CN116267361B - A method for preventing and controlling clubroot of cruciferous crops - Google Patents
A method for preventing and controlling clubroot of cruciferous crops Download PDFInfo
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- CN116267361B CN116267361B CN202310520963.3A CN202310520963A CN116267361B CN 116267361 B CN116267361 B CN 116267361B CN 202310520963 A CN202310520963 A CN 202310520963A CN 116267361 B CN116267361 B CN 116267361B
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protection of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/15—Leaf crops, e.g. lettuce or spinach
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
- A01N43/66—1,3,5-Triazines, not hydrogenated and not substituted at the ring nitrogen atoms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Botany (AREA)
- Plant Pathology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Pest Control & Pesticides (AREA)
- Toxicology (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Cultivation Of Plants (AREA)
Abstract
本发明公开了一种防治十字花科作物根肿病的方法，属于植物病害防治技术领域。所述方法包括在所述十字花科作物的根部施加甲基化转移酶抑制剂的步骤；所述甲基化转移酶抑制剂包括5‑氮杂胞苷。本发明向白菜和油菜植株中施加不同浓度的5‑AzaC，筛选出对植株正常生长影响不显著的药剂浓度，并以此浓度验证外源施加5‑AzaC对白菜和油菜根肿菌的防治效果，实验结果证实，5‑AzaC可明显降低感病植株的发病率和病情指数，且明显推迟根肿病的发病历程，对十字花科作物根肿病有显著的防治效果。本发明为植物根肿病病害提供新的防治途径。
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 disease.
Description
Technical Field
The invention relates to the technical field of plant disease control, in particular to a method for controlling clubroot of cruciferous crops.
Background
Clubroot is a serious soil-borne disease caused by brassica clubroot (Plasmodiophora brassicae), and has great negative influence on the yield of important cruciferous vegetables and oil crops such as Chinese cabbage, rape and the like. The brassica plasmodiophora is parasitic on the root of the cruciferae and can infect the plant in the whole growth period, the earlier the infection is, the heavier the attack is, the swelling of the root of the plant is finally caused, the growth is slow, and the overground part is dehydrated and wilted and even dies. At the same time, it can survive in soil for 15 years in the form of dormant spores. These characteristics also bring difficulties to the prevention and control of clubroot. The current chemical control method for clubroot is still limited to the application of traditional pesticides such as cyazofamid, fluazinam, chlorothalonil and the like.
In recent years, epigenetic inheritance has gradually proved to be one of the important mechanisms for responding to external stress. DNA methylation is a very important and common epigenetic modification in eukaryotes that is involved in many biological processes such as plant growth, response to stress, etc. For example, in the case of Arabidopsis, it was found that the whole genome showed hypomethylation after infection of Arabidopsis roots with nematodes, or that a slightly but widely differentially methylated region was found between Arabidopsis leaves and wild type after infection with Pseudomonas syringae PstDC 3000. It follows that plants do regulate the expression level of genes through apparent modification, thereby rapidly inducing immune responses in plants.
5-Azacytidine (5-AzaC) is a DNA methyltransferase inhibitor capable of reducing the methylation level of genomic DNA and activating the expression of certain silenced genes, and has been widely used in research on methylation of genomic DNA of animals and plants. In plant studies, 5-AzaC has been shown to have an effect on plant vernalization flowering, fruit ripening, secondary metabolite accumulation, and response to stress, but no application of 5-AzaC in controlling cruciferous crop clubroot has been reported.
Disclosure of Invention
The invention aims to provide a method for preventing and treating clubroot of cruciferous crops, which solves the problems in the prior art, and discovers that 5-AzaC can obviously reduce the incidence and disease index of infected plants, obviously delay the incidence course of clubroot, have obvious preventing and treating effects on the clubroot of cruciferous crops and provide a new preventing and treating way for the clubroot of plants.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides a method for preventing and treating clubroot of cruciferous crops, which comprises the step of applying a methyltransferase inhibitor to the roots of the cruciferous crops;
The methyltransferase inhibitor includes 5-azacytidine.
Further, the 5-azacytidine is applied at a concentration of 100. Mu.M.
Further, the cruciferous crops include chinese cabbage and canola.
The invention also provides a medicament for preventing and treating clubroot of cruciferous crops, which comprises 5-azacytidine.
Further, the medicament further comprises an auxiliary material.
Further, the auxiliary materials comprise any one or more of wetting agents, tackifiers, antifreezing agents and dispersing agents.
The invention discloses the following technical effects:
According to the invention, 5-AzaC with different concentrations is applied to cabbage and rape plants, the concentration of the medicament which has no obvious influence on normal growth of the plants is screened, and the control effect of exogenous application of 5-AzaC on cabbage and rape clubroot is verified by the concentration, and experimental results prove that 5-AzaC can obviously reduce the incidence and disease index of infected plants, obviously delay the incidence course of clubroot and have obvious control effect on cruciferous crop clubroot. The invention provides a new control way for plant clubroot diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of exogenous application of different concentrations of 5-AzaC on the normal growth of Peking New No. 3 of Chinese cabbage, wherein A is crown breadth, B is leaf width, C is leaf length, and D is plant height;
FIG. 2 shows the effect of exogenous application of different concentrations 5-AzaC on the normal growth of Westar of rape, wherein A is crown width, B is leaf width, C is leaf length, and D is plant height;
FIG. 3 shows the phenotype of the root of the Chinese cabbage Beijing No. 3 under the stress of the plasmodiophora brassicae;
FIG. 4 is statistics of the occurrence of the Chinese cabbage Beijing No. 3 clubroot under the stress of the clubroot, wherein the occurrence rate is A, the disease index is B, and the content of the clubroot is C;
FIG. 5 shows the Westar root phenotype of canola under stress of P.tumefaciens;
FIG. 6 shows statistics of the occurrence of Westar clubroot of rape under the stress of the clubroot bacteria A, B, disease index.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention performs screening comparison on genes involved in classical establishment of a DNA methylation pathway RdDM pathway in early transcriptome data, discovers that RdDM pathway genes are obviously up-regulated with the lapse of time after the Chinese cabbage is inoculated with the plasmodiophora, judges that the increase of DNA methylation degree possibly accompanies the inoculation of the Chinese cabbage, speculates that the resistance to the plasmodiophora can be improved after exogenously applied methyltransferase inhibitor 5-AzaC inhibits the increase of genome methylation of the Chinese cabbage, and particularly carries out the following research.
Example 1 verification of the Effect of 5-AzaC on plant Normal growth
1. Accelerating germination
Placing two pieces of filter paper in a culture dish, pouring a proper amount of distilled water, uniformly spreading Chinese cabbage seeds Beijing No. 3 (national ly-examined vegetable 2001007, national institute of agriculture and forestry sciences, beijing) and rape seeds Westar (cabbage type rape, national oil crop middle-stage warehouse, national institute of agricultural sciences, oil crop institute, etc.) on the filter paper, and accelerating germination in a dark warm environment for 2 days.
2. Sowing seeds
The sprouted sprouts are sowed in a 72-hole tray and managed according to the normal water and fertilizer conditions.
3. Exogenous application 5-AzaC
The initial 0d of 5-Azac is used after 20d sowing, and 5-AzaC of clear water, 50 mu M, 100 mu M, 150 mu M and 200 mu M concentration are respectively used at 0d, 7d and 14d by injection.
4. Physiological index measurement
To determine whether 5-AzaC exogenous application would affect normal plant growth, three groups of four treatments were each assayed for plant height, crown width, leaf length, leaf width-related physiological index 0d, 8d, 23d, 37d post application, three biological replicates were performed for each treatment. The plant height and the crown width are the height of the overground part and the plant unfolding width when the plant presents natural morphology. The leaf length and the leaf width are measured by selecting the outermost leaf of the plant, the leaf length refers to the length from the bottom of the leaf stalk to the highest point of the leaf margin, and the leaf width refers to the length of the widest position of the outer leaf.
5. Conclusion of the experiment
The results are shown in fig. 1 and 2, the effect of the applied concentration of 5-AzaC on the cabbage plants shows the S-shaped effect on plant height, leaf shape and the like, the plants with the applied concentrations of 50 mu M, 150 mu M and 200 mu M show dwarfing of the plants, the concentration of 100 mu M has no obvious effect, and the statistical difference is shown on the measured data. Thus, the present invention selects 5-AzaC at a concentration of 100. Mu.M for use in verifying its effect on plant clubroot.
Example 2 verification of the Effect of 5-AzaC on plant clubroot
1. Accelerating germination
Placing two pieces of filter paper in a culture dish, pouring a proper amount of distilled water, uniformly spreading the Chinese cabbage seeds Beijing Xin No. 3 and the rape seeds Westar on the filter paper, and accelerating germination in a dark and warm environment for 2 days.
2. Sowing seeds
The sprouted sprouts are sowed in a 72-hole tray and managed according to the normal water and fertilizer conditions. After 20d, the rhizomatous bacteria are inoculated.
3. Inoculating bacteria
(1) The preparation method of the plasmodiophora radicle bacterial liquid comprises weighing plasmodiophora radicle (the mycorrhiza is collected from Liaoning citizens) from a refrigerator at-40 ℃, identifying as physiological micro-species Pb4 of the plasmodiophora brassicae by an SCD identification system, filling the plasmodiophora radicle into a self-sealing bag, placing the self-sealing bag under normal temperature, then placing the mycorrhiza into a juicer which is soaked and sterilized in a potassium permanganate solution for 30min in advance, and adding distilled water for grinding. Placing the sheared clean 8 layers of gauze on the mouth of a sterilized wide-mouth bottle, pouring the bacterial liquid into the bottle for filtration, and collecting the bacterial liquid in a clean container for standby after uniform mixing.
(2) The concentration detection of the plasmodiophora comprises the steps of taking 10 mu L of bacterial liquid in a centrifuge tube, adding 2 mu L of aniline blue and 780 mu L of sterilized water, uniformly mixing, and standing for 2 minutes. Sterilizing the blood cell counting plate with 75% alcohol, wiping with a piece of mirror wiping paper, sucking 1 μl of mixed liquid drop on the blood cell counting plate, covering with a cover glass, counting the number of spores under an optical microscope, diluting to 1× 7/mL, and inoculating.
(3) Inoculating the rhizomatous bacteria liquid, namely inoculating the rhizomatous bacteria by adopting an injection method after 20d sowing, sucking the uniformly shaken bacteria liquid by using a10 mL syringe, and injecting 2mL of the rhizomatous bacteria liquid near the root of each seedling. 2mL of clean water was injected as a control group (CK).
4. Exogenous application 5-AzaC
100 Mu M5-AzaC is administered by injection at intervals of six hours after inoculation of the rhizomes for avoiding dilution of the rhizomes at 0d after inoculation of the rhizomes for 5-AzaC.
5. Statistics of morbidity and index of disease
Disease resistance investigation and statistics of plants are carried out at 0d, 8d, 23d and 37d after inoculation.
Incidence (%) = number of plants to be transplanted/total number of plants×100%;
Disease index = [ Σ (number of disease plants at each stage×value of disease grade)/(total number of investigation×highest value) ]×100%;
Grading the disease severity, namely, normal growth of the plant, no visible root nodule at the main root and no disease at the lateral root, namely, the root is not diseased, namely, 0 grade, the lateral root has nodules, namely, 1 grade, the lateral root has large nodules or the main root grows to generate nodules, namely, 2 grade, the normal growth of the plant is affected, and the main root has obvious large nodules, namely, 3 grade.
6. Identification of content level of root of Chinese cabbage 'Beijing Xin No. 3' relative to clubroot
(1) CTAB method for extracting plant root DNA
15 Plants are randomly selected at different time points (0, 8, 23 and 37 d), each group of plants with relatively close growth conditions are gently taken out from a 72-hole tray, the roots are carefully cleaned by clean water, and impurities such as peat, perlite and the like are not attached to root hairs. Collecting the cleaned root sample for later use.
The cleaned root tissue is put into a mortar to be ground into powder, then is transferred into a 2mL centrifuge tube, 600 mu L of CTAB buffer solution preheated in a constant temperature water bath kettle at a temperature of 65 ℃ is added, and the mixture is gently inverted and mixed uniformly. Then the centrifuge tube is inserted into the buoy and is mixed up and down for a plurality of times at intervals of 10 minutes in a water bath at 65 ℃ for 45-60 minutes. After the water bath 600. Mu.L of the extract (isoamyl alcohol: chloroform=1:24) was added to the fume hood, and after shaking up and down, immediately 12000rpm,4℃and centrifugation was carried out for 15min. mu.L of the upper aqueous phase was pipetted into a new 1.5mL centrifuge tube, 900. Mu.L of absolute ethanol was added and the mixture was allowed to stand at-20℃for 40min. After removal, the mixture was centrifuged at 12000rpm at 4℃for 10min, the supernatant was discarded, and 600. Mu.L of 70% ethanol was added. Centrifuge at 12000rpm,4 ℃ for 3min, discard supernatant and remove residual ethanol as much as possible, air dry in a fume hood. After air drying, 100-200 mu L of sterilized water is added, vortex oscillation is carried out, DNA is fully mixed, and standing and dissolution are carried out for 30min at normal temperature. Then, agarose gel electrophoresis is used for detecting the DNA quality, and an enzyme-labeled instrument is used for measuring the purity and the concentration of the DNA. The DNA solution was stored in a-20℃refrigerator for use.
(2) Real-time fluorescent quantitative PCR detection of DNA content of plasmodiophora
The real-time fluorescence quantification method is adopted, and the DNA content of the rhizomatous bacteria is calibrated by utilizing the expression quantity of Pb genes. The primers used were Pb-F5'-AAACAACGAGTCAGCTTGAATGC-3' and Pb-R5'-AGGACTTGGCTGCGGAT CAC-3'.
The PCR reaction system comprises:
TABLE 1
PCR procedure:
TABLE 2
7. Conclusion of the experiment
Exogenous administration of 5-AzaC delays the course of the clubroot disease.
In disease resistance investigation statistics, susceptible varieties Beijing No. 3 and Westar are serious in disease, the incidence rate almost reaches 100%, the incidence rate of 5-AzaC is about 60%, and the disease index is also obviously reduced. And the treatment group 5-AzaC had significantly delayed the course of the late clubroot compared to the Pb4 group (fig. 3, 4,5 and 6).
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (5)
1. A method of controlling clubroot in cruciferous crops comprising the step 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 for reducing the methylation level of the genome DNA of crucifer crops;
the 5-azacytidine was applied at a concentration of 100. Mu.M.
2. The method of claim 1, wherein the cruciferous crop comprises chinese cabbage and canola.
3. A medicament for controlling clubroot of cruciferous crops, characterized in that the medicament comprises 5-azacytidine;
The 5-azacytidine is used for reducing the methylation level of genomic DNA of crucifers.
4. A medicament according to claim 3, characterized in that it further comprises an auxiliary material.
5. The pharmaceutical formulation of claim 4, wherein the adjuvant comprises any one or more of a wetting agent, a viscosity increasing agent, an anti-freezing agent, and a dispersing agent.
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