Foliar spraying of aqueous garlic bulb extract stimulates growth and antioxidant enzyme activity in eggplant (Solanum melongena L.)

Muhammad Ali, CHENG Zhi-hui, Sikandar Hayat, Husain Ahmad, Muhammad lmran Ghani, LlU Tao

Department of Vegetable Sciences, College of Horticulture, Northwest A&F University, Yangling 712100, P.R.China

Abstract Recently, botanical extracts are gaining popularity as biostimulants in vegetable production. In present study, the effect of aqueous garlic bulb extract (AGE) was studied on the growth and physiology of eggplant grown in plastic tunnel. AGE was foliage sprayed with various frequencies, i.e., 0, S1 (once), S2 (twice) and S3 (three times) at two independent growth stages, pre- and post-transplant. The results showed that the treated plants exhibited stimulatory responses in growth and physiology in accord with the repetition of AGE spray and growth stages of the plants, respectively. A single foliage sprayed pre-transplant resulted in improved growth, i.e., plant morphology and biomass, and enhanced antioxidants enzymes (superoxide dismutase, SOD; peroxidase, POD), photosynthesis and chlorophyll abundance observed at vegetative, f irst f lowering and fruit setting stages, respectively. However, thrice application inhibited the plant growth and development and resulted in lipid peroxidation, i.e., increased malondialdehyde (MDA) content. In addition, the post-transplant application also showed growth stimulation and interestingly, an overall positive inf luence was observed with respect to the AGE application and no signif icant increase in the MDA content indicated the post-transplant seedlings responded well. Our f indings demonstrate that AGE can act as a biostimulant to enhance the eggplant growth in plastic tunnel production.

Keywords: aqueous garlic bulb extract, biostimulants, plant growth, malondialdehyde, Solanum melongena L.

1. lntroduction

The use of biostimulants, which are def ined as substances or materials other than nutrients and pesticides that can be used to regulate the physiological processes in plants to stimulate their growth, has increased over the last decade, with the global market projected to cross 2 billion USD by the year 2018 (Calvo et al. 2014). Biostimulants promote plant growth and development throughout the crop's life cycle, from the seed stage to mature plants, by improving metabolic eff iciency, resulting in increased yield and enhanced crop quality, and facilitating nutrient assimilation, translocation, and use, thereby increasing plant tolerance to and recovery from abiotic stresses.

Garlic has potent biological inf luence on plant growth in cropping systems (Dong et al. 2008; Cheng et al. 2011; Han et al. 2013), and can alleviate the problems associated with continuous cropping in eggplant (Wang et al. 2015), cucumber (Xiao et al. 2012, 2013), and pepper (Ahmad et al. 2013). However, the use of garlic-derived botanicals with antifungal potential is yet to be established, especially in horticultural practices, such as plastic tunnel farming systems, where production is sometimes limited due to microbial and fungal infections. To formulate effective biocontrol agents, bioactive compounds must be identif ied and purif ied, and their activity comprehensively be evaluated (Inderjit and Mukerji 2006).

Foliar application of aqueous garlic bulb extract (AGE) accelerates plant growth through the stimulation of photosynthetic pigments and soluble sugar content (Hammad 2008; Hanafy et al. 2012). External stressors can induce the production of reactive oxygen species (ROS) that act as early signals in the plant's defense response and as secondary messengers in subsequent reactions (Asada 2006; Wrzaczek et al. 2013; Yin et al. 2015). ROS are highly reactive and toxic, and cause cellular damage, including membrane disruption, lipid peroxidation, protein denaturation, and DNA mutation (Yamauchi et al. 2008; Celekli et al. 2013; Suleman et al. 2013). Plants have evolved highly eff icient enzymatic antioxidant defense systems for scavenging ROS and protecting cells from oxidative damage (Gill and Tuteja 2010; Talukdar 2013).

Growing awareness about the harmful effects of synthetic fertilizers and pesticides has stimulated interest in organic farming practices in the past decade (Gilden et al. 2010; Mostafalou and Abdollahi 2013; Sugeng et al. 2013). Garlic has been used in organic farming to enhance protection against a variety of diseases (Abd-El-Khair and Haggag 2007; Wei et al. 2011) and to stimulate plant growth (Mohamed and Akladious 2014; Al-Obady 2015). In addition, spraying of garlic extract increases fruit yield and quality (El-Hamied and El-Amary 2015). However, few studies have investigated the possibility of using garlic bulb extract to activate the antioxidant defense system in plants.

Superoxide dismutase (SOD) is the major antioxidant enzyme involved in the regulation of oxygen metabolism in plants. It acts as the f irst line of defense against ROS damage, controls lipid peroxidation, and reduces damage to the membrane system by scavenging· and converting it to hydrogen peroxide (H2O2) and O2(Huseynova et al. 2014; Shaf i et al. 2015). The highly toxic H2O2is then scavenged by catalase (CAT) and peroxidase (POD), and degraded by the POD through the oxidation of co-substrates, such as phenolic compounds and/or antioxidants, thereby mitigating its deleterious effects on cells (Asada 2006; Racchi 2013). POD activity is highly correlated with a wide range of plant physiological processes (Hiraga et al. 2001; Passardi et al. 2005; Kravi? et al. 2013), including cell wall construction and lignif ication, resistance to insects and pathogens, and wound healing (Moore et al. 2003; Almagro et al. 2009).

Malondialdehyde (MDA) is a decomposition product of the polyunsaturated fatty acid hydroperoxides generated from reactions with ROS (Tommasino et al. 2012). As a reactive aldehyde, MDA can serve as a biomarker of oxidative stress in an organism (Davey et al. 2005; Del et al. 2005). MDA bonds with molecules such as nucleic acids, proteins, and amino acids to form insoluble compounds that perturb cellular processes and inf luence a plant's normal growth and development (Huang et al. 2007).

Little is known of the eff icacy and mode of action of AGE as a biostimulator in plants. To address this issue, the present work is aimed to elucidate the effect of AGE applied at different frequencies and timing of foliar sprays on the eggplants grown under plastic tunnel conditions. Our study includes the growth and physiological characteristics related to stress, such as antioxidant enzyme activities and MDA content, to establish the bioactivity of AGE on the receiver plants.

2. Materials and methods

2.1. Experimental site

The study was performed between February and September, 2016, in a plastic tunnel at the Horticultural Experimental Station (34°17′N, 108°04′E) of Northwest A&F University, Yangling, Shaanxi Province, China. Under the plastic tunnels, the mean temperature during sample collection was 27.3°C, and the maximum and minimum temperatures were approximately 50°C (in summer) and -10°C (in winter), respectively. The plants were protected from excessive heat by shading and ventilation.

2.2. AGE preparation

Fresh garlic cultivar G025 of uniform size were selected from the Garlic Germplasm Unit, College of Horticulture, Northwest A&F University, and stored at -20°C until use. The aqueous extracts were prepared using the method described in our previous article (Hayat et al. 2016). Brief ly, the extracts were prepared with a concentration of 200 μg mL-1and administered on the eggplant at different growth stages. For each application, freshly prepared extracts were used to avoid possible degradation of the bioactivity.

2.3. Frequency of foliar spraying with AGE and experimental design

Eggplant (Solanum melongena L.) cultivar Taikong Qiewang was used to perform the experiment. Seeds were sown on February 10, 2016 in plastic trays and maintained in a growth chamber with 12 000 lux lighting at a temperature of (27±2)°C, 70% RH, and photoperiod of 16 h/8 h light/dark. For pre-transplant treatments, the foliage of 1-month-old seedlings in the trays was sprayed once with AGE. All plant surfaces were sprayed thoroughly with 30 mL of AGE until it began to drip off the plants. Spraying was continued at weekly intervals (March 11, 18 and 25) to achieve spraying frequencies of one, two, and three times. Control plants were sprayed with distilled water to maintain the same moisture content. Three days after the f inal application, the second leaf from the top was collected to measure antioxidant enzyme activity and MDA and chlorophyll contents, and the seedlings were transplanted into beds inside the plastic tunnel. Measurements were also made at f irst f lowering and fruiting stages.

For post-transplant treatments, seedlings exhibiting similar vigor were transplanted into beds (March 31). After they had overcome the transplantation shock, the plants were subjected to the same treatment as stated above. The experiment was a completely randomized design (CRD) with two-factor factorial arrangements with three replications. Each plot was 1.2 m×3.5 m, seedlings were spaced 50 cm apart, rows were spaced 80 cm apart, and each bed had two rows with seven seedlings per row. For eggplant, vine tying, pruning, and other farm management practices were done on a regular basis throughout the experiment. During appropriate growth stages, the eggplants were doublepole-trained, and the vine branches were suspended from nylon ropes.

2.4. Determination of SOD and POD activities and MDA content

Antioxidant enzyme activities and MDA content were evaluated following previously described protocols (Wang et al. 2015). Weighed leaf samples (0.5 g) were placed in a clean and pre-chilled mortar and ground with 2 mL of extraction buffer (0.05 mol L-1phosphate buffer, p H 7.8); the mixture was transferred to a centrifuge tube along with 6 mL of the same extraction buffer and centrifuged for 20 min at 10 000×g at 4°C. The extract (supernatant) obtained in this manner was used for all subsequent enzyme assays. The enzymatic activities and MDA content of the supernatant were determined for triplicate samples from each treatment.

Total SOD activity was determined based on the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT) (Gao 2006). The reaction mixture consisted of 1.5 mL of 0.05 mol L-1phosphate buffer (p H 7.8), 0.3 mL of 130 mmol L-1methionine, 0.3 mL of 0.75 mmol L-1NBT, 0.3 mL of 0.1 mmol L-1EDTA-Na2, 0.3 mL of 0.02 mmol L-1ribof lavin, 0.05 mL of enzymatic extract, and 0.25 mL of distilled water in a total volume of 3 mL. The reaction mixtures were exposed to f luorescent light (86.86 μmol m-2s-1) for 10-20 min (with the change in the color of the solution indicating the end of the reaction) before the absorbance was recorded at a wavelength of 560 nm (UV-3802, UNICO, MDN, USA). SOD activity was determined as 50% inhibition of NBT reduction caused by the superoxides generated from the reaction of the photo-reduced ribof lavin and oxygen. Total SOD activity was expressed as U g-1fresh leaf weight.

POD activity was estimated using the guaiacol method (Bestwick et al. 1998). The reaction mixture contained 90 mL of 0.2 mol L-1phosphate buffer (pH 7.0), 100 mL of 0.2% guaiacol, and 100 mL of 0.3% H2O2(v/v); 2.9 mL of the solution was placed in a cuvette and 0.1 mL of the enzyme extract was added. The change in the absorbance at a wavelength of 470 nm was recorded over 3 min at 30-s intervals, and the results are presented as U g-1fresh leaves min-1.

MDA content was determined using the thiobarbituric acid (TBA) assay (Vos et al. 1991). Then, 1.5 mL of the extract, prepared in the same manner used to prepare the extracts for the enzyme assays, was mixed with 2.5 mL of 0.5% (w/v) TBA, heated in boiling water for 10 min, and then cooled to allow the f locculate to sediment. After centrifugation at 7 888×g for 10 min, the supernatant was collected for spectrophotometric determination of the MDA content, based on the absorbance at 450 and 532 nm subtracted from the absorbance at 600 nm. MDA content was expressed as μmol per gram of fresh leaves.

2.5. Measurement of photosynthesis

Photosynthesis in the uppermost fully developed leaves was analyzed at the f irst f lowering stage, using an LI-6400XT photosynthesis system (LI-COR, Lincoln, NE, USA). The light-saturated net photosynthesis rate (Pn, in μmol m-2s-1), transpiration rate (E, in mmol m-2s-1), and stomatal conductance (Gs, in mol m-2s-1) were determined. Each treatment was subjected to the same bioassay conditions of (325±5) μL L-1CO2concentration and 5 mL min-1gas velocity.

2.6. Determination of chlorophyll content

Chlorophyll was extracted from the plant leaves following previously described protocols (Hemavathi et al. 2010) for triplicate samples from each treatment. Acetone (80%, v/v) was used as the solvent. Brief ly, 0.2 g fresh weight of fragmented leaf tissue was placed in a glass tube containing 10 mL of 80% acetone. The extracted liquid was maintained in the dark for 24 h, and the volume was adjusted to 20 mL with 80% acetone. Chlorophyll content was determined by measuring the spectrophotometric absorbance (spectrophotometer model 2100; Unico, Dayton, NJ, USA) at 470, 645, and 663 nm.

2.7. Soluble sugar content

The method described by Zhao et al. (2014) was followed to quantify the soluble sugar content in the eggplant fruit. Samples (0.5 g) were crushed in 5 mL of 80% ethanol and heated in a water bath at 80°C for 30 min. The samples were cooled at 25°C, and the extracts were centrifuged for 10 min at 3 500 r min-1. Total soluble sugars were determined by calculating the absorbance of the samples against those of glucose and anthrone (dissolved in H2SO4) with a spectrophotometer at 620 nm.

2.8. Measurement of eggplant growth and yields

Most morphological measurements were obtained on May 20, 2016, during the period of the most vigorous growth of the plant. We randomly selected seven plants from each replication, and 21 plants were evaluated for each treatment to measure the morphological parameters. Stem diameter and plant height were measured using electronic Vernier calipers (±0.01 mm) and measuring tape (±0.1 cm), respectively. Leaf area was calculated using ImageJ Software (Martin et al. 2013) based on measurements obtained from the digital images of the leaves at the same focal distance arranged on a white background (e.g., foam board) under bright light. Eggplant yield was calculated as the f irst ten harvests of edible mature fruits and was presented as an average value per plot (kg m-2). In September, the shoot and root fresh weights were recorded using an electronic balance immediately after the plants were uprooted. To record the dry weight, the samples were oven-dried at 70°C for 5-6 days until a constant dry weight was achieved.

2.9. Statistical analysis and preparation of illustrations

Differences between groups were evaluated by analysis of variance, and means were compared with the least signif icant difference test at a 0.05 level of signif icance. Correlation analyses were performed for the morphological and physiological traits as well as fruit yield by calculating Pearson's product-moment correlation (Pearson 1895). The SPSS statistical software program was used for all statistical calculations. The f igures were drawn using Origin Pro ver. 16.

3. Results

3.1. Morphology, soluble sugar content, and yield of eggplant

Plants showed distinct responses to pre- and post-transplant applications at various frequencies, with noticeable changes observed in the morphological parameters of the plants. As shown in Table 1, plant height was the maximum (44.9 cm) with a single pre-transplant treatment followed by three times of post-transplant AGE application (41.2 cm), whereas for the plants sprayed three times pre-transplantation, plant height decreased by 15% as compared to the respective control. A similar trend was observed for leaf area, i.e., in the plants treated with foliar AGE pre-transplantation, the largest leaf area was observed in the plants sprayed once (the leaf area increased by 25% that of the control), but the area was reduced in the plants sprayed three times (the leaf area decreased by 10% of that of the control). The maximum stem diameter (11.9 mm) was recorded with one-time foliar AGE application in the pre-transplanted plants and was signif icantly different from that of the control (10.9 mm), whereas for the plants sprayed three times post-transplantation, the maximum stem diameter of 11.3 mm was recorded compared to a stem diameter of 10 mm of the control plants. Regarding the soluble sugar contents and yield, the pre-transplanted eggplants treated once had the highest soluble sugar contents of 188%, and 23.6% higher yield than that of the control plants. Regarding the post-transplant treatments, three foliar applications increased the soluble sugar content to 112% and yield by 15.4% as compared to that in the control. Other morphological parameters such as shoot/root fresh and dry weights also revealed signif icant effects of AGE application in various frequencies, as shown in Table 2. Pre-transplanted plants treated one time with foliar AGE had the highest shoot and root fresh weights (622.0 and 127.5 g, respectively), respectively, whereas the lowest shoot and root fresh weights were observed for plants subjected to three foliar applications of AGE (312.6 and 62.4 g, respectively). Similar results were obtained for the shoot and root dry weights, where a single AGE spray on the pre-transplanted plants resulted in the maximum values of 245.3 and 31.4 g, respectively.

Table 1 Effect of the frequency of foliar spraying with aqueous garlic bulb extract (AGE) on morphology, soluble sugar content, and yield of eggplant

3.2. Effect of AGE on antioxidant enzyme levels and MDA content in eggplant leaves

Fig. 1-A shows that pre-transplanted plants treated once with foliar AGE application had higher SOD activity in all three growth stages as compared to their respective controls. In the post-transplant treatments, SOD activity increased signif icantly with three foliar AGE applications in the f lowering stage relative to that in the control. POD activity also differed in the three growth stages depending on whether AGE was applied pre- or post-transplantation (Fig. 1-B). POD activity was higher when the treatment was applied before than after transplantation; the highest activity was observed with two pre-transplantation applications compared to their respective control plants, whereas three times application had no signif icant difference compared to the control. When the post-transplanted plants were sprayed three times, POD activity was increased above that in the control plants in the f lowering and fruiting stages.

As shown in Fig. 1-C, compared with the control plants, the pre-transplanted plants treated once with AGE showed signif icantly reduced MDA content at vegetative and fruiting stages; in contrast, three applications increased the MDA content of the pre-transplanted plants compared to the control plants in the f lowering stage. Changes in the MDA content, although positively associated with AGE applications in the pre-transplanted plants, showed nonuniform patterns in the post-transplanted plants. In addition, in the post-transplanted plants, there were no signif icant changes in the MDA contents throughout plant development.

3.3. Effect of AGE on plant photosynthesis

Foliar AGE application on the pre- and post-transplanted plants signif icantly affected photosynthetic activity. Pnand Gswere the highest for the pre-transplanted plants treated once, whereas the lowest values of Pnwere observed in the pre-transplanted plants subjected to three AGE applications as compared to their respective controls (Fig. 2). Furthermore, Pn, E, and Gsincreased with three foliar applications of AGE in the post-transplanted plants as compared to in control plants. The intercellular CO2concentrations were the lowest in the pre-transplanted plants subjected to two AGE applications. Overall, a single foliar AGE application on the pre-transplanted plants grown in trays increased their Pnand Gsas compared to those in the control plants.

3.4. Effect of AGE on plant chlorophyll contents

Fig. 3 shows that the chlorophyll a, carotenoid and total chlorophyll content in the pre-transplanted plants was higher than that in the control group after a single application of AGE and decreasing below the control group content with three AGE applications in the f lowering stage. Moreover the Chl b content of plants treated three times with AGE application was lower than that in the control group; in the post-translation plants, the highest chlorophyll content (a, b, carotenoid, and total) was found in the plants subjected to three AGE applications compared to their respective control across all developmental stages, except for Chl b in the f lowering stage and total chlorophyll in the fruiting stage. In the plants treated once pre-transplantation, the chlorophyll contents was higher than those in the post-transplantation treated plants during the f lowering stage.

Table 2 Effect of the frequency of foliar spraying with aqueous garlic bulb extract (AGE) on the morphological parameters of eggplant

Fig. 1 Effect of pre- and post-transplant aqueous garlic bulb extract (AGE) foliar spray frequencies on the antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD) and malondialdehyde (MDA) content of eggplant leaves collected at different plant growth stages. AGE was applied to the leaves, pre- and post-transplant at weekly intervals, i.e., once (S1), twice (S2), and three times (S3). CK, control treatment. Data are mean±SE (n=6). Signif icant differences at P＜0.05 are denoted by different letters in the different growth stages.

Fig. 2 Effect of pre- and post-transplant aqueous garlic bulb extract (AGE) foliar spray frequencies on the net photosynthetic rate (P n), stomatal conductance (G s), intracellular CO2 concentration (C i), and transpiration rate (E). AGE was applied to leaves, pre- and post-transplantation, at weekly intervals, including once (S1), twice (S2), and three times (S3). CK, control treatment. Data are mean±SE (n=6). Different letters denote signif icant differences at P＜0.05.

Fig. 3 Effect of the frequency of foliar spraying with aqueous garlic bulb extract (AGE) on chlorophyll (a, b), carotenoid (car), and total chlorophyll (Chl (a+b)) contents of eggplant at different growth stages. AGE was applied to leaves, pre- and post-transplantation, at weekly intervals, including once (S1), twice (S2), and three times (S3). CK, control treatment. Data are mean±SE (n=6). Signif icant differences at P＜0.05 are denoted using different letters.

3.5. Correlations between morphological and physiological traits as well as fruit yield

We found that fruit yield was closely correlated with plant height (r=0.9754, P＜0.001), shoot dry weight (SDW) (r=0.9716, P＜0.01), Pn(r=0.9684, P＜0.01), and root fresh weight (RFW) (r=0.9604, P＜0.01), showing a slightly lower correlation with root dry weight (RDW) (r=0.9557, P＜0.01), shoot fresh weight (SFW) (r=0.9487, P＜0.001), soluble sugar content (r=0.9457, P＜0.01) and leaf area (r=0.9240, P＜0.001) (Table 3). In contrast, fruit yield was negatively correlated with MDA (r=-0.6545, P＜0.05). Plant height showed a signif icant positive correlation with Pn(r=0.9908, P＜0.01), SDW (r=0.9583, P＜0.01), RFW (r=0.9563, P＜0.01), and RDW (r=0.9516, P＜0.01), with a slightly lower correlation with SFW (r=0.9456, P＜0.01), soluble sugar content (r=0.9257, P＜0.01), leaf area (r=0.8854, P＜0.01), and total chlorophyll (r=0.8299, P＜0.01), whereas it was negatively correlated with MDA (r=-0.7386, P＜0.05). Pnwas positively correlated with RDW, RFW, SDW and SFW with respective correlation coeff icients of 0.9711 (P＜0.001), 0.9659 (P＜0.001), 0.9640 (P＜0.001), and 0.9580 (P＜0.001). The correlation coeff icient of SFW with RFW and SDW were 0.9832 (P＜0.001) and 0.9806 (P＜0.001) and those of soluble sugar content with SFW and SDW were 0.9768 (P＜0.001) and 0.9666 (P＜0.001), respectively. The results indicated that fruit yield of eggplant was strongly dependent on its morphological and physiological traits.

Table 3 Pearson's correlation coeff icient for determining the association between morphological, physiological parameters and fruit yield 1)

4. Discussion

Foliar spraying of AGE is a well-established practice, but the effects of the frequency and timing of foliar spray application have not yet been systematically investigated. In the present study, we found that the frequencies of foliar spraying with AGE, pre- and post-transplantation, had synergistic and antagonistic effects, respectively, on eggplant growth and the physiological characteristics related to stress, such as antioxidant enzyme activities and MDA content.

Pre-transplanted plants treated once with AGE foliar application increased eggplant height, leaf area, soluble sugar content, and plant yield, etc., whereas, three-time AGE foliar application had the opposite effect on plant height and yield under the tray condition. In contrast, posttransplantation treatment at three-times application under f ield conditions enhanced plant growth. This promotion and inhibition of growth in the subjected plants indicates the biological stimulatory effects of AGE at appropriate frequency and growth stages. Foliar application of AGE accelerates plant growth by stimulating the production of photosynthetic pigments and soluble sugars (Mohamed and Akladious 2014). Hanafy et al. (2012) reported that spraying Scheラera arboricola plants with garlic extract increased leaf area. Spraying pear trees with licorice and garlic extracts increased yield and fruit quality (El-Hamied and El-Amary 2015). Garlic contains at least 33 sulfur compounds, enzymes, vitamins B and C, minerals (such as Na, K, Zn, P, Mn, Mg, Ca, and Fe), carbohydrates, saponins, alkaloids, f lavonoids, and free sugars (such as sucrose, fructose, and glucose) (Otunola et al. 2010; Bhandari et al. 2014; El-Hamied and El-Amary 2015). Therefore, it offers a balanced source of nutrition for eggplant growth. Pre-transplantation treatment at higher frequencies under the tray condition suppressed the growth of young plants, this observation is in agreement with earlier reports where higher concentrations of garlic-derived compounds negatively affected crop growth (Cheng et al. 2011; Han et al. 2013). Garlic-derived organosulfur compounds particularly the allicin, diallyl disulf ides (DADS) and diallyl trisulf ide (DATS) (Jones et al. 2007), which are strong antioxidants (Leelarungrayub et al. 2006) and can actively react to the lipid bilayers (Gruhlke et al. 2015), therefore, might involve in signaling and altering the physiochemical of the receiver plants. AGE eff icacy was improved at higher spraying frequencies in posttransplantation treatment, indicating the increment in the growth condition of eggplant in f ield condition. Appropriate frequency as well as the correct growth stage of these compounds can actively participate in the promoted growth conditions of the receiver plants. Higher spraying frequency improves nutritional balance in eggplants indicating the allelochemical threshold level of AGE in f ield conditions. Ellis et al. (2004) reported that wheat plant size and canopy density have a signif icant effect on spray retention. A young crop may require less spraying than grown crops, as may those with a wider canopy and more foliage (Darwin 2015). Previous studies reported that allelochemicals exert biphasic effects, i.e., stimulatory or inhibitory effects, at low and high concentrations, respectively (Belz 2008; Hong et al. 2008; Cheng et al. 2016). Plant growth may be stimulated below a certain allelopathic threshold, above which there could be a severe growth reduction depending on the sensitivity of the species (Chon and Nelson 2010). It has been documented that at low concentrations, garlic promotes seed germination and seedling growth, whereas higher concentrations have the opposite effect (Xiao et al. 2012).

Chlorophyll is the core component of pigment protein complexes that play a major role in photosynthesis (Candan and Tarhan 2003; Fracheboud 2006). Any variation in chlorophyll content can alter photosynthetic rates. In the present study, we observed that chlorophyll a, carotenoid, and total chlorophyll contents increased signif icantly with a single pre-transplantation application of AGE at f lowering stage, which can be explained by the greater availability of nutrients. This is in agreement with a study reporting that spraying pear transplants with 4% garlic extract increased N and K content in the leaves, which is known to enhance chlorophyll levels (El-Hamied and El-Amary 2015). Carotenoids play a vital role in plant development, particularly during stress conditions (Bonet et al. 2016; Karppinen et al. 2016). However, we also observed a signif icant reduction in chlorophyll a, carotenoid, and total chlorophyll contents when AGE was applied at high frequencies during the pre-transplantation treatment under tray conditions at the f lowering stage (Fig. 3), indicating that inhibitive effects exerted on the receiver plants because of the application of AGE beyond threshold levels. These results strongly supported the results of previous studies that chlorophyll content is affected under stresses and various treatments may sometimes enhance growth and physiological conditions (Siddique and Ismail 2013; Chai et al. 2016; Khan et al. 2016; Yuyan et al. 2016). These results are in accordance with the f inding that shoot extracts of Artemisia judaica caused a dose-dependent reduction in the chlorophyll content of lettuce leaves (Zeng et al. 2009).

Compared with the control, a single foliar application of AGE on the pre-transplanted plants enhanced their Pn, and Gs. These results are in line with the observation that spraying of the foliage of pepper plants with natural extracts (yeast, garlic, and eucalyptus) or ascorbic acid increases the amount of photosynthetic pigments under drought stress (El-Ghinbihi and Hassan 2007). Recent studies showed that allelochemicals also signif icantly inf luenced photosynthesis like other environmental factors. These results strongly conf irm previous reports that garlic root exudates increased the chlorophyll content to enhance the absorption of light energy of tomato and hot pepper and which resulted in improved photosynthetic rate (Zhou et al. 2007). In posttransplantation, higher spray frequencies also showed stimulation of photosynthetic parameters in the eggplant and are therefore in strong agreement to previously stated results. A stimulated photosynthesis system ref lects an enhanced food factory (Wang et al. 2016; Slattery et al. 2017), which may have inf luenced the plant growth. Photosynthetic rate decreased with higher foliar spraying frequency under tray conditions at pre-transplantation. These f indings conf irmed the previous report that at higher concentrations (2.00 g L-1garlic; 15.0 mg L-1diallyl trisulf ide), the maximum effective quantum yield (Fv/Fm) and effective quantum yield (YII) were almost zero (Wang et al. 2016), implying that irreversible damage occurs only if the dose is above a critical value, which is consistent with our observations. Furthermore, increment in the activity of chlorophyllase under salt stress damages chlorophyll content and decreases stomatal conductance, photosynthetic activity, and modif ies source/sink relationship (Hashem et al. 2015; Sarwat et al. 2016). We also calculated Pearson correlation coeff icients, which clarif ied that there was a signif icant correlation between morphological physiological traits and fruit yield in eggplant (Table 3). These results suggest that foliar spray of AGE stimulates growth and enhances eggplant yield. The data showed the highest correlation of fruit production with plant height and photosynthetic rate which suggests the signif icance of these two parameters as promising indicators of plant productivity.

Our results also revealed signif icant variations in the antioxidant enzyme activities and MDA content of eggplant seedlings. AGE spraying stimulated the activities of the antioxidant enzymes. Moderate application activated antioxidants and possibly ROS, which led to enhanced plant growth; this was inhibited by a higher frequency of application. These f indings agree with those of Hayat et al. (2016), who found that AGE is biologically active inside cucumber seedlings and alters the defense mechanism of the plant, probably by activating reactive oxygen species at mild concentrations.

In the plants grown under tray conditions, a lower foliar spray rate, pre-transplantation, increased SOD activity relative to that in the control plants. In the current study, the changes observed in the levels of these antioxidants associated with the variations in the frequency of AGE spraying can be explained by ROS activation in the plants. ROS is a normal product of plant cellular metabolism; at high concentrations, they can damage biomolecules, whereas at low or moderate concentrations, they act as the second messengers in intracellular signaling cascades (Trchounian et al. 2016). In our experiment, SOD and POD expression was activated by stress, which probably caused an increase in the ROS levels in the eggplant seedlings; achieving a balance in the ROS levels could prevent lipid peroxidation, maintain cellular homeostasis (Das and Roychoudhury 2014), and consequently increase eggplant growth. Under physiological conditions, cells maintain a redox balance through the generation and elimination of ROS and reactive N species (Gupta et al. 2016). Garlic is an antioxidant that can modulate these processes (Banerjee et al. 2003).

Antioxidant enzyme activities and MDA content in eggplant are also dependent on the stage of plant growth. Increase in SOD and POD activities during the early stages of plant growth suggests an elevation in oxidative stress. Furthermore, MDA content reaches a maximum level during the fruit-bearing period, during which temperatures can exceed 50°C in the plastic tunnels (Wang et al. 2014). However, the physiological limit of eggplant is 35°C, and higher temperatures cause it to grow slowly and develop poorly.

In the present study, a single foliar application of AGE, pre-transplantation, under tray conditions decreased MDA content relative to that in the control plants, indicating that AGE can decrease membrane lipid peroxidation in eggplants, and that appropriate spraying frequency and timing can induce the activity of the protective enzymes (Fig. 1-C). Moreover, although there were f luctuations, the overall trends for SOD and POD activities were similar, with an initial increase followed by a decrease. In contrast, MDA, which is an important stress indicator in plants, showed the opposite trend, ref lecting the synergy of the protective enzyme system in eggplants (Wang et al. 2015). A single application of AGE, pre-transplantation, resulted in maximal SOD and POD activities. However, under tray conditions, eggplant growth was signif icantly inhibited by a higher spraying frequency, leading to stunted growth and a decrease in antioxidant levels, and a corresponding increase in MDA content. High concentrations of garlicderived compounds have been reported to negatively affect crop growth (Han et al. 2013), possibly due to a reduction in the activity of protective enzymes and an increase in MDA content. It is possible that ROS overproduction leads to an oxidative burst that causes membrane lipid peroxidation and an increase in MDA content (Savicka and Skute 2010).

5. Conclusion

The results of this study demonstrate that AGE alters the antioxidant mechanisms and enhances growth and yield in eggplants. A single foliar application, pretransplantation, modulated the antioxidative enzymes in eggplants to enhance their growth, whereas spraying three times caused lipid peroxidation and induced stress in the eggplant seedlings. However, after transplantation, a higher spraying frequency had no negative effects on plant growth. Furthermore, the preparation is not meticulous and has almost no hazardous side effects, and offers ecofriendly and greener production of the horticultural produce. Additional studies are needed to evaluate the most important bioactive constituents of AGE such as the allicin and DADS to identify the potent organosulfur component responsible for priming induce resistance against fungal pathogens.

Acknowledgements

This research was funded by the Shaanxi Provincial Science and Technology Innovation Project of China (2016KTCL02-01).