Plant growth and steviol glycosides as a ﬀ ected by foliar application of selenium, boron, and iron under NaCl stress in Stevia rebaudiana Bertoni

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Plant growth and steviol glycosides as a ﬀ ected by foliar application of selenium, boron, and iron under NaCl stress in Stevia rebaudiana Bertoni

Mehdi Aghighi Shahverdi, Heshmat Omidi

^⁎

, Seyed Jalal Tabatabaei

Department of Agronomy, Shahed University, Tehran, 331911865, Iran

A R T I C L E I N F O

Keywords:

Leaf mass ratio Leaf yield Micronutrients Salt-stress Selenium

Steviol glycosides accumulation

A B S T R A C T

Stevia(Stevia rebaudianaBert.) is well known for its high content of sweet steviol glycosides (SVglys) in its leaves (about 4–20% of dry weight) depending on the environmental, climatic, and growth conditions. The present study aimed to investigate the effects of foliar iron, selenium, and boron on stevia, irrigated with different concentrations of saline water. The experiment was conducted during two consecutive years in Firuzabad and Anzali, Iran. Results indicated that plant height, number of branches, leaf and biological yield and leaf mass ratio decreased by increasing the NaCl concentration of irrigation water. In both places, total SVglys content, and SVglys yield increased when the plants were irrigated with a 30 mM of saline water while higher salinities (90 mM) resulted in decreasing the traits. In addition, the foliar application of selenium alone and in combination with boron, and iron played a significant role in growth, yield parameters and SVglys accumulation including rebaudioside-A,-B and -C, Stevioside, and Dulcoside-A. Firuzabad had the highest mean values of vegetative traits, leaf yield, and SVglys yield in the second year of the study. Regarding Firuzabad, leaf yield and SVglys percentages had a compensatory relationship, which can play a role in the observed increase in the SVglys percentage under a moderate level of NaCl (30 mM) stress. Finally, the foliar application of selenium either alone or in combination with iron could alleviate the adverse effects of NaCl stress on the studied traits.

However, further studies are required to recognize the roles of selenium in SVglys accumulation and growth of stevia.

1. Introduction

Sugarleaf (Stevia rebaudianaBertoni) belonging to the Composite family is a perennial semi-shrub native to Brazil and Paraguay (South America) (Kumar-Pal et al., 2015; Tavarini et al., 2018). The im- portance of stevia herb is related to the presence of Steviol glycosides (SVglys) as natural sweeteners. These metabolites have a low caloric content and a natural sweetening power. Thus, there is a high demand for stevia herb in the food and pharmaceutical industries (Barbet- Massin et al., 2015b). The SVglys of the plant mainly includes Stevio- side (Stev: 4–13%), Rebaudioside-A (Reb-A: 2–4%), Rebaudioside-C (Reb-C: 1–2%), and Dulcoside-A (Dulc-A: 0.4-0.7%) (Gupta et al., 2016).

Salinity, deﬁned as a high content of soluble salts, is one of the most important environmental limiting factor which aﬀect crop yield worldwide (Zheng et al., 2013; Gharasallah et al., 2016). Sodium chloride (NaCl) is regarded as the dominant salt composition in the soil (Munns and Tester, 2008). Osmotic stress and)ionic toxicity are

considered as the most important eﬀects of salinity stress, which results in decreasing root absorption (Munns and Tester, 2008).Reis et al.

(2015)reported that irrigation with a saline water led to decreased stevia yield. In addition,Pandey and Chikara (2015)reported that low concentrations of NaCl lead to increased Stev and Reb-A.

The use of macro- and micronutrients is one of the strategies for coping with environmental stresses (Iqbal et al., 2015;Shahzadi et al., 2017). In addition, selenium (Se) is considered as a useful element for plant growth and production which plays an important role in physiological processes such as antioxidant activity (Sieprawska et al., 2015;Iqbal et al., 2015). Some studies have shown that Se is an essential element for human and animal, which plays some beneﬁcial roles in higher plants. However, the role of Se on morpho-physiological traits under salt stress needs further studies. Selenium application caused an increasing growth in mung bean and wheat under both stressed and non-stressed conditions (water and heat stress) (Malik et al., 2011;Iqbal et al., 2015;Shahzadi et al., 2017).

Micronutrients play an important role in growing and developing of

https://doi.org/10.1016/j.indcrop.2018.09.029

Received 23 April 2018; Received in revised form 9 September 2018; Accepted 12 September 2018

Abbreviations:SVglys, steviol glycoside; Dulc-A, dulcoside-A; Reb, rebaudioside; Se, selenium; Fe, iron; B, boron; LMR, leaf mass ratio; Stev, stevioside

⁎Corresponding author.

E-mail addresses:aghighi_sh@yahoo.com,omidi@shahed.ac.ir(H. Omidi).

T

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plants. As micronutrients, mainly Zn, B, Fe, and Mn are concerned with the nutrient management of medicinal plant (Moinudden et al., 2017).

The nutrients can be applied to crop plants in a variety of ways like seed treatment, soil and foliar application (Rehim et al., 2012). Foliar application of micronutrients is 6–20 times more useful than the soil application, which improves the nutrition (Arif et al., 2006).

Although the role of Se, Fe, and B, application in improving stevia performance is not well-documented, the worldwide demand for this plant is expected to increase. Some studies reported that the stevia plant can grow and produce in most climatic and soil conditions (Hajar et al., 2014). The leaf yield and second metabolic rate depend on the growing conditions, cultivar, and agronomic practices (Kumar-Pal et al., 2013).

Therefore, thisﬁeld study was designed to evaluate the role of Se, Fe, and B application (single or combined) under the salinity of irrigation water on growth and SVglys content in Anzali and Firuzabad during two consecutive years.

2. Materials and methods

2.1. Characteristics of soil, climate, and location

The present study was conducted in the experimental farms in Anzali (Guilan Province,−26 m above sea level, latitude of 37° 28′16″

N and longitude of 49° 27′44″E) and Firuzabad (Fars Province, 1386 m the above sea level, latitude of 28° 84′31″N and longitude of 52° 57′ 35″E) cities in Iran during two the growing seasons in 2014–15 and 2015–16 in order to evaluate the eﬀect of foliar application of nutrient solutions and salinity of irrigation water on plant growth and SVglys content in the stevia leaf. Anzali has Caspian climate in the north of Iran, while Firuzabad has a warm and dry climate in the southeast of Iran. Other meteorology data related to two growing seasons are pre- sented inFig. 1.Table 1demonstrates the results of soil analysis. It is worth noting that soil samples were collected in each experimental site at 0–30 cm depth. Based on the results of soil analysis, the supply of the elements (NPK) was conducted during planting.

2.2. Treatments and experimental design

These experiments were conducted based on split plot in a rando- mized complete block design (RCBD) with three repetitions. Main plots included four salinity of irrigation water levels (≤10 mM as control, 30 ± 5, 60 ± 5 and 90 ± 5 mM NaCl) with a plot size of 48 × 15 m (the distance between the plots was 2 m(. Each plot was divided into twenty-four 225 cm × 90 cm, subplots were eight spraying treatments (the distance between the subplots in each plot was 1 m). Subplots consisted of spraying eight fertilization levels (control, B, Se, Fe, B + Se, B + Fe, Se + Fe, and B + Se + Fe). Stevia plantlets (Stevia re- buadianaBertoni) were produced in sand beds through seed and three-

month-old seedlings were transplanted in thefields. The plants were transplanted manually to 45 × 45 cm spacing in order to achieve an approximate density of 50,000 plants per ha, which reflects the common practice in Iran steviafields.

After transplanting, the whole experimental area was irrigated with low EC water (≤1.81 dS/m) up to 15 days. Then, drip irrigation was applied in experiments and the proposed method ofReis et al. (2015), which was performed on stevia plant, was used in order to irrigate with salt ware (Fig. 2). The rate of daily water consumed was calculated to fill the soil profile of the farm to a depth of 0.5 m in terms offield capacity. Based on the calculations, the rate of water consumed per each salinity level (for 24 subplots) is about 200–250 liters per day depending on the location. In addition, in order to prevent salt accumulation in the soil (according to previous tests) after one week of irrigation with saline water, a single stage of irrigation with normal water (low EC) was carried out, in which case almost EC is within the pre- scribed treatments (Fronza and Folegatti, 2003; Allen et al., 2005;

Lavinia et al., 2008;Reis et al., 2015).

The results of some studies indicated that foliar application of Sequestraene 138 is used as Fe-source at 4 kg/ha, boric acid as B-source at 1.2 kg/ha, and sodium selenite as Se-source at 20 g/ha (Fe and B elements in concentration of 5 part per thousand (ppt) and Se in concentration of 2 ppt) (Baloch et al., 2008;Heidari et al., 2008;Feng et al., 2013;Hu et al., 2013). Spraying treatments were performed in plant growth and branching (V3) and plant re-growth (V4) (Zehtab-Salamasi

Fig. 1.Monthly mean temperature (°C), sunshine hours (h), rainfall (cm) and relative humidity (%) during two the growing seasons of 2014–15 and 2015–16 in Anzali and Firuzabad locations of Iran.

Table 1

Physical and chemical characteristics of the soil in two experimental locations (Anzali and Firuzabad, Iran) sampled on April 2014 and 2015 at 0–30 cm soil depth.

Characteristic Anzali Firuzabad

Sand (2–0.05 mm, %) 76 29.3

Silt (0.05–0.002 mm, %) 12 44.3

Clay (< 0.002 mm, %) 12 25.6

Soil texture Sandy Loam Loam

pH (H2O 1:2.5 soil: water suspension, McLean method)

7.13 7.44

Electrical conductivity in saturated extract (dS/m) 1.06 1.53 Cation exchange capacity (method BaCl2, pH 8.1,

mequiv/100 g)

8.34 8.23

Calcium carbonate (%) 32.8 42.5

Organic matter (%) 0.6 0.8

Nitrate of nitrogen (ppm) 1.95 1.98

Available phosphorus (Olsen method, ppm) 8.68 8.85 Exchangeable potassium (Thomas method, ppm) 311 321

Available iron (USDA-NRCS, ppm) 2.12 2.37

Available boron (USDA-NRCS, ppm) 0.7 0.9

Available manganese (USDA-NRCS, ppm) 3.13 3.98

Available copper (USDA-NRCS, ppm) 0.49 0.51

Available zinc (USDA-NRCS, ppm) 0.43 0.48

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et al., 2012;Wu et al., 2013).

2.3. Growing traits

In order to measure the growing traits such as plant height, number of branches per plant, leaf yield, biological (total biomass) yield, and leaf mass ratio (LMR), three plants were randomly selected from each experimental plot and accordingly the traits were measured. LMR is the biomass of the whole plant to the leaf yield, which is reported as a percentage. In other words, the harvest index was considered. Leaves were the aerial biomass of each plant and the stems were separated by hand and oven dried at 45 ± 3 °C for 48 h (Barbet-Massin et al., 2015a).

2.4. SVglys content and composition

The dried samples (hot air oven at 45 ± 3 °C for 48 h) were powdered by a mill machine (with 3 mm) in order to “measure SVgly compositions of leaf stevia. Thus, 100 mg of powdered samples was extracted with 10 mL water at 50 °C for 30 min. After centrifugation (7000 rpm, 5 min) andfiltration (0.2μm), 20μL of the resulting solu- tion was injected into a high-performance liquid chromatography system (HPLC) on a C18 column (250 × 4.6 mm inner diameter, 5μm particle size; Luna C18 Phenomenex, USA). In addition, SVglys were eluted by using an isocratic phase of ACN 31%: H2O (pH = 2.6, HCOOH) 69%, at 1 mL/minflow rate for 30 min. The SVglys compositions including Stev, Reb-A, Reb-B, Reb-C, and Dul-A were detected at 200 nm. Regarding quantification, Stev and Reb-A were used as an external standard. The results were expressed as a percentage per unit dry mass for the SVgly total content and as a percentage of SVglys total content for the SVglys proportions (Kumar-Pal et al., 2013; Barbet- Massin et al., 2015b)”.

2.5. Statistical analysis

All of the data obtained from Anzali and Firuzabad during two years were analyzed by SAS software (Statistical Analysis Software, 9.4). The two-factor-factorial ANOVA based on split-plot was separately pre- formed for each year to estimate the variance components of the effects of salinity and foliar application and their interactions (salinity × foliar application). Differences among the treatments were evaluated by LSD (least significant difference) only when the ANOVA F-test indicated the significance level of 0.05.

3. Results and discussion 3.1. Growth and yield characteristics

Based on the results inTables 2 and 3, the eﬀect of salinity of irrigation water was signiﬁcant on plant height, total branch, leaf and

biological yield, and LMR and the salinity of irrigation water caused a decrease in the rate of the traits. The highest plant height (60.5 cm), total branch (20.1 number per plant), leaf yield (2191.7 kg/ha) and biological yield (4813.7 kg/ha) achieved in the control level of irrigation water salinity in Firuzabad during the second year, which showed an increase of 43.26, 89.44, 89.67 and 82.98% respectively, compared to the total mean. However, the LMR was the highest (53.0%) at Anzali in the control level of irrigation water salinity during theﬁrst year.

Lower plant height (23.1 cm), total branch (3.7 number per plant), leaf yield (381.2 kg/ha), biological yield (1450.6 kg/ha) and LMR (25.3%) were in 90 mM salinity level at Anzali during thefirst year (Tables 2 and 3). The salinity treatment significantly reduced all investigated growth and yield components such as plant height, number branch, leaf yield, biological yield, and LMR. The plants treated with≤10 mM NaCl (control) significantly indicated the highest plant height, number branch, leaf yield, biological yield, and LMR. On the other hand, the least amount of these traits were observed in 90 mM salinity level. The reduction rates of leaf yield (economic yield in stevia) at salinity levels of 30, 60 and 90 mM were 443.2, 925.4, and 1306.22 kg/ha, respectively, compared to the control treatment. The reduction rates of biological yield in 90 mM was 2236.8 kg/ha, compared to the control treatment (≤10 mM NaCl). Since LMR is the result of the fraction biological yield to leaf yield, the average reduction of this trait is expected due to salinity stress and the results of the present study can confirm this. It has been reported that plant growth decreases under salt stress conditions due to the loss of photosynthesis related to the closure of plant stomata and the reduction of carbon uptake (Zheng et al., 2013). Some studies reported that the effect of salinity stress was significant on the dry and fresh weight of leaves, leaf area, and leaf number in different plant species (Ahmad et al., 2012; Sabra et al., 2012). Reducing the amount of water absorbed by the plant is due to the effects of salinity induced osmotic stress (Deinlein et al., 2014). It seems that stevia plant, like other herbs, is able to overcome the pro- blem of reducing water absorption by osmotic stress by adopting different strategies such as reducing evapotranspiration and stomatal conduction (Acosta-Motos et al., 2015; Cantabella et al., 2017). The impairment of element absorption from the soil is regarded as another secondary effect of NaCl stress since it heavily relies on the osmotic potential of water and plant. Further, some studies confirmed the results, which are consistent with the results of the present experiment (Tunçtürk et al., 2011;Talei et al., 2012).

The foliar application of the elements improved the growth and yield attributes of the plant. The highest total branch (18.2 number per plant), leaf yield (1954.5 kg/ha), and biological yield (4510.8 kg/ha) were observed in Se treatment in Firuzabad during the second year.

Furthermore, the integrated foliar application of B + Se + Fe in Firuzabad during the second year had the highest plant height (55.09 cm). However, the lowest rate of plant height (24.9 cm), total branch (4.6 number per plant), leaf yield (681.0 kg/ha), biological yield (1686.8 kg/ha) and LMR (35.9%) were observed in without foliar Fig. 2.General schema of drip irrigation system with four levels of saline water.

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application (control) in Anzali location during theﬁrst year (Tables 2 and 3). In this study, the yield and yield attributes of stevia such as plant height, number branch, leaf yield, biological yield, and LMR increased by foliar application of nutrients at both locations. In addition, Se application caused an increase in the number of branch, leaf yield, and biological yield.Shahzadi et al. (2017)reported that foliar application of Se could decrease oxidative stress, modulated photosynthetic pigments, and fertility while it increased the grain yield in wheat. The

results of the present study are in line with theﬁndings ofShahzadi et al. (2017). The results in the present study indicated that the crop was fertilized with the integrated application of B, Fe, and Se registered the maximum plant height at both locations during 2015 and 2016. It seems that a higher percentage of photosynthetic materials is applied to the stem of the plant by increasing plant hieght, leading to a decrease in the number of leaves per plant. Therefore, the integrated application of nutrients (B, Se, and Fe) was less eﬃcient than the Se spray application Table 2

Effect of different compounds selenium (Se), iron (Fe), and boron (B) on plant height and number of branch of stevia under NaCl stress (control, 30, 60, and 90 mM) in different experimental places (Anzali and Firuzabad, Iran).

Treatments Plant height (cm) Number of branch per plant

Anzali Firuzabad Anzali Firuzabad

2015 2016 2015 2016 2015 2016 2015 2016

Salinity level

NaCl≤10 mM 45.3 47.2 58.8 60.5 8.1 9.8 18.2 20.1

NaCl 30 ± 5 mM 40.1 43.5 53.8 54.6 7.3 8.2 15.3 17.1

NaCl 60 ± 5 mM 28.7 31.5 42.2 44.8 4.9 5.7 12.7 11.0

NaCl 90 ± 5 mM 23.1 24.9 37.2 38.8 3.7 4.4 10.0 13.3

St D ( ± ) 5.79 8.27 8.86 5.86 1.15 4.39 5.00 2.96

LSD (P= 0.05) 1.85 2.02 1.85 1.79 0.40 0.5 2.33 1.28

Foliar application

Control 24.9 28.4 38.4 40.1 4.6 5.4 13.4 14.4

B 30.9 32.8 44.4 44.8 6.0 6.6 14.7 15.1

Se 34.8 36.2 48.3 50.9 5.6 6.9 16.7 18.2

Fe 34.6 36.6 48.1 49.6 5.8 7.3 15.5 17.3

B + Se 37.2 38.9 50.7 52.3 6.5 7.5 12.5 14.0

B + Fe 36.9 39.5 50.4 51.7 7.0 8.3 14.5 15.6

Se + Fe 36.2 38.5 49.7 52.3 5.9 6.7 13.5 15.0

B + Se + Fe 40.5 43.7 54.0 55.9 6.7 7.6 11.5 13.4

St D ( ± ) 9.72 11.3 11.7 11.6 2.03 4.95 5.57 5.55

LSD (P= 0.05) 2.62 2.85 2.59 2.53 0.56 0.72 3.30 1.82

Total mean 42.23 10.61

Interaction eﬀect

LSD of S × F 5.25 5.71 5.25 5.07 1.13 1.45 6.60 3.64

St D: Standard division; LSD: Least signiﬁcant diﬀerence.

Table 3

Effect of different compounds selenium (Se), iron (Fe), and boron (B) on leaf yield, biological yield, and leaf mass ratio (LMR) of stevia under NaCl stress (control, 30, 60, and 90 mM) in different experimental places (Anzali and Firuzabad, Iran).

Treatments Leaf yield (kg/ha) Biological yield (kg/ha) LMR (%)

Anzali Firuzabad Anzali Firuzabad Anzali Firuzabad

2016 2015 2016 2015 2016 2015 2016 2015 2016 2015 2016 2015 2016

Salinity level

NaCl≤10 mM 20.1 1493.7 1537.2 2074.3 2191.7 2806.2 2899.7 4548.7 4813.7 53.0 52.8 45.5 45.6

NaCl 30 ± 5 mM 17.1 1048.3 1088.0 1663.3 1724.7 2159.1 2230.3 3726.7 3860.7 48.4 48.7 44.0 44.2

NaCl 60 ± 5 mM 11.0 682.8 711.6 1085.7 1115.0 1757.2 1830.5 2571.5 2765.7 38.7 38.8 41.4 40.1

NaCl 90 ± 5 mM 13.3 381.2 425.5 570.1 695.2 1450.6 1534.7 1540.3 1595.2 25.3 26.8 34.4 42.3

St D ( ± ) 2.96 126.5 339.5 388.0 371.1 190.7 737.6 842.8 763.2 3.09 4.54 4.55 5.62

LSD (P= 0.05) 1.28 25.6 26.4 149.3 83.2 34.4 43.1 298.6 114.0 1.07 1.4 1.16 1.23

Foliar application

Control 14.4 681.0 705.1 902.5 944.0 1686.8 1756.5 2205.0 2263.5 35.9 35.9 35.5 37.4

B 15.1 791.5 839.3 1323.9 1369.7 1906.2 2002.3 3047.7 3264.0 39.0 39.9 42.2 42.0

Se 18.2 881.9 932.1 1900.7 1954.5 2036.0 2104.1 4201.5 4510.8 40.9 42.0 44.5 43.2

Fe 17.3 916.3 952.0 1468.5 1615.2 2050.4 2132.6 3337.0 3552.3 42.3 42.5 43.1 45.2

B + Se 14.0 938.7 989.5 1132.4 1261.6 2107.3 2170.8 2664.8 2873.3 42.4 43.5 41.1 43.9

B + Fe 15.6 975.4 1009.4 1540.3 1621.2 2154.8 2258.0 3480.7 3574.2 43.3 43.0 41.9 44.7

Se + Fe 15.0 935.3 972.1 1206.9 1322.0 2084.6 2183.3 2813.8 2876.5 42.8 42.7 39.9 44.7

B + Se + Fe 13.4 1091.8 1125.0 1312.1 1365.3 2320.1 2383.0 3024.1 3155.9 44.5 44.9 42.2 43.5

St D ( ± ) 5.55 437.2 566.6 618.9 610.9 537.7 1018.5 1172.3 1171.4 11.3 9.31 8.63 8.50

LSD (P= 0.05) 1.82 36.32 37.40 211.1 117.7 48.6 61.0 422.3 161.2 1.52 2.03 1.64 4.22

Total mean 1155.5 2630.6 41.89

Interaction eﬀect

LSD of S × F 3.64 72.64 74.8 422.3 235.5 97.32 122.1 844.6 322.4 3.05 4.07 3.28 8.45

St D: Standard division; LSD: Least signiﬁcant diﬀerence.

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in term of leaf yield. Foliar application of Se, especially 4 ppm was ef- fective in increasing the plant height of wheat under stress condition (Shahzadi et al., 2017). In addition, the positive eﬀects of Se on growth and crop production have been reported in other plants such as wheat, wild cabbage, and melon (Hajiboland and Amjad, 2007; Yao et al., 2009;Hu et al., 2013). Further, the use of Se improves the absorption and transfer of other elements inside the plant, leading to an im- provement in growth and production (Shahzadi et al., 2017).

Based on the results of mean comparison of salinity stress and foliar application (interaction effects), an increase in the amount of NaCl in irrigation water decreased the average of traits such as plant height, number branch, leaf yield, and biological yield. Furthermore, the foliar application of nutrients caused an increase in the average of these traits and a decrease in the negative effects of salinity stress. The highest average of the four traits were achieved in control salinity level and foliar application of Se or integrated application of B, Se, and Fe. The results indicated that Firuzabad location has better growth conditions than Anzali location in terms of growth and yield attributes. In addition, regarding the comparison between the years of the implementa- tion experiment, the second year had more favorable results than those in thefirst year. The results indicated that the environmental conditions (soil and climates) of the site, especially during cultivation and growth, strongly affect the growth and yield attributes of the stevia plant. The plants, grown under Firuzabad location, produced the highest plant height, branch number, leaf yield, and biological yield, while the least mean of these traits was achieved in Anzali location. The results confirm the effects of environmental conditions on the yield and growth parameters of stevia. In this study, sunshine hours and temperature in Firuzabad location were more than those in Anzali location (Fig. 1).

Although relative humidity and rainfall were high in Anzali the growth and yield of stevia plant were less than those in Firuzabad due to the low sunshine hours and temperature in this location. In other words, a correlation should be established between climatic factors such as sunny hours, temperature, and rainfall, which are more favorable in Anzali than those in Firuzabad. Furthermore,Kumar-Pal et al. (2015) reported that“climatic factors signiﬁcantly inﬂuence the yield of stevia and CSIR-IHBT location was considered as the suitable among the three location in the case of dry leaf yield and secondary metabolite accumulation in leaves”. The solar radiation potential in the Anzali and Firuzabad regions were 2.8–3.8 and 5.2–5.4 kW h/(m²day), respectively. Having more favorable soil characteristics and higher levels of macro and micronutrients in the soil are regarded as some reasons for the preference of Firuzabad to Anzali locations. Many reports have suggested that light sources and quality directly stimulated the production of important secondary metabolites of stevia plant (Ahamd et al., 2015).

Regarding the year of conducting the experiment, the second year had the highest average of growth and yield attributes in both regions.

Kumar-Pal et al. (2015) reported higher total dry leaf yield in stevia grown as a perennial, compared to annul cycles in Palampur, India.

3.2. SVglys content

In addition, the results of the present study indicated that the SVglys composition (Reb-A, Stev, Reb-C, Dul-A, and Reb-B) plays a significant role in affecting salinity condition and foliar application. Further, these significant changes in the SVglys composition were observed between the years and locations in the present study (Tables 3 and 4). The highest percentage of Reb-A (3.13%), Stev (9.83%), Reb-C (0.23%), and Reb-B (0.48%) was achieved in 30 mM salinity level in Anzali location during the second year. As shown inTable 3 and 4the use of 90 mM NaCl had the lowest average for these traits in both year and location.

“The leaves are considered as the economic part of the stevia plant and the production of more leaf biomass with higher SVglys is the main criterion for crop performance (Kumar-Pal et al., 2014)”. The SVglys pathway is very active in the stevia leaves. Further, SVglys

accumulation in leaf was significantly improved by the moderate level of salinity in Firuzabad and Anzali. The results showed that the moderate salinity stress level (about 30 mM) is required to achieve the highest SVglys content and accumulation. The results were consistent with those ofPandey and Chikara (2015), in which the accumulation of SVglys was significantly higher, compared to the control plant under different concentrations of NaCl. Regarding the reason for the increase, we can refer to 30 mM NaCl concentration since gene transcription and SVgly accumulation is the result of plant reaction with increasing the concentration of NaCl which enhances their ROS scavenging capacity, compared to the control condition (Pandey and Chikara, 2015). In this regard, some other studies reported that the SVglys plays a role as os- moprotectant molecules and lead to an increase in the growth of plants exposed to stress condition (Geuns and Ceunen, 2013). The results of the present study and other previous studies indicate the positive effect of SVglys on osmotic adjustment in stevia leaf under salt stress conditions, which necessitates further research (Cantabella et al., 2017). The study results ofZheng et al. (2013)indicated that“NaCl could modulate the composition of SVglys in promoting the transformation of Stev to Reb-A”. Further,Gupta et al. (2014)reported that abiotic stress induced by the salts could significantly increase the concentration of SVglys. In another study, UGT76G1 gene played the key role in the transformation process (Mohamed et al., 2011). It was suggested that NaCl stress could enhance the transcription of UGT76G1 (Mohamed et al., 2011). Thus, the relationship between salinity stress and the transcription level of UGT76G1 should be highlighted in future studies.Aghighi Shahverdi et al. (2017), the low level of NaCl concentration leads to an increase in SVglys content and stevia is considered as a moderate NaCl tolerant plant”.

The integrated application of B, Se, and Fe in Anzali location during the second year had the highest average of Reb-A (2.93%), Stev (8.73%), Reb-C (0.23%), Dul-A (0.79%), and Reb-B (0.42%). Among foliar treatments, control (non-spraying) treatment had the lowest mean of the above-mentioned traits (Tables 3 and 4). In addition, the foliar application of nutrient solutions exerted a considerable eﬀect on Table 4

Effect of different compounds selenium (Se), iron (Fe), and boron (B) on rebaudioside-A (Reb-A) and stevioside (Stev) of stevia under NaCl stress (control, 30, 60, and 90 mM) in different experimental places (Anzali and Firuzabad, Iran).

Treatments Reb-A (%) Stev (%)

Anzali Firuzabad Anzali Firuzabad

2015 2016 2015 2016 2015 2016 2015 2016 Salinity level

NaCl≤10 mM 2.30 2.29 2.28 2.31 6.68 6.77 6.55 6.65 NaCl 30 ± 5 mM 3.08 3.13 3.10 3.10 9.68 9.83 9.52 9.72 NaCl 60 ± 5 mM 2.70 2.74 2.64 2.69 8.19 8.34 8.01 8.13 NaCl 90 ± 5 mM 2.01 2.29 1.98 2.01 4.34 4.49 4.31 4.37

St D ( ± ) 0.25 0.26 0.25 0.29 0.81 0.81 0.78 0.81

LSD (P= 0.05) 0.03 0.1 0.04 0.07 0.03 0.1 0.04 0.12 Foliar application

Control 2.26 2.25 2.20 2.20 6.13 6.22 9.07 6.13

B 2.28 2.22 2.32 2.28 6.51 6.57 6.25 6.26

Se 2.43 2.48 2.29 2.34 6.61 6.76 6.65 6.83

Fe 2.43 2.45 2.47 2.51 6.98 7.09 7.01 7.12

B + Se 2.65 2.69 2.53 2.58 7.30 7.44 7.26 7.37

B + Fe 2.63 2.68 2.66 2.72 7.65 7.76 7.60 7.75

Se + Fe 2.65 2.71 2.68 2.70 8.10 8.26 7.74 7.89

B + Se + Fe 2.86 2.93 2.85 2.85 8.51 8.73 8.21 8.41

St D ( ± ) 0.44 0.45 0.45 0.45 2.07 2.01 2.01 2.01

LSD (P= 0.05) 0.05 0.14 0.05 0.1 0.05 0.15 0.06 0.17

Total mean 2.53 7.26

Interaction eﬀect

LSD of S × F 0.11 0.28 0.11 0.20 0.10 0.30 0.13 0.35 St D: standard division; LSD: Least signiﬁcant diﬀerence.

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SVgly compounds such as Reb-A, Stev, Reb-B, Reb-C, and Dul-A, Reb-A to Stev ratio, and the total SVglys content and yield. In the present study, SVglys accumulated in the leaves at the concentration ranged from 7 to 15%. Most of the photosynthetic part of the leaves is allocated to the synthesis of this metabolite. Some studis reported that SVglys content depends on the cultivation practices of stevia, as well as the adoption of modern agro-techniques (Dus et al., 2006). However, SVglys compounds in the leaf were improved by the integrated foliar

application of B, Se, and Fe, which may be attributed to increased levels of photosynthetic pigments (Kumar-Pal et al., 2013). Based on the results of the present study, the integrated application of B, Se, and Fe had the highest Reb-A, Stev, Reb-B, Reb-C, Dul-A, as well as the total SVglys content.

In another study, the results indicated that carbohydrates are used to increase the amount of SVglys when the available nutrient is optimal (Barbet-Massin et al., 2015a). An enzyme encoded by UGT74G1 and Table 5

Effect of different compounds selenium (Se), iron (Fe), and boron (B) on steviol glycosides (SVglys) compounds of stevia under NaCl stress (control, 30, 60, and 90 mM) in different experimental places (Anzali and Firuzabad, Iran).

Treatments Reb-C (%) Dul-A (%) Reb-B (%)

2015 2016 2015 2016 2015 2016 2015 2016 2015 2016 2015 2016

Salinity level

NaCl≤10 mM 0.17 0.17 0.17 0.18 0.63 0.64 0.65 0.66 0.29 0.34 0.30 0.31

NaCl 30 ± 5 mM 0.23 0.23 0.22 0.22 0.78 0.79 0.80 0.81 0.47 0.48 0.45 0.46

NaCl 60 ± 5 mM 0.19 0.20 0.20 0.21 0.70 0.72 0.70 0.72 0.38 0.38 0.38 0.39

NaCl 90 ± 5 mM 0.14 0.14 0.13 0.13 0.61 0.62 0.59 0.61 0.23 0.24 0.22 0.23

St D ( ± ) 0.02 0.02 0.02 0.02 0.06 0.06 0.06 0.06 0.05 0.05 0.05 0.05

LSD (P= 0.05) 0.002 0.005 0.003 0.005 0.008 0.01 0.009 0.01 0.004 0.03 0.007 0.008

Foliar application

Control 0.15 0.16 0.17 0.17 0.62 0.63 0.62 0.64 0.29 0.30 0.28 0.29

B 0.16 0.16 0.16 0.17 0.67 0.68 0.66 0.68 0.30 0.36 0.28 0.29

Se 0.17 0.18 0.16 0.17 0.62 0.64 0.62 0.63 0.31 0.32 0.31 0.32

Fe 0.18 0.19 0.17 0.17 0.61 0.62 0.67 0.68 0.36 0.37 0.32 0.34

B + Se 0.18 0.19 0.18 0.19 0.71 0.72 0.70 0.70 0.32 0.34 0.36 0.37

B + Fe 0.19 0.19 0.19 0.20 0.71 0.73 0.75 0.76 0.39 0.39 0.36 0.37

Se + Fe 0.20 0.20 0.19 0.20 0.71 0.73 0.69 0.70 0.37 0.39 0.39 0.40

B + Se + Fe 0.23 0.23 0.21 0.21 0.77 0.79 0.78 0.79 0.40 0.42 0.41 0.42

St D ( ± ) 0.03 0.03 0.03 0.03 0.07 0.08 0.08 0.08 0.10 0.09 0.09 0.09

LSD (P= 0.05) 0.002 0.007 0.004 0.007 0.01 0.01 0.01 0.01 0.006 0.04 0.01 0.01

Total mean 0.18 0.68 0.34

Interaction eﬀect

LSD of S × F 0.005 0.01 0.008 0.01 0.02 0.03 0.02 0.03 0.01 0.04 0.02 0.02

St D: standard division; LSD: Least signiﬁcant diﬀerence; Reb-C: Rebaudioside-C; Dul-A: Dulcoside-A ; Reb-B: Rebaudioside-B.

Table 6

Effect of different compounds selenium (Se), iron (Fe), and boron (B) on Reb-A:Stev ratio, total SVglys, and SVglys yield of stevia under NaCl stress (control, 30, 60, and 90 mM) in different experimental places (Anzali and Firuzabad, Iran).

Treatments Reb-A:Stev ratio Total SVglys (%) SVglys yield (kg/ha)

2015 2016 2015 2016 2015 2016 2015 2016 2015 2016 2015 2016

Salinity level

NaCl≤10 mM 0.34 0.34 0.35 0.34 10.0 10.2 9.9 10.1 152.3 158.8 207.1 222.3

NaCl 30 ± 5 mM 0.31 0.31 0.32 0.32 14.2 14.4 14.1 14.3 150.7 158.6 235.6 248.7

NaCl 60 ± 5 mM 0.33 0.33 0.33 0.33 12.1 12.4 11.9 12.1 83.7 88.7 129.2 135.4

NaCl 90 ± 5 mM 0.46 0.45 0.46 0.46 7.3 7.5 7.2 7.3 28.8 33.1 41.3 51.2

St D ( ± ) 0.03 0.03 0.03 0.03 1.12 1.12 1.09 1.14 22.7 41.17 46.02 47.1

LSD (P= 0.05) 0.006 0.01 0.006 0.01 0.05 0.13 0.07 0.15 2.61 3.23 16.6 9.74

Foliar application

Control 0.39 0.38 0.38 0.37 9.4 9.5 9.3 9.4 68.4 71.1 87.6 91.6

B 0.36 0.35 0.39 0.38 9.9 10.0 9.6 9.7 82.6 88.2 131.9 134.4

Se 0.38 0.38 0.35 0.35 10.1 10.4 10.0 10.3 93.6 100.1 204.2 214.2

Fe 0.35 0.35 0.37 0.37 10.5 10.7 10.6 10.8 100.7 105.9 163.7 181.9

B + Se 0.38 0.38 0.35 0.36 11.1 11.4 11.0 11.2 109.1 116.4 127.1 142.6

B + Fe 0.36 0.36 0.35 0.35 11.5 11.7 11.5 11.8 116.7 122.6 194.0 206.6

Se + Fe 0.33 0.33 0.36 0.36 12.0 12.3 11.7 11.9 116.9 124.1 148.5 164.4

B + Se + Fe 0.34 0.34 0.35 0.35 12.7 13.0 12.4 12.7 143.2 149.3 169.7 179.5

St D ( ± ) 0.06 0.06 0.06 0.06 2.68 2.62 2.63 2.62 54.07 70.6 76.8 76.8

LSD (P= 0.05) 0.009 0.02 0.009 0.01 0.07 0.18 0.11 0.21 3.69 4.57 23.6 13.7

Total mean 0.36 10.94 132.84

Interaction eﬀect

LSD of S × F 0.01 0.04 0.01 0.03 0.15 0.35 0.22 0.43 7.38 8.98 47.22 27.5

St D: standard division; LSD: Least signiﬁcant diﬀerence.

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UGT76G1 was recognized for raising glucosylate steviolbioside and Stev to Stev and Reb-A, respectively (Fallah et al., 2017). It seems that the highest expression of both UGT74G1 and UGT76G1 genes which are involved in the synthesis of SVglys occurs in an integrated application of B, Se, and Fe.

Mean comparison of salinity stress and foliar application)interaction eﬀects(showed that an increasing trend was observed in the percentage of SVglys composition by increasing the amount of sodium chloride in irrigation water up to the 30 mM, and accordingly decreased the average of these traits as the highest salinity level (90 mM) had the lowest average. On the other hand, the foliar application of elements improved the increasing or decreasing rate due to salinity stress, leading to the highest average in the integrated application in B, Se, and Fe elements.

In addition, the Reb-A: Stev ratio, total SVglys, and SVglys yield were aﬀected by salinity stress. In this regard, the Reb-A: Stev ratio increased in 90 mM in both locations and the lowest mean of this trait was reported in 30 mM in Anzali. Further, the highest total SVglys (14.4%) and SVglys yield (248.7 kg/ha) were achieved in the 30 mM salinity level in Anzali and Firuzabad locations, respectively.

Furthermore, the lowest total SVglys (7.2%) and SVglys yield (28.2 kg/

ha) were reached in the 90 mM in Firuzabad and Anzali locations, respectively. Additionally, the integrated application of B, Se, and Fe in Anzali had the highest total SVglys (13.0%) while the highest SVglys yield (214.2 kg/ha) was reported in Se treatment in Firuzabad. The least average of total SVglys and SVglys yield was observed in the control treatment in both locations and years. In addition, the mean comparison of salinity stress and foliar application)interaction effect (indicated that the highest total SVglys were observed in 30 mM salinity and the integrated application of B, Se, and Fe and the lowest amount of this attribute was reported in 90 mM salinity and non-foliar application (control treatment). Further, the highest SVglys yield was observed in control level of salinity (≤10 mM) and the integrated application of B, Se, and Fe in Anzali location, while the highest SVglys yield was reported in 30 mM salinity and integrated application of Se and Fe in Firuzabad location. As illustrated inTables 3–6, the lowest total SVglys and SVglys yield were in 90 mM salinity and non-foliar application treatment. The findings indicated that the SVglys content and accumulation in the stevia leaves rely on the climatic conditions, agronomic practices, and soil characteristics (Munz et al., 2018). Further, the total SVglys content was more in Anzali than that of Firuzabad, while SVglys yield was more in Firuzabad than that of Anzali location. It is worth noting that the difference may be related to the fact that Anzali has a similar climate with high humidity as the origin of the plant, while fewer sunshine hours are available than those in Firuzabad location.

Thus, the total SVglys percentage was higher in Anzali location although the SVglys yield was lower than that of Firuzabad location since the leaf yield in this region was lower.Ladygin et al. (2008)reported that the accumulation of SVglys in stevia plant was related to the extent of the development of the chloroplasts membrane system and the photosynthetic pigments content. Furthermore, the variation in Stev accumulation in leaves due to location variation was rather high, compared to that of Reb-A. Thus, the accumulation of Stev is inﬂuenced by environmental and soil conditions. It was reported that Stev levels vary depending on the growing conditions and genotype (Staratt et al., 2002;Kumar-Pal et al., 2015).

4. Conclusion

Based on the increased utilization and interest in developing natural non-caloric sweeteners in recent decades, SVglys including high-sweet compounds extracted from the stevia leaves can be regarded as a good alternative for industrial sweeteners. The current study focused on growth, yield attributes, SVglys accumulation under the foliar application of B, Se, and Fe, as well as salt stress at diﬀerent growth conditions in Iran for increasing SVglys synthesis in the leaf. Based on the

results in the present study, growth and yield attributes such as plant height, number of branches, leaf yield, biological yield, and LMR and SVglys synthesis of stevia are strongly controlled by the exogenous supply of foliar application (B, Se, and Fe), NaCl stress and climatic conditions in Anzali and Firuzabad locations. The total SVglys content was more in Anzali than that of Firuzabad while SVglys yield was more in Firuzabad than that of Anzali location. Further, the climatic conditions of Firuzabad are more favorable compared to the Anzali location in terms of growth, leaf yield, and SVglys yield. These observations indicate that sunshine hours with humidity are very important for achieving an optimum yield of stevia, coupled with the temperature.

The integrated foliar application of B, Se, and Fe had the highest total SVglys compounds (Reb-A, Stev, Reb-B, Reb-C, and Dul-A) in Anzali location. Furthermore, the application of Se led to an increase in growth and yield parameters. The present study indicated that stevia plant can respond adaptively to moderate level of NaCl stress (30 mM) by decreasing its leaf yield while increasing the SVglys content in its leaves.

On the other hand, the foliar application of Se alone or integrated with Fe reduced the negative eﬀects of NaCl stress while improving the growth parameters of the stevia. However, further research is re- commended to recognize the role of Se in SVglys accumulation and growth of stevia.

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