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Introduction
The use of nanotechnology improves agricultural productivity by increasing the yield and nutritional quality of crops, as well as providing more excellent protection to the environment (Gondal and Tayyiba, 2022; Khairy et al., 2022). However, using metal nanoparticles can cause undesirable effects (Rizwan et al., 2017), as many of them are highly toxic and pollute the environment by harming the life of organisms (Vodyashkin, Kezimana, Vetcher and Stanishevskiy, 2022). Due to the above, there is a growing interest in ecologically friendly nanocomposites (Lira-Saldivar, Méndez, De los Santos and Vera, 2018).
In this regard, chitosan (poly β-(1,4)-N-acetyl-D-glucosamine, CS), a biopolymer, could be used for manufacturing nanocomposites with multiple advantages, including minimal toxicity and biodegradability (Chadha et al., 2022; Sangwan, Sharma, Wati and Mehta, 2023). NPsCS are very versatile and exhibit high stability and ease of preparation (Maluin and Hussein, 2020). These have multiple agricultural applications such as pesticides, herbicides, and insecticides and to obtain better quality food products with higher yields (Bandara, Du, Carson, Bradford and Kommalapati, 2020).
NPsCS can increase crop tolerance to biotic or abiotic stress (Wang et al., 2021). They are stimulants and potent inducers of antioxidant enzyme activity (Maluin and Hussein, 2020; Chandrasekaran, Kim, and Chun, 2020), decreasing the accumulation of reactive oxygen species (ROS) in plant cells, improving stress tolerance, growth, and yield of crops (Ishkeh et al., 2021); it also increases natural antioxidants in crops, which can generate benefits to human health (Ketnawa, Reginio, Thuengtung and Ogawa, 2022). On the other hand, tomato (Solanum lycopersicum L.) is the most produced and consumed vegetable worldwide and is considered a functional food because it is rich in fiber and contains a great variety of minerals, and bioactive compounds are beneficial to human health (Attia et al., 2021). These qualities make it an appropriate vegetable for biostimulation. Based on the above, this study aimed to determine the effect of foliar spraying with NPsCS on yield, enzyme activity, and bioactive compound content in tomato fruits.
Materials and Methods
Synthesis of chitosan nanoparticles
The NPsCS were synthesized at the Applied Chemistry Research Center in Saltillo, Coahuila, Mexico, by the ionic gelation method described by Kumaraswamy et al. (2018). The NPsCS have an average size of 111 ± 21 nm and a spherical shape, as previously reported by Ramírez-Rodríguez et al. (2021).
Growing conditions and plant material
The research was carried out in a tunnel-type greenhouse belonging to the Horticulture Department of the Universidad Autónoma Agraria Antonio Narro, in Buenavista, Saltillo, Coahuila, Mexico, located at 25° 21’ N and 101° 01’ W and an altitude of 1790 meters of altitude. Seeds of tomato F1 Hybrid (MARIANA, SAKATA®) were germinated in agricultural foam plates. Twenty days after sowing, the seedlings were transplanted in 10 L black polyethylene bags with Peat moss and perlite 2:1 (v:v). The planting density was six plants per square meter. Steiner’s (19841) nutrient solution was used for crop nutrition, applied through a drip irrigation system with three daily irrigations. In the first week after transplanting, irrigation was done with a 25% nutrient solution, the second week with a 50% solution, the third week with a 75% solution, and the fourth week after transplanting with a 100% Steiner solution. The average temperature was 22.4 °C, while the average photosynthetic active radiation was 677 μmol m-2 s-1, and the average relative humidity was 62%, monitored with a ThermoProTP359.
Treatments and experimental design
The NPsCS were prepared in deionized water with glycerin (reagent grade) and Bionex (Arysta®) as dispersing and coadjuvant agents, then sonicated for six minutes at an amplitude of 50% in an ultrasonic cleaner (Branson, 1510R-DTH), to obtain a concentration of 1 mg mL-1. Subsequently, from this solution, solutions were prepared and applied by foliar sprays at the following concentrations: 0 (distilled water), 0.05, 0.1, 0.1, 0.2, 0.4, and 0.8 mg mL-1, during the first hours of the morning (8:00 h), with four applications every 15 days during the crop cycle, starting 15 days after transplanting (DDT). The experiment was conducted in a completely randomized design with ten replicates per treatment, considering one plant as the experimental unit.
Sampling
At 100 DDT, ten fruits were harvested from each treatment and repetition, of uniform size and light red maturity, determining the physical quality of the fruit and the quantification of bioactive compounds.
Physical quality of the fruit
Tomato fruits were weighed on an analytical balance (Ohaus®, CS5000P). Fruit size was quantified by determining the polar and equatorial diameter using a digital vernier (ASK-500-196-30; Mintutoyo). Firmness was measured with an Extech penetrometer (FHT200). For this, the fruits were placed on a hard and fixed surface, recording the average of two measurements per fruit for each repetition and treatment.
Preparation of extracts for nutraceutical quality
Two g of fresh pulp was mixed in 10 mL of ethanol in a screw-capped plastic tube, which was placed in a rotary shaker (ATR Inc., EU) for six h at five °C and 20 rpm. Subsequently, the tubes were centrifuged at 3000 rpm for 5 min, and the supernatant was extracted for analytical tests (Preciado-Rangel, Troyo, Valdez, García and Luna, 2020).
Total phenolic compounds
Total phenol content was measured by the Folin-Ciocalteau method (Singleton, Orthofer, and Lamuela, 1999). 300 µL of the extract was mixed, and 1080 mL of distilled water and 120 µL of Folin-Ciocalteau reagent (Sigma-Aldrich, St. Louis MO, USA) were added, vortexing for 10 s. After 10 min, 0.9 mL of sodium carbonate (7.5% w/v) was added, vortexing for 10 s. The solution was allowed to stand at room temperature for 10 min. The solution was allowed to stand at room temperature for 30 min, and then its absorbance was read at 765 nm in a UV-vis spectrophotometer (VE-5100uv-VELAB). The phenol content was calculated by a standard curve using gallic acid as a standard (Sigma, St. Louis, Missouri, USA). Results were reported as mg gallic acid equivalent per 100 g fresh weight (mg equiv AG-100 g-1 fresh weight (FW).
Total flavonoids
The colorimetric method determined the total flavonoid content (Zhishen et al., 1999). For this purpose, 250 µL of the ethanolic extract was mixed with 1.25 mL of distilled water in a test tube, and then 75 µL of 5% NaNO2 solution was added. After 5 min, 150 µL of 10% AlCl3 + H2O solution was added and allowed to stand for another 6 min; then, a volume of 500 µL of 1 M NaOH plus an additional 275 µL of distilled water was added. All components were mixed by stirring in a vortex. The absorbance was measured immediately at 510 nm using a UV-vis spectrophotometer (VE-5100uv-VELAB). Results were expressed as mg quercetin equivalents per 100 g-1 fresh weight (mg equiv Q-100 g-1 FW).
Antioxidant capacity
The antioxidant capacity was determined with the in vitro DPPH+ method (Brand-Williams, Cuvelier and Berset, 1995). To determine antioxidant capacity, 50 µL of sample and 1950 µL of DPPH+ solution was mixed, and after 30 min of reaction, the absorbance of the mixture was read at 517 nm in a UV spectrophotometer (Genesys 10). The standard curve was fitted with Trolox (Aldrich, St. Louis, Missouri, USA). The results were reported as antioxidant capacity (µM equiv Trolox-100 g-1 FW).
Vitamin C
Vitamin C concentration was determined by the method of Klein and Perry (1982). For extraction, 10 mg of sample and 1 mL of H3PO4 (0.36 M) were added and centrifuged in an Ohaus Frontier FC5515 R centrifuge (Ohaus Corp., New Jersey, USA) at 5000 rpm for 10 min at four °C. Subsequently, 200 μL of the supernatant and 1 mL of 2.6-dichlorophenolindophenol (2.6 D) (0.09 M) were homogenized for subsequent reading in a Thermo Fisher G10S spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) at a wavelength of 515 nm. The results were expressed as mg g-1 dry weight (DW).
Lycopene
With 100 mg of lyophilized sample and 2 mL hexane, an extract was obtained and mixed in a vortex for 30 sec, then sonicated for 5 min and centrifuged at four °C for 10 min at 10 000 rpm. The supernatant was filtered and quantified at 472 nm. The concentration was obtained using the calibration curve previously plotted with the lycopene standard (Bunghez, Raduly, Doncea, Aksahin and Ion, 2011). A curve was performed with lycopene standard Sigma-Aldrich brand 98% purity. The results were expressed in milligrams per kilogram.
Preparation of extracts for total protein and enzyme activity
The enzymatic extract was obtained using the methodology of Ramos et al. (2010), which refers to the placement of 200 mg of lyophilized plant tissue (LABCONCO 2.5 freezone lyophilizer) in a 2 mL tube, with the addition of 20 mg of polyvinylpyrrolidone and 1.5 mL of phosphate buffer pH 7-7.2 (0.1 M), and then centrifuged at 12000 rpm for 10 min at four °C in a microcentrifuge (Labnet Int. Inc., PrismTM C2500-R). The supernatant was collected and filtered on a PVDF membrane with a pore size of 0.45 microns.
Total Protein
Total protein (TP) concentration was determined according to the Bradford colorimetric technique (Cheng, Wei, Sun, Tian and Zheng, 2016). The results were expressed in mg g-1 DW.
Superoxide dismutase
The superoxide dismutase (SOD) assay (EC 1.15.1.1) was performed using the commercial kit 19160 SOD (Sigma-Aldrich). Samples and blanks were assayed, then incubated at 37 ºC for 20 min. Finally, it was read at an absorbance of 450 nanometers.
Glutatión peroxidase
Glutathione peroxidase (GPX) activity (EC 1.11.1.9) was quantified following the methodology of Flohé and Günzler (1984). 200 μL of the extract, 400 μL of GSH (0.1 mM), and 200 μL of Na2HPO4 (0.067 M) were homogenized and placed in a water bath at 25 °C for 5 min. Then, 200 μL of H2O2 (1.3 mM) was added to react for 10 min. The reaction was stopped by the addition of 1 mL of trichloroacetic acid (1%) and then centrifuged in an Ohaus Frontier FC5515 R centrifuge (Ohaus Corp., New Jersey, USA) at 3000 rpm for 10 min at four °C. To determine GPX activity, 480 μL of the supernatant, 2.2 mL of Na2HPO4 (0.32 M), and 320 μL of DTNB (1 mM) were homogenized and subsequently read in a Thermo Fisher G10S spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) at a wavelength of 412 nm. Results were expressed as U g-1 PT, where U corresponds to mM reduced glutathione equivalents per milliliter per minute.
Statistical analysis
Bartlett’s test was run on the data obtained to test the homogeneity of variance, and the normality of the data was tested with the Kolmogorov-Smirnov and Shapiro-Wilk W tests. Subsequently, an analysis of variance was performed, and where a difference was detected between treatments, Fisher’s test (P ≤ 0.05) was used for the separation of means using the statistical program InfoStat version 2020 (Di Rienzo et al., 2020).
Results and Discussion
Fruit quality
Foliar spraying of NPsCS positively affected tomato fruits’ yield, weight, size, and firmness (Table 1). With the 0.4 and 0.8 mg mL-1 doses of NPsCS, the highest values were obtained in yield and fresh fruit weight concerning the control, with an increase of 25.9 and 42%, respectively. Fruit size was affected by the dose of 0.8 mg mL-1 NPsCS, the polar diameter of the fruit increased by 25.1%, and the equatorial diameter with 0.4 mg mL-1 NPsCS increased by 14.4%, both concerning the control. Tomato fruit firmness increased with 0.8 mg mL-1 NPsCS, increasing 36.9% concerning the control treatment. Chitosan has a high affinity towards plant cell membranes, resulting in enhanced reactivity in the plant system (Maluin and Hussein, 2020), allowing the penetration of NPs that increase cellular metabolic activity (Lin et al., 2009), thus managing to promote crop yield (Siskani, Seghatoleslami and Moosavi, 2015). NPsCS can stimulate plant growth by containing small amounts of nutrients such as C, O, N, and P, in addition to positively stimulating cell division and elongation, enzyme activation, and protein synthesis, leading to increased crop quality and productivity (Chakraborty et al., 2020; Prajapati et al., 2022). Parvin et al. (2019) reported that chitosan application increases fruit weight and size and, consequently, yield in Solanum lycopersicum L. The benefits of chitosan use are very diverse and range from a biostimulant effect by increasing the synthesis of growth regulators (Akhtar et al., 2022; Malerba and Cerana, 2016; Stasińska and Hawrylak, 2022); to improving nitrogen metabolism and as a consequence plant productivity Mondal et al. (2012) however, there is no single dose, as the plant response depends on the concentration, species and developmental stage of the plant (Balusamy et al., 2022; Hoang et al., 2022; Malerba and Cerana, 2016; Parvin et al., 2019), so further research is needed.
Table 1: Average values of yield (R), fruit weight (PF), polar diameter (DP), equatorial diameter (DE), and firmness of tomato fruits (F) by foliar application of NPsCS.
| NPsCS | R | PF | DP | DE | F | |
| mg mL-1 | g planta-1 | g | cm | N | ||
| 0 | 4198.2±71.79 b* | 88.50 ±36.89b | 67.56 ±11.94 bc | 47.40 ±7.32b | 56.60±17.80b | |
| 0.05 | 4221.1 ±150.7 b | 86.84 ±38.56 b | 68.39±13.32 abc | 46.69±7.52 b | 50.89±10.02 b | |
| 0.1 | 3942.6±108.97 c | 88.83 ±43.86b | 62.25±15.95c | 50.98 ±11.33ab | 73.75±22.38 a | |
| 0.2 | 4187.8±119.93 b | 91.69 ±30.27b | 68.86 ±8.32abc | 48.24±5.64 ab | 73.5±11.32 a | |
| 0.4 | 4367.4 ±72.70 a | 125.69 ±25.39a | 73.42 ±7.82ab | 54.26±4.58 a | 73.5±10.29 a | |
| 0.8 | 4437.2±54.53 a | 111.43 ±22.96ab | 77.93±8.19a | 45.87±6.00 ab | 77.5± 11.21a | |
*Mean values with different literals are significantly different (LSD P ≤ 0.05).
Bioactive compounds
Foliar spraying of NPsCS increased bioactive compounds (phenolics and flavonoids) and antioxidant capacity in tomato fruits (Table 2). The dose of 0.8 mg mL-1 of NPsCS increased antioxidant capacity by 81.3%, phenols by 6.6%, and flavonoids by 38.5% concerning the control treatment. Jahani, Behnamian, Dezhsetan, Karimirad and Chamani (2023) reported an increase in antioxidant capacity in Solanum lycopersicum L with the application of NPsCS. Similar responses are obtained with CS in Raphanus sativus L (Supapvanich, Anan and Chimsonthorn, 2019). Chandra et al. (2015) reported increased phenols and flavonoids in Camellia sinensis. Other investigations have reported increased content of these bioactive compounds due to NPsCS application in Oryza sativa L. (Divya, Thampi, Vijayan, Varghese and Jisha, 2020), Rosa damascena Mill. (Ali, Issa, Al-Yasi, Hessini and Hassan, 2022), in Triticum aestivum L. (Hajihashemi and Kazemi, 2022). The vitamin C content was affected by the NPsCS (Table 2). With the dose of 0.4 mg mL-1, the highest accumulation was achieved concerning the control. Similar results are reported by El Amerany et al. (2022), indicating that the application of CS increased the levels of natural antioxidants such as vitamin C, phytic acid, pantothenic acid, lycopene, and flavonoids. Vitamin C is important because it is a powerful antioxidant by trapping reactive oxygen species (ROS) and reversing or minimizing oxidative damage (Meena et al., 2020). It also plays an essential role in photosynthesis as an enzymatic cofactor (including the synthesis of ethylene, gibberellins, flavonoids, and anthocyanins) (Kawashima et al., 2015). Therefore, higher vitamin C accumulation will improve fruit quality (Paciolla et al., 2019). Lycopene synthesis was affected by NPsCS, with the dose of 0.05 mg mL-1 increased by 145% concerning the control (Table 2). Lycopene is a ROS-deactivating antioxidant (Imran et al., 2020). Other studies found that foliar application of CS increases lycopene content in Solanum lycopersicum L. (Parvin et al., 2019), so an increase of lycopene would also be expected due to foliar application of NPsCS. The results obtained by foliar application of NPsCS in the rise of bioactive compounds can be attributed to the fact that CS and NPsCS, in addition to having a biostimulant action, also have an elicitor activity that generates reactive oxygen species (ROS), which stimulate the biosynthesis of bioactive compounds such as enzymatic and non-enzymatic antioxidants (Ishkeh et al., 2021; Singh, 2016; Stasińska and Hawrylak, 2022; Malerba and Cerana, 2016; Wang et al., 2021). The above shows that NPsCS can be used in agriculture as a natural biostimulant because it favors the biosynthesis of bioactive compounds reflecting higher fruit quality (Paciolla et al., 2019).
Table 2: Effect of NPsCS foliar spray on bioactive compounds in tomato fruits.
| NPsCS | Total phenolic | Total flavonoids | Antioxidant capacity | Vitamin C | Lycopene |
| mg mL-1 | mg equiv GA·100 g−1 FW | mg equiv Q·100 g−1 FW | µmeq TROLOX 100 g-1 PF | - - - - - - - mg kg-1 - - - - - - - | |
| 0 | 21.04 ±4.53 ab* | 24.19±17.14 b | 30.45±4.41 b | 4982.5±39.48 ab | 47.72±10.93 c |
| 0.05 | 22.4 ±6.62 a | 31.71± 4.13 ab | 30.78±6.48 b | 4823.33±178.98 b | 117.19±11.22 a |
| 0.1 | 20.22±11.67 b | 34.71±7.39 ab | 50.50±14.74 a | 4872.3±137.9 ab | 79.72±30.07 bc |
| 0.2 | 22.21± 13.81 a | 30.41±9.56 ab | 55.06±13.63 a | 5030.00±60.83 a | 80.11±17.42 bc |
| 0.4 | 21.45 ±7.12 ab | 30.41±9.17 ab | 45.12±6.18 ab | 5030.00±123.56 a | 88.54±10.53 ab |
| 0.8 | 22.39±10.91 a | 40.45±6.12 a | 55.23±8.83 a | 5003.33±75.72 ab | 113.60±36.53 a |
*Mean values with different literals are significantly different (LSD P ≤ 0.05).
Enzymatic activity
Proteins were positively affected by the foliar application of NPsCS (Table 3); with the 0.05 mg mL-1 dose of NPsCS, the highest concentration of proteins was obtained. Previous studies have indicated that NPsCS application increases total amino acids (Balusamy et al., 2022) and proteins Hajihashemi and Kazemi (2022). This increase in protein content may be due to the N content in NPsCS, which plays an important role in protein synthesis (Behboudi et al., 2018), or to the stimulation of the plant defensive system conducive to stimulation of ROS and accumulation of proteins such as chitinase and activation of peroxidase, SOD and CAT enzymes (Bandara et al., 2020; Chun and Chandrasekaran, 2019; Li et al., 2015).
The highest dose of NPsCS (0.8 mg mL-1) produced the most increased SOD activity in tomato fruits (Table 3), with no modification in GPX enzyme activity. The stress caused by nanoparticles can produce ROS (Katiyar, Hemantaranjan and Singh, 2015), which modifies the activity of antioxidant enzymes, some transcription factors, and proteins involved in the stress response (López-Vargas et al., 2018). The elevated SOD activity caused by NPsCS could be responsible for ROS balance, degeneration, and scavenging to protect the plant from oxidative stress (Chun and Chandrasekaran, 2019). Faizan et al. (2021) report an increase in SOD activity in Solanum lycopersicumL. with the application of NPsCS. By increasing SOD activity, the accumulated H2O2 also decreased, maintaining GPX activity at a basal level (Hajihashemi and Kazemi, 2022), which could explain why there are no differences in GPX enzyme activity in tomato fruits treated with NPsCS.
Table 3: Effect of foliar application of NPsCS on the total protein content and enzymatic activity of SOD and GPX in tomato fruits.
| NPsCS | Enzyme activity (U) | ||
| Total protein | SOD | GPX | |
| mg mL-1 | g kg -1 | ||
| 0 | 2.46 ±0.61 b* | 1.48±0.10 ab | 1.51± 0.80 a |
| 0.05 | 4.59±0.52 a | 0.85±0.02 b | 1.78±0.17 a |
| 0.1 | 3.81 ±0.71ab | 1.57 ±0.41ab | 1.52±0.17a |
| 0.2 | 3.44 ±0.41ab | 1.05±0.33 ab | 1.59±0.38 a |
| 0.4 | 4.04 ±0.42 a | 1.89 ±0.56ab | 1.50±0.71 a |
| 0.8 | 3.56±0.38 ab | 2.34 ± 0.04 a | 1.60±0.20 a |
*Mean values with different literals are significantly different (LSD P ≤ 0.05).
Conclusions
Foliar spraying of NPsCS increased yield, bioactive compounds, and enzyme activity in tomato fruits. The medium dose (0.2 mg mL-1) optimized yield, fruit size, and weight, whereas the high dose (0.8 mg mL-1) increased the biosynthesis of bioactive compounds and enzyme activity in tomato fruits. The foliar application of NPsCS can be used as a natural biostimulant to increase yield and obtain functional foods.
