Efficient pine nematode disease resistant inducer based on salicylic acid and dicyclohexylamine as well as preparation method and application of efficient pine nematode disease resistant inducer

Abstract

The invention belongs to the technical field of forest tree disease control, and particularly discloses an efficient pine nematode disease resistant inducer based on salicylic acid and dicyclohexylamine as well as a preparation method and application thereof, and the efficient pine nematode disease resistant inducer mainly comprises salicylic acid, cyclohexanediamine, a synergist (such as organic silicon) and an environment-friendly solvent (such as ethanol or diethyl ether). Wherein salicylic acid and dicyclohexylamine are used as core active ingredients, and the resistance of pine trees to pine wood nematode diseases is remarkably improved by stimulating the immune defense system of the pine trees. The inducer has the advantages of being environmentally friendly, moderate in cost, free of residues and pollution and the like, is suitable for multiple fields such as forest control and seedling treatment, can effectively reduce the occurrence rate and loss of the pine wood nematode disease, and promotes sustainable development of forestry. Through a specific preparation process, all the components are uniformly mixed to prepare a solution which is easy to apply, so that efficient protection on pine trees is realized.

Description

Technical Field

[0001] The invention relates to the technical field of forest disease prevention and treatment, in particular to a highly effective pine tree anti-nematode disease inducer based on salicylic acid and dicyclohexylamine, and a preparation method and application thereof. Background Art

[0002] Pine wood nematode disease (PWD), also known as pine wilt disease and pine wilt, is a major quarantine forest disease caused by the pine wood nematode (Bursaphelenchus xylophilus) (Li Min et al., 2022).

[0003] The prevention and control of pine wood nematode disease has always attracted much attention at home and abroad. There are three main targets for the prevention and control of pine wood nematode disease: the first is the host pine tree itself; the second is the prevention and control of host nematodes; and the third is the prevention and control of insect vectors of nematodes. Among them, the prevention and control methods for diseased trees are mainly divided into three aspects: physical, chemical and biological. Physical control methods include felling, fumigating, crushing and burning infected trees to reduce the number of pine trees planted around the epidemic area and further prevent the spread of pine wilt disease in forest areas. The advantage of this control method is that the prevention and control of pine wilt disease is more thorough and effective in preventing the spread of pine wilt, but the disadvantages are also obvious, such as being destructive to forests and causing large economic losses. In addition, preventing and controlling PWD by burning trees can easily lead to uncontrollable forest fires; chemical methods include two angles. When targeting host nematodes, the development of pine wilt disease is controlled by injecting chemical synthetic insecticides into the trunks of infected trees or spraying insecticides into the epidemic area. The disadvantage of this method is that the use of nematicidal chemicals may have an adverse effect on the forest ecosystem; the chemical control method for pine trees themselves is to improve the stress resistance of pine trees themselves, thereby effectively resisting the damage of nematodes to plants. To improve the stress resistance of host plants, on the one hand, we can use genetic means to breed tree species with strong stress resistance, that is, highly resistant tree species to resist the infection of pine wood nematodes. On the other hand, we can also use traditional means to spray exogenous substances that induce plant resistance to improve plant resistance. The former can effectively solve the problem of pine wood nematode disease from the root, but its limitations are that on the one hand, the gene editing technology for pine wood is not yet mature, and on the other hand, the mining of stress resistance genes of different tree species is still imperfect. Moreover, the full gene sequencing of most pine wood varieties has not been completed, so it is difficult to breed highly resistant varieties through modern biological technology. It can be seen that by spraying plant resistance inducers to stimulate the plant's own immune defense system and improve resistance to pine wood nematode disease, green, environmentally friendly and sustainable disease prevention and control can be achieved.

[0004] At present, some researchers have improved the growth and development of pine wood by exogenous spraying, and have achieved remarkable results. Hu et al. (2023) used exogenous calcium to spray Masson pine seedlings. The results showed that exogenous calcium spraying can significantly promote the growth and development, photosynthesis and material metabolism of Masson pine seedlings, enhance cell wall formation and cell division, and activate the expression of genes related to calcium ion homeostasis and calcium signal transduction under high exogenous calcium levels. When Fan et al. (2021) sprayed Masson pine seedlings with exogenous brassinosteroids, it significantly promoted the development of xylem, increased the accumulation of endogenous plant hormones and the expression of related genes in the stem. Yuan et al. (2019) used different concentrations of exogenous plant hormones indole-3-acetic acid (IAA), gibberellin (GA3) and brassinosteroids (BR) to treat black poplar seedlings and found that the height of the seedlings sprayed with hormones was significantly increased compared with the control. At the same time, the treatment of IAA significantly increased the stem diameter and cellulose content of the seedlings. Wiszniewska et al. (2018) found that when exogenous spraying of plant hormones: gibberellin acid, jasmonic acid and brassinolide was applied to shrubs, the regulatory effect of nickel overdose response of shrubs was effectively alleviated. Exogenous JA stimulated the synthesis of active auxin and salicylic acid, which helped to enhance mitotic activity in explants.

[0005] In summary, the current prevention and control of pine wood nematode disease mainly involves physical, chemical and biological means to improve the stress resistance of infected wood. The addition of exogenous substances can effectively increase the production of cell signal factors, hormone synthesis and plant cell wall thickening in woody plants, thereby improving the growth and development of woody plants. Moreover, the physiological and biochemical reactions of woody plants improved by exogenous active substances are very similar to the defense mechanism produced by highly resistant pine wood varieties against pine wood nematodes. At present, although there are studies on spraying exogenous active substances to improve the stress resistance of woody plants, they are very rare, especially the research on improving the resistance of pine wood to pine wood nematode disease. The research and development of plant immune inducers for pine wood nematode disease is still in its infancy. Therefore, it is particularly important to select suitable active substances for spraying pine wood and explore their ability to improve the disease resistance of pine wood nematodes. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a highly effective pine anti-nematode disease inducer based on salicylic acid and dicyclohexylamine, and a preparation method and application thereof.

[0007] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0008] One of the purposes of the present invention is to provide a highly effective pine anti-nematode disease inducer based on salicylic acid and dicyclohexylamine, which is composed of the following components: salicylic acid, dicyclohexylamine, a synergist and a solvent, wherein the concentration of salicylic acid is 0.01mM-1.0mM, the concentration of dicyclohexylamine is 0.05mM-5mM, the volume percentage of the synergist is 0.5%-8%, and the solvent is in excess.

[0009] Furthermore, the solvent is ethanol or ether.

[0010] Furthermore, the synergist is silicone.

[0011] The functions of each component in the present invention are as follows:

[0012] Salicylic acid: As one of the main active ingredients, it utilizes its unique chemical structure characteristics to effectively stimulate the pine immune system and induce the expression of resistance genes.

[0013] Dicyclohexylamine: As the second main active ingredient, on the one hand, it is a competitive inhibitor of spermidine synthase, which can inhibit the reaction of spermidine synthase catalyzing putrescine (Put) to produce spermidine (Spd), stimulate the conversion of spermine to spermidine, thereby increasing the content of spermine and spermidine in plants and inducing plant resistance; on the other hand, as a chemical substance with a certain toxicity, it itself has a certain paralyzing effect on pests.

[0014] Synergist: used to improve the permeability and absorption efficiency of salicylic acid in pine trees and enhance the induction effect.

[0015] Solvent: Ensures uniform mixing of ingredients and facilitates application.

[0016] The second object of the present invention is to provide a method for preparing a highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine, comprising the following steps:

[0017] First, salicylic acid is added to an excess solvent and stirred thoroughly until it is completely dissolved; then, cyclohexanediamine and a synergist are added and stirred continuously until the mixture is uniformly mixed; finally, possible insoluble matter is removed by filtration to obtain a final inducer solution, wherein the concentration of salicylic acid is controlled to be 0.01 mM-1.0 mM, the concentration of dicyclohexanediamine is controlled to be 0.05 mM-5 mM, and the volume percentage of the synergist is controlled to be 0.5%-8%.

[0018] The third object of the present invention is to provide an application of a highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine in the prevention and treatment of pine nematode disease, comprising:

[0019] (1) Forest control: Spray the highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine on the trunks or roots of pine trees;

[0020] (2) Seedling treatment: Before transplanting pine seedlings, spray the leaves or soak the roots with a highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine.

[0021] Compared with the prior art, the inducer of the present invention is mainly composed of salicylic acid, cyclohexanediamine, a synergist (such as silicone) and an environmentally friendly solvent (such as ethanol or ether), wherein salicylic acid and dicyclohexylamine are used as core active ingredients, which significantly improve the resistance of pine trees to pine wood nematode disease by stimulating the immune defense system of pine trees themselves. The inducer of the present invention has the advantages of being green and environmentally friendly, moderate cost, no residue and no pollution, and is suitable for multiple fields such as forest prevention and control and seedling treatment, and can effectively reduce the incidence and loss of pine wood nematode disease and promote the sustainable development of forestry. Through a specific preparation process, the components are evenly mixed to form a solution that is easy to apply, thereby achieving efficient protection of pine trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention discloses the phenotypic results of pine seedlings sprayed with the highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine of the present invention.

[0023] Figure 2 The results of the analysis of the differential expression of genes in the cambium of Korean pine treated with an inducer with 0.1 mM salicylic acid as the main ingredient and without the addition of salicylic acid inducer.

[0024] Figure 3 Classification of differentially expressed genes in terms of molecular function, biological process and cellular composition.

[0025] Figure 4 The results of differential metabolite analysis between treatment with 0.1 mM salicylic acid as an inducer and treatment without adding salicylic acid.

[0026] Figure 5 The distribution of each level of metabolites in different treatments was detected for the treatment with 0.1 mM salicylic acid as an inducer and the treatment without adding salicylic acid as an inducer. DETAILED DESCRIPTION

[0027] 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 embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0028] Embodiment 1, spraying test

[0029] Test materials: The tested red pine was provided by the red pine planting base, and salicylic acid (CAS No. 69-72-7, relative molecular mass 138.12) and dicyclohexylamine (CAS No. 101-83-7, relative molecular mass 182.33) were used as active ingredients.

[0030] Test method:

[0031] 1. Prepare salicylic acid mother solution: weigh an appropriate amount of salicylic acid, dissolve it with a small amount of ethanol, and dilute to a 250 ml volumetric flask to obtain a 10 mM salicylic acid mother solution. Add 50 mM cyclohexanediamine to the mother solution.

[0032] 2. Prepare salicylic acid solutions of different concentrations: take 1 ml, 10 ml and 100 ml of the mother liquor respectively, add a predetermined amount of enhancer and double distilled water, and dilute to 1000 ml volumetric flask to obtain 0.01 mM, 0.1 mM and 1.0 mM salicylic acid solutions.

[0033] Test steps

[0034] Five treatment groups were set up, including CK (spraying double distilled water), 0.01SA, 0.1SA, 1.0SA and blank (no spraying), with 6 replicates in each group. 10 ml of the corresponding solution was sprayed in each replicate, and the growth and disease resistance changes of the pine trees were recorded and observed.

[0035] Example 2: Nematode inoculation test

[0036] Test methods

[0037] 1. Experimental materials: Pine wood nematodes were isolated and cultured from diseased tree species using the Baermann funnel method and inoculated into Korean pine seedlings.

[0038] 2. Inoculation process: Artificial bark grafting was used to inoculate pine wood nematodes on Korean pine treated with different concentrations of salicylic acid. Inoculation control and overall test control were set up to observe and record the incidence.

[0039] 3. Analysis of experimental results: Observations and photos were taken at different time points after inoculation, and samples were collected for transcriptome and metabolome analysis to evaluate the effects of salicylic acid inducers on pine disease resistance.

[0040] 4. Analysis of test results:

[0041] 1. Analysis of spraying phenotypic results

[0042] like Figure 1 As shown in the figure, 45 days after inoculation, the red pine needles treated with different treatments showed different degrees of yellowing and wilting, but the red pine treated with the inducer with 0.1mM salicylic acid as the main ingredient performed the best. It can be determined that spraying the inducer with 0.1mM salicylic acid as the main ingredient can significantly improve the resistance of red pine and prolong the onset time after the red pine is inoculated with nematodes.

[0043] 2. Transcriptome Analysis

[0044] Based on the above results, the blank control (no inoculation and no spraying), water control (no inducer of salicylic acid added), and cambium treated with 0.1 mM salicylic acid as inducer were used for transcriptome analysis.

[0045] The analysis results are as follows:

[0046] 2.1 Number of differentially expressed genes

[0047] The differential expression of genes in the cambium of Korean pine between the treatment with 0.1 mM salicylic acid as the main ingredient and the treatment without salicylic acid inducer was analyzed. A total of 611 differential genes were detected, including 449 up-regulated genes and 162 down-regulated genes; the differential expression of genes in the cambium of Korean pine between the treatment without spraying inducer and inoculation and the treatment without adding salicylic acid inducer was analyzed. A total of 1052 differential genes were detected, including 398 up-regulated genes and 654 down-regulated genes; the differential expression of genes in the cambium of Korean pine between the treatment without spraying inducer and inoculation and the treatment with 0.1 mM salicylic acid as the main ingredient was analyzed. A total of 1983 differential genes were detected, including 1688 up-regulated genes and 295 down-regulated genes, such as Figure 2 shown.

[0048] 2.2 Classification of differentially expressed genes in terms of molecular function, biological process, and cellular composition

[0049] like Figure 3 As shown, the molecular functions mainly involve antioxidant enzyme activity, catalytic enzyme activity, chemical attractant activity, electron transfer activity, metal chaperone activity, molecular function regulation, molecular converter activity, etc.; the biological processes mainly include biological regulation, cell component organization or biosynthesis, cell aggregation, cell activation, cellular process, developmental process, toxic immune system process, growth process, positioning, metabolic process, movement multicellular biological process, multi-biological process, negative regulation biological process, regulation of biological process, biological process regulation, biological process, reproductive process, response to stimulus rhythm process, establishment of positioning signal process; the cell composition includes cell connection, extracellular matrix, intercellular structure, internal cell structure, extracellular matrix, etc.

[0050] 3. Metabolomics analysis

[0051] Based on the above results, the blank control (no inoculation and no spraying), water control (no inducer of salicylic acid added), and cambium treated with 0.1 mM salicylic acid as inducer were used for transcriptome analysis.

[0052] 3.1 Analysis of differential metabolites

[0053] The results of differential metabolite analysis between treatment with 0.1 mM salicylic acid as inducer and treatment without salicylic acid added are as follows Figure 4As shown, a total of 2731 metabolites were detected, including 560 differential metabolites, of which 270 metabolites were up-regulated and 290 metabolites were down-regulated.

[0054] 3.2 Differential metabolite enrichment pathways

[0055] The 560 differential metabolites were mainly enriched in zeatin biosynthesis (2.13%), biosynthesis of hydroquinone and other terpenoid hydroquinones (6.38%), tyrosine metabolism (2.13%), thiamine metabolism (2.13%), biosynthesis of terpenoid backbone (6.38%), starch and sucrose metabolism (2.13%), sphingomyelin metabolism (2.13%), pyrimidine metabolism (2.13%), purine metabolism, phenylpropanoid metabolism, phenylalanine metabolism (4.26%), nucleotide metabolism (10.64%), monobactin biosynthesis (2.13%), metabolic pathways (68.83%), biosynthesis of isoquinoline alkaloids, glycine, serine and threonine metabolism (2.13%), glutathione metabolism (2.13%), biosynthesis of glucosinolates (2.13%), flavonoid biosynthesis Synthesis (10.64%), biosynthesis of flavonoids and flavonols (2.13%), biosynthesis of diterpenoids (2.13%), biosynthesis of unsaturated fatty acids (6.38%), biosynthesis of secondary metabolites (42.55%), biosynthesis of secondary metabolites of various plants (2.13%), biosynthesis of secondary metabolites (2.13%), biosynthesis of cofactors (8.51%), biosynthesis of amino acids (2.13%), β-alanine metabolism (2.13%), arginine biosynthesis (2.13%), arachidonic acid metabolism (2.13%), α-linolenic acid metabolism (2.13%), 2-oxoformate metabolism (2.13%), biosynthesis of aminoacyl-tRNA (4.26%), carbon metabolism (4.26%), ABC transporters (14.89%).

[0056] 3.3 Contents of differential metabolites in different treatments

[0057] The metabolites detected were divided into 7 levels, and the distribution of each level in different treatments is as follows Figure 5 As shown: there are 143 primary metabolites, 178 secondary metabolites, 238 tertiary metabolites, 123 quaternary metabolites, 228 fifth metabolites, 268 sixth metabolites, and 168 seventh metabolites. Among them, the treatment with the inducer with salicylic acid as the main ingredient has higher metabolite content, which is mainly distributed in the primary, fifth and seventh levels, mainly isoflavones and diterpenes.

[0058] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine, characterized in that: The invention is composed of the following components: salicylic acid, dicyclohexylamine, a synergist and a solvent, wherein the concentration of salicylic acid is 0.01mM-1.0mM, the concentration of dicyclohexylamine is 0.05mM-5mM, the volume percentage of the synergist is 0.5%-8%, and the solvent is excessive.

2. The highly effective pine anti-nematode disease inducer based on salicylic acid and dicyclohexylamine according to claim 1, characterized in that: The solvent is ethanol or ether.

3. The highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine according to claim 1, characterized in that: The synergist is silicone.

4. A method for preparing a highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: First, salicylic acid is added to an excess solvent and stirred thoroughly until it is completely dissolved; then, cyclohexanediamine and a synergist are added and stirred continuously until the mixture is uniformly mixed; finally, possible insoluble matter is removed by filtration to obtain a final inducer solution, wherein the concentration of salicylic acid is controlled to be 0.01 mM-1.0 mM, the concentration of dicyclohexanediamine is controlled to be 0.05 mM-5 mM, and the volume percentage of the synergist is controlled to be 0.5%-8%.

5. Use of the highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine according to any one of claims 1 to 3 in the prevention and treatment of pine nematode disease, characterized in that: include: (1) Forest control: Spray the highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine on the trunks or roots of pine trees; (2) Seedling treatment: Before transplanting pine seedlings, spray the leaves or soak the roots with a highly effective pine anti-nematode inducer based on salicylic acid and dicyclohexylamine.

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