline. Despite the decrease at 65 DAS, the AP of the plants were within a standard suitable for grafting.
The effect of salinity on TCAdc is presented in Figure 4A. The best adjustment of the data occurs in a linear equation with a decrease in the rootstock SOUZA, L. P. et al. submitted to greater CEa (3.1 dS m -1 ) of 38.30% (0.024 mm d -1 ). It is noted that this result can occurs due to the effect of salinity on the turgor pressure in the cells. The decrease in the water content in the tissues results in a decline in cell wall expansion, causing less plant growth (FREIRE et al., 2010;SOUZA et al., 2017). Similar behavior was observed for TCRdc ( Figure 4B) by the increase in saline levels, with a linear reduction in the order of 12.23% due to a unit increase in CEa. There was a total decrease of 37.91% in the TCRdc of plants subjected to irrigation with CEa of 3.1 dS m -1 in relation to irrigated plants with the lowest salinity (0.3 dS m -1 ). Irrigation with CEa water of 0.99 dS m -1 can be used in the production of cashew rootstocks due to the levels of DC and AP are within the standard cited by Serrano et al. (2013) which are 5.18 mm (DC) and 33.53 cm (AP). These values promote an acceptable average reduction of 10% over the TCAdc and TCRdc of the BRS 226 cashew rootstock.
TCRap was negatively and linearly affected by the salinity of the water ( Figure 4C). According to the regression equation, there is a decrease of 10.66% per unit increase in CEa, which means that plants irrigated with CEa water of 3.1 dS m -1 suffered reductions in this variable of 0.0067 cm cm -1 d -1 (29.86%) in relation to those irrigated with a level of 0.3 dS m -1 . It is noted that this reduction in plant growth was due to the lower consumption of water and / or nutrientes, which can be explained by the osmotic effect since the amount of saline in the soil has been increasing over time (TORRES et al., 2014).
Based on the summary of the analysis of variance (Table 3) we can observe that there was a significant effect of the interaction between the factors (salinity of the irrigation water and different concentrations of proline) for fresh and dry leaf phytomass. However, for fresh and dry stem phytomass there was a significant effect only of the irrigation water salinity factor. For dry root phytomass there was an effect in isolation of the factors salinity and concentration of proline at 65 days after sowing (Table 3).
FFF and FSF were significantly affected by the interaction between salinity of irrigation water and doses of proline. According to the regression equations ( Figure 5A and B) we can verify that the plants submitted to 7.3 mM and 12 mM proline and irrigated with 0.3 dS m -1 CEa reached the largest fresh and dry phytomasses (9.97 g and 2.51 g plant -1 ). However. the lowest production of FFF of 4.87 g and FSF 1.  (FFF) and 15.13 % (FSF). This type of behavior demonstrates that the reduction of the photosynthetic process in saline treatment is not only due to the partial closure of the stomatal opening but also due to a damage to the cell structure responsible for CO 2 assimilation. This cell structure damage can be possibly caused by reduction in osmotic-water potential and accumulation of ions above the tolerated range. These changes can affect the accumulation of fresh and dry phytomass of plants grown under salt stress conditions (LACERDA et al., 2012).
The behavior of the FFC and FSC variables as a function of the salinity levels of the irrigation water is shown in Figure 6. We can observe that variables values reduced with the increase of the CEa levels. Maximum values of 6.31 and 1.69 g per plant were found in the ECa of 0.3 dS m -1 and the lowest values of 4.15 g per plant (FFC) and 0.97 g per plant (FSC) were found in the ECa of 3.1 dS m -1 . This decrease in the production of phytomass can be associated with the photosynthetic rate and the energy deviation intended for growth. This energy deviation may have been used for the activation and maintenance of metabolic activity associated with adaptation to salinity such as maintaining the integrity of membranes; for synthesis of organic solutes for osmoregulation and / or protection of macromolecules and regulation of ionic transport and distribution in various organs within cells (SOUZA et al., 2016). Regarding the effects of salinity on the dry root phytomass of the BRS 226 cashew rootstock ( Figure 7A), we can verify a linear reduction with the increase in the electrical conductivity of irrigation water. There was a decrease in this variable of 17.27% due to a unit increase in CEa. This result is equivalent to a reduction of 48.35% (0.70 g per plant) of plants subjected to 3.1 dS m -1 when compared to those that were under the lowest salinity level of (0.3 dS m -1 ).
The reduction in the biomass accumulation may be the result of mechanisms for adjusting to salt stress conditions. These mechanisms include SALINE STRESS AND EXOGENOUS APPLICATION OF PROLINE IN CASHEW ROOTSTCK Figure 5. Fresh leaf phytomass -FFF (A) and dry leaf phytomass-FSF (B) of BRS 226 cashew rootstock as a function of the interaction between water salinity (CEa) and doses of proline at 65 days after sowing changes in the ion balance, water potential, mineral nutrition, stomatal closure, photosynthetic efficiency and carbon allocation (SILVA et al., 2008). According to Figure 7B for FSR at 65 DAS, we can observe a better adjustment of the data in quadratic regression due to the increase in proline concentrations, whose highest FSR value of 1.09 g per plant was obtained when the plants were under the concentration of 8 mM proline. This result may be associated with the ability of a given dose of proline to provide osmotic adjustment without causing tissue damage. Therefore, a protective role was developed through the accumulation of nitrogenous organic compounds, reflecting an osmoregulatory mechanism (LARCEDA et al., 2014).

CONCLUSION
• Irrigation with slightly saline water up to 0.85 dS m -1 can be used for the production of BRS 226 Planalto cashew rootstock.
• 8 mM proline concentration provides greater value for dry roots phytomass of BRS 226 Planalto cashew rootstock.
• The increase in concentrations of proline applied via leaf does not attenuate the deleterious effects of salinity on height growth rate and leaf phytomass of the BRS 226 cashew rootstock.