CN115997768A - Plant immunity induced resistance agent for preventing pine wood nematode disease and preparation method thereof - Google Patents
Plant immunity induced resistance agent for preventing pine wood nematode disease and preparation method thereof Download PDFInfo
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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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Abstract
The invention discloses a plant immunity inducer for preventing pine wood nematode disease and a preparation method thereof, wherein the immunity inducer comprises chitosan oligosaccharide, a synergistic agent and a solvent; the synergistic agent is organic silicon, the solvent is an organic solvent, and the types of the organic solvents in the solvent are at least one; the preparation method comprises the following steps: the method comprises the following steps: firstly quantifying a solvent, and adding chitosan oligosaccharide into the solvent for dissolution after the solvent is in a certain proportion; step two: adding a quantitative synergist into the dissolved medicament obtained in the last step, and uniformly stirring to obtain an immunity induction agent; the plant immunity inducer comprises chitosan oligosaccharide as an active ingredient, and the chitosan oligosaccharide acts on pine trees and can induce the expression of related resistance genes, so that the disease resistance of the pine trees is improved.
Description
Technical Field
The invention relates to the technical field of forest disease control, in particular to a plant immunity inducer for preventing pine wood nematode disease and a preparation method thereof.
Background
Pine wood nematode disease, also called pine tree wilting disease, is a very large destructive disease which is parasitic in pine tree bodies by pine wood nematodes Bursaphelenchus xylophilus (Steiner & Buhrer) and causes rapid death of trees, and belongs to an important national quarantine object, which is called as a smokeless forest fire. Since pine wood nematode disease is first discovered in 1982, china is gradually expanded to a plurality of provinces, causes huge loss of forestry economy and forest ecology and serious damage of natural landscapes, and forms serious threat to pine forests in vast suitable areas of China.
The existing pine wood nematode disease epidemic prevention adopts an integrated prevention and control strategy which takes the cleaning of dead pine trees as core measures and takes the medium insect medicament prevention and control, perforation and drug injection and the like as auxiliary measures, and the important point is that the transmission path of the pine wood nematode disease is cut off, and the method does not relate to how to excite the immunity of the pine wood nematode disease to the pine wood nematode disease.
The plant immunity inducer is also called as plant vaccine, is a new field of vaccine engineering technology after human vaccine and animal vaccine, and is a new practice for scientists to scientifically control plant diseases and insect pests on the basis of revealing the relation theory of plant, plant diseases and insect pests and biological pesticides. The plant immunity inducer or the metabolite thereof has no direct bactericidal, insecticidal or antiviral activity or has very low activity, but can stimulate the immune defense system of plants so as to promote the plants to generate corresponding resistance. Compared with traditional pesticides, the plant immunity inducer has the characteristics of broad spectrum, low toxicity, no drug resistance risk and the like. At present, the corresponding products have been developed for the plant immunity inducer for partial crops, but the development of the plant immunity inducer for pine wood nematode disease is still in blank zone at present.
Chitosan oligosaccharide is a polysaccharide which is a main product degraded by chitosan or chitin through deacetylation/depolymerization of chemical hydrolysis or enzymolysis processes. It has been reported that chitosan oligosaccharide induces resistance of arabidopsis to pseudomonas tomato DC3000 and tobacco mosaic virus; the fluorinated chitosan oligosaccharide derivative can effectively kill plant root knot nematode in vitro and has good control effect on plant root knot nematode in vivo in cucumber.
Disclosure of Invention
The invention aims to provide a plant immunity inducer for preventing pine wood nematode disease and a preparation method thereof, which are used for solving the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a plant immunity inducer for preventing pine wood nematode disease comprises chitosan oligosaccharide, synergist and solvent; the synergist is organic silicon, the solvent is an organic solvent, and the types of the organic solvents in the solvent are at least one.
Specifically, the mass percentages of the components are as follows: 0.1 to 3 percent of chitosan oligosaccharide, 0.05 to 0.1 percent of synergist and the balance of solvent; the solvent is one or more of styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine.
The preparation method of the plant immunity inducer comprises the following steps:
step one: firstly quantifying a solvent, and adding chitosan oligosaccharide into the solvent for dissolution after the solvent is in a certain proportion;
step two: and adding the quantitative synergist into the dissolved medicament obtained in the last step, and uniformly stirring to obtain the immunity-inducing and resisting agent.
Specifically, in the first step, specific proportion determination is performed by experimental comparison, and differences generated by the method and the common treatment mode are compared.
Compared with the prior art, the invention has the beneficial effects that:
(1) The plant immunity inducer comprises chitosan oligosaccharide as an active ingredient, and the chitosan oligosaccharide acts on pine trees and can induce the expression of related resistance genes, so that the disease resistance (including pine wood nematode disease) of the pine trees is improved.
(2) The application of the chitosan oligosaccharide in improving pine immunity resistance is realized by exciting pine autoimmunity, is not easy to generate drug resistance, has broad spectrum and has good development and utilization prospects.
(3) The chitosan oligosaccharide mentioned in the application has the characteristics of pure nature, no pollution and the like, and accords with the sustainable development concept.
Drawings
FIG. 1 shows the onset of pine wood nematodes inoculated with the attractant of the present invention after treatment with the immune attractant.
FIG. 2 is a comparison of five-year-old masson pine after application of the elicitor of the present invention.
FIG. 3 shows the expression levels of the resistance genes associated with the use of the elicitors of the present invention on masson pine, wherein the signs indicate that the immune elicitor treatment differs significantly from the control treatment at a level of difference P > 0.05.
Detailed Description
The technical solutions in the comparative examples of the present invention will be clearly and completely described below with reference to the drawings in the comparative examples of the present invention, and it is apparent that the described comparative examples are only some of the comparative examples of the present invention, not all of the comparative examples. All other comparative examples obtained by a person of ordinary skill in the art without making any inventive effort based on the comparative examples in the present invention are within the scope of the present invention.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
In the invention, a plant immunity inducer for preventing pine wood nematode disease comprises chitosan oligosaccharide, a synergistic agent and a solvent; the synergist is organic silicon, the solvent is an organic solvent, and the types of the organic solvents in the solvent are at least one.
Specifically, the mass percentages of the components are as follows: 0.1 to 3 percent of chitosan oligosaccharide, 0.05 to 0.1 percent of synergist and the balance of solvent; the solvent is one or more of styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine.
The preparation method of the plant immunity inducer comprises the following steps:
step one: firstly quantifying a solvent, and adding chitosan oligosaccharide into the solvent for dissolution after the solvent is in a certain proportion;
step two: and adding the quantitative synergist into the dissolved medicament obtained in the last step, and uniformly stirring to obtain the immunity-inducing and resisting agent.
Specifically, in the first step, specific proportion determination is performed by experimental comparison, and differences generated by the method and the common treatment mode are compared.
Specific examples of immune elicitors are as follows:
example 1:
the mass percentages of the components are as follows: 0.1% of chitosan oligosaccharide, 0.05% of synergist and 99.85% of solvent.
In the second step of the preparation process, the stirring time is 10min.
Example 2:
the mass percentages of the components are as follows: 3% of chitosan oligosaccharide, 0.1% of synergist and 96.9% of solvent.
In the second step of the preparation process, the stirring time is 10min.
Example 3:
the mass percentages of the components are as follows: 1.5% of chitosan oligosaccharide, 0.08% of synergist and 98.42% of solvent.
In the second step of the preparation process, the stirring time is 10min.
Example 4:
the mass percentages of the components are as follows: 1.5% of chitosan oligosaccharide, 0.08% of synergist and 98.42% of solvent.
In the second step of the preparation process, the stirring time is 15min.
Comparative example one is as follows:
disease conditions of four-year old masson pine seedlings inoculated with pine wood nematodes after immune-induced resistance treatment (1) test crop: 4-year-old pinus massoniana seedlings with average seedling height of 95cm and average ground diameter of 1.5cm. The preparation method comprises the following steps: the immune elicitor was formulated according to the proportions shown in table 1.
TABLE 1
(3) The test method comprises the following steps:
cutting off the top of the trunk of the seedling, inserting a 1 milliliter-specification pipette head at the cutting-off position, and sealing the trunk and the pipette head by using a sealing film. 1 ml of the prepared immune antigen is added into a straw connected with a trunk, a sealing film is used for sealing, and 10 pinus massoniana seedlings are treated at each concentration. A control group is additionally arranged, and the liquid medicine of the control group is formed by ddH 2 O replaces chitosan.
After 30 days of immune attractant treatment, all pinus massoniana seedlings were artificially inoculated with pine wood nematodes. And inoculating 10000 pine wood nematodes into each pinus massoniana seedling by adopting a bark grafting method. Observations were made 1 week after inoculation, and the disease state was recorded.
As shown in Table 2 and figures 1 and 2, the immune attractant with different chitosan oligosaccharide concentrations can reduce the disease susceptibility of the pinus massoniana seedlings after being inoculated with pine wood nematodes. The immune resistance inducer with the concentration of 1% of chitosan oligosaccharide has the optimal effect, and compared with a control group, the disease susceptibility of pine wood nematodes after pine wood nematodes are inoculated with seedlings of masson pine for 7 weeks is reduced by 50%.
Table 2:
comparative example 2 3 post-treatment transcriptome analysis of Pinus massoniana seedlings
(1) Test crop: the seedlings of the 3-year-old masson pine have the average seedling height of 90cm and the average ground diameter of 1.2cm.
(2) The test method comprises the following steps: cutting off the top of the trunk of the seedling, inserting a 1 milliliter-specification pipette head at the cutting-off position, and sealing the trunk and the pipette head by using a sealing film. 1 ml of 1% chitosan (Aladin, shanghai, china) was added to a straw attached to the trunk, sealed with a sealing film, and the control group was ddH-treated 2 And (3) O treatment. The test group and the control group were each provided with 3 biological replicates, each replicate treated 1 pinus massoniana seedling. Needle leaves of Pinus massoniana seedlings were collected after 7 days of treatment, flash frozen with liquid nitrogen and then stored at-80 ℃. Samples were extracted with RNA, reverse transcribed and analyzed by BGISEQ-500 platform (Beijing genomics institute, shenzhen, china) sequencing.
By comparative analysis, 501 differentially expressed genes were found between the test group and the control group. Wherein the number of up-regulated differentially expressed genes is 251 and the number of down-regulated differentially expressed genes is 250. And the chitosan oligosaccharide induced the expression of 31 plant resistance related genes of Pinus massoniana (as shown in Table 3). These induced plant resistance related genes belong to the RLP, TNL, N, NL, CNL, CN, T and RPW8-NL domain classes.
TABLE 3 Chitosan oligosaccharide induced expression 31 plant resistance related genes of Pinus massoniana
(1) Test crop: as in comparative example one.
(2) The preparation method comprises the following steps: as in comparative example one.
(3) The test method comprises the following steps: cutting off the top of the trunk of the seedling, inserting a 1 milliliter-specification pipette head at the cutting-off position, and sealing the trunk and the pipette head by using a sealing film. 1 ml of the prepared immune antigen is added into a straw connected with a trunk, a sealing film is used for sealing, and 10 pinus massoniana seedlings are treated at each concentration. A control group is additionally arranged, and the liquid medicine of the control group is formed by ddH 2 O replaces chitosan.
After 7 and 30 days of immune elicitor treatment, 5 pinus massoniana seedlings were taken from each group, needle leaves were collected, flash frozen with liquid nitrogen, and then stored at-80 ℃. The total RNA of masson pine was extracted using a column plant total RNA extraction purification kit (Shanghai, china) according to the manufacturer's instructions. The integrity of the RNA samples was checked by 1% agarose gel electrophoresis and RNA concentration was determined using a Multiscan GO microplate spectrophotometer (Thermo Fisher Scientific, massachusetts, usa) with a mu Drop ultra-micro detection plate.
cDNA was prepared from total RNA using the easy script One-Step gDNA Removal and cDNA Synthesis SuperMix kit kit (full gold, beijing, china) according to the manufacturer's protocol.
Gene-specific primers for amplification of the internal reference gene u2af and 6 plant resistance genes were designed on the Primer3web (version 4.1.0) (see Table 4).
qPCR amplification was performed on a LineGene 4800 fluorescent quantitative PCR detection system (Hangzhou Bo, zhejiang, china) according to the protocol provided by the PefectStart-Green-qPCR-Supermix kit (full gold, beijing, china). The program is set as follows: 94 ℃ for 30s;94℃for 5s,60℃for 30s,40 cycles.
The specificity of the SYBR green PCR signal was confirmed by the melting curve at 60℃to 94℃and u2af was the reference gene.
The relative expression level of the gene was determined by the 2- ΔΔct method.
TABLE 4 primers for qPCR amplification
The results are shown in figure 3, and the results show that after the pinus massoniana seedlings are treated by the immune induction agent for 7 days and 30 days, the expression of 6 plant resistance genes is up-regulated, but the up-regulation significance degree is closely related to the content of chitosan oligosaccharide in the immune induction injection agent. Wherein, the up-regulation difference of the plant resistance genes of the masson pine seedlings treated by 0.1 percent and 0.5 percent of chitosan oligosaccharide is not large, and the up-regulation of the plant resistance genes of the masson pine seedlings treated by 1 percent of chitosan oligosaccharide is more obvious. Upregulation of the resistance gene enhanced resistance of masson pine to pine wood nematode disease, which was consistent with the results of pine wood onset after pine wood nematode inoculation in comparative example 1.
It will be apparent to those skilled in the art that the present invention is not limited to the details of the above-described exemplary comparative examples, but that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The comparative examples are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity only, and those skilled in the art will recognize that the embodiments of the present disclosure as a whole, and that other embodiments may be suitably combined to form one or more embodiments as would be apparent to one of skill in the art.
Claims (8)
1. A plant immunity inducer for preventing pine wood nematode disease is characterized by comprising chitosan oligosaccharide, a synergistic agent and a solvent; the synergist is organic silicon, the solvent is an organic solvent, and the types of the organic solvents in the solvent are at least one.
2. The plant immunity-inducing agent for preventing pine wood nematode disease according to claim 1, wherein the mass percentages of the components are as follows: 0.1 to 3 percent of chitosan oligosaccharide, 0.05 to 0.1 percent of synergist and the balance of solvent.
3. The plant immunity-inducing agent for preventing pine wood nematode disease according to claim 2, wherein the mass percentages of the components are as follows: 0.1% of chitosan oligosaccharide, 0.05% of synergist and 99.85% of solvent.
4. The plant immunity-inducing agent for preventing pine wood nematode disease according to claim 2, wherein the mass percentages of the components are as follows: 3% of chitosan oligosaccharide, 0.1% of synergist and 96.9% of solvent.
5. The plant immunity-inducing agent for preventing pine wood nematode disease according to claim 2, wherein the mass percentages of the components are as follows: 1.5% of chitosan oligosaccharide, 0.08% of synergist and 98.42% of solvent.
6. A plant immunity-inducing agent for preventing pine wood nematode disease according to claim 1, wherein the solvent is one or more of styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine.
7. A method for preparing a plant immunity-inducing agent for preventing pine wood nematode disease, which is characterized by comprising the following steps: firstly quantifying a solvent, and adding chitosan oligosaccharide into the solvent for dissolution after the solvent is in a certain proportion;
step two: and (3) adding the quantitative synergist into the dissolved medicament obtained in the step one, and uniformly stirring to obtain the immunity inducer.
8. The method of claim 4, wherein in the first step, specific ratio determination is performed by experimental comparison, and the difference between the generated agent and the common treatment mode is compared.
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