Application of small molecule compound SPD in prevention of hyphantria cunea pests of poplars

Abstract

The invention belongs to the technical field of insect-resistant research, and particularly relates to application of a small-molecule compound SPD to resistance of hyphantria cunea pests of poplars. Comprising the following steps: (1) taking a plurality of populus tomentosa plant seedlings growing for 3 months in a greenhouse; (2) adding water into the spermidine solution in proportion, and respectively diluting the spermidine solution into working concentrations of 0.5 mM, 1.0 mM, 2.0 mM and 4.0 mM; (3) taking the diluted spermidine solution as a treatment group and an aqueous solution group or a control group, respectively spraying the leaves of the poplar twice a day, continuously spraying for 3 days at the interval of 6 hours, and then culturing in a greenhouse; and (4) 36 hours after the last time of spraying, taking the seventh leaf and the contrast, and carrying out a hyphantria cunea feeding experiment. According to the method, a new green protection strategy is provided for the artificial forest to cope with the fall webworm pests, pesticide dependence is reduced, and agriculture and forestry and sustainable development are promoted.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of pest resistance research, and particularly relates to application of a small molecule compound SPD in poplar resistance to Hyphantria cunea. BACKGROUND

[0002] The description of the background of the application belongs to the related art related to the application, and is only used for describing and facilitating the understanding of the content of the application, and should not be understood as the applicant's explicit recognition or presumption that the application is the prior art on the date of the first filing of the application.

[0003] Hyphantria cunea, a lepidopteran insect originating from North America, has rapidly spread since its introduction to Asia in the mid-20th century and has become one of the most important quarantine pests worldwide. It has been identified as a serious invasive alien species in China and is listed in the National Forestry Quarantine Pest List and the Entry Plant Quarantine Pest List, receiving high attention at the national level. To date, the pest has spread to more than 14 provinces (including autonomous regions and municipalities) in China, affecting more than 611 counties and posing a significant threat to forest ecology, urban greening, and agricultural and forestry economies.

[0004] The lack of effective natural enemies in new habitats allows Hyphantria cunea to rapidly increase in number, and control measures often lag behind the speed of pest outbreaks. The larvae of Hyphantria cunea have a strong feeding tendency, and when they reach the third instar, they can consume all the leaves of various broadleaf trees such as poplar, willow, elm, and mulberry in a few days in high-density areas, leaving only the main veins. Such severe leaf loss not only significantly reduces the area of trees for photosynthesis, disrupting the normal physiological processes and growth cycle of trees, but also leads to deterioration of tree health and reduced resistance, making them more susceptible to secondary diseases or pests, ultimately resulting in the death of large areas of forest and severely affecting urban landscapes, shelterbelt systems, and even the ecological balance of the entire region. Hyphantria cunea has strong reproductive capacity and adult moths can migrate long distances through flight or pupae transported with nursery stock. These characteristics further increase the difficulty of control. Therefore, there is an urgent need to develop an efficient, environmentally friendly, and sustainable comprehensive management strategy to address this increasingly serious ecological challenge.

[0005] Chemical control of the leaf-feeding pest, Malacosoma neustria, is fast-acting and convenient, but has obvious limitations. First, broad-spectrum insecticides kill not only M. neustria, but also natural enemy insects such as ladybugs, grasshoppers, and parasitic bees, disrupting the ecological balance and weakening the ability to control pests naturally. Second, chemical pesticides can pollute the soil, water, and air, especially when applied near urban greenbelts, residential areas, or water sources, posing environmental and health risks. Third, long-term single use of chemical pesticides can lead to pest resistance, reducing control effectiveness and forcing the use of increasing amounts of pesticides, creating a vicious cycle. Therefore, relying solely on direct chemical killing methods cannot achieve sustainable management, and there is an urgent need to develop compounds that interact with plants and activate endogenous insect resistance substances to improve containment efficiency and reduce their spread and harm. SUMMARY

[0006] The application provides a use of spermidine in enhancing plant resistance to M. neustria. By applying exogenous spermidine, the plant jasmonic acid defense signaling pathway is activated, significantly improving the plant's resistance to leaf-feeding pests. This method provides a new green protection strategy for plantations to cope with M. neustria pest damage, reducing pesticide dependence and promoting sustainable development in agriculture and forestry.

[0007] The application of a small molecule compound SPD in poplar resistance to M. neustria pest damage includes the following steps: (1) Take several 3-month-old Populus tomentosa seedlings grown in a greenhouse; (2) Dilute the spermidine solution with water in proportion to obtain working concentrations of 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM; (3) Use the diluted spermidine solution as the treatment group, and the water solution group as the control group, respectively, to spray the poplar leaves, spray twice a day with a 6-hour interval, and continuously spray for 3 days, then place them in the greenhouse for cultivation; (4) After 36 hours of the last spraying, take the 7th leaf and the control together for M. neustria feeding experiments.

[0008] Further, the spermidine solution has a concentration of 0.1 M, and is stored in a 4℃ refrigerator before use to avoid repeated freezing and thawing.

[0009] Further, the dilution operation in step (2) is performed in a clean bench using a calibrated pipette and sterile centrifuge tubes; the diluted working solution is prepared and used immediately, or stored at 4℃ for a short period of time in the dark.

[0010] Furthermore, the poplar seedlings grown in the greenhouse for 3 months are poplar seedlings that have grown normally under controlled greenhouse conditions, are 3 months old, have similar height and number of leaves, are free from pests and diseases, and have uniform growth. The controlled greenhouse conditions are a temperature of 25 ± 2℃, a relative humidity of 60–70%, and a photoperiod of 16 h light / 8 h darkness.

[0011] Furthermore, in step (3), the spraying of poplar leaves focuses on covering the 5th to 8th fully unfolded functional leaves from the top of the plant each time, ensuring that both sides of the leaves are evenly moistened but without obvious droplet accumulation. Spray twice a day, once in the morning and once in the evening, with a 6-hour interval between the two treatments; continue for 3 days, for a total of 6 sprays.

[0012] Furthermore, 36 hours after the final spraying of spermidine solution, the 7th functional leaf of poplar plants treated with distilled water, 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine working solutions were collected. Each treatment leaf was placed individually in a covered transparent plastic feeding container with a bottom diameter of 24 cm and a height of about 10 cm, arranged in order of concentration gradient to avoid cross-interference. Forty synchronously developing early 4th instar fall webworm larvae, with a body length of about 12–15 mm, were placed in each container. Before the experiment, high-resolution photographs were taken of each group of leaves and larvae. After feeding continuously for 16 hours under standard environmental conditions, photographs were taken again. The standard environment was 25 ± 1℃, relative humidity 65% ​​± 5%, and photoperiod 16L:8D. The leaves were then removed, and the leaf area loss rate before and after feeding was calculated to quantify the insect resistance effect induced by different concentrations of spermidine.

[0013] Furthermore, poplar leaves sprayed with distilled water and 2.0 mM spermidine solution were continuously fed to fall webworm larvae. Fresh leaves were replaced daily to ensure consistent intake. Feeding continued until the larvae completed feeding and pupating. After the adults emerged, they paired up and laid eggs. The egg masses were collected and cultured under the same environmental conditions. The hatching time, hatching rate, and hatching synchronicity of the eggs were observed and recorded regularly to assess the potential impact of exogenous spermidine on the growth, development, and reproductive capacity of fall webworm mediated by the host plant.

[0014] Furthermore, newly hatched fall webworm larvae were reared in the laboratory under artificial feed until the end of the second instar. Larvae with consistent growth at the end of the second instar were selected and transferred to a feeding system using fresh leaves of Populus tomentosa treated with distilled water or 2.0 mM spermidine solution as their food source. The fresh leaves were replaced daily to ensure consistent intake. After 48 hours of continuous feeding, the changes in larval body length were observed and recorded. The body length and weight of larvae in the control group and the 2.0 mM spermidine-treated group were measured before and after feeding, and their growth was calculated and statistically analyzed.

[0015] The embodiments of the present invention have the following beneficial effects: Treating Populus tomentosa leaves with spermidine (SPD) significantly enhances the plant's resistance to the fall webworm. This method not only provides a green and efficient protection strategy against the fall webworm for the important afforestation species Populus tomentosa, but can also be extended to other ecological forest trees susceptible to fall webworm damage, such as poplar, willow, elm, black locust, and paulownia, or economic tree species such as apple, pear, hawthorn, peach, plum, apricot, cherry, and jujube. Given the fall webworm's wide host range, high reproductive capacity, and severe damage, this technology has significant application value in reducing dependence on chemical pesticides, improving the health of ecological protection forests, and promoting sustainable forestry development. Attached Figure Description

[0016] Figure 1 Here is the chemical structural formula of spermidine (SPD).

[0017] Figure 2 Exogenous application of spermidine (SPD) enhanced the antifeedant effect of Populus tomentosa against the fall webworm. (A) Populus tomentosa seedlings of uniform growth at 3 months of age under controlled greenhouse conditions were treated with distilled water and 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine solutions as working solutions for foliar spraying. Each time, the 5th to 8th fully unfolded functional leaves from the top of the plant were sprayed twice a day, once in the morning and once in the evening, with a 6-hour interval between the two treatments, for 3 consecutive days. 36 hours after the last application of spermidine solution, the 7th leaf was used for feeding experiments; (B) Statistical analysis of leaf area loss rate showed that 24 hours after feeding, the leaf area loss rate of poplar leaves treated with 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine solutions was reduced by 95.9%, 96.9%, 44.2%, and 5.8%, respectively, compared with the wild type.

[0018] Figure 3 Exogenous application of spermidine (SPD) enhanced the resistance of Populus to the fall webworm (Populus tomentosa) and inhibited embryonic development. (A) Adult fall webworms; (B) Fall webworm larvae were continuously fed with Populus tomentosa leaves sprayed with distilled water (as a control) and 2.0 mM spermidine solution. The hatching speed of egg masses after larvae completed feeding, pupation, and mating was observed. (C) Comparison of embryonic hatching speed of fall webworms: After hatching and laying eggs, the embryonic development speed of fall webworms continuously fed with Populus tomentosa leaves sprayed with 2.0 mM spermidine solution was significantly inhibited, and prolonged by 25.4% compared with the control group.

[0019] Figure 4Exogenous application of spermidine (SPD) enhanced the resistance of Populus to the fall webworm and inhibited larval development. (A) Fall webworm larvae were fed with feed until the end of the second instar; (B) Fall webworm larvae were continuously fed with poplar leaves sprayed with distilled water (as a control) and 2.0 mM spermidine solution, and the changes in body length of the fall webworm larvae were observed after 48 h; (C) Statistical analysis of the changes in body length and weight of fall webworm larvae in the control and treatment groups. Detailed Implementation

[0020] The present application will be further described below with reference to the embodiments.

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, in the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Different embodiments can be substituted or combined, and for those skilled in the art, other implementation methods can be obtained based on these embodiments without creative effort.

[0022] Endogenous metabolic small molecule compounds in leaves play a crucial role in plant insect defense. They can inhibit phytophagous pests through direct toxicity, antifeedant effects, or interference with insect digestion and development. The synthesis of these metabolites is often induced by pests and regulated by hormone pathways such as jasmonic acid, and is an important component of plant innate immunity.

[0023] The applicant's research found that in plants, spermidine is generated by the combination of putrescine and a propylamine group under the catalysis of spermidine synthase (SPDS). It is a core intermediate in the polyamine metabolic pathway, participating in the regulation of various physiological processes such as cell proliferation, differentiation, senescence, and stress response. Spermine possesses significant anti-aging and cytoprotective functions. Studies have shown that exogenous application or endogenous accumulation of spermidine can activate autophagy, clearing damaged organelles and misfolded proteins, thereby delaying cell senescence, extending the lifespan of model organisms, and exhibiting protective effects in models of neurodegenerative diseases, cardiovascular diseases, and metabolic disorders. In plants, spermidine not only regulates growth and development, such as flowering time, fruit ripening, and seed germination, but also plays an important role in responding to abiotic stresses such as drought, salinity, and high temperature. Notably, spermidine also plays a crucial defensive role against biotic stresses such as pests and diseases, regulating plant resistance to pests and diseases and exerting important defensive functions.

[0024] Current research indicates that spermidine, as a naturally occurring endogenous polyamine, is readily biodegradable in the environment and exhibits extremely low toxicity to water, soil, and non-target organisms. Its concentrations used in agriculture and food are typically low, and no significant ecological risks have been reported. Currently, high-purity spermidine has achieved low-cost industrial production, driving its rapid application in areas such as agricultural pest and disease control stimulants.

[0025] Therefore, using spermidine to enhance plant insect resistance has significant advantages: as a natural polyamine, it can activate plant defense pathways, induce the synthesis of insect-resistant metabolites, and enhance resistance to pests; at the same time, it is safe, non-toxic, and easily degradable, aligning with the principles of green agriculture. This strategy helps reduce the use of chemical pesticides, improves the health and yield of agricultural and forestry crops, and is of great significance for the development of sustainable ecological agriculture.

[0026] The application of a small molecule compound, SPD, in the resistance of poplar trees to the fall webworm pest includes the following steps: (1) Take several Populus tomentosa seedlings that have been grown in the greenhouse of Beijing Forestry University for 3 months; (2) Purchase 0.1 M (100 mM) spermidine solution (product number 05292-1ML-F, CAS number 124-20-9, purchase website https: / / www.sigmaaldrich.cn / CN / zh / product / sial / 05292) directly from Sigma-Aldrich, and then dilute it with water according to the ratio to make working concentrations of 0.5 mM, 1.0 mM, 2.0 mM and 4.0 mM respectively; (3) Spray poplar leaves with diluted spermidine solution (treatment group) and aqueous solution (control group), mainly spraying the 5th to 8th leaves, spraying twice a day with a 6-hour interval between spraying, for 3 consecutive days, and then place them in a greenhouse for cultivation; (4) 36 hours after the last spray, the 7th leaf was taken together with the control and fed to the American white moth.

[0027] First, spermidine solution was purchased from Sigma-Aldrich (Shanghai). After dilution with water, working solutions of 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM were prepared. Poplar trees that were 3 months old and growing in a greenhouse were sprayed on the leaves, mainly the 5th to 8th leaves. The spraying was done twice a day with a 6-hour interval between sprays for 3 consecutive days. 36 hours after the last spray, the 7th leaf was taken and fed to the fall webworm along with the control group. After 16 hours of feeding, the leaf area loss rate was calculated to analyze the resistance to fall webworm.

[0028] The spermidine (SPD) used in this experiment has the chemical name N-(3-aminopropyl)-1,4-butanediamine and the molecular formula C7H10. 19 N3, CAS Registry No. 124-20-9, is a high-purity standard, purchased directly from Sigma-Aldrich (Shanghai, China), product code 05292-1ML-F, 1 mL, provided as an aqueous solution (concentration 0.1 M). Before use, the stock solution should be stored at 4°C to avoid repeated freeze-thaw cycles. During the experiment, the stock solution was serially diluted with sterile distilled water (ddH2O) according to the required final concentration to prepare working solutions of spermidine at 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM. All dilution operations were performed in a laminar flow hood using calibrated pipettes and sterile centrifuge tubes to ensure accurate concentrations and prevent contamination. The prepared working solutions were used immediately or stored at 4°C in the dark for a short period (not exceeding 24 hours) to maintain their chemical stability and biological activity for subsequent spraying treatment on Populus tomentosa leaves.

[0029] As a preferred embodiment, Populus tomentosa seedlings that were growing normally under controlled greenhouse conditions (temperature 25 ± 2℃, relative humidity 60–70%, photoperiod 16 h light / 8 h dark), were 3 months old, had similar plant height and number of leaves, were free from pests and diseases, and exhibited uniform growth were selected as experimental materials. The plants were randomly divided into five groups, and foliar spraying was performed using distilled water (ddH2O, as a blank control) and 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine aqueous solutions as working solutions. Each spray focused on covering the 5th to 8th fully unfolded functional leaves from the top of the plant, ensuring uniform wetting of both sides of the leaves without significant droplet accumulation. Spraying was performed twice daily, once in the morning and once in the evening, with a 6-hour interval between treatments (e.g., 9:00 AM and 3:00 PM) to maintain an effective concentration on the leaf surface and simulate a continuous induction state. The treatment regimen involved six sprayings over three consecutive days to fully activate the plant's internal defense response. All operations were conducted under identical environmental conditions to avoid interference from external factors such as light, temperature, and humidity, providing a stable and repeatable experimental basis for subsequent determinations of insect resistance or physiological indicators.

[0030] As a preferred embodiment, 36 hours after the last spraying of spermidine solution, the 7th functional leaf (counted from top to bottom) of poplar plants treated with distilled water (control), 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine working solutions were collected to ensure that the leaves were at a consistent developmental stage and free from mechanical damage and pests. Leaves of each treatment were placed individually in a covered transparent plastic feeding container with a bottom diameter of 24 cm and a height of approximately 10 cm, arranged in order of concentration gradient to avoid cross-interference. Forty synchronously developing early-stage 4th instar fall webworm larvae (approximately 12–15 mm in length) were placed in each container. High-resolution photographs of the leaves and larvae were taken before the experiment began; photographs were taken again after 16 hours of continuous feeding under standard environmental conditions (temperature 25 ± 1℃, relative humidity 65% ​​± 5%, photoperiod 16L:8D). The leaves were then removed, and image analysis software (such as ImageJ or LeafArea) was used to calculate the leaf area loss rate before and after feeding, thereby quantifying the insect resistance effect induced by different concentrations of spermidine. The entire experiment was set up with 6 biological replicates (i.e., leaves from 6 independent plants, 6 containers per treatment), and the data were used to statistically analyze the effects of different spermidine treatments on the feeding behavior of the fall webworm and the indirect insect resistance of poplar.

[0031] As a preferred embodiment, poplar leaves sprayed with distilled water (as a control) and 2.0 mM spermidine solution were continuously fed to fall webworm larvae. Fresh leaves were replaced daily to ensure consistent intake. Feeding continued until the larvae completed feeding and pupating. After the adults emerged, they paired up and laid eggs. The egg masses were collected and cultured under the same environmental conditions (e.g., temperature 25±1℃, relative humidity 50%, photoperiod 16L:8D). The hatching time, hatching rate, and hatching synchronicity of the eggs were observed and recorded regularly to assess the potential impact of exogenous spermidine on the growth, development, and reproductive capacity of fall webworm mediated by the host plant.

[0032] In a preferred embodiment, newly hatched fall webworm larvae were reared under laboratory conditions with artificial feed until the end of the second instar to ensure consistent initial developmental status. Second instar larvae with consistent growth were selected and transferred to a feeding system using fresh leaves of Populus tomentosa treated with distilled water (as a control) or 2.0 mM spermidine solution as a food source. The leaves were replaced daily to ensure consistent intake. After 48 hours of continuous feeding, changes in larval body length were observed and recorded. The body length and weight of larvae in the control group and the 2.0 mM spermidine-treated group were measured before and after feeding, and their growth was calculated and statistically analyzed. The results showed that exogenous spermidine treatment significantly inhibited the elongation and weight gain of fall webworm larvae, indicating that spermidine effectively weakened the pest's growth and development by inducing a host plant defense response. Exogenous application of spermidine (SPD) can enhance Populus tomentosa resistance to fall webworm and inhibit larval development.

[0033] Figure 2 Exogenous application of spermidine (SPD) enhanced the antifeedant effect of Populus tomentosa against the fall webworm. (A) Populus tomentosa seedlings of uniform growth at 3 months of age under controlled greenhouse conditions were treated with distilled water and 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine solutions as working solutions for foliar spraying. Each time, the 5th to 8th fully unfolded functional leaves from the top of the plant were sprayed twice a day, once in the morning and once in the evening, with a 6-hour interval between the two treatments, for 3 consecutive days. 36 hours after the last application of spermidine solution, the 7th leaf was used for feeding experiments; (B) Statistical analysis of leaf area loss rate showed that 24 hours after feeding, the leaf area loss rate of poplar leaves treated with 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine solutions was reduced by 95.9%, 96.9%, 44.2%, and 5.8%, respectively, compared with the wild type.

[0034] Figure 3 Exogenous application of spermidine (SPD) enhanced the resistance of Populus to the fall webworm (Populus tomentosa) and inhibited embryonic development. (A) Adult fall webworms; (B) Fall webworm larvae were continuously fed with Populus tomentosa leaves sprayed with distilled water (as a control) and 2.0 mM spermidine solution. The hatching speed of egg masses after larvae completed feeding, pupation, and mating was observed. (C) Comparison of embryonic hatching speed of fall webworms: After hatching and laying eggs, the embryonic development speed of fall webworms continuously fed with Populus tomentosa leaves sprayed with 2.0 mM spermidine solution was significantly inhibited, and prolonged by 25.4% compared with the control group.

[0035] Figure 4 Exogenous application of spermidine (SPD) enhanced the resistance of Populus to the fall webworm and inhibited larval development. (A) Fall webworm larvae were fed with feed until the end of the second instar; (B) Fall webworm larvae were continuously fed with poplar leaves sprayed with distilled water (as a control) and 2.0 mM spermidine solution, and the changes in body length of the fall webworm larvae were observed after 48 h; (C) Statistical analysis of the changes in body length and weight of fall webworm larvae in the control and treatment groups.

[0036] The above results indicate that treating Populus tomentosa leaves with spermidine (SPD) significantly enhances the plant's resistance to the fall webworm. This method not only provides a green and efficient protection strategy against the fall webworm for the important afforestation species Populus tomentosa, but can also be extended to other ecologically vulnerable trees such as poplar, willow, elm, black locust, and paulownia, or economically important trees such as apple, pear, hawthorn, peach, plum, apricot, cherry, and jujube. Given the fall webworm's wide host range, high reproductive rate, and severe damage, this technology has significant application value in reducing dependence on chemical pesticides, improving the health of ecological protection forests, and promoting sustainable forestry development.

[0037] It should be noted that the above embodiments can be freely combined as needed. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a small molecule compound SPD in the resistance of poplar trees to fall webworm pests, characterized in that, Includes the following steps: (1) Take a number of poplar seedlings that have been grown in a greenhouse for 3 months; (2) The spermidine solution was diluted with water according to the proportion to make working concentrations of 0.5 mM, 1.0 mM, 2.0 mM and 4.0 mM respectively; (3) The diluted spermidine solution was used as the treatment group, the aqueous solution group and the control group. Poplar leaves were sprayed twice a day, with a 6-hour interval between the two sprays, for 3 consecutive days. Then they were placed in a greenhouse for cultivation. (4) 36 hours after the last spray, the 7th leaf was taken together with the control and fed to the American white moth.

2. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that, The concentration of spermidine solution is 0.1 M. Before use, the spermidine solution should be stored in a refrigerator at 4°C to avoid repeated freeze-thaw cycles.

3. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that... The dilution operations in step (2) are all performed in a clean bench, using calibrated pipettes and sterile centrifuge tubes; the diluted working solution is prepared and used immediately, or stored at 4°C for a short period in the dark.

4. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that, Populus tomentosa seedlings grown in a greenhouse for 3 months are Populus tomentosa seedlings that have grown normally under controlled greenhouse conditions, are 3 months old, have similar height and number of leaves, are free from pests and diseases, and have uniform growth. The controlled greenhouse conditions are a temperature of 25 ± 2℃, a relative humidity of 60–70%, and a photoperiod of 16 h light / 8 h darkness.

5. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that, In step (3), the spraying of poplar leaves should focus on covering the 5th to 8th fully unfolded functional leaves from the top of the plant each time, ensuring that both sides of the leaves are evenly moistened but without obvious droplet accumulation. Spray twice a day, once in the morning and once in the evening, with a 6-hour interval between the two treatments; continue for 3 days, for a total of 6 sprays.

6. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that, Thirty-six hours after the final spraying of spermidine solution, the seventh functional leaf of poplar plants treated with distilled water, 0.5 mM, 1.0 mM, 2.0 mM, and 4.0 mM spermidine working solutions were collected. Each treatment leaf was placed individually in a covered transparent plastic feeding container with a bottom diameter of 24 cm and a height of about 10 cm, arranged in order of concentration gradient to avoid cross-interference. Forty synchronously developing early fourth instar fall webworm larvae, with a body length of about 12–15 mm, were placed in each container. High-resolution photographs of the leaves and larvae were taken before the experiment. After feeding continuously for 16 hours under standard environmental conditions (temperature 25 ± 1℃, relative humidity 65% ​​± 5%, photoperiod 16L:8D), the leaves were then removed, and the leaf area loss rate before and after feeding was calculated to quantify the insect resistance effect induced by different concentrations of spermidine.

7. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that, American white moth larvae were continuously fed with poplar leaves sprayed with distilled water and a 2.0 mM spermidine solution. Fresh leaves were replaced daily to ensure consistent intake. Feeding continued until the larvae finished feeding and pupating. After the adults emerged, they paired up and laid eggs. The egg masses were collected and cultured under the same environmental conditions. The hatching time, hatching rate, and hatching synchronicity of the eggs were observed and recorded regularly to assess the potential impact of exogenous spermidine on the growth, development, and reproductive capacity of the American white moth mediated by the host plant.

8. The application of the small molecule compound SPD according to claim 1 in the resistance of poplar to fall webworm pests, characterized in that, Newly hatched fall webworm larvae were raised in the laboratory under artificial feed until the end of the second instar. Larvae of the end of the second instar with consistent growth were selected and transferred to a feeding system using fresh leaves of Populus tomentosa treated with distilled water or 2.0 mM spermidine solution as the food source. The fresh leaves were replaced daily to ensure consistent intake. After 48 hours of continuous feeding, the changes in larval body length were observed and recorded. The body length and weight of larvae in the control group and the 2.0 mM spermidine treatment group were measured before and after feeding, and their growth was calculated and statistically analyzed.

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