The Effects of Foliar Spray of Chitosan Nanoparticles on Tomato Resistance Against of Cuscuta Campestris Yunck

2.1. Plants Material and Treatments

Tomato (Solanum lycopersicum L.) seeds and Dodder (Cuscuta campestris Yunck) were sterilized by 10% sodium hypochlorite for 5 minutes and planted together in a pot on a perlite bed in growth chamber with 16 h light (350 mol photons m^˗2s^˗1) at 26°C and 8 h dark at 20°C, both at 50% humidity. After germination of seeds and successful infection with the Dodder, the plants were irrigated 100% by Hoagland for 4 weeks. During this time, the plants were sprayed once a week with 10 ml of 50 kilodalton (kDa) (50 mg ml^˗1) chitosan (Sigma-Aldrich CAS-No 9012-76-4) and distilled water was used as a control.

2.2. Determination of Total Protein Concentration

Protein content was determined by the method of Bradford utilizing bovine serum albumin as the standard [14].

2.3. Enzymes Assay

Plants material was homogenized in 3 ml of 0.05 M sodium phosphate buffer (pH 7.8), including 1 mM EDTA and 2% (w/v) PVP. The homogenate was centrifuged at 10,000 × g for 10 min at 4 ̊ C. Supernatant was used for enzyme activity and protein content assays. All steps in the preparation of the enzyme extract were carried out at 4°C [15].

SOD activity assay was based on the method described by Giannopotitis and Ries [16]. One unit of enzyme activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of nitro blue tetrazolium reduction measured at 560 nm.

Ascorbate peroxidase (APX) activity was determined according to Nakano and Asada [17], the reaction mixture in a total volume of 2 ml consisted of 25 mM (pH 7.0) sodium phosphate buffer, 0.1 mM EDTA, 0.25 mM ascorbate, 1.0 mM H₂O₂ and 100µl enzymes extract [18]. H₂O₂-dependent oxidation of ascorbate was followed by a decrease in the absorbance at 290 nm.

Catalase (CAT) activity was done according to Cakmak and Marschner. The reaction mixture in a total volume of 2 ml contained 25 mM sodium phosphate buffer (pH= 7.0), 10 mM H₂O₂. The reaction was initiated by the addition of 100 µl of enzyme extract and activity was determined by measuring the initial rate of disappearance of H₂O₂ at 240 nm.

Peroxidase (POD) activity was measured using modification of the procedure of McAdam et al. [19]. Guaiacol was used as the substrate. POD activity was measured in a reaction mixture (3 ml) that contained 0.1 ml enzyme extract, 12 mM H₂O₂, and 7.2 mM guaiacol in 50 mM phosphate buffer, pH 5.8. The kinetics of the reaction were followed at 470 nm.

PAL activity was assayed by an adaptation of the methods of Zucker [20] and McCallum and Walker [21]. The assay mixture consisted of 0.06 M Na borate buffer, pH 8.8, and crude enzyme. The reaction was started by the addition of 11 mM L-phenylalanine. Tubes were incubated at 30°C for 60 min and the reaction was stopped by the addition of 35% (w/v) trifluoroacetic acid (TFA). Tubes were then centrifuged for 5 min at 5000rpm to pellet the denatured protein. Phenylalanine ammonia lyase (PAL) was enzymatic, and a sample extract was boiled and assayed.

PPO activity was assayed as described by Nicoli et al. with some modifications [22]. Optimum activity was reached using SDS at 2% (data not shown). The assay mixture consisted of 30 mM caffeic acid in 100 mM buffer (Na₂HPO₄/KH₂PO₄), pH= 7.0, through which air was bubbled for 5 min. Catalase (420 units) from bovine liver (Fluka) was added in 0.1 ml H₂O to prevent peroxidation of the substrate. The assay was initiated by the addition of enzyme extract. Polyphenol oxidase (PPO) activity was measured by the change in A₃₇₀ of the assay mixture (30°C) based on the measurement of the disappearance of caffeic acid by enzymatic oxidation. To determine whether the reaction was enzymatic, the sample extract was boiled and assayed.

Total phenol content was estimated by the method suggested by using Folin–Ciocalteu reagent (1 N) and the absorbance was measured at 650 nm against each blank [23]. The content of phenol was calculated from the standard curve obtained from different concentration of catechol and expressed as mg g ^˗1 FW.

2.4. Lignin Quantification

Lignin was quantified by the thioglycolic acid (TGA) method described by Lange et al. [24] and adapted by Torre et al. [25]. Tubes containing about 100 mg of plant material were treated with 1 ml of methanol, shaken for 1 h, and centrifuged at 10,000 g for 10 min. The supernatant was removed and the pellet was treated successively with 1.5 ml of the following solvents: methanol, 1 M NaCl, 1% (w/v) Sodium dodecyl sulfate (SDS), deionized water and chloroform: methanol (1:1). Finally, the pellet was resuspended in 1.5 ml of tert-butyl methyl ether and lyophilized. 10 milligrams of the resulting powder was resuspended in 1 ml of 2 M HCl and 0.2 ml of thioglycolic acid and incubated at 95 ̊ ^C for 4 h. The solution was centrifuged at 10,000 g for 10 min and the resulting pellet was washed three times with deionized water, resuspended in 1.5 ml of 0.5 M NaOH and shaken for 12 h. The samples were then centrifuged at 10,000 g for 20 min, and the supernatant was mixed with 0.3 ml of 12 M HCl and incubated for 4 h at 4 ̊ C. After centrifugation for 40 min, the resulting pellets were dried using a speed vacuum concentrator and resuspended in 1 ml of 0.5 M NaOH. The A₂₈₀ was measured and the concentration was calculated with a previously determined lignin standard made with pure lignin from Sigma-Aldrich.

2.5. SDS-PAGE Technique

The total cell proteins were subjected to Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in gel slabs 1 mm thick (3.5 cm, 4% stacking and 15.5 cm, 12% resolving gels) as mentioned by Laemmli [26]. Electrophoresis was carried out with a discontinuous buffer system in a UVP vertical electrophoresis chamber (Cambridge, UK). The gel was run at 30 mA until the bromophenol blue marker had reached the bottom of the gel. The molecular weight of proteins was calculated on the basis of comparison with the following standards (Thermo Scientific Page Ruler Protein Ladder SDS-PAGE Standards, Fermentas, molecular weight range 10-200 kilodalton). After electrophoresis, the gels were stained for 6 h in 0.01% (w/v) Coomassie Brilliant Blue R-250. Finally, the gels were destained in a methanol acetic acid-water (3:1:6) mixture until protein bands became clearly visible.

3.1. Biomass

In this study, the biomass of the Dodder-infected tomato plant was significantly reduced compared to the not-infected plant. Chitosan foliar spray increased biomass in both infected and non-infected plants, indicating an improvement in plant conditions (Fig. 1). Chitosan has a positive effect on plant growth and productivity and increases plant yield by improving plant condition, especially in stressful conditions [27]. Not-infected with Dodder (Control), data represent the mean ± SD with n =4 for each treatment, * letters show significantly different values after one-way ANOVA followed by Duncan’s multiple range test (p ˂ 0.05).

3.2. Proteins Concentrations

In this study, the application of chitosan caused protein concentrations in not-infected and Dodder-infected plants increase which indicates an increase in the expression of defense enzymes and pathogenesis-related (PR) genes/proteins (Fig. 2). Studies have shown that the use of chitosan increases the concentration of proteins in plants that are related to the increased expression of defense system enzymes and genes or proteins related to pathogenicity.

Generally, the activity of antioxidant enzymes increased in Dodder-infected plants compared to control, and in plants that were both infected and treated with chitosan, the enzyme activity was much higher (Fig. 3). The activity of antioxidant enzymes increases under stressful conditions and reduces the oxidative damage by decreasing active oxygen species and increasing plant resistance. Chitosan, as an elicitor, stimulates the plant's defense system and thus increases the plant's resistance to the pathogen [28, 29].

3.3. Phenolic Metabolism

Phenylalanine ammonia lyase (PAL) activity was significantly increased in infected plants compared to control. This increase in enzyme activity was also observed in plants treated with chitosan. However, in plants that were both infected and treated with chitosan, the enzyme activity was much higher. In the case of PPO, the activity increased in infected plants and the application of chitosan made the enzyme more active in both infected and non-infected plants. The same pattern was observed for phenolic and lignin compounds (Fig. 4). Biostimulants such as chitosan cause the production of phenolic compounds including lignin. Phenolic compounds can act as antioxidants by removing ROS from plant cells [30-32].

3.4. SDS PAGE Analysis

The findings of SDS-PAGE showed that the levels of 2 protein bands of molecular masses 200 kDa and 60 kDa gradually increased in Dodder-infected plants treated with chitosan. We suggested that these proteins may be related to pathogenesis. In brief, the findings of SDS-PAGE revealed that several proteins are involved in plant defense response. However, in the treatment of chitosan alone and also in infected plants that have not been treated with chitosan, the intensity of these bands is less (Fig. 5).