The experiment was conducted under field condition during 2021 in Tartus suburb (Syria), on Santa Teresa citrus lemon young trees (4 years old), young trees of Santa Teresa were brought from the nursery, and planted in field in Tartus suburb, and let to grow normally until they reached 4 years old, during this time, nutrients like nitrogen, phosphor and potassium were added to the soil. The young trees were irrigated when needed.

Different chitosan concentrations were applied four times in February by 15 days interval. Treatments were: T0 Control (treated with distilled water), T1 Chitosan 200 ppm, T2 Chitosan 300 ppm, T3 Chitosan 400 ppm, and Tween-20 was added (2 ml/l).

Complete random design was used, and one young tree was considered as replicate, repeated 3 times.

Fresh leaves affected by CLM larval, were washed with distilled water and kept frozen until analysis at Tishreen university labs., 2 g was homogenized in 7 ml cold potassium phosphate buffer (pH = 7, M = 0.1), and centrifuged at 10,000 rpm for 10 min at 4˚C, and the supernatant was used to determine POD and PPO activity. All steps of the extraction procedure were carried out at 1˚C - 4˚C. PPO and POD activity was determined using a spectrophotometric method. Blank sample contained the same mixture solution without enzyme extract.

Peroxidase activity was determined using guaiacol as hydrogen donor, according to [39]: 200 μL of the supernatant was added to 3.5 ml of potassium phosphate buffer, 200 μL of (0.25% v/v) guiacol and 200 μL 0.1 M H₂O₂, the reaction was followed calorimetrically at 470 nm in spectrophotometer (JASCO-Japan) for 3 minutes. POD activity was expressed as increasing in absorbance at 470 nm min⁻¹ mg⁻¹ protein.

1.95 mL of Phosphate buffer solution (0.1 M, pH 7), 1 mL of 0.1 M catechol as a substrate and 50 μL of the enzyme extract were pipetted into a test tube and mixed thoroughly. The mixture was rapidly transferred to water path (25˚C). The absorbance at 410 nm was recorded continuously for 5 min. PPO activity was expressed as increasing in absorbance at 410 nm (ΔA/min/gFW), according to [40] [41].

Fresh infected leaves were washed with distilled water, and 2 g was homogenized in 15 ml of 80% (v/v) ethanol and centrifuged for 15 min at 10,000 rpm. The extract was infiltrated, and the residue was extracted again with 80% (v/v) ethanol, centrifuged again for 15 min at 10,000 rpm. 40 μl of extract was added to 200 μl Folin-Ciocalteu reagent + 3.16 ml distilled water and 600 μl of 20% Na₂CO₃. Then, the mixture was shaken and kept in dark for 1.5 h. The absorbance at 720 nm (at 25˚C) was recorded with spectrophotometer, using glass cuvettes at 720 nm (25˚C) and Catechol standard curves (from 0 - 400 mg /l). Blank tube contained 40 μl of methanol. Total phenols were expressed as milligrams of Catechol acid equivalents per gram of fresh extract, according to [42].

Using (Scale Master Pro-China) devise, leaves with full tunnels (the larva reached up the pupae) or leaves with dead larva tunnels were used.

Using the formula:

Number of tunnels/number of injured leaves.

The number of tunnel was counted on each injured leaf, on both sides, on all branches.

Analysis of variance (One way ANOVA) was applied using the Gen stat 12 statistical program, and differences between means were evaluated with Duncan’s test (LSD 0.05).

POD activity was affected by chitosan treatments, T2 (CH 300 ppm) enhanced significantly enzymatic activity (0.533 mm⁻¹ mg⁻¹ protein), compared to control (0.237 mm⁻¹ mg⁻¹ protein), followed by T1 (CH 200 ppm) (0.483 mm⁻¹ mg⁻¹ protein), T3 (CH 400 ppm) (0.341 mm⁻¹ mg⁻¹ protein), Figure 1.

Plant under biotic stress start to generate ROS, this is an effective mechanism against the pathogenic infection, which induce oxidative stress, that could generate specific genes, responsible on antioxidant response [43], by activating antioxidant enzymes (such as Superoxide dismutase (SOD), Peroxidase (POX) and Catalase (CAT)) responsible on scavenging ROS.

According to [28], chitosan increased the accumulation of H₂O₂, improved the activities of phenylalanine ammonialyase and chitinase, improved as well

transcription of defense related genes (b-1,3-glucanase and chitinase) and accumulation of pathogen related protein (PR1). The increase of POD activity in our results, may be due to the increase of H₂O₂ concentration, as a result of CLM attack in lemon leaves, this is considered as a major plant defense mechanism [44] [45] [46].

These results are in consistent with [47], which demonstrated that treatment of wheat seedling with Oligochitosan promoted POD, CAT and SOD.

PPO activity was increased by Chitosan treatments, T1 (CH 200 ppm) enhanced PPO activity (1.394 ΔA/min/gFW) compared to control (1.325 ΔA/min/g FW), while other treatment didn’t record any enhancement in PPO activity, Figure 2.

PPOs are known to oxidize phenols to quinones, which is implicated in pest resistance.

It was found that feeding ability of cotton bollworm (Helicoverpa armigera) on foliage transgenic tomato, which contain an overexpressing PPO was less than those feeding on non-transformed tomato, which indicate that tomato PPO plays an important role in cotton bollworm resistance [48].

PPO catalyze two distinct reactions: the o-hydroxylation of monophenols to o-diphenols and the oxidation of o-dihydroxy phenols to o-diquinones [49], these quinones have a high activity and are quickly polymerized leading to form brown and black pigments that affect the quality and nutritive value of fresh plants [50] [51].

Quinones as well at pH < 4 submit to reactions that results semiquinone leading to ROS generation, which may perform as part of signal transduction pathways for establishment of plant immunity [52]. Reference [53] found that H₂O₂ is able to encourage several defense genes, including PPO and proteinase inhibitors I and II, that could lead to improved levels of disease and insect resistance.

Figure 2 showed that chitosan (T1) improved PPO activity compared to the control and other treatments, which is in accord with [54], who indicated an increase in PPO and POD enzymes activity in tomato fruit. Reference [55] showed as well, that treatment with chitosan increased PPO and POD activity in palm roots.

Total phenolic content in lemon leaves was stimulated by chitosan treatment, T2 (CH300 ppm) compared to the control (5.975 mg/g; 5.373 mg/g) respectively, while TPC in other treatment T1, T3 were not affected, Figure 3.

Chitosan is known to regulate gens, which are responsible on phenylalanine ammonia-lyase (PAL) biosynthesis [56], this enzyme controls phenolic biosynthesis [57]. In our results, chitosan at 300 ppm stimulated TPC in lemon leaves, referring to Figure 2, chitosan decreased PPO activity at the same concentration, this indicates that phenolics didn’t oxidize by PPO and accumulated in the leaves. T1 (200 ppm) stimulated PPO (Figure 2), phenols content didn’t increase, it is possible that PPO oxidize phenols to quinone or other products. Chitosan at (400 ppm T3) decreased PPO activity (Figure 2), and phenolic didn’t affect, it is possible that there are other factors that influenced TPC.

This result is in agreement with [58], who indicated chitosan enhanced cytosolic H⁺, oxidative bursts and phytoalexins (such as terpenoids, isoflavones, alkaloids and phenolics).

Chitosan application at different concentrations decreased significantly tunnel length, the heights reduction in tunnel length was at T3 (CH 400 ppm), followed by T1 and T2 (49.75, 61.5, 66.2 mm) respectively, compared to the control (89.2 mm), Figure 4.

The effect of chitosan may be due to the reduction in larva diet [34]. Chitosan effect on phenolic content may have an influence on the nature of cell compounds which may be not favorable to the larva nutrition, it is possible as well that chitosan has toxic effects, this results in accord with [33], and with [59] who showed that chitosan increased phytoalexins compounds which play a major role in plant defense.

Chitosan treatment was effective in reducing damage density, T1 CH 200 ppm was the most effective in damage density reduction (1.067), compared to the control (4.423). T2 CH 300 ppm and T3 CH 400 ppm were effective as well (2.28 - 2.323) alternatively, Figure 5.

Damage density results of tunnel numbers and not of tunnel length. Chitosan treatments decreased tunnel length which may due to the reduction of larva nutrition; this may reflect on larva activity and as consequence tunnel numbers and damage density. Reference [60] showed that PPO and POD enzymes oxidize phenolic compound to quinones, which may interact with leaves proteins, and affect the amino acids availability. Amino acids may be converted to alkaloids (which lower the proteins nutritional value in plants) which may affect the insects feeding [61] [62]. Other studies indicated that quinones have a toxic effect on herbivores [63]. Chitosan stimulated PPO and POD activities which explain its effect on damage density.