A method for preventing and controlling clubroot of cruciferous crops

Abstract

The present invention discloses a method for preventing and treating root knot disease of cruciferous crops, and belongs to the technical field of plant disease prevention and control. The method includes the step of applying a methyltransferase inhibitor to the root of the cruciferous crop; the methyltransferase inhibitor includes 5-azacytidine. The present invention applies different concentrations of 5-AzaC to cabbage and rape plants, screens out the concentration of the agent that has no significant effect on the normal growth of the plant, and verifies the control effect of exogenously applied 5-AzaC on cabbage and rape root knot fungi at this concentration. The experimental results confirm that 5-AzaC can significantly reduce the incidence rate and disease index of susceptible plants, and significantly delay the course of root knot disease, and has a significant control effect on root knot disease of cruciferous crops. The present invention provides a new control method for plant root knot diseases.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, in particular to a method for preventing and controlling clubroot disease of cruciferous crops. Background Art

[0002] Clubroot is a serious soil-borne disease caused by Plasmodiophora brassicae, which has a huge negative impact on the yield of important cruciferous vegetables and oil crops such as Chinese cabbage and rapeseed. Plasmodiophora brassicae specifically parasitizes the roots of cruciferous plants and can infect plants throughout the entire growth period. The earlier the infection, the more severe the disease, which eventually causes the plant roots to swell, grow slowly, and the aboveground parts to wilt due to water loss, and even die. At the same time, it can survive in the soil in the form of dormant spores for 15 years. These characteristics also bring difficulties to the prevention and control of clubroot. At present, chemical control methods for clubroot are still limited to the application of traditional pesticides such as cyazofamid, fluazinam and thiophanate-methyl.

[0003] In recent years, epigenetics has gradually been proven to be one of the important mechanisms for responding to external stress. DNA methylation is a very important and common epigenetic modification in eukaryotes, which is involved in many biological processes such as plant growth and development, and response to stress. For example, in the study of Arabidopsis, it was found that after the roots of Arabidopsis were infected with nematodes, the whole genome showed low methylation; or after the leaves of Arabidopsis were infected with Pseudomonas syringae Pst DC3000, slight but extensive differential methylation regions were found between it and the wild type. This shows that plants do regulate the expression level of genes through epigenetic modifications, thereby quickly inducing plant immune responses.

[0004] 5-AzaC is a DNA methyltransferase inhibitor that can reduce the level of genomic DNA methylation and activate the expression of certain silent genes. It has been widely used in the study of DNA methylation in animal and plant genomes. In plant research, 5-AzaC has been shown to have an effect on plant vernalization and flowering, fruit ripening, accumulation of secondary metabolites, and response to stress, but there is no report on the application of 5-AzaC in the prevention and control of clubroot in cruciferous crops. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preventing and treating clubroot of cruciferous crops to solve the problems existing in the above-mentioned prior art. The present invention finds that 5-AzaC can significantly reduce the incidence and disease index of susceptible plants and significantly delay the onset of clubroot, and has a significant preventive and therapeutic effect on clubroot of cruciferous crops, providing a new preventive and therapeutic approach for plant clubroot diseases.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preventing and controlling clubroot of cruciferous crops, comprising the steps of applying a methyltransferase inhibitor to the roots of the cruciferous crops;

[0008] The methyltransferase inhibitors include 5-azacytidine.

[0009] Furthermore, the applied concentration of 5-azacytidine is 100 μM.

[0010] Furthermore, the cruciferous crops include cabbage and rapeseed.

[0011] The present invention also provides a medicament for preventing and treating clubroot disease of cruciferous crops, wherein the medicament comprises 5-azacytidine.

[0012] Furthermore, the medicament further comprises excipients.

[0013] Furthermore, the auxiliary materials include any one or more of a wetting agent, a viscosity increasing agent, an antifreeze agent and a dispersant.

[0014] The present invention discloses the following technical effects:

[0015] The present invention applies different concentrations of 5-AzaC to cabbage and rape plants, screens out the concentration of the agent that has no significant effect on the normal growth of the plants, and verifies the control effect of exogenously applied 5-AzaC on cabbage and rape root knot fungi at this concentration. The experimental results confirm that 5-AzaC can significantly reduce the incidence rate and disease index of susceptible plants, and significantly delay the course of root knot disease, and has a significant control effect on root knot disease of cruciferous crops. The present invention provides a new control method for plant root knot disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 Effects of exogenous application of different concentrations of 5-AzaC on the normal growth of Chinese cabbage Beijing New 3; A: crown width; B: leaf width; C: leaf length; D: plant height;

[0018] Figure 2 Effects of exogenous application of different concentrations of 5-AzaC on the normal growth of rapeseed Westar; A: crown width; B: leaf width; C: leaf length; D: plant height;

[0019] Figure 3The root phenotype of Chinese cabbage Beijing New 3 under the stress of plasmodium flavescentiae;

[0020] Figure 4 The statistics of the incidence of Beijing Xin No. 3 clubroot disease in Chinese cabbage under the stress of clubroot fungi; A: incidence; B: disease index; C: content of clubroot fungi in the roots;

[0021] Figure 5 This is the root phenotype of rapeseed Westar under the stress of plasmodium fumigatus;

[0022] Figure 6 The statistics of Westar clubroot disease in rapeseed under the stress of clubroot fungus A: incidence rate; B: disease index. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] In the early transcriptome data, the present invention screened and compared the genes involved in the classic RdDM pathway for establishing the DNA methylation pathway, and found that after the Chinese cabbage was inoculated with root-knot fungi, the RdDM pathway genes had a significant up-regulation in expression over time. It was thus judged that the inoculation of the Chinese cabbage with the fungi might be accompanied by an increase in the degree of DNA methylation. It was speculated that after the exogenous application of the methyltransferase inhibitor 5-AzaC to inhibit the increase in the whole genome methylation of the Chinese cabbage, the resistance to root-knot fungi might be improved, and the following research was carried out.

[0029] Example 1 Verification of the effect of 5-AzaC on normal plant growth

[0030] 1. Germination

[0031] Place two filter papers in a culture dish, pour in an appropriate amount of distilled water, evenly spread Chinese cabbage seeds Beijing New No. 3 (National Approved Vegetable No. 2001007, Vegetable Research Center of Beijing Academy of Agricultural and Forestry Sciences) and rapeseed seeds Westar (Brassica napus variety, National Oil Crop Germplasm Medium-term Bank, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences) on the filter paper, and place in a dark and warm environment for germination for 2 days.

[0032] 2. Planting seeds

[0033] Sow the stimulated seedlings in 72-hole plug trays and manage them according to normal water and fertilizer conditions.

[0034] 3. Exogenous administration of 5-AzaC

[0035] The starting day for the application of 5-Azac was 20 days after sowing, and 5-AzaC at concentrations of 50 μM, 100 μM, 150 μM, and 200 μM were injected on days 0, 7, and 14, respectively.

[0036] 4. Physiological index measurement

[0037] To determine whether the exogenous application of 5-AzaC would affect the normal growth of plants, the physiological indicators related to plant height, crown width, leaf length and leaf width were measured for three groups of plants and four treatments at 0d, 8d, 23d and 37d after application, and three biological replicates were performed for each group of plants and each treatment. Plant height and crown width refer to the height of the aboveground part and the expansion range of the plant when the plant presents a natural form. Leaf length and leaf width are measured from the outermost leaf of the plant. Leaf length refers to the length from the bottom of the petiole to the highest point of the leaf edge, and leaf width refers to the length at the widest position of the outer leaf.

[0038] 5. Experimental Conclusion

[0039] The results are as follows Figure 1 , Figure 2As shown, the effect of the applied concentration of 5-AzaC on the cabbage plants showed its "S" effect on plant height, leaf shape, etc., and the plants applied with 50μM, 150μM, and 200μM concentrations showed dwarfing of the plants, and the concentration of 100μM had no significant effect, showing statistical differences in the measured data. Therefore, the present invention selects 5-AzaC with a concentration of 100μM to verify its effect on plant clubroot.

[0040] Example 2 Verification of the effect of 5-AzaC on plant clubroot

[0041] 1. Germination

[0042] Place two filter papers in a culture dish, pour in an appropriate amount of distilled water, evenly spread Chinese cabbage seeds Beijing New No. 3 and rapeseed seeds Westar on the filter paper, and place in a dark and warm environment for germination for 2 days.

[0043] 2. Planting seeds

[0044] The stimulated seedlings were sown in 72-hole plug trays and managed under normal water and fertilizer conditions. After 20 days, the root knot fungus was inoculated.

[0045] 3. Inoculation

[0046] (1) Preparation of root-knot fungus liquid: Weigh the root-knot fungus roots (the roots were collected from Xinmin, Liaoning, and identified as Brassica root-knot fungus physiological race Pb4 by the SCD identification system) from a -40°C refrigerator, put them into a ziplock bag and place them at room temperature. Then put the roots into a juicer that had been previously disinfected with potassium permanganate solution for 30 minutes, add distilled water and grind them. Place the clean 8-layer yarn cut on the mouth of a disinfected wide-mouth bottle, pour the liquid into it and filter it, mix it evenly, and collect it in a clean container for later use.

[0047] (2) Detection of root knot fungus concentration: Take 10 μL of bacterial solution in a centrifuge tube, add 2 μL of aniline blue and 780 μL of sterile water, mix well and let stand for 2 minutes. Disinfect the blood cell counting plate with 75% alcohol, then wipe it dry with lens paper, take 1 μL of the mixed solution and drop it on the blood cell counting plate, cover it with a coverslip, and then count the number of spores under an optical microscope and dilute it to 1×10 7 / mL and then inoculate.

[0048] (3) Inoculation of root-knot fungus solution: 20 days after sowing, root-knot fungus was inoculated by injection. After the shaken solution was sucked up with a 10 mL syringe, 2 mL of root-knot fungus solution was injected near the root of each seedling. The control group (CK) was injected with 2 mL of clean water.

[0049] 4. Exogenous administration of 5-AzaC

[0050] 100 μM 5-AzaC was injected at 0, 7, and 14 days after inoculation. To avoid dilution of the root-knot bacteria solution at 0 days after inoculation, 5-AzaC was injected at intervals of six hours after inoculation of the root-knot bacteria.

[0051] 5. Statistics of morbidity and disease index

[0052] The disease resistance of the plants was investigated and statistically analyzed at 0d, 8d, 23d and 37d after inoculation.

[0053] Incidence rate (%) = number of diseased plants / total number of plants × 100%;

[0054] Disease index = [∑ (number of diseased plants at each level × value of the disease level) / (total number of plants surveyed × highest level value)] × 100%;

[0055] Disease severity classification: if the plant grows normally and there are no visible nodules on the main root and lateral roots, that is, the roots are not diseased, it is recorded as level 0; if there are small nodules on the lateral roots, it is recorded as level 1; if there are large nodules on the lateral roots or small nodules on the main root, it is recorded as level 2; if the normal growth of the plant is affected and there are obvious large nodules on the main root, it is recorded as level 3.

[0056] 6. Identification of relative clubroot bacteria content in the roots of Chinese cabbage "Beijing New No. 3" susceptible to clubroot

[0057] (1) CTAB method to extract plant root DNA

[0058] Randomly select 15 plants with similar growth and development conditions at different time points (0, 8, 23, and 37 days) in each group, gently remove the plants from the 72-hole tray, and carefully wash the roots with clean water. Do not let the roots adhere to impurities such as peat and perlite. Collect the cleaned root samples for later use.

[0059] Put the cleaned root tissue into a mortar and grind it into powder, then transfer it to a 2mL centrifuge tube, add 600μL of CTAB buffer solution preheated in a 65℃ constant temperature water bath, and gently invert to mix. Then insert the centrifuge tube into the float and bathe it in 65℃ water bath for 45-60min, inverting it upside down several times every 10min to mix it. After the water bath, add 600μL of extraction solution (isoamyl alcohol: chloroform = 1:24) in the fume hood, shake it up and down to mix it, and immediately centrifuge it at 12000rpm, 4℃ for 15min. Pipette 450μL of the upper aqueous phase into a new 1.5mL centrifuge tube, add 900uL of anhydrous ethanol, and let it stand in a -20℃ refrigerator for 40min. After taking it out, centrifuge it at 12000rpm, 4℃ for 10min, discard the supernatant, and add 600μL of 70% ethanol. Centrifuge at 12000rpm, 4℃ for 3min, discard the supernatant and remove the residual ethanol as much as possible, and dry in the fume hood. After drying, add 100-200μL sterile water, vortex and oscillate, mix the DNA thoroughly, and let it stand at room temperature for 30min to dissolve. Then use agarose gel electrophoresis to detect the quality of DNA, and use an enzyme marker to determine the purity and concentration of DNA. The DNA solution is stored in a -20℃ refrigerator for future use.

[0060] (2) Real-time fluorescence quantitative PCR detection of root knot fungus DNA content

[0061] The real-time fluorescence quantitative method was used to calibrate the DNA content of root knot fungi using the expression level of the Pb gene. The primers used were as follows: Pb-F: 5'-AAACAACGAGTCAGCTTGAATGC-3'; Pb-R: 5'-AGGACTTGGCTGCGGAT CAC-3'.

[0062] PCR reaction system composition:

[0063] Table 1

[0064]

[0065] PCR procedure:

[0066] Table 2

[0067]

[0068]

[0069] 7. Experimental Conclusion

[0070] Exogenous application of 5-AzaC delayed the course of clubroot disease.

[0071] In the disease resistance survey, the susceptible varieties Beijing New No. 3 and Westar were seriously susceptible to the disease, with an incidence rate of almost 100%, while the incidence rate after the application of 5-AzaC was around 60%, and the disease index was also significantly reduced. In addition, the course of the late clubroot disease in the 5-AzaC treatment group was significantly delayed compared to the Pb4 group ( Figure 3 , Figure 4 , Figure 5 and Figure 6 ).

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preventing and treating clubroot of cruciferous crops, characterized in that: The method comprises the steps of applying a DNA methyltransferase inhibitor to the roots of the cruciferous crop; The DNA methyltransferase inhibitor is 5-azacytidine; The 5-azacytidine is used to reduce the methylation level of genomic DNA in cruciferous crops; The applied concentration of 5-azacytidine was 100 μM.

2. The method according to claim 1, characterized in that The cruciferous crops include cabbage and rapeseed.

3. A medicament for preventing and treating clubroot of cruciferous crops, characterized in that: The agent comprises 5-azacytidine; The 5-azacytidine is used for reducing the genomic DNA methylation level of cruciferous crops.

4. The medicament according to claim 3, characterized in that The medicament further comprises excipients.

5. The medicament according to claim 4, characterized in that The auxiliary materials include any one or more of a wetting agent, a tackifier, an antifreeze agent and a dispersant.

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