Impaginato 495 Adv. Hort. Sci., 2019 33(4): 495­501 DOI: 10.13128/ahsc­8127 Effects of foliar application of glycine and glutamine amino acids on growth and quality of sweet basil Y. Aghaye Noroozlo 1, M.K. Souri 1 (*), M. Delshad 2 1 Department of Horticultural Sciences, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran. 2 Departments of Horticulture, Faculty of Agriculture, University of Tehran, Karaj‐Iran. Key words: ascorbic acid, biofortification, biostimulation, fertilization, foliar feeding, nutrient uptake, proline. Abstract: Amino acids have diverse roles in plant metabolism, and recently amino acid based fertilizers have been used largely in crop production systems. Despite most of these new fertilizers are formulated for foliar application; how­ ever, morphophysiological responses of crops to amino acids application have not yet been well documented. In the present study, foliar application of glycine or glutamine in different concentrations of 0 (distilled water), 250, 500, 1000 mg·L­1, as well as a treatment of 250 mg·L­1 glycine + 250 mg·L­1 glutamine were evaluated on growth of sweet basil (Ocimum basilicum L.) plants. The results showed that foliar application of glycine or glutamine at 1000 mg·L­1 showed no improvement in comparison with control for all traits except for leaf L­ascorbic acid concentration that showed the highest value under these amino acids treatments. However, foliar application of these amino acids at 250 and particularly 500 mg·L­1 showed promising effect on sweet basil growth. Plant shoot fresh and dry weight, leaf area, leaf SPAD value, and leaf chloro­ phyll content were improved by foliar application of 500 mg·L­1 glycine or gluta­ mine in comparison with control plants. Foliar application of amino acids increased leaf nitrogen (glutamine 250 and 500 mg·L­1), potassium (glycine 250 mg·L­1), magnesium (glutamine 250 or 500 mg·L­1, glycine 250 mg·L­1, glycine + glutamine; 250 + 250 mg·L­1), iron (glycine and glutamine at 500 mg·L­1 and glut­ amine at 250 mg·L­1), and zinc (glycine and glutamine at 250 or 500 mg·L­1), whereas the increase in leaf nutrients caused by other treatments was not sig­ nificantly different from control plants. Leaf calcium concentration was not changed by amino acid treatments. The results indicate that foliar application of moderate to low concentrations of glutamine or glycine can improve sweet basil growth. 1. Introduction Basil (Ocimum basilicum L.) is a high marketable value vegetable, which is consumed as a fresh aromatic ingredient and included in cooked dishes. Increasing importance is being given to this produce due to its remarkable quality and nutritional features (Morano et al., 2017). (*) Corresponding author: mk.souri@modares.ac.ir Citation: NOROOZLO Y.A., SOURI M.K., DELSHAD M., 2019 ­ Effects of foliar application of Glycine and Glutamine amino acids on growth and quality of sweet basil. ­ Adv. Hort. Sci., 33(4): 495­501. Copyright: © 2019 Noroozlo Y.A., Souri M.K., Delshad M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 9 December 2018 Accepted for publication 8 July 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(4): 495­501 496 Chemical fertilizers have an inevitable role in food production of agricultural crops; however, during last decades the soil fertility and quality have been adversely affected by many fertilization strategies (Souri and Hatamian, 2019). This is mainly due to the low efficiency of applied fertilizers which can even cause yield reduction upon increasing supply (Ercolano et al., 2015). In some situations and partic­ ularly under adverse climatic conditions, foliar instead of soil application (Golubkina et al., 2018), or chelated forms instead of simple chemical forms of fertilizers (Garcia et al., 2011; Marschner, 2012; Souri and Aslani, 2018) can improve nutrient uptake and use efficiency of applied fertilizers. Each year, agricul­ ture production needs a large amount of expensive nitrogenous fertilizers that generally have low uptake efficiency rates and result in both plant (Caruso et al., 2011) and environmental pollution (Souri and Hatamian, 2019). Amino acids are being incorporated for more than two decades into fertilizer formula­ tions to improve fertilizers use efficiency (Abdul­ Qados, 2009; Souri, 2016). Foliar application of amino acids can have benefi­ cial effects on plant growth and production (Sadak et al., 2015; Shams et al., 2016; Hussain et al., 2018; Souri and Aslani, 2018). It has been shown that foliar application of arginine amino acid has improved shoot growth and grain yield of wheat in saline and non saline conditions (Abdul­Qados, 2009; Rizwan et al., 2017). Better growth and higher biomass produc­ tion via application of various amino acid chelates of iron or zinc has been also reported on some agro­ nomic and horticultural crops (Khan et al., 2012; El Sayed et al., 2014). Similarly, foliar application of a mix of amino acids at 500 and 700 ppm increased plant height, plant fresh and dry weights, leaf N con­ centration, yield of leaves and leaf soluble carbohy­ drates in celery (Shehata et al., 2011). Glycine is the simplest amino acid and is used largely for production of chelated fertilizers in form of aminochelates (Souri, 2016), whereas effect of foliar application of glutamine has not been well doc­ umented so far. In the present study, the effect of foliar application of different concentrations of glycine and glutamine has been evaluated on growth characteristics of sweet basil plants. 2. Materials and Methods This study was conducted under greenhouse con­ ditions and from 20 May till the end of July 2017. The soil used in experiment had a loamy texture and moderate levels of nutrients. The soil physiochemical characteristics are presented in Table 1. 4 L black plastic pots (30 cm height and 20 cm diameter) were filled with about 4 kg dry soil and watered at 80% of its soil field capacity (FC). Three days later 40 seeds of sweet basil (Ocimum basilicum L.) from a local population (Karaj landrace) were sown in 1 cm depth of the soil. Two weeks after germination plants were reduced to 10 uniform seedlings per pot. The plants were irrigated daily based on 80% soil field capacity. The temperature was on average 28±5°C, the light intensity 200 µmole.m­2.s­1, and the air humidity about 70­75%. Different concentrations of two amino acids of glycine and glutamine including 0, 250, 500 and 1000 mg·L­1, as well as a treatment of 250 mg·L­1 glycine + 250 mg·L­1 glutamine were sprayed on sweet basil plants. Treatments and pots were arranged in com­ pletely randomized design and each treatment con­ tained three replications. Each pot represented one replication and contained 10 plants. Each concentra­ tion of glycine or glutamine was sprayed on plant leaves five times during two­month growth period, and the first spray was done two weeks after seedling emergence. The remaining sprays were done in one week interval. Distilled water was sprayed on control plants. Spray treatments were done in the early morning, one hour after sunrise and with a portable sprayer by which the upper and lower surface of leaves were sprayed. A total amount of 80­90 mL solution of each treatment was sprayed on plants per pot. Plants were harvested two months after emer­ gence and before flowering stage. The average height of ten plants in each pot was measured by a ruler before harvest. The leaf SPAD value was mea­ sured by a portable SPAD meter (Model SPAD­502 Texture EC (dS.m­1) pH O.C. (%) Total N (mg.kg­1) Available P (mg.kg­1) Available K (mg.kg­1) Fe (mg.kg­1) Zn (mg.kg­1) Lime (%) Loamy­Sand 1.79 7.62 0.78 750 11.61 385 5.5 1.6 7.3 Table 1 ­ Physico­chemical characteristics of soil used for the experiment Noroozlo et al. ‐ Effects of glycine and glutamine amino acids on sweet basil 497 Plus Illinois, USA) performing 30 readings (on middle part of leaves) per pot. The plant leaf area of three randomly selected plants per pot was measured by leaf area meter (Delta­T Devices Ltd, England). Plants were cut at soil surface and shoot fresh weight was measured by a precise digital scale. Fresh shoots of each pot were transferred to an oven at 65°C for 48 h, and thereafter plant dry weight was measured accordingly. Leaf chlorophyll and carotenoids were determined using acetone extraction of 0.5 g fresh leaves following the methods of Khan et al. (2012) and Kałużewicz et al. (2018). For determination of leaf L­ascorbic acid (vitamin C), 5 g of fresh leaf tissue were crushed in a porcelain mortar in 10 mL metaphosphoric acid 6%, and then the juice was transferred into a 25 mL tube and centrifuged at 4000 rpm for 10 min. 5 mL of the supernatant was transferred into an Erlenmeyer flask, and added with 20 mL of metaphosphoric acid 3%. The extract was then titrated using di­chloro phenol indophenol reagent until appearance of a rosa color. The amount of vitamin C (mg 100 g­1 FW) was calculated accord­ ing to a standard curve of L­ascorbic acid concentra­ tions of 0, 25, 50, 2100 and 200 mg·L­1 following Souri and Aslani (2018). Nitrogen concentration of leaves was determined by the Kjeldahl method, K using flame photometry, Mg, Ca, Fe and Zn by atomic absorption spectrophotometer. Data were analyzed by analysis of variance using SPSS 16 and comparison of means was performed using LSD test at P≤0.05 probability level. 3. Results and Discussion In the present study a soil with moderate levels of nutrient elements was used and no further applica­ tion of chemical fertilizers was applied. The results showed that plant height was significantly increased by foliar application of 250 mg·L­1 glutamine com­ pared to control plants (Table 2). Application of both amino acids of glycine and glutamine significantly increased the leaf concentration of chlorophyll a (at 250 or 500 mg·L­1) and chlorophyll b (at 500 mg·L­1) compared to control plants (Table 2). Total chloro­ phyll concentration in leaves was significantly increased than control by glutamine or glycine at 500 mg·L­1 or glutamine at 250 mg·L­1 (Table 2). Leaf carotenoids concentration increased and decreased upon foliar application of 1000 mg·L­1 glycine and 1000 mg·L­1 glutamine compared to control plants, respectively (Table 2). The plant leaf SPAD value (Fig. 1) was not significantly changed under different con­ centrations of glutamine or glycine compared to con­ trol plants. However, foliar application of 1000 mg·L­1 glycine reduced leaf SPAD value of plants compared to some amino acid treatments (Fig. 1). This could be due to damage of high glycine concentration to basil leaves, as the same finding was also reported by Fig. 1 ­ Leaf SPAD value of sweet basil plants under foliar appli­ cation of glycine or glutamine amino acids. Comparison of means was performed at P≤0.05 probability level of LSD test. Table 2 ­ Plant height and leaf pigments concentration of sweet basil plants under foliar application of glycine and glutamine amino acids ­ 250, 500 and 1000 indicates their concentration as mg·L­1. ­ Comparison of means was performed at P≤0.05 probability level of LSD test. Treatments Plant height (cm) Chl a (mg.g­1 FW) Chl (mg.g­1 FW) Total Chl (mg.g­1 FW) Carotenoids (mg.g­1 FW) Control (distilled water) 46±5 bc 10.5±1.4 d 5.6±0.8 b 16.1±1.3 bc 1.7±0.3 b Gly1000 44±4 c 11.9±3.2 cd 6.0±1 b 18.0±4.1 bc 2.3±0.1 a Glu1000 43±10 c 10.3±1 d 5.2±0.9 b 15.5±1.4 c 1.2±0.1 c Gly500 55±15 abc 18.3±1.3 ab 9.0±2 a 27.3±3.4 a 1.5±0.2 bc Glu500 56±10 ab 19±2.4 a 9.2±1.3 a 28.2±3.6 a 1.4±0.1 bc Gly250 53±11 abc 17.4±0.7 ab 7.8±0.9 ab 25.2±1.4 a 1.6±0.5 b Glu250 62±13 a 15.2±2.7 bc 5.0±0.9 b 20.2±3.6 b 1.4±0.3 bc Gly250+Glu250 50±30 b 13.1±0.5 c 5.2±3.7 b 18.3±3.6 bc 1.5±0.3 bc Adv. Hort. Sci., 2019 33(4): 495­501 498 Fahimi et al. (2016) on cucumber plants. The increase in plant height and leaf greenness can be due to ben­ eficial effects of glycine and particularly glutamine on leaf pigmentation (Sadak et al., 2015; Fahimi et al., 2016). Amino acids can have stimulation effect on plants growth, and in this respect, it has been shown that application of amino acids can enhance chloro­ phyll content and leaf greenness of plants (Kielland, 1994; Souri et al., 2017; Kałużewicz et al., 2018), probably due to higher protein biosynthesis and reduction in chlorophyll degradation rates (Rainbird et al., 1984; Souri and Hatamian, 2019). Better nutri­ ents use efficiency and their enhanced translocation play also an important role in this regard (Marschner, 2012). Application of aminolevulinic acid (ALA), as a common precursor to tetrapyrrole compounds found in chlorophyll and hemes, improved spinach plant growth and increased the concentration of photosyn­ thetic pigments (Smoleń et al., 2010). In the present study, plant leaf area (Fig. 2) was increased by foliar application of both glycine and glutamine at 500 mg·L­1 compared to control plants. Both glycine and glutamine at either concentration of 250 or 500 mg·L­1 increased shoot fresh weight (Fig. 3); whereas plant shoots dry weight was increased only under foliar application of 500 mg·L­1 glycine or glutamine compared to control plants. Leaf area expansion is a function of interacting various external and internal factors that results in increased cell divi­ sion or particularly cell enlargement. Various amino acids like glutamine and glycine are required compo­ nents for normal cell growth and expansion (Marschner, 2012; Souri, 2016). The increase in plant biomass caused by foliar application of moderate lev­ els of glutamine or glycine could be due to the latter stimulation effects on plant growth. The beneficial effects of foliar application of amino acids on plant growth, yield and quality of the product has been shown for some crops (Keutgen and Pawelzik, 2008; Amin et al., 2011; Khan et al., 2012; Sadak et al., 2015). In addition, amino acids can enhance nutrient uptake and act as precursors for certain plant hor­ mones (Marschner, 2012; Souri and Hatamian, 2019). Foliar application of different concentrations of a mix 17 different amino acids after 45 and 60 days from sowing showed promising effects on bean plant growth and productivity under salinity conditions (Sadak et al., 2015). A similar effect was also obtained in bean plants under field conditions (Souri and Aslani, 2018). Foliar application of zinc­lysine sig­ nificantly increased the photosynthesis, grain yield, enzyme activities and Zn contents in different wheat plant tissues (Rizwan et al., 2017). Multiple sprays of a mix of amino acids (0.5 mL·L­1) at different growth stages of grapevines particularly at flowering stage, increased growth characteristics and various compo­ nents of fruit yield and quality compared to control (Khan et al., 2012). Leaf L­ascorbic acid (vitamin C) was not signifi­ cantly affected by foliar application of glycine or glut­ amine compared to control plants; however, the highest leaf ascorbic acid content was recorded in plants treated with 1000 mg·L­1 glycine or glutamine (Fig. 4). Foliar application of amino acids significantly increased leaf nutrient concentration (except calci­ um) under some specific concentrations of glycine or glutamine (Table 3). Leaf nitrogen was significantly increased by glutamine spray either at 250 or 500 mg·L­1, whereas leaf potassium concentration was significantly increased by glycine at 250 mg·L­1 com­ pared to control plants. Foliar application of gluta­ mine either at 250 or 500 mg·L­1, and foliar applica­ tion of glycine at 250 mg·L­1 significantly increased Fig. 2 ­ Plant leaf area of sweet basil under foliar application of glycine or glutamine amino acids. Comparison of means was performed at P≤0.05 probability level of LSD test. Fig. 3 ­ Shoot fresh and dry weights of sweet basil plants under foliar application of glycine or glutamine amino acids. Comparison of means was performed at P≤0.05 probabi­ lity level of LSD test. Noroozlo et al. ‐ Effects of glycine and glutamine amino acids on sweet basil 499 adverse climatic conditions. Moreover, amino acids or their fertilizer products can be considered as “semi­intelligent fertilizers” (Souri, 2016). They are natural ligands with a friendlier effect on environ­ mental issues and best candidates to partially replace fertilizer application, and to relief chemical fertilizers pressure in many cropping systems. Amino acids are also a preferential source of nitrogen for plant nutrition. It has been shown that partial replacement of nitrate by amino acid applica­ tion can have beneficial effects on plant growth and production (Marschner, 2012; Sadak et al., 2015; Souri et al., 2017). Amino acids are the intermediate compounds in nitrogen assimilation and play key roles in plant cell metabolism, as they are the main form of nitrogen translocation through phloem to growing parts (Marschner, 2012; Kolota et al., 2013). Nitrate in the soil is very vulnerable to leaching and gaseous emissions (Kolota et al., 2013); whereas the mandatory application of reduced forms of nitrogen under organic farming (Caruso et al., 2012) or the advisable supply of ammonium and amino acids can prevent these effects and reduce nitrate accumula­ tion in plant tissues (Cao et al., 2010; Souri et al., 2017). Total concentration of amino acids and leaf protein concentration significantly increase by foliar application of single or a mix of amino acids (Marschner, 2012; Sadak et al., 2015). Moreover, it has been shown that foliar or soil application of amino acids or their chelated nutrients can improve quality parameters of plants (Koksal et al., 1999; Keutgen and Pawelzik, 2008; Shaheen et al., 2010; Smoleń et al., 2010; El Sayed et al., 2014; Souri et al., 2017). Application of glutamic acid enhanced some quality parameters of Chinese chive plants (Cao et al., 2010). Similarly, foliar spray of proline and trypto­ phan enhanced growth, yield and fruit quality and leaf magnesium concentration compared to control plants. Leaf iron concentration was significantly increased by foliar application of glutamine either at 250 or 500 mg·L­1, or by foliar application of glycine at 500 mg·L­1 compared to control plants. Similarly, leaf zinc concentration was significantly increased by foliar application of glycine or glutamine either at 250 or 500 mg·L­1 compared to control plants (Table 3). Foliar spray of 250­500 mg·L­1 of both amino acids significantly increased leaf nutrient concentrations. The most significant effect of amino acids on plants is reportedly the increased plant nutrient uptake (Koksal et al., 1999; Abdul­Qados, 2009; Garcia et al., 2011). Various amino acids can act as ligand for nutri­ ents particularly micronutrient metal ions, so they can protect metal nutrients from harmful reactions (Souri and Hatamian, 2019). The structure of amino acids allows the molecule to act as acid or base, depending on medium pH, thus helping to improve the fertility management of soils particularly under Table 3 ­ Leaf nutrient concentrations of sweet basil plants under foliar application of glycine and glutamine amino acids ­ 250, 500 and 1000 indicates their concentration as mg·L­1. ­ Comparison of means was performed at P≤0.05 probability level of LSD test. Treatments N (% DW) K (% DW) Mg (% DW) Ca (% DW) Fe (mg.kg­1 DW) Zn (mg.kg­1 DW) Control (distilled water) 2.1±0.2 b 1.5±0.2 b 0.21±0.04 d 1.5±0.2 ab 49±6 c 33±3 c Gly1000 2.13±0.4 b 1.6±0.1 ab 0.19±0.03 d 1.3±0.4 b 45±7 c 33±2 c Glu1000 2.4±0.3 ab 1.5±0.3 b 0.23±0.04 cd 1.5±0.2 ab 52±10 bc 35±2 bc Gly500 2.3±0.4 ab 1.7±0.2 ab 0.27±0.04 bcd 1.6±0.2 ab 61±10 ab 40±4 ab Glu500 2.7±0.5 a 1.8±0.4 ab 0.36±0.06 a 1.9±0.5 a 66±8 a 42±6 a Gly250 2.5±0.2 ab 1.9±0.3 a 0.29±0.05 abc 1.7±0.2 ab 53±6 abc 41±4 ab Glu250 2.6±0.3 a 1.8±0.3 ab 0.33±0.04 ab 1.7±0.3 ab 62±5 ab 42±5 a Gly250+Glu250 2.5±0.3 ab 1.7±0.1 ab 0.30±0.06 abc 1.8±0.1 a 57±9 abc 38±6 abc Fig. 4 ­ Leaf L­ascorbic acid (vitamin C) concentration of sweet basil plants under foliar application of glycine or glutami­ ne amino acids. Comparison of means was performed at P≤0.05 probability level of LSD test. 500 Adv. Hort. Sci., 2019 33(4): 495­501 minimized cracked fruit percentage of pomegranates (El Sayed et al., 2014). It has also been shown that under heavy metal pollution of soil, application of amino acids can reduce the heavy metal content of plant tissues (Bashir et al., 2018). In the present study, foliar application of gluta­ mine resulted in better growth traits than glycine spraying. This effect can be mainly due to the well­ known role of glutamine in nitrogen assimilation and plant metabolism (Marschner, 2012). Enhancement of glutamine synthetase has been shown to reduce the severity of ammonium toxicity in tomato plants (Forde and Clarkson, 1999; Marschner, 2012). In addition to the amino and carboxyl groups, amino acids have a side chain or R group attached to the α­ carbon. Each amino acid has unique characteristics arising from the size, shape, solubility, and ionization properties of its R group. As a result, the side chains of amino acids exert a profound effect on the struc­ ture and biological activity of proteins (Souri, 2016; Souri and Hatamian, 2019). Among various amino acids used as foliar spray on bean plants, it was found that glutamic acids and arginine were the most effective on plant growth biostimulation (Sadak et al., 2015). It has been revealed that amino acids are effective components in nutrient use efficiency as well as in detoxification of toxins and heavy metals in plants that can significantly increase their antioxidant capacity and tolerance to stressful conditions. For many decades, soil fertility and quality was adversely affected by continues application of chemical fertiliz­ ers and adverse climatic conditions. Fertilization strategies have actively contributed in enhancing soil salinity that nowadays is a global challenge in many countries. Biostimulation effect of amino acids on nutrient uptake and plant growth can reduce fertiliz­ er application rates and detrimental effects of fertil­ ization on environment and food quality. However, in the present study low to moderate levels of glycine or glutamine (250­500 mg·L­1) had beneficial effects on sweet basil growth, while both amino acids at 1000 mg·L­1 were less or not effective on plant growth characteristics. 4. Conclusions In the present study, the growth of sweet basil plants was improved by foliar application of 250 or 500 mg·L­1 of glycine or glutamine amino acids, whereas application of 1000 mg·L­1 of these two amino acids resulted in no difference from control plants. There were significant better effect on plant height, leaf SPAD value, leaf area, shoot fresh and dry weights, and nutrient uptake with foliar application of glycine and particularly glutamine at 500 mg·L­1. Leaf vitamin C was highest in plants treated with 1000 mg·L­1 amino acid spray. Foliar application of glutamine was more effective than glycine on many growth traits and nutrients uptake. 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