CN116267361A

CN116267361A - Method for preventing and treating clubroot of cruciferous crops

Info

Publication number: CN116267361A
Application number: CN202310520963.3A
Authority: CN
Inventors: 李晓楠; 郑敬一; 朴钟云; 魏金康
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2023-05-10
Filing date: 2023-05-10
Publication date: 2023-06-23

Abstract

The invention discloses a method for preventing and treating clubroot of cruciferous crops, and belongs to the technical field of plant disease prevention and treatment. The method comprises the step of applying a methyltransferase inhibitor to the roots of the crucifer crop; the methyltransferase inhibitor includes 5-azacytidine. 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 the 5-AzaC can obviously reduce the incidence and disease index of infected plants, obviously delay the incidence course of clubroot and has obvious control effect on cruciferous crop clubroot. The invention provides a new control way for plant clubroot diseases.

Description

Method for preventing and treating clubroot of cruciferous crops

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 study of Arabidopsis thaliana, it was found that after infection of Arabidopsis thaliana roots with nematodes, the whole genome showed hypomethylation; or after infection of Arabidopsis thaliana leaves with Pseudomonas syringae PstDC 3000, a slight but extensive differential methylation region was found to occur from the wild type. 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 that reduces the level of genomic DNA methylation and activates 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 is 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, has obvious preventing and treating effects on the clubroot of cruciferous crops, and provides 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 the 5-AzaC can obviously reduce the incidence and disease index of infected plants, obviously delay the incidence course of clubroot and has 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 is a graph showing the effect of exogenous application of 5-AzaC at different concentrations on the normal growth of Peking New No. 3 Chinese cabbage; a: a crown web; b: leaf width; c: leaf length; d: plant height;

FIG. 2 is a graph showing the effect of exogenous application of 5-AzaC at various concentrations on the normal growth of Westar in canola plants; a: a crown web; b: leaf width; c: leaf length; d: 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; a: incidence of disease; b: index of condition; c: the content of root plasmodiophora radicis;

FIG. 5 shows the Westar root phenotype of canola under stress of P.tumefaciens;

FIG. 6 is statistics A of the disease states of Westar clubroot of rape under stress of clubroot: incidence of disease; b: index of disease.

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 vegetable examination 2001007, national institute of agriculture and forestry, beijing city, academy of sciences of agriculture and forestry, vegetable research center) and rape seeds Westar (cabbage type rape, national oil crop mid-term stock, national institute of agricultural sciences of oil crops) 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 administration of 5-AzaC

The initial 0d of 5-Azac was used after 20d sowing, and 5-Azac was administered at concentrations of 50. Mu.M, 100. Mu.M, 150. Mu.M, and 200. Mu.M in clear water, 50. Mu.M, 100. Mu.M, and 14d by injection, respectively.

4. Physiological index measurement

To determine if 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 after application, and 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 FIGS. 1 and 2, the effect of the applied concentration of 5-AzaC on the cabbage plants showed an "S" effect on plant height, leaf profile, etc., the application of 50. Mu.M and 150. Mu.M, 200. Mu.M concentration showed dwarfing of the plants, 100. Mu.M concentration did not show significant effect, and statistical differences were 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 validation 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) Preparation of the plasmodiophora radicis bacterial liquid: weighing the mycorrhizal tumefaciens (the mycorrhizal is collected from Liaoning citizens) from a refrigerator at the temperature of-40 ℃, identifying the mycorrhizal tumefaciens as a physiological micro-seed Pb4 of the brassica tumefaciens by an SCD identification system, filling the mycorrhizal into a self-sealing bag, placing the self-sealing bag under the normal temperature condition, then placing the mycorrhizal into a juicer which is soaked in a potassium permanganate solution in advance and disinfected for 30min, 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) Detecting the concentration of the rhizopus). 10. Mu.L of the bacterial liquid was placed in a centrifuge tube, and 2. Mu.L of aniline blue and 780. Mu.L of sterilized water were added to the tube, followed by mixing and standing for 2 minutes. Sterilizing the blood cell counting plate with 75% alcohol, wiping with a piece of mirror paper, sucking 1 μl of mixed liquid drop onto the blood cell counting plate, covering with a cover glass, counting spore number under an optical microscope, and diluting to 1×10 ⁷ Inoculating bacteria after/mL.

(3) Inoculating the rhizopus bacteria liquid: after 20d of sowing, the rhizomatous bacteria is inoculated by adopting an injection method, and after the uniformly shaken bacterial liquid is sucked by a 10mL syringe, 2mL of rhizomatous bacterial liquid is injected to the root of each seedling. 2mL of clean water was injected as a control group (CK).

4. Exogenous administration of 5-AzaC

100. Mu.M of 5-AzaC was administered by injection at intervals of six hours after inoculation of 0d, 7d, and 14d to avoid dilution of the plasmodiophora radiata bacterial solution at 0 d.

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: the plants grow normally, no visible root nodule exists in the main root and the lateral root, namely, the root is not diseased, and the grade is marked as grade 0; the lateral root has small tumors and is marked as grade 1; the lateral root grows with a large tumor or the main root grows with a small tumor, which is marked as grade 2; the normal growth of the plants is affected, and the main roots have obvious large tumors which are marked as grade 3.

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.

Placing the cleaned root tissue into a mortar, grinding to powder, transferring to a 2mL centrifuge tube, adding 600 mu L of CTAB buffer solution preheated in a constant-temperature water bath kettle at a temperature of 65 ℃, and gently inverting and uniformly mixing. Then the centrifuge tube is inserted into the buoy and is mixed up and down for a plurality of times at intervals of 10min in the water bath of 65 ℃ for 45-60 min. 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, the mixture was immediately centrifuged at 12000rpm at 4℃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 in a refrigerator. After removal, the mixture was centrifuged at 12000rpm at 4℃for 10 minutes, 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, and 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 further use.

(2) Real-time fluorescent quantitative PCR (polymerase chain reaction) detection of DNA (deoxyribonucleic acid) 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 were used as follows: pb-F:5'-AAACAACGAGTCAGCTTGAATGC-3'; pb-R:5'-AGGACTTGGCTGCGGAT CAC-3'.

The PCR reaction system comprises:

TABLE 1

PCR procedure:

TABLE 2

7. Conclusion of the experiment

Exogenous administration of 5-AzaC delayed the course of 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 application is about 60%, and the disease index is also obviously reduced. And the incidence of the post-clubroot disease was significantly delayed in the 5-AzaC treated group compared to the Pb4 group (FIGS. 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

1. A method of controlling clubroot in cruciferous crops comprising the step of applying a methyltransferase inhibitor to the roots of the cruciferous crop;

the methyltransferase inhibitor includes 5-azacytidine.

2. The method of claim 1, wherein the 5-azacytidine is applied at a concentration of 100 μm.

3. The method of claim 1, wherein the cruciferous crop comprises chinese cabbage and canola.

4. A medicament for controlling clubroot of cruciferous crops, characterized in that the medicament comprises 5-azacytidine.

5. The medicament of claim 4, further comprising an adjuvant.

6. The pharmaceutical formulation of claim 5, wherein the adjuvant comprises any one or more of a wetting agent, a viscosity increasing agent, an anti-freezing agent, and a dispersing agent.