Effect of salicylic acid isolated from three native willow species in combination with Bacillus thuringiensis B. on the oak leaf roller, Tortrix viridana L.

Introduction and Objective: The oak leaf roller, Tortrix viridana L., is one of the most important pests in oak forests in Iran. This pest tops the list of domestic and quarantine forest pests and its larvae cause heavy damage to oak trees by feeding on the reproductive buds and leaves of trees. Considering the destructive and harmful effects of chemical pesticides, especially biological control using natural enemies and plant compounds as alternative methods, have been used as integrated pest management solutions. Among the biological factors, the insect pathogen, Bacillus thuringiensis B. has a special place due to its many advantages such as specific effect on the larvae of lepidoptera order, insignificant effect on the environment and non-target organisms, as well as the ease of integrating with other control methods in the management of agricultural and forest pest control. Studies have shown that this bacterium exhibits synergistic properties when combined with specific plant tannins. The organic acids like tannic acid, when mixed with B. thuringiensis, can serve as synergists in controlling various pest larvae. Willow tree species, due to their salicin content in the bark, possess medicinal and industrial properties. Salicin, as a plant tannin, can be effective when combined with B. thuringiensis in controlling oak leaf rollers. Additionally, salicylic acid, due to its phenolic composition, is one of the essential growth factors in plants. This substance, as a secondary metabolite, is produced in plants. The role of salicylic acid as a critical compound in plant response to abiotic stresses has been well established. Salicylic acid is water-soluble and acts as an antioxidant, playing a vital role in plant responses to non-living stresses such as drought, cold, heavy metals, heat, and osmotic stress. It seems to enhance metabolic responses, affecting plant photosynthetic parameters and water relations. Therefore, this study was conducted to extract and measure salicylic acid in babylonian willow (Salix babylonica), white willow (S. alba), and goat willow (S. caprea) and assess its impact in combination with B. thuringiensis on the oak leaf roller, T. viridana.
Materials and Methods: The bark of babylonian willow, white willow, and goat willow was collected, dried, and powdered in spring and summer. Sample extraction was carried out using a soxhlet apparatus, and the extract purification was done using a rotary device. Salicylic acid extraction was performed according to the TAPPI standard using ethanol as a solvent. Subsequently, the lethal concentrations (LC₅₀) of B. thuringiensis obtained from bioassays were combined with salicylic acid concentrations of 1, 3, 5, 7, and 9% from the willow species and applied to third and fourth instar oak leaf roller larvae. In this experiment, insects treated were considered dead if no movement was observed upon close approach with a hot needle to their legs and thoracic appendages after 48 hours. Each treatment in this research was replicated three times.
Findings: The results showed that the total extractable material percentages in the bark of babylonian, white and goat willow were 23.55, 36.25, and 20.75%, respectively. The salicylic acid content in the bark extract of babylonian, white, and goat willow was found to be 15.20, 20.10, and 14.70%, respectively. The mortality rate (LC₅₀) of B. thuringiensis on third and fourth instar oak leaf roller larvae 48 hours after treatment was 659.77 and 742.45 ppm, respectively. The mortality rates of third and fourth instar oak leaf roller larvae in combination with 1, 3, 5, 7, and 9% salicylic acid extracted from babylonian, white and goat willow along with B. thuringiensis 's LC₅₀ showed significant differences. Higher percentages of salicylic acid led to an increase in mortality of third instar oak leaf roller larvae in all three willow species. The results of combining salicylic acid extract with B. thuringiensis at all concentrations showed that the combination of salicylic acid extracted from white willow with B. thuringiensis had the highest mortality rate compared to the other two treatments on third instar larvae. In fourth instar larvae, the combination of salicylic acid extracted from white willow with the LC₅₀ concentration of bacteria was higher in three concentrations, 5, 7, and 9%, compared to the other two treatments.
General Conclusion: The results of this study demonstrated that B. thuringiensis have insecticidal effects on oak leaf roller larvae and when combined with salicylic acid extracted from three willow species (babylonian willow, white willow, and goat willow), they exhibit better control effects on the mortality of this pest, with the best effectiveness observed with salicylic acid extracted from white willow. Based on the results of this research, it is possible to utilize the bark of various willow species, especially white willow, for extracting salicylic acid and employing it in combination with B. thuringiensis in managing this important forest pest.