Application and method for improving wheat stripe rust resistance by exogenous substance treatment
By spraying wheat seedlings with a 500 μmol/L luteolin solution, the germination of stripe rust spores was inhibited, solving the problem of green control of wheat stripe rust and achieving efficient and safe disease control.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YUNNAN AGRICULTURAL UNIVERSITY
- Filing Date
- 2025-02-06
- Publication Date
- 2026-07-21
Smart Images
Figure CN119655130B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to, but is not limited to, the field of plant protection technology, and particularly relates to the application and method of improving wheat stripe rust resistance through exogenous substance treatment. Background Technology
[0002] Control of wheat stripe rust mainly relies on disease-resistant breeding and the use of fungicides. However, due to pathogen variation and the irrational use of resistance genes, single resistance genes have become ineffective, posing a significant challenge to disease-resistant breeding. Furthermore, the use of fungicides can lead to increased drug resistance in pathogens, and in the context of green pest control, the use of chemical agents is not the optimal choice. Therefore, developing safer, greener, and more effective measures is an urgent problem to be solved in the control of wheat stripe rust.
[0003] Luteolin belongs to the flavonoid family and is widely found in plants. It possesses antioxidant and antibacterial activities, playing a crucial role in plant stress response. For example, under drought stress, luteolin significantly accumulates in drought-resistant citrus, scavenging reactive oxygen species. Under biotic stress, luteolin accumulates more rapidly and significantly in anthracnose-resistant sorghum varieties, and luteolin treatment inhibits anthracnose germination. A significant increase in luteolin content was detected in thrips-resistant carrot varieties, and feeding luteolin reduced the growth rate and survival rate of thrips larvae. Luteolin also exhibits a strong ability to inhibit the growth of Fusarium graminearum. In summary, luteolin plays an important role in plant disease resistance responses. Therefore, studying the function of luteolin in wheat stripe rust resistance is of practical significance for the development of green control measures for wheat stripe rust and the research on its resistance mechanisms.
[0004] Existing technologies and limitations: Luteolin plays an important role in plant disease resistance, but different microorganisms have different tolerances to luteolin. Currently, there are no studies reporting the antibacterial effect of luteolin on stripe rust fungi or its function in wheat resistance to stripe rust. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention provides an application and method for improving wheat stripe rust resistance through exogenous substance treatment, thereby solving the problems described in the background section. This invention discovers that treatment with an exogenous substance can inhibit the germination of stripe rust spores. Applying this substance to wheat leaves during the seedling stage can improve wheat's resistance to stripe rust and slow down the occurrence of wheat stripe rust.
[0006] This invention is achieved through an application of exogenous substance treatment to improve wheat stripe rust resistance, wherein the exogenous substance is luteolin.
[0007] Furthermore, an exogenous substance at a concentration of 250 μmol / L can inhibit the germination of stripe rust spores.
[0008] Furthermore, seedling wheat refers to wheat that has grown to the two-leaf-one-heart stage.
[0009] Furthermore, exogenous substances were applied to wheat leaves during the seedling stage, prior to stripe rust infection.
[0010] Furthermore, before application, the treatment steps are as follows: when the wheat grows to the two-leaf-one-heart stage, spray the wheat three times with an exogenous substance, once a day. After the leaves have absorbed the substance, inoculate with stripe rust fungus.
[0011] Furthermore, exogenous substances were sprayed onto wheat at a concentration of 500 μmol / L.
[0012] Another object of the present invention is to provide a method for improving wheat stripe rust resistance through exogenous substance treatment, comprising: S1. Preparation and application of luteolin solution: Weigh 57.3 mg of luteolin (purchased from E. En Chemical Technology Co., Ltd.), completely dissolve it in 20 mL of anhydrous ethanol to prepare a 10 mmol / L luteolin ethanol solution, and add deionized water to bring the volume to 400 mL, preparing a final concentration of 500 μmol / L luteolin solution. When the stripe rust-susceptible material Yun 0402 grows to the two-leaf-one-heart stage, spray the upper and lower surfaces of the leaves evenly with the 500 μmol / L luteolin solution once a day for a total of three sprays.
[0013] S2. Detection of endogenous luteolin content: Weigh 0.1 g of wheat leaves pretreated with 500 μmol / L luteolin, grind with liquid nitrogen, add 0.5 mL of 80% methanol aqueous solution, vortex mix, ultrasonically extract for 30 min, centrifuge at 12000 rpm for 10 min, take the supernatant, and detect it using ultra-high performance liquid chromatography (UHPLC) by Vanquish (Thermo, USA) and high resolution mass spectrometry (QExactive, Thermo, USA).
[0014] S3. Stripe rust inoculation and material culture: Wheat pretreated with 500 μmol / L luteolin was inoculated with stripe rust. Stripe rust CYR32 and talc were mixed at a ratio of 1:20. A certain amount of inoculum was applied evenly to both sides of the leaves using a small brush. The plants were first placed in an incubator at 12℃ and 80% humidity and cultured in the dark for 24 hours. The subsequent conditions were set as follows: temperature 12℃, humidity 80%, 16 hours of light, 8 hours of darkness, and cultured for about 2 weeks.
[0015] S4. WGA staining and histological observation: Leaves were cut at 48 and 168 hours after inoculation and immersed in 12 mL KOH (1 mol), with 2 μL Tween added. The mixture was incubated overnight at 37°C. The leaves were then stained for 1 hour in Tris (50 mmol, pH 7.5) solution containing 20 μg / mL wheat lectin WGA-FITC. The leaf area was then observed under a fluorescence microscope, and the infection area was quantified using ImageJ software.
[0016] S5. Phenotypic observation: Investigate the disease status of plants 17 days after inoculation and take photos for record.
[0017] S6. Stripe rust biomass detection: Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The stripe rust internal reference gene in the DNA template was detected by real-time fluorescent PCR. PstEF1α ) and wheat internal reference gene ( TaEF-1α The CT value of the stripe rust fungus and wheat DNA were used to calculate the relative amount of stripe rust fungus biomass.
[0018] First, the beneficial effects of this invention are as follows: This invention utilizes 250 μmol / L exogenous luteolin to culture stripe rust fungi. The results show that, compared with the control group, luteolin significantly inhibited the germination of stripe rust fungi spores. After pretreating wheat leaves with 500 μmol / L luteolin and inoculating them with stripe rust fungi, the results, based on the detection of endogenous luteolin content, histological observation of infected leaves, plant phenotypic observation, and stripe rust fungi biomass, showed that, compared with the control group, wheat leaves pretreated with 500 μmol / L luteolin exhibited significantly increased endogenous luteolin content, significantly reduced stripe rust-infected area, significantly reduced wheat plant leaf spores, and significantly decreased stripe rust fungi biomass. Therefore, 500 μmol / L luteolin can inhibit stripe rust fungi infection in wheat, slow down the occurrence of wheat stripe rust, and provide a certain reference for the research on control measures and disease resistance mechanisms of wheat stripe rust.
[0019] Secondly, this invention can be applied to the prevention and control of wheat stripe rust, and can be developed as a raw material for green pesticides.
[0020] Wheat stripe rust is a significant disease affecting wheat yield and quality. Current control measures mainly rely on the breeding of resistant varieties and the application of chemical pesticides. However, the development of resistant varieties is time-consuming and suffers from resistance decline, while the long-term use of chemical pesticides may cause environmental pollution and carries the risk of increased pesticide resistance in pathogens. Therefore, finding an efficient, safe, and practical method for stripe rust control has become an urgent problem to be solved in the current technological field.
[0021] This invention provides a technical solution for improving wheat stripe rust resistance by using the exogenous substance luteolin. By spraying luteolin during the wheat seedling stage (two-leaf and one-heart stage), it can not only significantly inhibit the germination of stripe rust spores, but also slow down the occurrence of stripe rust and reduce dependence on chemical pesticides, thus providing a new approach for the green control of wheat stripe rust.
[0022] Through scientific verification, this invention has found that luteolin can significantly inhibit the germination of stripe rust spores and effectively reduce the infection rate of the pathogen. In particular, after spraying a 500 μmol / L luteolin solution, the number of spores on wheat stripe rust-infected leaves was significantly reduced (by 52.9%), and the control effect was significantly better than that of traditional chemical agents.
[0023] Unlike chemical pesticides, luteolin is a natural plant extract that is environmentally friendly and less likely to induce antibiotic resistance in pathogens. This invention is simple to operate; only a few applications to the leaves of wheat seedlings are needed to achieve highly effective control of stripe rust. This method is characterized by high safety, ease of operation, and suitability for large-scale application, significantly improving the sustainability and economic benefits of wheat stripe rust control. Attached Figure Description
[0024] Figure 1 This describes the inhibition of the germination rate of stripe rust fungus by exogenous luteolin in Example 1 of this invention. Figure 2 This refers to the content of endogenous luteolin in wheat leaves after pretreatment with exogenous luteolin in Example 2 of this invention. Figure 3 This is the histological observation of wheat pretreated with exogenous luteolin and inoculated with stripe rust fungus in Example 2 of the present invention; Figure 4 This is the wheat stripe rust disease incidence on day 17 after pretreatment with exogenous luteolin and inoculation with stripe rust fungus, as provided in Example 2 of this invention. Figure 5 This is the biomass detection of wheat leaves after pretreatment with exogenous luteolin and inoculation with stripe rust fungus on day 17, according to Example 2 of this invention. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0026] This invention provides an application of exogenous substance treatment to improve wheat stripe rust resistance, wherein the exogenous substance is luteolin.
[0027] Furthermore, an exogenous substance at a concentration of 250 μmol / L can inhibit the germination of stripe rust spores.
[0028] Furthermore, seedling wheat refers to wheat that has grown to the two-leaf-one-heart stage.
[0029] Furthermore, exogenous substances were applied to wheat leaves during the seedling stage, prior to stripe rust infection.
[0030] Furthermore, before application, the treatment steps are as follows: when the wheat grows to the two-leaf-one-heart stage, spray the wheat three times with an exogenous substance, once a day. After the leaves have absorbed the substance, inoculate with stripe rust fungus.
[0031] Furthermore, exogenous substances were sprayed onto wheat at a concentration of 500 μmol / L.
[0032] Another object of the present invention is to provide a method for improving wheat stripe rust resistance through exogenous substance treatment, comprising: S1. Preparation and application of luteolin solution: Weigh 57.3 mg of luteolin (purchased from E. En Chemical Technology Co., Ltd.), completely dissolve it in 20 mL of anhydrous ethanol to prepare a 10 mmol / L luteolin ethanol solution, and add deionized water to bring the volume to 400 mL, preparing a final concentration of 500 μmol / L luteolin solution. When the stripe rust-susceptible material Yun 0402 grows to the two-leaf-one-heart stage, spray the upper and lower surfaces of the leaves evenly with the 500 μmol / L luteolin solution once a day for a total of three sprays.
[0033] S2. Determination of endogenous luteolin content: Weigh 0.1 g of wheat leaves pretreated with 500 μmol / L luteolin, grind with liquid nitrogen, add 0.5 mL of 80% methanol aqueous solution, vortex mix, ultrasonically extract for 30 min, centrifuge at 12000 rpm for 10 min, take the supernatant, and detect it using an ultra-high performance liquid chromatograph (Vanquish, Thermo, USA) and a high resolution mass spectrometer (QExactive, Thermo, USA).
[0034] S3. Stripe rust inoculation and material culture: Wheat pretreated with 500 μmol / L luteolin was inoculated with stripe rust. Stripe rust CYR32 and talc were mixed at a ratio of 1:20. A certain amount of inoculum was applied evenly to both sides of the leaves using a small brush. The plants were first placed in an incubator at 12℃ and 80% humidity and cultured in the dark for 24 hours. The subsequent conditions were set as follows: temperature 12℃, humidity 80%, 16 hours of light, 8 hours of darkness, and cultured for about 2 weeks.
[0035] S4. WGA staining and histological observation: Leaves were cut at 48 and 168 hours after inoculation and immersed in 12 mL KOH (1 mol), with 2 μL Tween added. The mixture was incubated overnight at 37°C. The leaves were then stained for 1 hour in Tris (50 mmol, pH 7.5) solution containing 20 μg / mL wheat lectin WGA-FITC. The leaf area was then observed under a fluorescence microscope, and the infection area was quantified using ImageJ software.
[0036] S5. Phenotypic observation: Investigate the disease status of plants 17 days after inoculation and take photos for record.
[0037] S6. Stripe rust biomass detection: Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The stripe rust internal reference gene in the DNA template was detected by real-time fluorescent PCR. PstEF1α ) and wheat internal reference gene ( TaEF-1α The CT value of the stripe rust fungus and wheat DNA were used to calculate the relative amount of stripe rust fungus biomass.
[0038] Example 1: Exogenous luteolin inhibits the germination of stripe rust spores. 1. Method 1.1. Preparation of luteolin solution Weigh 57.3 mg of luteolin (purchased from E. En Chemical Technology Co., Ltd.), completely dissolve it in 20 mL of anhydrous ethanol to prepare a luteolin ethanol solution with a concentration of 10 mmol / L. Take 2.5 mL of the luteolin ethanol solution and add deionized water to make up to 100 mL to prepare a luteolin solution with a final concentration of 250 μmol / L.
[0039] 1.2. Stripe rust germination test A certain mass of stripe rust spores was weighed and evenly placed in petri dishes containing 250 μmol / L luteolin solution and water (control group), respectively, and incubated in the dark at 12℃ for 6 hours. The germination status of the spores was observed under a microscope and the germination rate was calculated. Three fields of view were counted for each treatment, and approximately 150 stripe rust spores were counted in each field of view.
[0040] 2. Results The germination rate of *Strombus stripe rust* spores treated with water was 19%, while the germination rate of *Strombus stripe rust* spores treated with 250 μmol / L luteolin solution was 5%, significantly reducing the germination rate of *Strombus stripe rust* spores. Figure 1 This demonstrates that osmanthus extract solution inhibits the normal germination of stripe rust spores.
[0041] Example 2: Exogenous luteolin slows down the occurrence of wheat stripe rust. Yun 0402 is a local wheat germplasm resource in Yunnan Province. Based on four consecutive years of field resistance identification results for mature wheat stripe rust, Yun 0402 exhibits a stable high susceptibility to the disease. Seedling-stage stripe rust resistance identification also showed that Yun 0402 is highly susceptible to stripe rust. Therefore, this invention uses the Yunnan local wheat germplasm Yun 0402 as the research material and employs the method described herein to study the function of exogenous luteolin in wheat resistance to stripe rust.
[0042] 1. Method 1.1. Preparation and application of luteolin solution Weigh 57.3 mg of luteolin (purchased from E. En Chemical Technology Co., Ltd.), completely dissolve it in 20 mL of anhydrous ethanol to prepare a 10 mmol / L luteolin ethanol solution, and then add deionized water to bring the volume to 400 mL, preparing a final luteolin solution with a concentration of 500 μmol / L. When the stripe rust-susceptible material Yun 0402 reaches the two-leaf-one-heart stage, spray the upper and lower surfaces of the leaves evenly with the 500 μmol / L luteolin solution once a day for a total of three sprays.
[0043] 1.2. Detection of endogenous luteolin content Weigh 0.1 g of wheat leaves pretreated with 500 μmol / L luteolin, grind with liquid nitrogen, add 0.5 mL of 80% methanol aqueous solution, vortex to mix, extract by sonication for 30 min, centrifuge at 12000 rpm for 10 min, collect the supernatant, and detect it using an ultra-high performance liquid chromatograph (Vanquish, Thermo, USA) and a high resolution mass spectrometer (Q Exactive, Thermo, USA).
[0044] 1.3. Inoculation and Culture of Stripe Rust Fungi Wheat pretreated with 500 μmol / L luteolin was inoculated with stripe rust fungus. Stripe rust fungus CYR32 and talc powder were mixed at a ratio of 1:20. A certain amount of the fungal inoculum was applied evenly to both sides of the leaves using a small brush. The plants were first placed in an incubator at 12℃ and 80% humidity and cultured in the dark for 24 hours. The subsequent conditions were set as follows: 12℃, 80% humidity, 16 hours of light, 8 hours of darkness, and cultured for about 2 weeks.
[0045] 1.4. WGA staining and histological observation Leaves were cut at 48 and 168 hours after inoculation and immersed in 12 mL KOH (1 mol), with 2 μL Tween added. The mixture was incubated overnight at 37°C. The leaves were then stained with Tris (50 mmol, pH 7.5) solution containing 20 μg / mL wheat lectin WGA-FITC for 1 hour. The leaves were then removed and the mycelial infection area was observed under a fluorescence microscope. The infection area was quantified using ImageJ software.
[0046] 1.5. Phenotypic Observation The disease incidence of the plants was investigated 17 days after inoculation, and photos were taken for record-keeping.
[0047] 1.6. Stripe rust biomass detection Leaves were harvested 17 days after inoculation, and DNA was extracted using the CTAB method. The presence of the stripe rust internal reference gene in the DNA template was detected by real-time fluorescent PCR. PstEF1α ) and wheat internal reference gene ( TaEF-1α The CT value of the stripe rust fungus and wheat DNA were used to calculate the relative amount of stripe rust fungus biomass.
[0048] 2. Results Compared with the control group, wheat pretreated with 500 μmol / L luteolin showed a significant increase in endogenous luteolin content. Figure 2 ), at 48 hours and 168 hours after infection by stripe rust fungus, the area of mycelial infection was significantly reduced ( Figure 3 ), 17 days after infection with stripe rust, compared with the control group, the number of stripe rust spore masses on the leaves was significantly reduced by 52.9% ( Figure 4 The biomass of stripe rust fungi decreased significantly by 48.2%. Figure 5 This study demonstrates that exogenous luteolin plays a role in wheat's resistance to stripe rust infection and can slow down the occurrence of stripe rust.
[0049] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for improving wheat stripe rust resistance through exogenous substance treatment, characterized in that, include: S1. Preparation and application of luteolin solution: Weigh 57.3 mg of luteolin, completely dissolve it in 20 mL of anhydrous ethanol to prepare a 10 mmol / L luteolin ethanol solution, and add deionized water to make up to 400 mL to prepare a luteolin solution with a final concentration of 500 μmol / L; When the stripe rust susceptible material Yun 0402 grows to the two-leaf-one-heart stage, spray the upper and lower surfaces of the leaves evenly with the 500 μmol / L luteolin solution once a day for a total of three sprays; S2. Detection of endogenous luteolin content: Weigh 0.1 g of wheat leaves pretreated with 500 μmol / L luteolin, grind with liquid nitrogen, add 0.5 mL of 80% methanol aqueous solution, vortex mix, extract by ultrasonication for 30 min, centrifuge at 12000 rpm for 10 min, take the supernatant, and detect it using ultra-high performance liquid chromatography (Vanquish) and high resolution mass spectrometry (Q Exactive). S3. Stripe rust inoculation and material culture: Wheat pretreated with 500 μmol / L luteolin was inoculated with stripe rust. Stripe rust CYR32 and talc were mixed at a ratio of 1:
20. A certain amount of inoculum was applied evenly to both sides of the leaves using a small brush. The plants were first placed in an incubator at 12℃ and 80% humidity and cultured in the dark for 24 hours. The subsequent conditions were set as follows: temperature 12℃, humidity 80%, 16 hours of light, 8 hours of darkness, and cultured for about 2 weeks. S4. WGA staining and histological observation: Leaves were cut at 48 and 168 hours after inoculation and immersed in 12 mL KOH with 2 μL Tween added. The leaves were then incubated overnight at 37°C. Subsequently, the leaves were stained in Tris solution containing 20 μg / mL wheat lectin WGA-FITC for 1 hour. The leaves were then removed and the mycelial infection area was observed under a fluorescence microscope. The infection area was quantified using ImageJ software. S5. Phenotypic observation: Investigate the disease incidence of plants 17 days after inoculation and take photos for record-keeping; S6. Stripe rust biomass detection: Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The stripe rust internal reference gene in the DNA template was detected by real-time fluorescent PCR. PstEF1α and wheat internal reference gene TaEF-1α The CT value was used to calculate the relative amount of stripe rust fungus and wheat DNA as stripe rust fungus biomass.