Composition for preventing and treating poplar leaf rust and method for preventing and treating poplar leaf rust

Abstract

The invention belongs to the field of plant protection, and provides a poplar leaf rust control composition and a poplar leaf rust control method. The prevention and treatment composition for poplar leaf rust comprises 30-65 [mu] M of abscisic acid and 130-270 [mu] M of sodium nitroprusside. A prevention and control method for poplar leaf rust comprises the steps that an abscisic acid solution and a sodium nitroprusside solution are prepared, the concentration of the abscisic acid ranges from 30 mu M to 65 mu M, and the concentration of the sodium nitroprusside ranges from 150 mu M to 270 mu M; in the poplar young leaf period, the abscisic acid and sodium nitroprusside solution is sprayed to the back faces of poplar leaves; after spraying, covering the young leaves of the poplar with black plastic bags, and removing the black plastic bags after 24 hours, or selecting the spraying time at nightfall or at night. The composition for preventing and treating poplar leaf rust and the prevention and treatment method for poplar leaf rust can be sprayed on poplar young leaves, and the prevention and treatment problem of poplar leaf rust is solved.

Description

Technical Field

[0001] This invention relates to the field of plant protection, and more particularly to a composition for controlling poplar leaf rust and a method for controlling poplar leaf rust. Background Technology

[0002] Poplar leaf rust not only damages the economic benefits of poplar trees but also leads to ecological degradation. Leaf rust reduces the dry weight of poplar clones by 29-32% and their volume by 31-42%, severely weakening their economic output. Therefore, strengthening the control of *Melampsora larici-populina* is of great significance for the prevention and control of poplar leaf rust, the protection of the ecological environment, and the promotion of sustainable forestry development in my country. *Melampsora larici-populina* is one of the most widespread rust fungi infecting poplar leaves. As an obligate parasitic pathogen, its infection process exhibits typical binucleate mycelial development characteristics. The fungus's urediniospores are spread by air currents and invade leaf tissue through stomata during the spring leaf expansion period. The hyphae expand in the intercellular spaces of the mesophyll cells and form haustoria, directly absorbing nutrients from the host. As the infection progresses, orange-red rust spore masses gradually appear on the underside of the lesions. The leaves develop rust spots, wither, and suffer a significant loss of photosynthetic area. A single lesion can release tens of thousands of rust spores, which can lead to the death of large areas of poplar trees in severe cases.

[0003] For the control of poplar leaf rust, timely spraying at the initial stage of leaf infection is generally chosen. Currently, the main pesticides used in my country for controlling poplar leaf rust are chemical agents such as Bordeaux mixture, triadimefon, carbendazim, and zineb. However, this still does not meet the needs of practical application. There is a need to develop green control agents that are more environmentally friendly and have better control effects on poplar leaf rust, achieving a dual improvement in both control efficiency and ecological safety.

[0004] In existing technology one, physical methods are used to reduce the initial source of infection. For example, in early spring, diseased buds need to be removed manually, and the process must be carried out simultaneously with the use of sealed plastic bags to prevent the spread of spores; in winter, diseased leaves need to be thoroughly removed and burned in a concentrated manner to reduce the overwintering population of pathogens. This technology is limited by labor costs and operational precision in manual removal, while burning may cause forest fire risks and is not feasible for large-scale forest stands.

[0005] In existing technology two, fungicides are sprayed to control poplar leaf rust. Common methods include spraying with 65% zineb at a dilution of 500 times or triadimefon wettable powder at a dilution of 1500 times, or using sodium chlorate at a dilution of 200 times, once every two weeks. Zineb is a protective fungicide that inhibits the enzyme activity of the pathogen, forming a physical barrier. Triadimefon, also known as triadimefon, is a systemic fungicide that inhibits the synthesis of ergosterol in the pathogen, possessing both protective and curative effects. However, zineb is toxic to fish and aquatic organisms and may cause fatal pollution to nearby waters; frequent use of triadimefon increases the resistance of poplar rust fungus, and it is toxic to pollinating insects such as bees, so it should not be used during the poplar flowering period. Sodium chlorate (sodium chlorate, 200 times dilution) interferes with the respiration of the pathogen and is a protective and weakly systemic fungicide; long-term use alone can easily lead to the development of antibodies against poplar rust fungus.

[0006] For example, some publicly available technologies for controlling poplar leaf rust involve first spraying with a 1:125-1:170 Bordeaux mixture (copper sulfate: quicklime = 1:1, water 125-170 parts), followed by a second application of 0.3-0.5 Baume lime-sulfur mixture (calcium polysulfide). Bordeaux mixture releases copper ions to disrupt the pathogen's cell membrane and enzyme activity, forming a protective film on the leaf surface to prevent rust spore invasion. Lime-sulfur mixture releases polysulfides and hydrogen sulfide, directly killing the uredinia on the underside of the leaves and sealing stomata to inhibit spore spread. However, the copper ions in Bordeaux mixture can pollute the soil around the poplar trees, and lime-sulfur mixture can acidify the soil. Furthermore, the high alkalinity of these substances during spraying can easily irritate the skin, cause respiratory damage if inhaled, and may release hydrogen sulfide gas, irritating the eyes and respiratory tract. The combined use of Bordeaux mixture and Baume lime-sulfur mixture can easily lead to blackening and withering of leaf tips during the young leaf stage of poplar trees, making it unsuitable for use on poplar seedlings.

[0007] For example, patent application number 201510085939 discloses the use of 50% thiram wettable powder at a dilution of 500 times, or 25% triadimefon wettable powder at a dilution of 500 times, or 80% carbendazim wettable powder at a dilution of 800 times. Carbendazim belongs to the benzimidazole class of fungicides, which controls poplar leaf rust by inhibiting β-tubulin synthesis; however, long-term use can easily induce resistance in rust fungi. Thiram, a thiocarbamate protectant, works by interfering with the metabolism of pathogens; repeated use can easily induce resistance in rust fungi.

[0008] Science Popularization China also published information on how to prevent and control poplar leaf rust: ① Avoid planting deciduous trees and poplars in close proximity. ② Chemical control: Bordeaux mixture, chlorothalonil, 50% carbendazim (500x dilution), 80% zineb (500x dilution), 50% thiophanate-methyl (800x dilution), 70% methyl thiophanate (1000x dilution), 50% thiophanate-methyl (500x dilution), lime sulfur (0.3 Baume), sodium thiophanate-methyl (200-500x dilution), and triadimefon (1500x dilution) all have relatively good effects in controlling this disease. ③ Select and breed rust-resistant tree species. However, thiophanate-methyl / methyl thiophanate-methyl are benzimidazole fungicides that are converted into carbendazim in plants, exerting their effect by inhibiting the synthesis of β-tubulin in the pathogen. Carbendazim-resistant strains of poplar leaf rust have been reported, and long-term use can easily lead to the screening of resistant populations, resulting in a significant decrease in control efficacy.

[0009] Paper: Field Efficacy Trials of Triadimefon and Diniconazole on Uromyces transversalis (Theum) Wint. This study investigated the control effects of triadimefon EC, 12.5% ​​tebuconazole WP, and 50% thiophanate-methyl WP on poplar rust using *Populus rubra* (Hebei poplar) as the test material. Results showed that 20% triadimefon EC at 800–1200 times dilution and 12.5% ​​tebuconazole WP at 1500–3000 times dilution both exhibited good control effects on poplar rust, with efficacy exceeding 92.3%, significantly superior to 50% thiophanate-methyl WP at 600 times dilution. However, tebuconazole, which works by inhibiting ergosterol synthesis in the pathogen, simultaneously interferes with gibberellin synthesis in the plant, leading to slowed shoot growth and thickened leaves, making it unsuitable for use on poplar seedlings; furthermore, it can induce resistance.

[0010] In summary, the existing technology 2, which involves the application of chemical agents, carries the risk of drug resistance, and frequent spraying can easily cause environmental pollution. Improper application of chemical fungicides can lead to safety hazards and economic losses, disrupting the natural ecological balance of agriculture. Avoiding close-range interplanting of deciduous trees and poplars is also an effective control measure, but this measure cannot be implemented for established poplar forests. Breeding rust-resistant tree species is indeed an effective measure to eradicate poplar leaf rust, but it requires significant investment and a long timeframe; replacing large areas of established poplar forests with new rust-resistant species is also impractical.

[0011] Therefore, there is an urgent need for a composition for controlling poplar leaf rust and a method for controlling poplar leaf rust. Summary of the Invention

[0012] The purpose of this invention is to provide a composition for preventing and controlling poplar leaf rust and a method for preventing and controlling poplar leaf rust, which can be sprayed on young poplar leaves to solve the problem of poplar leaf rust control.

[0013] To solve the above-mentioned technical problems, the composition and method for controlling poplar leaf rust provided by the present invention are implemented as follows:

[0014] A composition for controlling poplar leaf rust comprises: 30-65 μM abscisic acid and 130-270 μM sodium nitroprusside.

[0015] Abscisic acid (ABA) is a naturally occurring hormone in plants, while sodium nitroprusside is a common exogenous nitric oxide donor. Poplar trees do not develop resistance to either substance and are environmentally friendly. ABA and sodium nitroprusside can be applied together to young poplar leaves without harming them. They inhibit stomatal opening and closing in poplar leaves, blocking the invasion pathway of poplar rust fungi, thereby preventing poplar leaf rust. Furthermore, the application of ABA and sodium nitroprusside increases lignin in poplar leaves, providing a denser physical defense system against rust fungi invasion; the increased chitinase activity in the leaves indicates that the poplar defense system is activated, further preventing rust fungi from invading poplar cells.

[0016] Optionally, the composition for controlling poplar leaf rust comprises: 50 μM abscisic acid and 200 μM sodium nitroprusside.

[0017] Optionally, the composition for controlling poplar leaf rust comprises: 40 μM abscisic acid and 150 μM sodium nitroprusside.

[0018] Optionally, the composition for controlling poplar leaf rust comprises: 60 μM abscisic acid and 250 μM sodium nitroprusside.

[0019] A method for controlling poplar leaf rust includes: preparing a solution of abscisic acid and sodium nitroprusside, wherein the concentration of abscisic acid is between 30 μM and 65 μM, and the concentration of sodium nitroprusside is between 150 μM and 270 μM; spraying the abscisic acid and sodium nitroprusside solution onto the underside of poplar leaves during the young leaf stage; covering the young poplar leaves with a black plastic bag after spraying, removing the bag after 24 hours, or spraying at dusk or night.

[0020] This invention involves covering poplar leaves with black plastic bags after spraying abscisic acid and sodium nitroprusside solution, or spraying at dusk or nighttime to prevent sodium nitroprusside from decomposing in light.

[0021] Optionally, the abscisic acid and sodium nitroprusside solution is sprayed onto the back of the poplar leaves 3 to 5 times at a time.

[0022] Optionally, the concentration of abscisic acid is 50 μM, and the concentration of sodium nitroprusside is 200 μM.

[0023] Optionally, the concentration of abscisic acid is 30 μM, and the concentration of sodium nitroprusside is 270 μM.

[0024] Optionally, the concentration of abscisic acid is 65 μM, and the concentration of sodium nitroprusside is 130 μM.

[0025] Application of combined abscisic acid and sodium nitroprusside in the control of poplar leaf rust.

[0026] Applying abscisic acid or sodium nitroprusside alone to poplar leaves will not effectively control poplar leaf rust.

[0027] The present invention has the following beneficial effects:

[0028] By combining exogenous application of abscisic acid (ABA) and sodium nitroprusside (SNP), stomatal opening in poplar leaves was inhibited, leaf lignin content was increased, and chitinase activity in leaves was enhanced, thus constructing a poplar rust resistance regulation system based on the combined use of plant hormones and signaling molecules.

[0029] In the experiment of this invention, (1) treatment with abscisic acid and sodium nitroprusside alone reduced the stomatal aperture of infected poplar leaves by 18% and 12% respectively compared with the control (Rust-), ​​while the combined treatment reduced the stomatal aperture of infected poplar leaves by 24% compared with the control (Rust-), ​​which was significantly better than the effect of single application and effectively blocked the stomatal invasion channel of pathogens; (2) the number of rust spores in the control group reached 101, and the rust infection area reached 97%. Treatment with abscisic acid and sodium nitroprusside alone reduced the number of rust spores on infected poplar leaves by 81% compared with the control group (Rust+). The rust infection area was reduced by 74% (area ratio reduced to 26%) and 59% (area ratio reduced to 40%) compared to the control (Rust+), respectively. The combined treatment reduced the number of rust spores on the infected poplar leaves by 90% (number reduced to 10) compared to the control (inoculated with rust fungus), and the rust infection area was reduced by 93% (area ratio reduced to 4%) compared to the control (inoculated with rust fungus), which was significantly better than the effect of single application; (3) After rust infection, the content of lignin in the poplar leaves reached 229.5 mg·g when abscisic acid and sodium nitroprusside were applied alone. -1 and 192.1 mg·g -1 The combined treatment caused a sharp increase in the lignin content of infected poplar leaves, reaching 300.85 mg / g. -1 Compared to the treatment groups treated with abscisic acid and sodium nitroprusside alone, the chitinase activity was increased by 31% and 57%, respectively. Similarly, after infection with Populus tomentosa, the chitinase activity in leaves treated with abscisic acid alone reached 292.46 IU·g⁻¹. -1 The enzyme activity after treatment with sodium nitroprusside alone reached 268.9 IU·g. -1The combined treatment resulted in the highest chitinase activity (345.68 IU·g) in the leaves of infected poplar trees. -1 Compared with the single treatment groups of abscisic acid and sodium nitroprusside, the efficacy was increased by 15% and 22.0%, respectively. This scheme achieves enhanced disease resistance in poplar through the synergistic regulation of plant hormones and signaling molecules, providing a technical solution for forest biological control that combines theoretical innovation with industrial application value.

[0030] The composition for controlling poplar leaf rust of the present invention, compared with the first prior art, only requires spraying, which can now be achieved by drones, saving time and effort; compared with the second prior art, it can be sprayed on young poplar leaves without harming them, and both substances are common in plants, are harmless to the environment, and plants will not develop resistance to them.

[0031] The composition for controlling poplar leaf rust of the present invention, when sprayed on poplar leaves, inhibits the opening and closing of stomata on poplar leaves, preventing rust fungus from invading. After spraying the composition of the present invention, the lignin content in the leaves increases significantly, providing physical defense for the leaves against rust fungus invasion. After spraying the composition of the present invention, the chitinase activity in poplar leaves increases significantly, causing fungal cell wall disintegration, hyphae breakage, or spore rupture. Chitin triggers the PTI cascade reaction, amplifies the defense signal, activates systemic resistance, and strengthens the cell wall of poplar leaves. Attached Figure Description

[0032] Figure 1 This is a graph showing the changes in stomatal size (A), stomatal conductance (B), and transpiration rate (C) for each treatment in Example 1; Note: Results are shown as mean ± standard error, stomatal size (n=100), stomatal conductance (n=3), and transpiration rate (n=3); According to the Tukey test results of ANOVN analysis, different lowercase letters indicate significant differences (P<0.05);

[0033] Figure 2 Example 1: Poplar leaf leaf infection with rust spores 14 days after infection; Note: (A) represents the leaf phenotype; (BC) represents the number of rust spores and the proportion of rust spore area to leaf area. Rust, rust alone; A+R, exogenous application of ABA and rust; S+R, exogenous application of SNP and rust; A+S+R, exogenous application of ABA, SNP, and rust; Scale bar 1cm. Results are shown as mean ± standard deviation, n=3; according to the Tukey test results of ANOVN analysis, different lowercase letters indicate significant differences (P<0.05);

[0034] Figure 3 This refers to the lignin content of poplar leaves treated in Example 1; Note: Results show average value ±

[0035] Standard deviation, n=3; According to the Tukey test results of ANOVN analysis, different lowercase letters indicate significant differences (P<0.05);

[0036] Figure 4 This refers to the chitinase activity of poplar leaves treated in Example 1; Note: Results show average values.

[0037] ± standard deviation, n=3; according to the Tukey test results of ANOVN analysis, different lowercase letters indicate significant differences (P<0.05). Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0039] It should be noted that the core components of the poplar leaf rust control composition of this invention are abscisic acid and sodium nitroprusside. However, if other substances are added, it will not affect the combined effect of abscisic acid and sodium nitroprusside in controlling poplar leaf rust, or the combined effect of abscisic acid and sodium nitroprusside in controlling poplar leaf rust will still be achieved. Therefore, the scheme of adding other substances is also within the protection scope of this invention.

[0040] Abscisic acid (ABA) is one of the five major classes of natural hormones in plants, named for its initial association with leaf and fruit abscission. However, later research revealed that its primary function is actually to regulate plant stress responses and growth retardation (such as dormancy), with its relationship to the abscission process being relatively minor.

[0041] Sodium nitroprusside is a potent and fast-acting vasodilator primarily used to treat hypertensive emergencies and heart failure. It works by releasing nitric oxide (NO), activating guanylate cyclase, increasing intracellular cyclic guanosine monophosphate (cGMP) levels, leading to relaxation of vascular smooth muscle, thereby achieving a vasodilatory effect.

[0042] To date, there are no reports on the inhibitory and control effects of combined application of abscisic acid and sodium nitroprusside on poplar leaf rust fungi, especially larch-poplar rust fungi.

[0043] The following detailed embodiments illustrate the composition and method for controlling poplar leaf rust according to the present invention.

[0044] Example 1. Laboratory verification of the combined application of abscisic acid and sodium nitroprusside to prevent rust fungal infection.

[0045] 1.1 Cultivation of female poplar cuttings

[0046] Female poplar cuttings were taken from Ledu County, Qinghai Province, China (36°48′N, 102°40′E). The cuttings were 10cm long and 0.5cm in diameter. They were transplanted into plastic pots and cultured in a 1:1 mixture of potting soil and vermiculite, supplemented with Hogrange nutrient solution (pH 6.0). The cuttings were kept in a greenhouse (65% relative humidity, average temperature 25℃, 20000 Lux illuminance, 12h·d light). -1 Fifteen Populus tomentosa plants with uniform growth, cultivated in a greenhouse for two months, were selected for indoor controlled experiments.

[0047] 1.2 Design Processing Group

[0048] The treatment groups were set up as shown in Table 1. Distilled water (control group CK), 50 μM MABA (abscisic acid) solution, 200 μM SNP (sodium nitroprusside) solution, and 50 μM MABA and 200 μM SNP solution were prepared in advance in four 75 mL spray bottles. The solutions were evenly sprayed onto the underside of the second to fifth fully unfolded leaves at the top of the poplar plant. Each poplar plant was sprayed five times, with each sprayer containing 125 μL. There were four treatment groups, with 15 poplar plants sprayed in each group.

[0049] Table 1 Processing Group Settings

[0050]

[0051] 1.3 Inoculating poplar cuttings with Populus spp. rust spores

[0052] Inoculation experiments were conducted using freshly harvested larch-poplar rust spores. A suspension of rust spores (approximately 1.5 × 10⁻⁶) was used. 6 Spores were transferred to a 75ml spray bottle (1mL distilled water per spore) and evenly sprayed onto the leaves of the treatment groups shown in Table 1, as shown in Table 2. Each poplar plant was sprayed 5 times, with each spray consisting of 125μL. There were four treatment + inoculation groups, with 10 poplar plants sprayed in each group.

[0053] Table 2 Vaccination experimental group

[0054]

[0055] After spraying, cover the plants with black PET plastic bags to protect them from light and maintain high humidity. Remove the plastic bags after 24 hours, and the plants will continue to grow in the greenhouse.

[0056] 1.4 Treatment of poplar leaves inoculated with *Platycladus mukorossi* spores

[0057] Six hours after inoculation, leaves were harvested. The leaves were decolorized with a decolorizing solution (ethanol:acetic acid = 3:1 v:v) and stained with Coomassie brilliant blue solution for 3 minutes. After rinsing off excess dye with water, the leaves were stored in a stock solution (acetic acid:glycerol:water = 5:20:75 v:v:v) and observed under a Leica upright microscope. For each treatment, 10-15 leaves from ≥6 plants were examined in Tables 1 and 2. The stomatal length and width were measured using ImageJ software (https: / / imagej.nih.gov / ij / ), and the stomatal size was expressed as width / length, referred to as stomatal aperture.

[0058] Figure 1 Results showed that poplar leaves with stomata decreased in size after infection, by 7% compared to the control group (uninoculated with rust fungus).

[0059] After application of abscisic acid, stomatal aperture decreased further, by 10% compared to the control (inoculated with rust fungus). Sodium nitroprusside application did not significantly reduce stomatal aperture compared to the control (inoculated with rust fungus). However, the combined application of abscisic acid and sodium nitroprusside reduced poplar leaf stomatal aperture to a minimum of 0.23, a 21% reduction compared to the control (inoculated with rust fungus), representing effects two and four times greater than abscisic acid and sodium nitroprusside treatments alone, respectively.

[0060] 1.5 Determination of stomatal conductance and transpiration rate of leaves

[0061] Using a portable photosynthesis system, stomatal conductance and transpiration rate were measured on the third fully expanded leaf (from the tip) of a poplar plant 6 hours after rust fungus infection. Three plants were randomly selected for each treatment, and measurements were taken between 09:00 and 11:30 AM. The instrument settings were as follows: leaf temperature 25℃, photosynthetic photon flux density 1500 μmol / m². -2 s -1 The CO2 cylinder provides a constant CO2 concentration (400 μmol / mol). -1 The flow rate was 500 μmol / s. -1 .

[0062] Pore ​​conductance results as follows Figure 1 As shown in Figure B, the stomatal conductance of poplar leaves generally decreased after infection. However, the stomatal conductance of leaves treated with abscisic acid and sodium nitroprusside decreased by 28% and 23% respectively compared to the control (inoculated with rust fungus). When abscisic acid and sodium nitroprusside were used in combination, the stomatal conductance of infected poplar leaves showed a sharp and significant decrease, with a reduction of up to 77%, far exceeding the reduction of the single treatment group.

[0063] transpiration rate Figure 1Results showed that the transpiration rate of poplar leaves decreased after infection. In the groups treated with abscisic acid and sodium nitroprusside alone, the transpiration rate of leaves decreased by 19% and 21% respectively compared with the control (inoculated with rust fungus). When abscisic acid and sodium nitroprusside were used in combination, the transpiration rate of poplar leaves after infection decreased by 72%, which was significantly higher than that of the groups treated with abscisic acid and sodium nitroprusside alone.

[0064] The results show that abscisic acid and sodium nitroprusside have a strong synergistic effect in regulating the stomatal conductance of susceptible poplar. The combined application of the two can more effectively inhibit stomatal opening, reduce stomatal conductance, block pathogen infection channels, and thus significantly reduce leaf transpiration rate.

[0065] Therefore, although this embodiment only uses larch-poplar rust spores for inoculation, it is applicable to the prevention and control of all rust fungi, because rust fungi invade through stomata and form haustoria in the host cells to obtain nutrients, such as rust fungi like *Pseudomonas maculatus* and *Ustilago maydis*. In this invention, the combined application of abscisic acid and sodium nitroprusside can inhibit the opening of leaf stomata, reduce the stomatal conductance of leaves, and block the infection channels of pathogens.

[0066] Furthermore, since the infection pathways of sugarcane rust pathogens Puccinia kuehnii and Puccinia nigra are similar to those of poplar rust, the composition of this invention can also be used to control sugarcane rust pathogens Puccinia kuehnii and Puccinia nigra.

[0067] 1.6 Poplar rust spore infection status 14 days after infection

[0068] Fourteen days after inoculation, the infected leaves were photographed, and leaves from three plants were selected. ImageJ software was used to calculate the area and number of infected pathogens. Figure 2 The results showed that the number of rust spores on leaves treated with abscisic acid (A+R) was reduced by 81% and the infected area was reduced by 74% compared with the control (inoculated with rust fungus). Similarly, spraying sodium nitroprusside alone (S+R) could also inhibit rust fungus infection, reducing the number of rust spores by 77% and the infected area by 59%. However, the combined application of abscisic acid and sodium nitroprusside after infection (A+S+R) reduced the number of rust spores by 90% and the infected area by 93% compared with the control, indicating that this combined treatment method has a more significant effect in reducing rust fungus infection.

[0069] 1.7 Combined application of abscisic acid and sodium nitroprusside promotes lignin accumulation in poplar leaves.

[0070] The lignin content of *Populus tomentosa* leaves 7 days after inoculation was determined using a lignin content assay kit (Beijing Solarbio), with three biological replicates per group. Results are as follows: Figure 3As shown, after infection with rust fungus, the lignin content in the leaves of Populus tomentosa was significantly higher than that of the control (uninoculated with rust fungus), with an increase of up to 142%. Even with rust fungus infection, application of abscisic acid or sodium nitroprusside alone promoted lignin accumulation in Populus tomentosa leaves, with contents reaching 229.5 mg / g. -1 and 192.1 mg·g -1 Compared to the control (inoculated with rust fungus), the lignin content increased by 21% and 6% respectively. When abscisic acid and sodium nitroprusside were applied in combination, the lignin content of infected poplar leaves increased dramatically, reaching 300.85 mg / g. -1 Compared with the treatment groups treated with abscisic acid and sodium nitroprusside alone, the lignin accumulation rate was increased by 31% and 57%, respectively. This result indicates that abscisic acid and sodium nitroprusside have a synergistic effect in inducing lignin accumulation in poplar leaves. The combined application of the two can more strongly promote lignin accumulation, thereby constructing a denser physical defense system for poplar.

[0071] 1.8. Combined application of abscisic acid and sodium nitroprusside enhances chitinase activity in poplar trees.

[0072] Chitinase activity in *Populus tomentosa* leaves 14 days after inoculation was measured using an ELISA kit (Nanjing Jiancheng), with three biological replicates per group. Results are as follows: Figure 4 As shown, after Populus tomentosa was infected with the disease, the chitinase activity in the leaves treated with abscisic acid alone reached 292.46 IU·g. -1 (International Unit (IU): Defined by international organizations as the amount of enzyme that catalyzes the conversion of 1 micromole (μmol) of substrate per minute under specific conditions (25°C, optimal pH, etc.), i.e., 1 IU = 1 μmol / min). This represents a 21% increase compared to the control (inoculated with rust fungus). Sodium nitroprusside alone also promoted chitinase activity, reaching 268.9 IU·g. -1 Compared to the control (inoculated with rust fungus), the activity was increased by 11%, but not as significantly as the abscisic acid treatment. When abscisic acid and sodium nitroprusside were applied in combination, the chitinase activity in infected poplar leaves reached a maximum of 345.68 IU·g. -1 Compared with the single treatment groups of abscisic acid and sodium nitroprusside, the efficacy was increased by 15% and 22.0% respectively, indicating that the combined application of abscisic acid and sodium nitroprusside has a better control effect on poplar disease.

[0073] Enhanced chitinase activity in poplar trees is an important marker of activated plant defense systems, playing a crucial role, especially in combating fungal pathogens such as rust fungi. Chitin is a core component of the cell wall of rust fungi. Chitinase can specifically hydrolyze the β-1,4 glycosidic bonds of chitin, leading to fungal cell wall disintegration, hyphal breakage, or spore rupture. After degrading the fungal cell wall, chitinase releases chitin oligosaccharides (such as chitosan hexasaccharide), which can serve as pathogen-associated molecular patterns (PAMPs). These oligosaccharides are recognized by receptors on the poplar cell membrane (such as LYK5 / CERK1), triggering a PTI (PAMP-Triggered Immunity) cascade reaction that amplifies defense signals. Chitinase can also indirectly activate systemic resistance and enhance the reinforcement of poplar leaf cell walls.

[0074] Example 2: An experiment was conducted on the combined application ratio of abscisic acid and sodium nitroprusside.

[0075] Based on the concentrations of ABA (abscisic acid) and SNP (sodium nitroprusside) used in combination as determined in Example 1, an orthogonal experiment was designed, as shown in Table 3.

[0076] Group ABA (μM) SNP(μM) G1 33.6 132.8 G2 33.6 267.2 G3 66.4 132.8 G4 66.4 267.2 G5 38 152 G6 38 248 G7 62 152 G8 62 248 G9 50 200

[0077] Then, referring to the cultivation of female poplar cuttings in Example 1.1 and the easy preparation and spraying methods in Example 1.2, solutions G1 to G9 were prepared respectively, and solutions G1 to G11 were sprayed on the back of the leaves of the poplar plants, with 5 poplar plants in each group sprayed.

[0078] Then, referring to Example 1.3, poplar cuttings were inoculated with Populus rust spores and then sprayed on the poplars that had been sprayed with the solutions from Groups G1 to G9.

[0079] After 14 days of cultivation, following the method described in 1.6 of Example 1, leaves from three plants were selected, and the area and number of pathogen infections were calculated using ImageJ software. The number of rust spore masses and the ratio of rust spore area to leaf area for groups G1 to G9 are shown in Table 4.

[0080] Table 4. Number of rust spore masses and rust spore area / leaf area for groups G1–G9

[0081]

[0082]

[0083] As shown in Table 4, group G4 had the highest ABA and SNP content, but the decrease in the number of spore masses and the area ratio was not significant, indicating that higher ABA and SNP content is not necessarily better. Group G1 had the lowest ABA and SNP content; although the decrease in the number of spore masses and the area ratio was acceptable, it did not achieve the effect required by this invention. Group G8 also failed to meet the requirements of this invention. This invention selected groups G2, G3, G5, G6, and G7, which showed better results. Therefore, the concentration range of abscisic acid in the poplar leaf rust control composition of this invention was determined to be 30 μM–65 μM, and the concentration range of sodium nitroprusside was determined to be 130 μM–270 μM.

[0084] Then, experiments were conducted on the boundary values ​​of the above concentration range. Experimental groups were set up, and the experimental methods in 1.1, 1.2, 1.3 and 1.6 of Example 1 were also followed. Leaves from three plants were selected, and ImageJ software was used to calculate the pathogen infection area and number, the number of rust spore heaps and the rust spore area / leaf area ratio of groups G1 to G9, and the experimental groups and their corresponding number of rust spore heaps and rust spore area / leaf area ratios are shown in Table 5.

[0085]

[0086] Although the concentration ratios of G10 to G13 in Table 5 are not as effective as ABA 50μM and SNP 200μM, they can still meet the requirements of this invention.

[0087] Example 3 Outdoor Test

[0088] Female poplar trees were selected from Lihuaping, Pengzhou City, Sichuan Province, China (30°54'~31°2N, 103°40'~104°10E). This area also contains larch forests, and poplars are susceptible to rust fungus infection, resulting in poplar leaf rust disease. Generally, spraying with the composition of this invention on uninfected poplar leaves before the end of May, when the spores of *Pseudomonas spp.* on the larch trees are not yet mature and cannot begin to spread, and the poplar leaves are in their early growth stage, can effectively reduce the infection rate of poplar leaves without affecting the growth of the poplar trees. When spraying outdoors, it is best to choose the evening when it is almost dark or at night to ensure that the sodium nitroprusside has sufficient time to be absorbed by the leaves.

[0089] The specific measures are as follows: From mid-April to the end of May, the composition of this invention is sprayed every week. This invention uses 50 μM ABA and 200 μM SNP, and 40 μM ABA and 150 μM SNP, with each group sprayed on three poplar trees. In July and August, the poplar forest is inspected, and a very small number of leaves on the poplars at the edge of the forest that have not been completely sprayed are infected with rust fungus.

[0090] This invention employs a combination of abscisic acid (ABA) and sodium nitroprusside (SNP), which is pre-sprayed onto young poplar leaves to prevent rust fungal infection. Since abscisic acid (ABA) is a naturally occurring hormone in plants, it does not harm young poplar leaves; similarly, sodium nitroprusside (SNP) is a common exogenous nitric oxide donor and also does not harm young poplar leaves. Spraying the young poplar leaves with this composition prevents rust fungal infection. Furthermore, this composition increases lignin and chitinase levels in poplar leaves, enhancing the poplar's disease resistance.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for controlling poplar leaf rust, characterized in that, It contains: 30-65 μM abscisic acid and 130-270 μM sodium nitroprusside.

2. The composition for controlling poplar leaf rust according to claim 1, characterized in that, It contains: 50 μM abscisic acid and 200 μM sodium nitroprusside.

3. The composition for controlling poplar leaf rust according to claim 1, characterized in that, Contains: 40 μM abscisic acid and 150 μM sodium nitroprusside.

4. The composition for controlling poplar leaf rust according to claim 1, characterized in that, It contains: 60 μM abscisic acid and 250 μM sodium nitroprusside.

5. A method for controlling poplar leaf rust, characterized in that, include: Prepare a solution of abscisic acid and sodium nitroprusside, wherein the concentration of abscisic acid is between 30 μM and 65 μM, and the concentration of sodium nitroprusside is between 150 μM and 270 μM; spray the abscisic acid and sodium nitroprusside solution onto the underside of poplar leaves during the young leaf stage; after spraying, cover the young poplar leaves with a black plastic bag and remove it after 24 hours, or choose to spray at dusk or at night.

6. The method for controlling poplar leaf rust according to claim 5, characterized in that, The abscisic acid and sodium nitroprusside solution is sprayed onto the back of the poplar leaves, and the same poplar leaf is sprayed 3 to 5 times at a time.

7. The method for controlling poplar leaf rust according to claims 5 and 6, characterized in that, The concentration of abscisic acid is 50 μM, and the concentration of sodium nitroprusside is 200 μM.

8. The method for controlling poplar leaf rust according to claims 5 and 6, characterized in that, The concentration of abscisic acid is 30 μM, and the concentration of sodium nitroprusside is 270 μM.

9. The method for controlling poplar leaf rust according to claims 5 and 6, characterized in that, The concentration of abscisic acid is 65 μM, and the concentration of sodium nitroprusside is 130 μM.

10. Application of combined application of abscisic acid and sodium nitroprusside in the control of poplar leaf rust.

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