Application and method for improving wheat stripe rust resistance through allogenic material treatment

Abstract

The invention belongs to but not limited to the technical field of plant protection, and particularly relates to application and method for improving wheat stripe rust resistance through allogenic material treatment, 250 micromoles of luteolin is used for culturing stripe rust fungus spores, germination of the stripe rust fungus spores is remarkably inhibited, 500 micromoles of luteolin is used for pretreating wheat leaves, spraying is carried out once a day, spraying is carried out three times in total, and the yield of the wheat stripe rust resistance is increased. After the leaves absorb the luteolin, the puccinia striiformis is inoculated, and the result shows that the endogenous luteolin content of the wheat plants treated by the luteolin is increased, the infection area of the puccinia striiformis is remarkably reduced, the number of puccinia striiformis spores on the leaves is remarkably reduced, and the biomass of the puccinia striiformis is remarkably reduced. The invention proves that the occurrence of wheat stripe rust can be inhibited by exogenous luteolin treatment, and a certain reference is provided for stripe rust disease-resistant mechanism research and prevention and control measures.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical field of plant protection, and in particular relates to an application and method of improving wheat stripe rust resistance by treating with exogenous substances. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the oldest crops in cultivation history and is widely cultivated around the world. It is the staple food of more than half of the world's population. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the main disease of wheat. 88% of wheat cultivars in the world are susceptible to stripe rust infection, resulting in a loss of 5.47 million tons of wheat production each year, seriously affecting wheat quality and yield.

[0003] The prevention and control of wheat stripe rust mainly relies on disease-resistant breeding and the use of fungicides. However, due to the variation of pathogens and the irrational use of disease-resistant genes, single disease-resistant genes have become ineffective, which has brought great challenges to disease-resistant breeding. The use of fungicides will increase the resistance of pathogens, and in the context of green prevention and control, the use of chemical agents is not the best choice. Therefore, developing safer, greener and more effective measures is an urgent problem to be solved in the prevention and control of wheat stripe rust.

[0004] Luteolin belongs to the flavonoids and is widely found in plants. It has antioxidant activity and antibacterial effects and plays an important role in plant adversity. For example, under drought stress, luteolin significantly accumulates in drought-tolerant citrus and plays a role in scavenging reactive oxygen; under biological stress, luteolin accumulates more rapidly and significantly in sorghum anthracnose-resistant varieties, and luteolin treatment inhibits the germination of anthracnose fungi; a significant increase in luteolin content was detected in carrot thrips-resistant varieties, and feeding luteolin reduced the growth rate and survival rate of thrips larvae; luteolin also has a strong ability to inhibit the growth of Fusarium graminearum. In summary, luteolin plays an important role in plant disease resistance. Therefore, studying the function of luteolin in wheat stripe rust resistance has certain practical significance for the development of green prevention and control measures for wheat stripe rust and the study of disease resistance mechanisms.

[0005] Existing technology and its shortcomings: Luteolin plays an important role in plant disease resistance, but different microorganisms have different tolerance to luteolin. So far, no research has reported the antibacterial effect of luteolin on stripe rust and its function in wheat resistance to stripe rust. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides an application and method of improving wheat stripe rust resistance by treating with an exogenous substance, so as to solve the problems existing in the above-mentioned background technology. The present invention finds that treating with an exogenous substance can inhibit the germination of stripe rust spores, and applying the substance to wheat leaves at the seedling stage can improve the resistance of wheat to stripe rust and slow down the occurrence of wheat stripe rust.

[0007] The present invention is achieved by using an exogenous substance to improve wheat stripe rust resistance, wherein the exogenous substance is luteolin.

[0008] Furthermore, an exogenous substance could inhibit the germination of stripe rust spores at a concentration of 250 μmol.

[0009] Furthermore, seedling wheat refers to wheat that has grown to the two-leaf and one-heart stage.

[0010] Furthermore, the exogenous substances are applied to wheat leaves at the seedling stage before the stripe rust infection.

[0011] Furthermore, before the application time, the treatment steps are as follows: when the wheat grows to the two-leaf and one-heart stage, the wheat is sprayed with an exogenous substance three times, once a day, and after the leaves absorb it, stripe rust is inoculated.

[0012] Furthermore, the exogenous substances were sprayed on wheat at a concentration of 500 μmol.

[0013] Another object of the present invention is to provide a method for improving wheat stripe rust resistance by treating with exogenous substances, comprising:

[0014] S1. Preparation and application of luteolin solution: Weigh 57.3 mg of luteolin (purchased from Eon Chemical Technology Co., Ltd.), completely dissolve in 20 mL of anhydrous ethanol, prepare a luteolin ethanol solution with a concentration of 10 mmol, and add deionized water to 400 mL to prepare a luteolin solution with a final concentration of 500 μmol. When the stripe rust susceptible material Yun0402 grows to the two-leaf one-heart stage, spray the front and back of the leaves evenly with 500 μmol of luteolin solution, once a day, for a total of three times.

[0015] S2. Detection of endogenous luteolin content: Weigh 0.1 g of wheat leaves pretreated with 500 μmol 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-performance liquid chromatography Vanquish (Thermo, USA) and high-resolution mass spectrometer QExactive (Thermo, USA).

[0016] S3. Stripe rust inoculation and material culture: Wheat pretreated with 500 μmol luteolin was inoculated with stripe rust. The stripe rust CYR32 and talcum powder were mixed in a ratio of 1:20. A certain amount of the fungus was dipped with a small brush and evenly applied on the front and back of the leaves. The plants were first placed in an incubator at a temperature of 12°C and a humidity of 80% for 24 hours in the dark. The subsequent conditions were set to a temperature of 12°C, a humidity of 80%, 16 hours of light, and 8 hours of darkness, and the culture was carried out for about 2 weeks.

[0017] S4. WGA staining and histological observation: Leaves were cut at 48 and 168 hours after inoculation, immersed in 12 mL KOH (1 mol), added with 2 μL Tween, and cultured at 37°C overnight. The leaves were then stained in a Tris (50 mmol, PH 7.5) solution containing 20 μg / mL wheat agglutinin WGA-FITC for 1 hour. The leaves were taken out and the mycelial infection area was observed under a fluorescence microscope. The infection area was quantified using Image J software.

[0018] S5. Phenotypic observation: Investigate the disease status of the plants 17 days after inoculation and take photos to record.

[0019] S6. Stripe rust biomass detection: Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The CT values ​​of the stripe rust reference gene (PstEF1α) and the wheat reference gene (TaEF-1α) in the DNA template were detected by real-time fluorescence PCR, and the relative amounts of stripe rust and wheat DNA were calculated as the stripe rust biomass.

[0020] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0021] First, the beneficial effect of the present invention is that the present invention uses 250 μmol of exogenous luteolin to cultivate stripe rust, and the results show that compared with the control group, luteolin significantly inhibits the germination of stripe rust spores. After pre-treating wheat leaves with 500 μmol of luteolin, stripe rust is inoculated. According to the results of endogenous luteolin content detection, infected leaf histological observation, plant phenotype observation, and stripe rust biomass detection, compared with the control group, the endogenous luteolin content of wheat leaves pre-treated with 500 μmol of luteolin is significantly increased, the area infected by stripe rust is significantly reduced, the spores of wheat plant leaves are significantly reduced, and the stripe rust biomass is significantly reduced. It can be seen that 500 μmol of luteolin can inhibit the infection of stripe rust to wheat, slow down the occurrence of wheat stripe rust, and provide a certain reference for the prevention and control measures of wheat stripe rust and the study of disease resistance mechanism.

[0022] Second, the present invention can be applied in the prevention and treatment of wheat stripe rust, and luteolin can be developed as a raw material for green pesticides.

[0023] Wheat stripe rust is an important disease that affects wheat yield and quality. Existing prevention and control measures mainly rely on the selection of disease-resistant varieties and the spraying of chemical agents. However, the development cycle of disease-resistant varieties is long and there is a problem of resistance decline, while the long-term use of chemical agents may cause environmental pollution and increase the risk of bacterial resistance. Therefore, how to find an efficient, safe and highly operable method for stripe rust prevention and control has become an urgent problem to be solved in the current technical field.

[0024] The present invention provides a technical solution for improving wheat stripe rust resistance by utilizing an exogenous substance, luteolin. By spraying luteolin at the wheat seedling stage (two leaves and one heart stage), not only can the germination of stripe rust spores be significantly inhibited, but also the occurrence of stripe rust can be slowed down, and dependence on chemical pesticides can be reduced, thereby providing a new idea for the green prevention and control of wheat stripe rust.

[0025] Through scientific verification, the present invention found that luteolin can significantly inhibit the germination of stripe rust spores and effectively reduce the infection rate of the pathogen. Especially after spraying 500 μmol of luteolin solution, the number of spores infected on wheat stripe rust leaves was significantly reduced (reduced by 52.9%), and the prevention and control effect was significantly better than traditional chemical agents.

[0026] Unlike chemical pesticides, luteolin is a natural plant extract that is environmentally friendly and not prone to causing drug resistance. The present invention is simple to operate and only requires spraying the leaves of wheat seedlings a few times to achieve efficient prevention and control of stripe rust. The solution is highly safe, easy to operate, and suitable for large-scale promotion and application, significantly improving the sustainability and economic benefits of wheat stripe rust prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the inhibition of the germination rate of stripe rust by exogenous luteolin in Example 1 provided in the embodiments of the present invention;

[0028] Figure 2 The content of endogenous luteolin in wheat leaves after pretreatment with exogenous luteolin according to Example 2 provided in the embodiments of the present invention;

[0029] Figure 3 This is the histological observation of wheat pretreated with exogenous luteolin and inoculated with stripe rust in Example 2 provided in the embodiments of the present invention;

[0030] Figure 4 This is the incidence of wheat stripe rust on the 17th day after pretreatment with exogenous luteolin and inoculation with stripe rust fungi according to Example 2 of the present invention;

[0031] Figure 5 This is the stripe rust biomass detection result of wheat leaves pretreated with exogenous luteolin and inoculated with stripe rust on the 17th day in Example 2 provided in the examples of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] The invention provides application of exogenous substance treatment for improving wheat stripe rust resistance, wherein the exogenous substance is luteolin.

[0034] Furthermore, an exogenous substance could inhibit the germination of stripe rust spores at a concentration of 250 μmol.

[0035] Furthermore, seedling wheat refers to wheat that has grown to the two-leaf and one-heart stage.

[0036] Furthermore, the exogenous substances are applied to wheat leaves at the seedling stage before the stripe rust infection.

[0037] Furthermore, before the application time, the treatment steps are as follows: when the wheat grows to the two-leaf and one-heart stage, the wheat is sprayed with an exogenous substance three times, once a day, and after the leaves absorb it, stripe rust is inoculated.

[0038] Furthermore, the exogenous substances were sprayed on wheat at a concentration of 500 μmol.

[0039] Another object of the present invention is to provide a method for improving wheat stripe rust resistance by treating with exogenous substances, comprising:

[0040] S1. Preparation and application of luteolin solution: Weigh 57.3 mg of luteolin (purchased from Eon Chemical Technology Co., Ltd.), completely dissolve in 20 mL of anhydrous ethanol, prepare a luteolin ethanol solution with a concentration of 10 mmol, and add deionized water to 400 mL to prepare a luteolin solution with a final concentration of 500 μmol. When the stripe rust susceptible material Yun0402 grows to the two-leaf one-heart stage, spray the front and back of the leaves evenly with 500 μmol of luteolin solution, once a day, for a total of three times.

[0041] S2. Detection of endogenous luteolin content: Weigh 0.1 g of wheat leaves pretreated with 500 μmol 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-performance liquid chromatography Vanquish (Thermo, USA) and high-resolution mass spectrometer QExactive (Thermo, USA).

[0042] S3. Stripe rust inoculation and material culture: Wheat pretreated with 500 μmol luteolin was inoculated with stripe rust. The stripe rust CYR32 and talcum powder were mixed in a ratio of 1:20. A certain amount of the fungus was dipped with a small brush and evenly applied on the front and back of the leaves. The plants were first placed in an incubator at a temperature of 12°C and a humidity of 80% for 24 hours in the dark. The subsequent conditions were set to a temperature of 12°C, a humidity of 80%, 16 hours of light, and 8 hours of darkness, and the culture was carried out for about 2 weeks.

[0043] S4. WGA staining and histological observation: Leaves were cut at 48 and 168 hours after inoculation, immersed in 12 mL KOH (1 mol), added with 2 μL Tween, and cultured at 37°C overnight. The leaves were then stained in a Tris (50 mmol, PH 7.5) solution containing 20 μg / mL wheat agglutinin WGA-FITC for 1 hour. The leaves were taken out and the mycelial infection area was observed under a fluorescence microscope. The infection area was quantified using Image J software.

[0044] S5. Phenotypic observation: Investigate the disease status of the plants 17 days after inoculation and take photos to record.

[0045] S6. Stripe rust biomass detection: Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The CT values ​​of the stripe rust reference gene (PstEF1α) and the wheat reference gene (TaEF-1α) in the DNA template were detected by real-time fluorescence PCR, and the relative amounts of stripe rust and wheat DNA were calculated as the stripe rust biomass.

[0046] Example 1 Exogenous luteolin inhibits the germination of stripe rust spores

[0047] 1. Methods

[0048] 1.1. Preparation of luteolin solution

[0049] 57.3 mg of luteolin (purchased from Eon Chemical Technology Co., Ltd.) was weighed and completely dissolved in 20 mL of anhydrous ethanol to prepare a luteolin ethanol solution with a concentration of 10 mmol. 2.5 mL of the luteolin ethanol solution was added to deionized water to make the volume to 100 mL to prepare a luteolin solution with a final concentration of 250 μmol.

[0050] 1.2. Stripe rust germination test

[0051] A certain mass of stripe rust spores was weighed and evenly placed in a culture dish containing 250 μmol of luteolin solution and water (control group), and cultured in the dark at 12°C for 6 hours. The germination state of the spores was observed under a microscope and the germination rate was calculated. Three fields of view were counted for each treatment, and about 150 stripe rust spores were counted in each field of view.

[0052] 2. Results

[0053] The germination rate of stripe rust spores treated with clean water was 19%, and the germination rate of stripe rust spores treated with 250 μmol luteolin solution was 5%, which significantly reduced the germination rate of stripe rust spores ( Figure 1 ), proving that luteolin solution inhibited the normal germination of stripe rust spores.

[0054] Example 2 Exogenous luteolin slows down the occurrence of wheat stripe rust

[0055] Yun 0402 is a local wheat germplasm resource in Yunnan. According to the results of the resistance identification of wheat stripe rust in the field for four consecutive years, Yun 0402 has a stable high sensitivity to stripe rust. The stripe rust resistance identification at the seedling stage showed that Yun 0402 showed a high sensitivity to stripe rust. Therefore, the present invention uses the Yunnan local wheat germplasm Yun 0402 as the research material and uses the inventive method to study the function of exogenous luteolin in wheat resistance to stripe rust.

[0056] 1. Methods

[0057] 1.1. Preparation and application of luteolin solution

[0058] Weigh 57.3 mg of luteolin (purchased from Eon Chemical Technology Co., Ltd.) and completely dissolve it in 20 mL of anhydrous ethanol to prepare a 10 mmol luteolin ethanol solution, and add deionized water to 400 mL to prepare a 500 μmol luteolin solution. When the stripe rust susceptible material Yun0402 grows to the two-leaf one-heart stage, spray the front and back of the leaves evenly with 500 μmol luteolin solution once a day for a total of three times.

[0059] 1.2. Detection of endogenous luteolin content

[0060] 0.1 g of wheat leaves pretreated with 500 μmol luteolin were weighed, ground with liquid nitrogen, added with 0.5 mL of 80% methanol aqueous solution, vortexed, ultrasonically extracted for 30 min, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for detection using ultra-performance liquid chromatography Vanquish (Thermo, USA) and high-resolution mass spectrometer QExactive (Thermo, USA).

[0061] 1.3. Stripe rust inoculation and material culture

[0062] Wheat pretreated with 500 μmol luteolin was inoculated with stripe rust. The stripe rust CYR32 and talcum powder were mixed in a ratio of 1:20. A certain amount of the fungus was dipped with a small brush and evenly applied to the front and back of the leaves. The plants were first placed in an incubator at a temperature of 12°C and a humidity of 80% for 24 hours in the dark. The subsequent conditions were set to a temperature of 12°C, a humidity of 80%, 16 hours of light, and 8 hours of darkness for about 2 weeks.

[0063] 1.4. WGA staining and histological observation

[0064] The leaves at 48 and 168 hours after inoculation were cut and immersed in 12 mL KOH (1 mol), 2 μL Tween was added, and cultured at 37°C overnight. The leaves were then stained in a Tris (50 mmol, PH 7.5) solution containing 20 μg / mL wheat agglutinin WGA-FITC for 1 hour. The leaves were taken out and the mycelial infection area was observed under a fluorescence microscope. The infection area was quantified using Image J software.

[0065] 1.5. Phenotypic observation

[0066] The disease conditions of the plants were investigated 17 days after inoculation and photographed and recorded.

[0067] 1.6. Stripe rust biomass detection

[0068] Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The CT values ​​of the stripe rust reference gene (PstEF1α) and the wheat reference gene (TaEF-1α) in the DNA template were detected by real-time fluorescence PCR, and the relative amounts of stripe rust and wheat DNA were calculated as the stripe rust biomass.

[0069] 2. Results

[0070] Compared with the control group, the endogenous luteolin content of wheat pretreated with 500 μmol luteolin was significantly increased ( Figure 2 ), at 48 hours and 168 hours after stripe rust infection, the mycelium infection area was significantly reduced ( Figure 3), 17 days after the stripe rust infection, the stripe rust spore piles on the leaves were significantly reduced by 52.9% compared with the control group ( Figure 4 ), the biomass of stripe rust was significantly reduced by 48.2% ( Figure 5 ), proving that exogenous luteolin plays a role in wheat resistance to stripe rust infection and can slow down the occurrence of stripe rust.

[0071] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. An application of exogenous substance treatment to improve wheat stripe rust resistance, characterized in that: The exogenous substance is luteolin.

2. The use of exogenous substance treatment to improve wheat stripe rust resistance as claimed in claim 1, characterized in that: Exogenous substances can inhibit the germination of stripe rust spores at a concentration of 250 μmol.

3. The use of exogenous substance treatment to improve wheat stripe rust resistance as claimed in claim 1, characterized in that: Seedling wheat refers to wheat that has grown to the two-leaf and one-heart stage.

4. The use of exogenous substance treatment to improve wheat stripe rust resistance as claimed in claim 1, characterized in that: The exogenous substances were applied to wheat leaves at the seedling stage before the stripe rust infection.

5. The use of exogenous substance treatment to improve wheat stripe rust resistance as claimed in claim 1, characterized in that: Before application, the treatment steps are as follows: when wheat grows to the two-leaf and one-heart stage, spray the wheat with an exogenous substance three times, once a day, and after the leaves absorb it, inoculate it with stripe rust.

6. The use of exogenous substance treatment to improve wheat stripe rust resistance as claimed in claim 1, characterized in that: Exogenous substances were sprayed on wheat at a concentration of 500 μmol.

7. A method for improving wheat stripe rust resistance by treating with exogenous substances, characterized in that: include: S1. Preparation and application of luteolin solution: Weigh 57.3 mg of luteolin and completely dissolve it in 20 mL of anhydrous ethanol to prepare a luteolin ethanol solution with a concentration of 10 mmol, and add deionized water to make the volume to 400 mL to prepare a luteolin solution with a final concentration of 500 μmol; when the stripe rust susceptible material Yun0402 grows to the two-leaf and one-heart stage, spray the front and back of the leaves evenly with 500 μmol of luteolin solution, once a day, for a total of three times; S2. Detection of endogenous luteolin content: Weigh 0.1 g of wheat leaves pretreated with 500 μmol 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 Vanquish (Thermo, USA) and high-resolution mass spectrometer Q Exactive (Thermo, USA); S3. Stripe rust inoculation and material culture: Wheat pretreated with 500 μmol luteolin was inoculated with stripe rust. Stripe rust CYR32 and talcum powder were mixed in a ratio of 1:

20. A certain amount of fungus was dipped into a small brush and evenly applied on the front and back of the leaves. The plants were first placed in an incubator at a temperature of 12°C and a humidity of 80% for 24 hours in the dark. The subsequent conditions were set to a temperature of 12°C, a humidity of 80%, 16 hours of light, and 8 hours of darkness for about 2 weeks. S4. WGA staining and histological observation: Cut the leaves at 48 and 168 hours after inoculation, soak them in 12 mL KOH, add 2 μL Tween, and culture them at 37°C overnight. Then, stain the leaves in Tris solution containing 20 μg / mL wheat agglutinin WGA-FITC for 1 hour. Take out the leaves and observe the mycelial infection area under a fluorescence microscope. Use Image J software to quantify the infection area. S5. Phenotypic observation: 17 days after inoculation, investigate the disease status of the plants and take photos to record; S6. Stripe rust biomass detection: Leaves were cut 17 days after inoculation, and DNA was extracted using the CTAB method. The CT values ​​of the stripe rust reference gene (PstEF1α) and the wheat reference gene (TaEF-1α) in the DNA template were detected by real-time fluorescence PCR, and the relative amounts of stripe rust and wheat DNA were calculated as the stripe rust biomass.

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