Salicylic acid relieves salt stress damage on basil growth

ABSTRACT Salinity is one of the main challenges in agriculture, especially in arid and semi-arid regions. Salicylic acid application may be a strategy to mitigate the harmful effects of salt stress on plant growth. Basil (Ocimum basilicum ) is a medicinal, spice, and ornamental plant grown around the world that has its growth reduced by salt stress. Therefore, the objective was to evaluate salicylic acid in mitigating the damage caused by salt stress in ‘Cinnamon’ basil. Growth was evaluated at 90 days after the start of irrigation with saline water. Salicylic acid application attenuates the harmful effects of salt stress on the number of leaves, number of inflorescences, leaf dry mass, inflorescence dry mass, root dry mass, seed mass, leaf area, leaf area ratio, specific leaf area, leaf mass ratio, root mass ratio, inflorescence mass ratio, Dickson quality index, sclerophilia index, robustness quotient and shoot root ratio. Salt stress decreased plant height, stem diameter, stem dry mass, and stem mass ratio of basil ‘Cinnamon’. Salicylic acid can be used to lessen the harmful effects of salt stress on basil ‘Cinnamon’.

The high salts content in irrigation water is a limiting factor for plant development, which can compromise the growth and physiology of plants (Nóbrega et al., 2021).
The decrease in plant growth under salt stress is caused by the osmotic effect that reduces the water and essential elements absorption, causing nutritional imbalance, oxidative stress, and specific ions toxicity (Ahammed et al., 2018;Silva et al., 2019;Naveed et al., 2020).
The use of techniques to mitigate the harmful effects of salinity in plants is necessary.Salicylic acid can alleviate the harmful effects of salt stress by adjusting physiological and biochemical mechanisms (Kim et al., 2018).Foliar application of salicylic acid decreased lipid peroxidation caused by excess NaCl and improved plant growth.This behavior is related to a decrease in NaCl and an increase in Ca and Mg in the plant, in addition to the increase in the activity of the antioxidant system enzymes (SOD, CAT, Toshik Iarley da Silva et al. GPX, etc.) (Joseph et al., 2010).Salicylic acid is used in the formulation of some biostimulants used in agriculture, as a growth promoter and plant resistance inducer.In addition, this phytohormone acts as an effective inducer of genes related to stress-linked proteins (Angooti & Nourafcan, 2015).
Several studies are performed with basil plants under salt stress and salicylic acid (Elhindi et al., 2017;Silva et al., 2019;Kahveci et al., 2021); however, few studies have been performed about such effects on the growth of 'Cinnamon' basil.Therefore, the objective was to evaluate salicylic acid in mitigating the damage caused by salt stress in 'Cinnamon' basil.

MATERIAL AND METHODS
The experiment was performed in a greenhouse at the Universidade Federal da Paraíba, Areia, Brazil.The experimental design was in randomized blocks, using an incomplete factorial scheme generated through the central composite design, with five electrical conductivities of irrigation water 1.3,3.25,5.2,and 6.0 dS m -1 ) and five concentrations of salicylic acid (SA -0, 0.29, 1.0, 1.71, and 2.0 mM - Silva et al., 2019).Totaling nine combinations (Table 1), with five repetitions and two plants per plot, with 90 plants analyzed at the end of the experiment.
Basil seeds were sowed in 162-cell polyethylene trays that were filled with soil and commercial organic compost (1:1, v:v).Basil seeds were obtained from the basil germplasm bank of the Universidade Federal de Sergipe.Seedlings were transplanted 25 days after sowing into 5 dm 3 pots with soil and 100 g of poultry manure.Saline waters were prepared by adding of NaCl, CaCl 2 .2H 2 O, and Mg-Cl 2 .6H 2 O (7:2:1, v:v), respectively, to water (0.5 dS m -1 ).
Salicylic acid concentrations were prepared with 30% ethyl alcohol and Tween 80 (0.05%).Distilled water and Tween 80 (0.05%) were used as control.The plants were sprayed with 10 mL per plant of each treatment, every week for 21 days.Salt stress was applied 30 days after sowing along with salicylic acid applications.
Basil growth was evaluated 90 days after the start of saline water irrigation (DAI).A graduated ruler was used to measure plant height (PH); calipers were used to measure stem diameter (Ds); the number of leaves (NL) was measured manually.The plants were divided into root, stem, leaves and inflorescences and dried in an oven with forced air circulation at 65 ºC.Then, the plant parts were weighed on a precision scale (0.001 g).Seeds were separated from the inflorescences and weighed on a precision scale (0.001 g) to obtain the seed mass.
Leaf area (LA) was obtained by averaging the product of length and width of 20 leaves and the leaf area form factor (0.6775).Leaf area ratio (LAR) was obtained using the formula: leaf area (LA)/total dry mass (TDM).The specific leaf area (SLA) was obtained using the formula: LA/leaf dry mass (LDM).The leaf mass ratio (LMR) was obtained using the formula: leaf dry mass/TDM.The stem mass ratio was obtained using the formula: stem dry mass/ TDM.The root mass ratio (RMR) was obtained using the formula: root dry mass/TDM.The inflorescence mass ratio (IMR) was obtained using the formula: inflorescence dry mass/TDM.The sclerophilia index was obtained using the formula: LDM/LA.The robustness quotient was obtained Salicylic acid relieves salt stress damage on basil growth using the formula: plant height/stem diameter.Dickson quality index was obtained using the formula: TDM/((PH/ Ds) + (shoot dry mass/root dry mass)).The root/shoot ratio was obtained using the formula: root dry mass/shoot dry mass.
Data were subjected to the normality test (Shapiro-Wilk) and homogeneity of variances (Bartlett) and to the regression analysis and, when the interaction between the factors was significant, response surface graphs were created, with the equations being generated by the rsm package (Lenth, 2009) and by the GA package (Scrucca, 2013).A canonical variables analysis with confidence ellipses (p ≤ 0.01) were performed to study the interrelationship between variables and factors using the candisc package (Friendly & Fox, 2021).The statistical program R (R Core Team, 2021) was used to perform the statistical analysis.

RESULTS AND DISCUSSION
The summary of the analysis of variance (ANOVA) is shown in Table 2.The mean values per treatment for each variable are shown in Table 3.
The increase in salt stress reduced the plant height and stem diameter, with losses of 71.90 and 63.22%, respectively, when comparing the lowest and highest ECw (Figures 1A and 1C).Increasing the dose of salicylic acid increased plant height and stem diameter, with an increase of 109.02 and 89.87%, respectively, when comparing the lowest and highest doses (Figures 1B and 1D).Salicylic acid reduced the harmful effects of salt stress on the number of leaves and number of inflorescences, with the highest values (671 and 104) observed at ECws 0.52 and 0.91 dS m -1 and 1.98 and 1.99 mM of salicylic acid, respectively (Figures 1E and F).
The negative effect of salt stress on plant height and stem diameter is related to the decrease in CO 2 assimilation due to stomatal limitation and/or impairment in metabolic processes, which can lead to inhibition of cell division and expansion, as observed in pistachio (Pistacia vera L.) (Rahneshan et al., 2018).Furthermore, this behavior may be related to the negative effects of high salinity that can cause hyperionic and hyperosmotic stress, causing reduced plant growth and even death (Wang et al., 2018a).This action was also observed by Silva et al. (2022)  The increase in salinity of the irrigation water decreased the stem dry mass with losses of 33.05% when comparing the lowest and highest ECw (Figure 2A).Salicylic acid reduced this variable up to the dose of 1.71 mM, with an increase in the dose of 2.0 mM (Figure 2B).Salicylic acid attenuated the harmful effects of salt stress on leaf area and leaf area ratio, with the highest values (2657.57cm 2 and 34.87 cm 2 g -1 ) observed at ECws 0.54 and 5.99 dS m -1 and 1.98 and 0.25 mM of salicylic acid, respectively (Figures 3A and 3B).Salicylic acid also mitigated the harmful effects of salt stress on specific leaf area and leaf mass ratio, with the highest values (328.98 cm 2 g -1 and 0.23 g g -1 ) observed in ECws 5.99 and 0.51 dS m -1 and 0.86 and 0.0069 mM of salicylic acid, respectively (Figures 3C and D).The highest stem mass ratio (0.33 g g -1 ) was observed with ECw of 2.64 dS m -1 (Figure 3E) and the lowest (0.29 g g -1 ) was observed with 1.17 mM Toshik Iarley da Silva et al.
of salicylic acid (Figure 3F), above this dose, there was an increase for this variable.Salicylic acid reduced the harmful effects of salt stress on root mass ratio and inflorescence mass ratio, with the highest values (0.075 and 0.939 g g -1 ) observed with ECws 0.53 and 5.99 dS m -1 and 0.025 and 1.99 mM of salicylic acid, respectively (Figures 3G and 3H).
Salicylic acid alleviated the deleterious effects of salt stress on leaf area, leaf area ratio, specific leaf area, leaf mass ratio, root mass ratio, and inflorescence mass ratio of basil plants, possibly because this phytohormone promotes the plant growth under salt stress, as observed in C. sativus (Miao et al., 2020).In addition, this phytohormone may increase the osmotic adjustment capacity of plants, improving their stress defense mechanisms, in addition to increasing the production of phytohormones to adjust physiological processes and reduce the harmful effects of stress (Khan et al., 2018;Malik et al., 2021).
Thus, this phytohormone allows biomass accumulation in plants, even under stress.Salicylic acid increased the absorption of water, nutrients, root growth, and photosynthesis, contributing to the increase in the dry mass of plants under stress in a study performed with cucumber seedlings (Miao et al., 2020).4E).
The harmful effects of salt stress on the Dickson quality index, sclerophilia index, robustness quotient, and root/shoot ratio were mitigated by the salicylic acid.This behavior may be related to the action of salicylic acid as a regulator of the synthesis and osmolytes of the enzyme that is linked to growth and photosynthesis, acting mainly against the deleterious effects of salinity (Khanam & Mohammad, 2018).In addition, phytohormones act on gene signaling and expression, increasing plant tolerance to the effects of salt stress (Arif et al., 2020).Salicylic acid relieves salt stress damage on basil growth in basil plants grown under salt stress.The beneficial effect of salicylic acid on plant height and stem diameter and attenuating salt stress on the number of leaves and number of inflorescences is related to the effect of this phytohormone in the regulation of several defense mechanisms, especially the maintenance of ionic balance and photosynthetic activity and detoxification of reactive oxygen species (ROS), as observed in maize (Zea mays L.) (Tahjib-Ul-Arif et al., 2018).

Figure 1 :
Figure 1: Plant height (A and B), stem diameter (C and D), number of leaves (E) and number of inflorescences (F) of Ocimum basilicum 'Cinnamon' under salt stress (ECw) and treated with salicylic acid.ns, *, ** -Not significant, significant at p ≤ 0.05 and at p ≤ 0.01 by F test, respectively.

Figure 2 :
Figure 2: Stem dry mass (A and B), leaf dry mass (C), inflorescence dry mass (D), root dry mass (E) and seed mass (F) of Ocimum basilicum 'Cinnamon' under salt stress (ECw) and treated with salicylic acid.ns, *, ** -Not significant, significant at p ≤ 0.05 and at p ≤ 0.01 by F test, respectively.

Figure 3 :
Figure 3: Leaf area (A), leaf area ratio (B), specific leaf area (C), leaf mass ratio (D), stem mass ratio (E and F), root mass ratio (G) and inflorescence mass ratio (H) of Ocimum basilicum 'Cinnamon' under salt stress (ECw) and treated with salicylic acid.ns, *, ** -Not significant, significant at p ≤ 0.05 and at p ≤ 0.01 by F test, respectively.

Table 1 : Combinations generated by the central composite design
Electrical conductivity of irrigation water (ECw); salicylic acid (SA).

Table 3 :
Mean values per treatment of Ocimum basilicum 'Cinnamon' under salt stress (ECw) and treated with salicylic acid.Mean ± standard deviation Toshik Iarley da Silva et al. addition, salicylic acid attenuated the harmful effects of salt stress on root/shoot ratio, with the highest value (0.082) observed with ECw 0.56 dS m -1 and 0.0059 mM of salicylic acid (Figure