阅读说明：本技术 一种线虫提取物及其应用 (Nematode extract and application thereof ) 是由 李世友 赵福强 王萍 李帅强 韩月泠 于 2021-06-11 设计创作，主要内容包括：本发明属于农药领域,具体涉及一种线虫提取物及其活性化合物在作为杀线虫剂的应用。线虫提取物是以线虫自身为原料提取获得的,该提取物中含有一系列具有杀线虫活性的化合物。所述线虫提取物,其活性化合物、其盐或酯中的一种或几种,能与农业上可接受的溶剂和/或辅料混合,形成杀线虫制剂,作为保护植物抵抗线虫侵染或危害中的杀线虫剂。(The invention belongs to the field of pesticides, and particularly relates to a nematode extract and application of an active compound thereof as a nematicide. The nematode extract is obtained by extracting nematodes as raw materials, and contains a series of compounds with nematicidal activity. The nematode extract, one or more of its active compounds, salts or esters, can be mixed with agriculturally acceptable solvents and/or adjuvants to form a nematicidal formulation as a nematicide in protecting plants against nematode infestation or damage.)

1. A nematode extract characterized by: the nematode extract is an extract obtained by extracting nematodes by using the nematodes as raw materials.

2. The nematode extract of claim 1, wherein: the nematodes are all nematodes of phylum nematoda.

3. Use of a nematode extract according to claim 1 as a nematicide.

4. Use according to claim 3, characterized in that: the nematicide contains one or more of an extract obtained by extracting the nematodes serving as raw materials, active substances in the extract and analogues of the active substances in the extract.

5. Use according to claim 4, characterized in that: the active substance in the extract contains one or more of fatty acid compounds, benzene compounds, nitrogen compounds, polyalcohol compounds or amino acid compounds, and their salts or esters.

6. Use according to claim 4, characterized in that: the analogue of the active substance in the extract is a compound shown as a formula I,

in the formula (I), the compound is shown in the specification,

R₁、R₂and R₄Each independently of the others is hydrogen, -OH, -COOH or C₁-C₄Alkyl groups of (a); r₃Selected from hydrogen, -OH or-COOR'; r' is selected from hydrogen or C₁-C₄Alkyl groups of (a);

n₁selected from 0 to 22; n is₂Selected from 0 to 1; n is₃Selected from 1 to 6;

and, except when R is₁、R₂、R₄And R' are simultaneously hydrogen, R₃is-OH, n₁Is 4, n₂Is 0, n₃Is 1;

or, a salt or ester of a compound of formula (la).

7. Use according to claim 4, characterized in that: the active substance analogue compound in the extract is oleic acid, methyl oleate, ethyl oleate, linoleic acid, methyl linoleate, ethyl linoleate, azelaic acid, monomethyl azelate, dimethyl azelate, monoethyl azelate, diethyl azelate, n-pelargonic acid, methyl pelargonate and/or ethyl pelargonate.

8. A nematicide characterized by: the active ingredients of the nematicide contain one or more of extracts obtained by extracting the nematodes as raw materials, active substances in the extracts and analogues of the active substances in the extracts.

9. The nematicide of claim 8, wherein: the nematicide is a mixture of an active ingredient and an agriculturally acceptable solvent, wherein the active ingredient accounts for 0.00001 to 99.9 percent of the weight of the insecticide; the solvent is one or more of water or organic solvent.

10. The nematicide of claim 8, wherein: the active substance in the extract contains one or more of fatty acid compounds, benzene compounds, nitrogen compounds, polyalcohol compounds or amino acid compounds, and their salts or esters.

11. The nematicide of claim 8, wherein: the analogue of the active substance in the extract is a compound shown as a formula I,

in the formula (I), the compound is shown in the specification,

R₁、R₂and R₄Each independently of the others is hydrogen, -OH, -COOH or C₁-C₄Alkyl groups of (a); r₃Selected from hydrogen, -OH or-COOR'; r' is selected from hydrogen or C₁-C₄Alkyl groups of (a);

n₁selected from 0 to 22; n is₂Selected from 0 to 1; n is₃Selected from 1 to 6;

and, except when R is₁、R₂、R₄And R' are simultaneously hydrogen, R₃is-OH, n₁Is 4, n₂Is 0, n₃Is 1;

or, a salt or ester of a compound of formula (la).

12. The nematicide of claim 10, wherein: the fatty acid compound is fatty acid, hydroxy fatty acid or dibasic acid; wherein the fatty acid contains one or more of oleic acid, stearic acid, linoleic acid, arachidonic acid, ethyl myristate, cis-8, 11, 14-eicosatrienoic acid, cis-11, 14-eicosadienoic acid, 14-eicosenoic acid, alpha-eleostearic acid, nervonic acid, arachidic acid, erucic acid, lignoceric acid, n-pelargonic acid, n-capric acid, cis 13,16, 19-docosatrienoic acid or salts or esters of the above substances;

the hydroxy fatty acid contains (15Z) -9,12,13-trihydroxy-15-octadecenoic acid, 2-hydroxypentanoic acid, (9Z) -12, 13-dihydroxy-octadecenoic acid, 3-hydroxymyristic acid, 20-hydroxyeicosa-5Z, 8Z,11Z, 14Z-tetraenoic acid, Corchfaritty acid F, 16-hydroxypalmitic acid, 2-hydroxymyristic acid, 10-hydroxy-2-decenoic acid or one or more of salts or esters of the above substances;

the dibasic acid contains one or more of methylmalonic acid, azelaic acid, 2-isopropylmalic acid, citraconic acid or salts or esters of the above substances;

the benzene compound contains one or more of 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, ferulic acid, vanillic acid, gentisic acid, benzoic acid, monobutyl phthalate, phenyllactic acid, homogentisic acid, 4-hydroxyphenylpyruvic acid, dihydrobenzofuran-2-carboxylic acid, 2, 5-di-tert-butylhydroquinone, bisphenol B or salts or esters of the substances;

the polyalcohol compound contains erythritol or one or more of salts or esters thereof;

the nitrogen-containing compound contains one or more of norsalsoline, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2-picolinic acid, 5-carboxyindole-3-acetic acid, 1-isopropenyl-2, 3-dihydro-1H-benzo [ D ] imidazole-2-ketone, urocanic acid, ureidopropionic acid or D-pantothenic acid or salts or esters of the above substances;

the amino acid compound contains one or more of glutamic acid, leucine, phenylalanine, tyrosine methyl ester, L-tyrosine, aspartic acid, tryptophan, N-acetyl-DL-norvaline or salts or esters of the above substances.

13. A nematicidal composition formulation comprising: the composition comprises an active component A and an active component B, wherein the active component A and the active component B are mixed according to the weight part ratio of 1:100-100: 1; the active component A is one or more of an extract obtained by extracting the nematode serving as a raw material, an active substance in the extract and an analogue of the active substance in the extract; the active component B is one or more of other nematocides, insecticides, acaricides and bactericides.

14. The use of a compound in the preparation of a nematicidal formulation, wherein: the compound is one or more of a compound shown in a formula I, 2-hydroxy valeric acid and 3-hydroxy myristic acid;

in the formula (I), the compound is shown in the specification,

R₁、R₂and R₄Each independently of the others is hydrogen, -OH, -COOH or C₁-C₄Alkyl groups of (a); r₃Selected from hydrogen, -OH or-COOR'; r' is selected from hydrogen or C₁-C₄Alkyl groups of (a);

n₁selected from 0 to 22; n is₂Selected from 0 to 1; n is₃Selected from 1 to 6;

and, except when R is₁、R₂、R₄And R' are simultaneously hydrogen, R₃is-OH, n₁Is 4, n₂Is 0, n₃Is 1;

or, a salt or ester of a compound of formula (la).

15. The use of a compound according to claim 14 in the preparation of a nematicidal formulation, wherein: the compound is one or more of oleic acid, methyl oleate, ethyl oleate, linoleic acid, methyl linoleate, ethyl linoleate, azelaic acid, monomethyl azelate, dimethyl azelate, monoethyl azelate, diethyl azelate, n-pelargonic acid, methyl pelargonate and ethyl pelargonate.

Technical Field

The invention belongs to the field of pesticides, and particularly relates to a nematode extract and application thereof as a nematode killing preparation.

Background

Nematodes, phyla nematoda, are ubiquitous in fresh water, seawater, and land, are a group of animals with a diversity next to insects, and currently there are over 28,000 species recorded, including 256 families, genus 2271, and a large number of species that have not yet been named. In 1743, after wheat grain nematodes were found in wheat grains for the first time by a scientist needleham in the United kingdom, different reports on various nematodes appeared in each region of the world one after another. In 1993, the dietes et al classed the nutritional groups of nematodes into 8 groups according to their feeding characteristics, esophageal structure and predation methods, among which Plant-parasitic nematodes (PPN), fungal nematodes (Fu), bacteriophagous nematodes (Ba) and omnivores-predators (Op) are important nutritional groups in the soil ecosystem. Such as plant parasitic nematodes, pine wood nematodes (Bursaphelenchus xylophilus), Meloidogyne incognita (Meloidogyne incognita), and Heterocystis sojae (Heterodera glycines Ichimohe); heterodera feeding nematodes, primitive Bursaphelenchus (Trichoderma primativus), and Panagrellus redivivus. The classification status, morphological characteristics, breeding methods and the like of the nematodes are very different, and the main differences are shown in table 1 below.

TABLE 1 differences between the various nematodes

Pine wood nematode (Bursaphelenchus xylophilus), a common pine parasitic nematode, is spread by vector insects such as Monochamus alternatus, and further causes pine wood nematode disease. The host mainly comprises Pinus plants such as Pinus thunbergii, Pinus densiflora and Pinus massoniana. Pine infected by pine wood nematode has yellow brown or red brown needle, and the whole plant dies due to withering. Pine nematode disease occurs in Mexico, the United states, Canada, Japan, Korea, and other countries. China discovers the disease for the first time in Shanling in Nanjing in 1982, then forms several centers of attack in Shandong, Anhui, Guangdong, Zhejiang and the like in succession, and spreads all around, so that local areas of the provinces are subjected to parallel flow and disaster formation, a large number of pine trees die, the pine wood nematode disease spreads and spreads to 18 666 counties (urban areas) in 18 provinces in China, 1671.9 ten thousand mu occur in 2019, 1946.7 thousand dead pine trees are killed, the death of 9 hundred million mu Pinaceae forests in China is threatened directly, and great damage is brought to forest production and ecological safety in China. The pine trees lost due to the pine wilt disease in China still reach billions of plants, and the direct economic loss and the loss of ecological service value are billions of yuan.

Root-knot nematodes (melodogyne spp.) are a highly obligate type of phytopathogen nematode that is omnivorous. In 1855, Berkeley first discovered the existence of root-knot nematodes in rhizosphere tumors of cucumbers, which are one of pathogenic organisms harmful to vegetables in facilities and are widely distributed around the world. Up to now, more than 80 kinds of root-knot nematodes are reported all over the world, 29 kinds are reported in China, the main range is very wide, and about more than 3000 kinds of crops are discovered through investigation and parasitized by the root-knot nematodes, wherein the crops comprise vegetables, food crops, economic crops, fruit trees, ornamental plants, weeds and the like. Cyst nematodes are a class of plant parasitic nematodes that occur in many countries of the world and are of economic importance. They cause yield losses in many important crops, including cereal crops, rice, potato, and soybean, among others. The most serious of them belongs to Soybean Cyst Nematodes (SCNs), which are obligate soybean parasitic nematodes and are destructive pests of soybeans all over the world. Mainly distributed in northeast China, inner Mongolia, Henan, Hebei, Jiangsu and other places. It is estimated that the economic losses due to soybean cyst nematodes are as high as $ 12 billion per year in china.

Primitive burr nematodes (Trichodorus primativus) are soil-borne nematodes that occur mostly in sandy and sandy soils, and occasionally in clay. Directly harming the roots by eating. The nematode is a transmission mediator of TRV (tobacco rattle virus), which causes tomato vascular bundle malformation, and it also transmits PEBV (pea early brown virus), which damages pea crops and affects alfalfa, cucumber, tobacco, beet, causing beetroot malformation. Can also be remotely transmitted through bulbous flowers and seedlings. Mainly distributed in the temperate zone. Most of the reports concerned are from europe.

At present, the prevention and treatment of the nematodes mainly adopt a chemical prevention and treatment method, namely, a high-efficiency chemical insecticide is adopted. The active ingredients mainly comprise abamectin, fosthiazate, methyl isoxathion, fenamiphos, methyl bromide, aldicarb, amino-oligosaccharin, dazomet, cartap, metam, carbofuran, carbosulfan and carbosulfan, most of the products are organic phosphorus products, the toxicity is high, most medicaments have a general inhibition effect on the pine wood nematodes parasitized in woody plants of Pinaceae and the like, and if the mortality rate of the abamectin to the pine wood nematodes under the recommended use concentration is less than 30%. In addition, nematodes are also prone to develop resistance to these broad-spectrum synthetic drugs, and these insecticides are mostly highly toxic, highly toxic or highly residual pesticides, which cause severe pollution to humans, the environment, microorganisms, water resources, etc., and thus the application of chemical insecticides is greatly limited. With the improvement of the environmental protection consciousness of human, the search of a novel biological insecticide which is safe, efficient, low in toxicity and environment-friendly from the nature is a research hotspot. The existing research shows that ethanol extracts of folium artemisiae argyi, paederia scandens, sophora alopecuroides, cynanchum komarovii, asarum and purple stem have strong poisoning activity on the root-knot nematodes of bananas, and methanol extracts of medicinal plants such as mint, chinaberry, polygonum hydropiper, pokeberry root, horseweed herb, sweet wormwood herb, red sage root and the like have good poisoning activity on soybean cyst nematodes, but the extracts only have poisoning activity on specific nematodes, and the active ingredients and the action mechanism and the practical application of the active substances for killing nematodes need to be further researched.

At the same time, it can be shown to a considerable extent that the thread-fighting action of pesticides and/or compounds of the same class against different nematodes is not analogizable, based on the marked differences in classification, main morphology, mode of propagation, most importantly in pesticide or compound resistance, between pine wood nematodes and other nematodes (such as root knot, primary bur and whole tooth complex nematodes). By using a 5-HT3 receptor antagonist MDL-72222 with nematode killing activity as a pilot, through changing the position of an aza-bicyclo bridge chain and introducing a pesticide active group thiadiazole or thiazolyl, 61 unreported 3-azabicyclo [3.2.1] octane aryl thiadiazole/thiazole compounds are designed and synthesized, and 42 of the 3-azabicyclo [3.2.1] octane aryl thiadiazole/thiazole compounds are subjected to activity experiments (in vitro nematode tests) on meloidogyne incognita and bursaphelenchus xylophilus respectively, and the results show that: 6 compounds in total have certain inhibitory activity on the pine wood nematode; under different concentrations, the 14 compounds with the inhibitory activity on the root-knot nematode are different from the compounds with the inhibitory activity on the pine wood nematode; i.e. the anti-linear activity of the same class of even the same compound against different nematodes is not analogized.

Therefore, the application of the biological prevention and control method with operability such as the nematode extract and the like to control the nematodes is a consensus of scholars at home and abroad, and the research, development and application of the novel high-efficiency biological reasonable nematicide is a trend of the development of nematode prevention and control.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a nematode extract and application thereof as a nematicidal agent.

In order to achieve the purpose, the invention adopts the technical scheme that:

a nematode extract is an extract obtained by extracting nematodes by using the nematodes as raw materials.

The nematodes are all nematodes of phylum nematoda.

The nematodes are nematodes of the class Thymophia.

The nematode is nematode of the family Plectridae of the order Plectranthoides of the class Pectinophorales of the class Thymophyceae.

The nematodes are pine wood nematodes (Bursaphelenchus xylophilus), pine wood nematodes (B.mcronatus), cryptogra praecox nematodes (Cryptohediones propofol), cylindrographa pinellii (Cylindlorylenchenpini), Bursaphelenchus xylophilus (B.nanjaci), Trichophythrix pinicola (B.pinasteri), Trichophythus lyratus (B.leoni) and Trichostrongylus arboricus (B.silvestris).

The nematode is preferably a pine wood nematode (Bursaphelenchus xylophilus).

The extract is a substance formed by directionally obtaining and/or concentrating one or more target components (namely, components with nematicidal effect) in a raw material through a physical and chemical extraction and separation process according to the requirement of the nematicidal application of the extracted final substance. Including those obtained by extraction methods known in the art and combinations of extraction methods known in the art; the extraction method includes but is not limited to solvent treatment (water, ethyl acetate, alcohol, acetone, oil, etc.), solid phase extraction, supercritical CO extraction₂Thermal or ultrasonic extraction, subjecting to pressure or centrifugal force, or other chemical and/or mechanical processes to obtain the desired component from the raw material. Thus, the term "extract" as used herein includes crude extracts, active components and active monomers.

The "crude extract" and "crude extract" in the invention both refer to a mixture of a target component with a lower concentration and a non-target component with a higher concentration obtained by extracting and separating raw materials through a solvent (alcohol such as ethanol and isopropanol, water, ethyl acetate and the like) or biological enzymolysis.

The "active ingredient" as referred to herein means an extract (relative to a crude extract) containing a target component at a higher concentration obtained by further treatment such as extraction, chromatography, distillation, sublimation, crystallization and precipitation, ion exchange, chromatographic separation, centrifugal separation, electrodialysis, electrolytic separation, electromagnetic separation, adsorptive separation, magnetic separation, etc., with respect to the crude extract.

The "active monomer" of the present invention refers to a target compound (i.e., an active compound having nematicidal effect) in the "active component".

The active substance refers to one or more active monomers (namely active compounds with nematicidal effect) obtained from the extract.

The analogue is a compound which takes one or more active monomers (namely active compounds with nematicidal effect) obtained from the extract as parent compounds, (1) the compound has similarity with the parent compounds in chemical structure; (2) a compound having a core structural feature of a parent compound; (3) the basic structure of the parent compound is retained and the parent compound is substituted with a similar atom or group instead of a carbon or other atom.

The nematode extract is obtained by taking nematodes as raw materials and extracting the nematodes with an organic solvent.

The extraction method can be one or more of squeezing hair, extracting, supercritical extraction, water extraction, and solvent extraction.

Adding the nematodes into an organic solvent, leaching for 12-36h, performing ultrasonic treatment, and performing suction filtration and reduced pressure distillation to obtain an extract; adding methanol into the extract, centrifuging, and filtering the supernatant to obtain the final product.

The organic solvent is alcohol; preferably anhydrous ethanol.

Application of nematode extract as nematicide is provided.

The nematicide contains one or more of an extract obtained by extracting the nematodes serving as raw materials, active substances in the extract and analogues of the active substances in the extract.

The use of said nematicide as a means to protect a plant against infestation or damage by nematodes; in particular as nematicides in the protection of plants against infestation or damage by pine wood nematodes.

The use of the pesticide as a nematicide to protect a plant or plant propagation material against damage by nematodes, against re-spread by transportation or against re-hazard; in particular as nematicides to protect plants or plant propagation material against damage caused by pine wood nematodes, against re-spread by transportation or against re-damage.

The active substance in the extract contains one or more of fatty acid compounds, benzene compounds, nitrogen-containing compounds, polyalcohol compounds or amino acid compounds, and salts or esters of the compounds (the compounds are fatty acid compounds, benzene compounds, nitrogen-containing compounds, polyalcohol compounds or amino acid compounds).

The fatty acid compound is a compound which consists of three elements of carbon, hydrogen and oxygen, has carboxyl on a carbon chain and can have hydroxyl on the carbon chain; wherein the carbon chain may be saturated or unsaturated.

The "salt of the foregoing material" or "salt of the compound of the general formula" according to the present invention is a salt in which hydrogen in a hydroxyl group or a carboxyl group in an active compound is substituted with a metal ion to form a corresponding salt. Furthermore, hydrogen in hydroxyl or carboxyl in the active compound is replaced by metal ions such as Na, K and the like to form corresponding salts.

The "ester of the foregoing substance" or "ester of the compound of the general formula" in the present invention is a corresponding product formed by esterification of a carboxyl group in an active compound with an alcohol.

Further, the fatty acid compound is fatty acid, hydroxy fatty acid or dibasic acid; wherein the fatty acid contains one or more of oleic acid, stearic acid, linoleic acid, arachidonic acid, ethyl myristate, cis-8, 11, 14-eicosatrienoic acid, cis-11, 14-eicosadienoic acid, 14-eicosenoic acid, alpha-eleostearic acid, nervonic acid, arachidic acid, erucic acid, lignoceric acid, n-pelargonic acid, n-capric acid, cis 13,16, 19-docosatrienoic acid or salts or esters of the above substances; preferably one or more of oleic acid, linoleic acid, ethyl myristate, n-nonanoic acid or salts or esters of the foregoing; more preferably one or more of oleic acid, ethyl myristate, n-nonanoic acid, or salts or esters of the foregoing.

The hydroxy fatty acid contains one or more of 6-hydroxy caproic acid, (15Z) -9,12,13-Trihydroxy-15-octadecenoic acid, 2-hydroxy valeric acid, (9Z) -12, 13-dihydroxy-octadecene-9-oic acid, 3-hydroxy myristic acid, 20-hydroxy eicosa-5Z, 8Z,11Z, 14Z-tetraenoic acid, Corchoriftty acid F, 16-hydroxy palmitic acid, 2-hydroxy myristic acid, 10-hydroxy-2-decenoic acid or salts or esters of the above substances; preferably one or more of 2-hydroxypentanoic acid, 3-hydroxymyristic acid, 16-hydroxypalmitic acid, 2-hydroxymyristic acid, 10-hydroxy-2-decenoic acid, or salts or esters of the foregoing; more preferably 2-hydroxy valeric acid, 3-hydroxy myristic acid, 16-hydroxy palmitic acid, 2-hydroxy myristic acid or one or more of the salts or esters of the aforementioned substances.

The dibasic acid contains one or more of methylmalonic acid, azelaic acid, 2-isopropylmalic acid, citraconic acid or salts or esters of the above substances; preferably one or more of methylmalonic acid, azelaic acid or salts or esters of the foregoing; more preferably azelaic acid and one or more of its salts or esters, such as monomethyl azelate, dimethyl azelate, monoethyl azelate and/or diethyl azelate.

The benzene compound contains one or more of 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, ferulic acid, vanillic acid, gentisic acid, benzoic acid, monobutyl phthalate, phenyllactic acid, homogentisic acid, 4-hydroxyphenylpyruvic acid, dihydrobenzofuran-2-carboxylic acid, 2, 5-di-tert-butylhydroquinone, bisphenol B or salts or esters of the substances; preferably 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, ferulic acid, benzoic acid, butyl phthalate, phenyllactic acid or one or more of salts or esters of the above substances; more preferably 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, benzoic acid, butyl phthalate, homogentisic acid or one or more of the salts or esters of the above substances.

The nitrogen-containing compound contains one or more of norsalsoline, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2-picolinic acid, 5-carboxyindole-3-acetic acid, 1-isopropenyl-2, 3-dihydro-1H-benzo [ d ] imidazole-2-ketone, urocanic acid or ureidopropionic acid or salts or esters of the above substances; preferably one or more of norsalsoline, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2-picolinic acid, 5-carboxyindole-3-acetic acid or salts or esters of the foregoing.

The polyalcohol compound contains erythritol, and one or more of salts and esters thereof.

The amino acid compound contains one or more of glutamic acid, leucine, phenylalanine, tyrosine methyl ester, L-tyrosine, aspartic acid, tryptophan, D-pantothenic acid/vitamin B5, N-acetyl-DL-norvaline or salts or esters of the above substances.

The analogue of the active substance in the extract is a compound shown as a formula I,

in the formula (I), the compound is shown in the specification,

R₁、R₂and R₄Each independently of the others is hydrogen, -OH, -COOH or C₁-C₄Alkyl groups of (a); r₃Selected from hydrogen, -OH or-COOR'; r' is selected from hydrogen or C₁-C₄Alkyl groups of (a);

n₁selected from 0 to 22; n is₂Selected from 0 to 1; n is₃Selected from 1 to 6;

and, except when R is₁、R₂、R₄And R' are simultaneously hydrogen, R₃is-OH, n₁Is 4, n₂Is 0, n₃Is 1;

or, a salt or ester of a compound of formula (la).

Furthermore, the analogue of the active substance in the extract is a compound shown in a formula I, and the compound is preferably one or more of oleic acid, methyl oleate, ethyl oleate, linoleic acid, methyl linoleate, ethyl linoleate, azelaic acid, monomethyl azelate, dimethyl azelate, monoethyl azelate, diethyl azelate, n-pelargonic acid, methyl nonanoate, ethyl nonanoate and the like.

The compounds can also be obtained naturally, commercially or by preparation according to the prior art.

A nematicide contains one or more of extract obtained by extracting nematodes, active substances in the extract, and analogues of the active substances in the extract.

The nematicide is a mixture of an active ingredient and an agriculturally acceptable solvent, wherein the active ingredient accounts for 0.00001 to 99.9 percent of the weight of the insecticide; the solvent is one or more of water or organic solvent.

The active ingredient is preferably 0.00005 to 99%, more preferably 0.0005 to 30%, and even more preferably 0.00005 to 15% by weight of the insecticide.

Further, the organic solvent is one or more of a polar solvent or a nonpolar solvent.

Further, the polar solvent includes anisole, dimethyl sulfoxide (DMSO), esters, amides, ketones, alcohols, glycols and derivatives thereof, pyrrolidones and derivatives thereof, and the like.

Further, the esters are one or more of butyl acetate, sec-butyl acetate, benzyl benzoate, butyl benzoate, 2-ethoxypropyl acetate, propylene glycol acetate, tributyl phosphate and the like.

Further, the amide is N, N-dialkyl alkylamide, preferably fatty acid dimethyl amide, more preferably one or more of N, N-dimethyl octanoyl amide, N-dimethyl decanoyl amide, N-methyl-2-pyrrolidone, N-ethyl-2-pyrrolidone (NEP), N-octyl-2-pyrrolidone (NOP) and N-methyl valerolactam.

Furthermore, the ketone is one or more of butanone, 2-heptanone, cyclohexanone, acetophenone and the like, and acetophenone derivatives such as 4-methoxyacetophenone and the like.

Further, the alcohol is one or more of butanol, cyclohexanol, benzyl alcohol, etc., diacetone alcohol, such as 4-hydroxy-4-methyl-2-pentanone, n-octanol, 2-ethylhexanol, etc.

Further, the dihydric alcohol and the derivative thereof are one or more of polyethylene glycol, glycerol, propylene glycol monomethyl ether acetate, dipropylene glycol monomethyl ether, propylene glycol monophenyl ether and the like.

Furthermore, the pyrrolidone and the derivatives thereof are one or more of N-octyl pyrrolidone, N-ethyl pyrrolidone, N-dodecyl pyrrolidone and the like.

Further, the polar solvent is preferably one or more of DMSO, N-ethyl-2-pyrrolidone, N-octyl-2-pyrrolidone, acetophenone, 2-heptanone, cyclohexanone, benzyl alcohol, 2-ethylhexanol, fatty acid dimethylamide or N, N-dimethyloctanamide and N, N-dimethyldecanamide.

Further, the nonpolar solvent is one or more of aromatic petroleum derivatives (aromatic hydrocarbons) or vegetable oil; the vegetable oil is coconut oil, palm kernel oil, palm oil, soybean oil, rapeseed oil, corn oil and methyl ester or ethyl ester of the above oil.

The solvent is preferably water.

The nematicide is a mixture of an active ingredient and an agriculturally acceptable solvent, and can also comprise agriculturally acceptable auxiliary materials; wherein the active ingredient accounts for 0.00001-99.9% of the weight of the nematicide.

The active ingredient is preferably 0.00005 to 99%, more preferably 0.0005 to 30%, even more preferably 0.00005 to 15% by weight of the nematicide.

Further, the auxiliary materials comprise agriculturally acceptable auxiliary agents and/or carriers.

Further, the auxiliary agent includes a wetting agent, a dispersing agent, a thickening agent, an antifreezing agent, an antifoaming agent, a film forming agent, a preservative and/or an emulsifier.

The active substance in the extract contains one or more of fatty acid compounds, benzene compounds, nitrogen-containing compounds, polyalcohol compounds or amino acid compounds and salts or esters of the compounds.

The fatty acid compound is fatty acid, hydroxy fatty acid or dibasic acid; wherein the fatty acid contains one or more of oleic acid, stearic acid, linoleic acid, arachidonic acid, ethyl myristate, cis-8, 11, 14-eicosatrienoic acid, cis-11, 14-eicosadienoic acid, 14-eicosenoic acid, alpha-eleostearic acid, nervonic acid, arachidic acid, erucic acid, lignoceric acid, n-pelargonic acid, n-capric acid, cis 13,16, 19-docosatrienoic acid or salts or esters of the above substances; preferably one or more of oleic acid, linoleic acid, ethyl myristate, n-nonanoic acid or salts or esters of the foregoing; more preferably one or more of oleic acid, ethyl myristate, n-nonanoic acid, or salts or esters of the foregoing.

The hydroxy fatty acid contains one or more of 6-hydroxy caproic acid, (15Z) -9,12,13-Trihydroxy-15-octadecenoic acid, 2-hydroxy valeric acid, (9Z) -12, 13-dihydroxy-octadecene-9-oic acid, 3-hydroxy myristic acid, 20-hydroxy eicosa-5Z, 8Z,11Z, 14Z-tetraenoic acid, Corchoriftty acid F, 16-hydroxy palmitic acid, 2-hydroxy myristic acid, 10-hydroxy-2-decenoic acid or salts or esters of the above substances; preferably one or more of 2-hydroxypentanoic acid, 3-hydroxymyristic acid, 16-hydroxypalmitic acid, 2-hydroxymyristic acid, 10-hydroxy-2-decenoic acid, or salts or esters of the foregoing; more preferably 2-hydroxy valeric acid, 3-hydroxy myristic acid, 16-hydroxy palmitic acid, 2-hydroxy myristic acid or one or more of the salts or esters of the aforementioned substances.

The dibasic acid contains one or more of methylmalonic acid, azelaic acid, 2-isopropylmalic acid, citraconic acid or salts or esters of the above substances; preferably one or more of methylmalonic acid, azelaic acid or salts or esters of the foregoing; more preferably azelaic acid and one or more of its salts or esters, such as monomethyl azelate, dimethyl azelate, monoethyl azelate or diethyl azelate.

The benzene compound contains one or more of 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, ferulic acid, vanillic acid, gentisic acid, benzoic acid, monobutyl phthalate, phenyllactic acid, homogentisic acid, 4-hydroxyphenylpyruvic acid, dihydrobenzofuran-2-carboxylic acid, 2, 5-di-tert-butylhydroquinone, bisphenol B or salts or esters of the substances; preferably 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, ferulic acid, benzoic acid, butyl phthalate, phenyllactic acid or one or more of salts or esters of the above substances; more preferably 2-methyl benzoic acid, salicylic acid, o-methyl salicylic acid, benzoic acid, butyl phthalate, homogentisic acid or one or more of the salts or esters of the above substances.

The nitrogen-containing compound contains one or more of norsalsoline, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2-picolinic acid, 5-carboxyindole-3-acetic acid, 1-isopropenyl-2, 3-dihydro-1H-benzo [ d ] imidazole-2-ketone, urocanic acid or ureidopropionic acid or salts or esters of the above substances; preferably one or more of norsalsoline, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, 2-picolinic acid, 5-carboxyindole-3-acetic acid or salts or esters of the foregoing.

The polyalcohol compound contains erythritol, and one or more of salts and esters thereof.

The amino acid compound contains one or more of glutamic acid, leucine, phenylalanine, tyrosine methyl ester, L-tyrosine, aspartic acid, tryptophan, D-pantothenic acid/vitamin B5, N-acetyl-DL-norvaline or salts or esters of the above substances.

The analogue of the active substance in the extract is a compound shown as a formula I,

in the formula (I), the compound is shown in the specification,

R₁and R₂And R₄Each independently of the others is hydrogen, -OH, -COOH or C₁-C₄Alkyl groups of (a); r₃Selected from hydrogen, -OH or-COOR'; r' is selected from hydrogen or C₁-C₄Alkyl groups of (a);

n₁selected from 0 to 22; n is₂Selected from 0 to 1; n is₃Selected from 1 to 6;

and, except when R is₁、R₂、R₄And R' are simultaneously hydrogen, R₃is-OH, n₁Is 4, n₂Is 0, n₃Is 1; or, a salt or ester of a compound of formula (la).

Furthermore, the analogue of the active substance in the extract is a compound shown in a formula I, and the compound is preferably one or more of oleic acid, methyl oleate, ethyl oleate, linoleic acid, methyl linoleate, ethyl linoleate, azelaic acid, monomethyl azelate, dimethyl azelate, monoethyl azelate, diethyl azelate, n-pelargonic acid, methyl nonanoate, ethyl nonanoate and the like.

The compounds can be natural, commercially available or prepared according to the prior art.

Furthermore, the analogues of the active substances in the extract are fatty acid compounds in the formula I except fatty acids, hydroxy fatty acids and dibasic acids described in Table 1. Such as: linolenic acid, isopropyl dodecanoate, monomethyl 4, 4-dimethyl-1, 7-heptanedioate, 2, 3-enaminodioic acid, 2-hydroxy-4-ethylpentanoic acid; the compounds can be natural or synthesized artificially.

A nematicidal composition preparation comprises an active component A and an active component B, wherein the active component A and the active component B are mixed according to the weight part ratio of 1:100-100: 1; the active component A is one or more of an extract obtained by extracting the nematode serving as a raw material, an active substance in the extract and an analogue of the active substance in the extract; the active component B is one or more of other nematocides, insecticides, acaricides and bactericides.

The preparation can be injection, spray, fumigant, coating agent or nanocrystallization preparation. Wherein, the nano preparation can be prepared into a compound nano preparation by adopting a wet medium grinding method, and the nano particle size is 50-500nm, preferably 300-500 nm.

The application of a compound in preparing a nematicidal preparation, wherein the compound is one or more of a compound shown as a formula I, 2-hydroxy valeric acid and 3-hydroxy myristic acid;

in the formula (I), the compound is shown in the specification,

R₁、R₂and R₄Each independently of the others is hydrogen, -OH, -COOH or C₁-C₄Alkyl groups of (a); r₃Selected from hydrogen, -OH or-COOR'; r' is selected from hydrogen or C₁-C₄Alkyl groups of (a);

n₁selected from 0 to 22; n is₂Selected from 0 to 1; n is₃Selected from 1 to 6;

and, except when R is₁、R₂、R₄And R' are simultaneously hydrogen, R₃is-OH, n₁Is 4, n₂Is 0, n₃Is 1;

or, a salt or ester of a compound of formula (la).

Further, the compound is preferably one or more of oleic acid, methyl oleate, ethyl oleate, linoleic acid, methyl linoleate, ethyl linoleate, azelaic acid, monomethyl azelate, dimethyl azelate, monoethyl azelate, diethyl azelate, n-pelargonic acid, methyl pelargonate and ethyl pelargonate.

A method of protecting a plant or its propagation material against damage caused by nematodes, said method comprising applying to the plant or its locus one or more of an extract obtained by extraction of the nematode itself as a starting material, an active substance in the extract, an analogue of the active substance in the extract, or a formulation thereof.

The method comprises applying one or more of an extract obtained by extracting nematodes themselves as a raw material, an active substance in the extract, an analogue of the active substance in the extract or the preparation to the plant material after nematode infestation for four weeks.

In the above-mentioned nematicides, the nematodes are all nematodes of the phylum nematoda.

The nematodes are nematodes of the class Thymophia.

The nematode is nematode of the family Plectridae of the order Plectranthoides of the class Pectinophorales of the class Thymophyceae.

The nematodes are pine wood nematodes (Bursaphelenchus xylophilus), pine wood nematodes (B.mcronatus), cryptogra praecox nematodes (Cryptohediones propofol), cylindrographa pinellii (Cylindlorylenchenpini), Bursaphelenchus xylophilus (B.nanjaci), Trichophythrix pinicola (B.pinasteri), Trichophythus lyratus (B.leoni) and Trichostrongylus arboricus (B.silvestris).

The nematode is preferably a pine wood nematode (Bursaphelenchus xylophilus).

The invention has the beneficial effects that:

(1) the nematode extract and the active substances in the extract are obtained by extracting the nematode per se and are further used for killing the nematode per se; does not contain any additive or exogenous compound, not only has high efficiency, but also is safer and nontoxic.

(2) The nematode extract obtained by the extraction of the invention and the active substances in the extract can be used around plant materials infected by nematodes by methods of spraying, soaking, brushing and the like.

(3) The compound is used as a nematicide, so that the harm of nematodes, particularly pine wood nematodes, can be effectively controlled, and the compound can be extracted by the nematodes and is easy to obtain; meanwhile, the compound can be combined with other compounds with insecticidal action, nematocides, insecticides, acaricides, bactericides and the like, such as abamectin, matrine, fosthiazate and amino-oligosaccharin, so that the activity can be obviously improved; the preparation of the invention is beneficial to subsequent market popularization and application.

Drawings

FIG. 1 is a graph showing the effect of azelaic acid (DMSO/Tween aqueous system) provided in example 3 of the present invention on indoor poisoning LC50 of Bursaphelenchus xylophilus for 24 h.

FIG. 2 is a graph showing the effect of azelaic acid (water system) provided in example 3 of the present invention on indoor poisoning LC50 of Bursaphelenchus xylophilus for 24 h.

FIG. 3 is a graph showing the effect of diethyl azelate (in an aqueous system) on indoor poisoning of Bursaphelenchus xylophilus LC50 for 24h, provided in example 4 of the present invention.

FIG. 4 is a graph showing the effect of dimethyl azelate (water system) provided in example 4 of the present invention on indoor poisoning of Bursaphelenchus xylophilus LC50 for 24 h.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and the described embodiments are only a part of the present invention, and not all of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of crude extract of Bursaphelenchus xylophilus and analysis of its chemical composition

1 test source of Bursaphelenchus xylophilus and expanded culture of Bursaphelenchus xylophilus

Weighing 300g of dry corn kernels with complete shapes, soaking the dry corn kernels in clear water at normal temperature for 2 days, then subpackaging the dry corn kernels into 10 conical bottles with the volume of 250mL, sealing the bottles with a breathable sealing film after subpackaging, and placing the bottles into a moist heat autoclave for sterilization at 120 ℃ for 30 minutes. After the sterilization is finished, after the strain is naturally cooled to the room temperature, inoculating a proper amount of Botrytis cinerea under the aseptic condition, inoculating 3000-plus-one heads of pine wood nematodes to each conical flask after the strain is cultured in an artificial climate box at 25 ℃ and 70% RH in a dark place for 5-7 days, and continuously culturing in the artificial climate box at 25 ℃ and 70% RH in the dark place for 8-15 days to obtain a large amount of pine wood nematodes.

2 preparation of crude extract of Bursaphelenchus xylophilus

After the nematodes were washed out of the wall of each flask with 200mL of distilled water, the eluate was collected in a 250mL plastic centrifuge cup, centrifuged at 2000r/min at room temperature for 10min, the supernatant was discarded, and the precipitate was collected. Transferring the nematodes separated by the Bellman funnel method into a 500mL conical flask, adding absolute ethyl alcohol until the alcohol concentration is 85%, soaking for 24h, performing ultrasonic treatment at 50 ℃ for 30min, performing suction filtration, and removing the ethyl alcohol by reduced pressure evaporation at 60 ℃. Adding methanol into the extract, centrifuging at room temperature of 2000r/min for 2min, collecting supernatant to obtain crude extract, filtering with 0.45 μm needle filter, and storing at 4 deg.C.

3 analysis of crude extract of Bursaphelenchus xylophilus by LC/MS/MS

Analyzing the crude extract of the pine wood nematode by LC/MS/MS to obtain the active components in the extract. The analysis conditions were as follows:

the instrument comprises the following steps: the Thermo Scientific Vanqish ultra-performance liquid chromatography system comprises a binary high-pressure gradient pump, an automatic sample injector, a column incubator and a vacuum degasser, and is coupled with a Thermo Scientific Q active Orbitrap mass spectrometer for mass spectrometry, and a Q-active host system comprises an H-ESI II source, an automatic injection pump and a switching valve. Data were analyzed by Thermo Scientific Compound discover software. The column was a Water Atlantis T3 column (2.1m m X150 mm, 3 μm).

LC/MS/MS analysis: the chromatographic mobile phase was acetonitrile, 0.1% (v/v) aqueous formic acid. Gradient elution is carried out on acetonitrile and acid water, the flow rate is 0.3ml/min, the elution time is 40min, the column temperature is 25 ℃, the sample injection amount is 3 mu L, a double-electric-spray ionization source works in a positive and negative ion mode to obtain a full-scanning mass spectrum of 50-1200m/z, the scanning rate is 1.0 spectrum/second, the source gas temperature is set to be 350 ℃, the additional source parameters are set to be 40 and 15 respectively for the sheath layer and the auxiliary gas flow, and the scavenging flow is 0; the spraying voltage is 3.0 kV; the capillary temperature was 325 ℃; the S-lens rf level is 60V. Data were collected from Massharer Data Acquisition V.04 and analyzed by Thermo Scientific Compound distributor software, the results of which are shown in the Table below.

As a result: as seen in the table below, the Bursaphelenchus xyfolus contains mainly long-chain fatty acids, fatty acids with hydroxyl and double bonds, bicarbonic acids, benzoic acid and its analogues, and nitrogen-containing compounds.

TABLE 2 Bursaphelenchus xyfolus extract LC/MS/MS analysis

Example 2 Activity assay of crude extract of Bursaphelenchus xylophilus on Bursaphelenchus xylophilus

Preparation of the medicament: the crude nematode extract prepared in example 1 was prepared as a 10g/L aqueous solution using a DMSO test system (4% DMSO + 15% Tween 80) as a blank control.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 3 poisoning effect of crude extract of Bursaphelenchus xylophilus on Bursaphelenchus xylophilus

As can be seen from the table above, the crude extract of the pine wood nematodes has a certain insecticidal activity on the pine wood nematodes (24h, the corrected mortality rate of the nematodes reaches 94.51%), and has a certain development prospect in the development of nematicide products.

Example 3 Activity test of monomer of active ingredient contained in extract of Bursaphelenchus xylophilus against Bursaphelenchus xylophilus

Experiment I, Activity of azelaic acid on pine wood nematode and LC50 determination

(I) Activity experiment of azelaic acid (DMSO/Tween aqueous System) on Bursaphelenchus xylophilus

Preparing a medicament:

the following experimental groups were formulated: preparing azelaic acid aqueous solution with different concentrations by using azelaic acid (commercially available) through aqueous solution containing DMSO and Tween 80; wherein, the DMSO content in the aqueous solution is 4%, and the Tween 80 content is 15%.

Experimental groups:

1) 10mL of azelaic acid aqueous solution with the concentration of 7 g/L;

2) 10mL of azelaic acid aqueous solution with the concentration of 5 g/L;

3) 10mL of azelaic acid aqueous solution with the concentration of 3 g/L;

4) 10mL of azelaic acid aqueous solution with the concentration of 2.5 g/L;

5) 10mL of 1.25g/L azelaic acid aqueous solution;

6) 10mL of azelaic acid aqueous solution with the concentration of 0.625 g/L;

7) 10mL of azelaic acid aqueous solution with the concentration of 0.025 g/L;

8) 10mL of azelaic acid aqueous solution with the concentration of 0.0125 g/L;

9) 10mL of azelaic acid aqueous solution with the concentration of 0.00625 g/L;

10) 10mL of azelaic acid aqueous solution with the concentration of 0.00375 g/L;

control group: the experimental group solvent was used as a blank control.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 4 poisoning effect of azelaic acid (aqueous system containing DMSO/Tween) on Bursaphelenchus xyfolus at different working concentrations

Fitting an equation to the poisoning activity of azelaic acid (water system containing DMSO/Tween) on pine wood nematodes under different concentrations for 24h to obtain a figure 1; as can be seen from FIG. 1, azelaic acid (aqueous system containing DMSO/Tween 80) has a certain poisoning activity against Bursaphelenchus xyfolus, and LC50 for Bursaphelenchus xyfolus in 24h in the room is 3.43 g/L.

(II) azelaic acid target specificity assay

Common nematocide abamectin and methylamino abamectin benzoate on the market are selected as positive control agents, azelaic acid is selected as a treatment agent, and clear water is selected as a blank control.

The method comprises the steps of selecting 60 coleoptera, which are bred in an indoor environment and have consistent development stages and similar individuals, as non-target insects, dividing the insects into 6 groups, repeating the treatment for two times, and respectively spraying a dissolution system of 1% azelaic acid (a water system containing DMSO/Tween 80), 1% abamectin and 1% emamectin benzoate (emamectin benzoate) on tested insects. And after 2h, counting the dead insect rate of each group.

The experimental results are as follows:

TABLE 5 treatment results of willow herb leaf beetles treated with different treatment agents

And (4) experimental conclusion: compared with the conventional nematicide products sold in the market, azelaic acid has stronger target specificity and higher safety to non-target insects (willow blue leaf beetle).

Activity experiment of (III) azelaic acid (water system) on pine wood nematode

Preparing a medicament:

the following experimental group of agents were prepared by mixing azelaic acid (commercially available) with distilled water to prepare aqueous solutions of azelaic acid of different concentrations, which were 1g/L, 0.9g/L, 0.8g/L, 0.7g/L, 0.6g/L, and 0.5g/L, respectively. Distilled water was used as a blank control.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

The experimental results are as follows:

TABLE 6 poisoning effect of azelaic acid (aqueous System) on Bursaphelenchus xyfolus

Fitting an equation to the poisoning activity of the pine wood nematodes under different concentrations of azelaic acid (water system) for 24h to obtain a figure 2; as can be seen from FIG. 2, azelaic acid (water system) has a good poisoning activity against Bursaphelenchus xyfolus, and LC50 for Bursaphelenchus xyfolus in 24h in the chamber is 0.55 g/L. Experiment on Activity of different agents of di-2-hydroxyvaleric acid and 3-hydroxymyristic acid on Bursaphelenchus xylophilus

2-hydroxyvaleric acid was prepared as a 10g/L solution in sterile water, 3-hydroxymyristic acid was prepared as a 10g/L solution in DMSO test system (water with 4% DMSO + 15% Tween 80), and the experimental group solvent was used as a blank (DMSO test system, sterile water). The preparation method and the medicament treatment method of the nematode suspension are the same as those of the first experiment, and the total number and the dead number of the nematodes are observed and recorded under a microscope for 24 hours under a 40-fold optical microscope for olympics CX33, and the results are as follows.

TABLE 7 poisoning effect of different agents on Bursaphelenchus xylophilus

As can be seen from the above table, both 2-hydroxyvaleric acid and 3-hydroxymyristic acid have a certain poisoning activity against Bursaphelenchus xylophilus at 10 g/L. Experiment III, 6-Hydroxyhexanoic acid, methylmalonic acid, malic acid or citraconic acid different agents for activity experiment of pine wood nematode

6-hydroxy caproic acid is prepared into 10g/L solution by DMSO test system (water containing 4% DMSO + 15% Tween 80), and methyl malonic acid, malic acid and citraconic acid are prepared into 10g/L solution by sterilized water, and experimental group solvent is used as blank control (DMSO test system, sterilized water). The preparation method and the medicament treatment method of the nematode suspension are the same as those of the first experiment, and the total number and the dead number of the nematodes are observed and recorded under a microscope for 24 hours under a 40-fold optical microscope for olympics CX33, and the results are as follows.

TABLE 8 poisoning effect of different agents on Bursaphelenchus xylophilus

As can be seen from the above table, 6-hydroxycaproic acid has almost no poisoning activity against pine wood nematodes at 10g/L, and methylmalonic acid, malic acid and citraconic acid have weak poisoning activity against pine wood nematodes at 10 g/L.

Example 4 Activity of azelaic acid esters/Potassium azelate on Bursaphelenchus xylophilus

Experiment I, Activity experiment of diethyl azelate on Bursaphelenchus xylophilus

1. Activity experiment of diethyl azelate/DMSO system on pine wood nematode

Preparation of the medicament: diethyl azelate (commercially available) was prepared as a 10g/L solution using a DMSO test system (water containing 4% DMSO + 15% Tween 80) as a blank control.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 9 poisoning effect of diethyl azelate/DMSO on Bursaphelenchus xyfolus

As can be seen from the above table, diethyl azelate (DMSO/Tween system) has a certain insecticidal activity against Bursaphelenchus xyfolus (24h, insecticidal activity up to 91.1%).

2. Activity experiment of diethyl azelate/water system on pine wood nematode

Preparation of the medicament: diethyl azelate (commercially available) was prepared as a blank control in aqueous solution of diethyl azelate at 1g/L, 0.7g/L, 0.5g/L, 0.3g/L, 0.1g/L, 0.07g/L, 0.05g/L, 0.03g/L and 0.01g/L with water.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 10 poisoning effect of diethyl azelate/water on Bursaphelenchus xyfolus

Fitting an equation to the poisoning activity of the pine wood nematodes under different concentrations of diethyl azelate (water system) for 24h to obtain a graph 3; as can be seen from FIG. 3, the LC50 of diethyl azelate (water system) for 24h against Bursaphelenchus xyfolus is 0.222g/L, which indicates that diethyl azelate has better nematocidal activity against Bursaphelenchus xyfolus.

Experiment II, Activity experiment of dimethyl azelate on pine wood nematode

Preparation of the medicament: dimethyl azelate (commercially available) was prepared into aqueous solutions of 0.01g/L, 0.03g/L, 0.05g/L, 0.07g/L, 0.1g/L, and 0.2g/L dimethyl azelate, respectively, using water as a blank control.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 11 poisoning effect of dimethyl azelate/water on Bursaphelenchus xyfolus

Test concentration (g/L)	Total insect number	Number of dead insects	Total insect number	Number of dead insects	Corrected mortality (%)
						0.2	52	52	56	56	100±0
0.1	40	40	68	66	98.53±2.08
						0.07	38	21	50	33	60.63±7.59
0.05	38	5	50	9	15.58±3.42
						0.03	28	2	32	3	8.26±1.58
0.01	17	0	23	0	0

Fitting an equation to the poisoning activity of the dimethyl azelate (water system) on the pine wood nematodes under different concentrations for 24h to obtain a graph 4; as can be seen from FIG. 4, dimethyl azelate (water system) has a good poisoning activity against Bursaphelenchus xyfolus, and LC50 for Bursaphelenchus xyfolus in 24 hours in a room is 0.0656 g/L.

Experiment III, Activity experiment of dipotassium azelate on pine wood nematode

Preparation of the medicament: dipotassium azelate was formulated into a 10g/L solution using a DMSO test system (water containing 4% DMSO + 15% Tween 80) as a blank control.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 12 poisoning effect of dipotassium azelate on Bursaphelenchus xylophilus

As can be seen from the above table, the dipotassium azelate has a certain insecticidal activity on the pine wood nematodes (24h, the insecticidal activity reaches 82.2%), which indicates that the salt-forming form of the pine wood nematode nematocide may also have a certain nematocidal activity on the nematodes.

Example 5 synergistic Effect of azelaic acid Compound pesticide on Bursaphelenchus xylophilus poisoning Activity

Preparing a medicament:

group 1. azelaic acid was prepared as a 3g/L aqueous solution with DMSO test system (water containing 4% DMSO + 15% Tween 80);

group 2. matrine is prepared into 0.6mg/L aqueous solution by sterilized water;

group 3, preparing the abamectin into 18mg/L aqueous solution by using sterilized water;

group 4. Compound 1 is prepared by adding azelaic acid and matrine into sterilized water, wherein the final concentration of azelaic acid is 3g/L, and the final concentration of matrine is 0.6 mg/L;

group 5. the compound medicament 2 is prepared by adding azelaic acid and abamectin into sterilized water, wherein the final concentration of the azelaic acid is 3g/L, and the final concentration of the abamectin is 0.6 mg/L;

group 6.DMSO assay system (water containing 4% DMSO + 15% tween 80) was blank control;

preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 13 synergism of azelaic acid built pesticide on Bursaphelenchus xylophilus poisoning Activity

Group of	Number of dead insects	Total insect number	Number of dead insects	Total insect number	Nematode mortality (%)	Corrected mortality (%)
							1	72	136	80	149	53.3％	51.0
2	67	161	53	155	38.0％	35.0
							3	65	177	50	162	33.9	30.7
4	142	168	118	153	81.0	80.0
							5	97	149	109	156	67.5	65.9
6	7	158	8	168	4.6	0

TABLE 14 synergistic effect of azelaic acid combination pesticide on Bursaphelenchus xylophilus poisoning activity

And (4) conclusion: as can be seen from the above table, azelaic acid, abamectin and matrine have obvious synergistic effect.

Example 6 preparation method of azelaic acid nanometer preparation and Activity experiment on Bursaphelenchus xylophilus

1) The preparation method of the azelaic acid nano preparation comprises the following steps:

0.1g to 0.5g of Tween 80 and PVP K30 are respectively added into 100ml of deionized water and dissolved in a magnetic stirrer at 500r/min to form a water phase. Weighing 0.3g of azelaic acid, adding the azelaic acid into the solution, uniformly mixing, transferring the mixture into a WG-0.3L medium grinder, selecting zirconia beads with the diameter of 0.2mm as grinding media, filling the medium into the cavity of the grinder by 80 percent, grinding the mixture for 30min at 2500r/min, equally dividing the ground nanosuspension into 4 parts, respectively placing the nanosuspension at the temperature of 52 ℃ for 0.5h, 2d, 6d and 12d, and then measuring and counting the particle size, the polydispersity and the azelaic acid decomposition rate of the nanosuspension, wherein detailed data are shown in a table.

TABLE 15 nanometer suspension indexes

Standing at 52 deg.C	Average particle diameter (d.nm)	Polydispersity	Decomposition rate of azelaic acid
				0.5h	385.2±49.08	0.09±0.05	1.15％
2d	378.5±33.44	0.09±0.06	1.31％
				6d	393.5±42.69	0.13±0.05	2.51％
12d	433.1±33.82	0.19±0.09	2.99％

The average grain diameter of the azelaic acid is less than 500nm after the azelaic acid is ground by the wet medium and stored for 12 days at 25 ℃, and the polydispersity PDI of the azelaic acid is less than 0.25, which shows that the solution grains not only reach the nano level, but also the distribution of the azelaic acid in the nano suspension is narrow, and the system uniformity is better. In the wet medium grinding process, due to the fact that a certain decomposition rate exists in the effective components caused by repeated collision of the grinding medium, the decomposition rate of the azelaic acid in the nanometer suspension is less than 5% through high performance liquid chromatography quantitative analysis, the GB/T19136-plus 2003 'determination method for pesticide thermal storage stability' stipulates that the pesticide passes through a thermal storage chemical stability test, and the qualification is that the decomposition rate of the effective components of the drug is less than 5%. In conclusion, the azelaic acid nanosuspension prepared by a medium grinding method has the advantages that the average particle size of the active ingredients, the dispersibility of the active ingredients in the system, the stability of the active ingredients and the like meet the requirements, so that the method can be used for preparing qualified azelaic acid nanocrystallization preparations.

2) The obtained azelaic acid nano preparation is utilized to measure the poisoning activity of the pine wood nematode:

preparing a medicament: taking the prepared azelaic acid nano suspension with the effective component concentration of 3g/L, preparing the azelaic acid solution with the concentration of 3g/L and without nano treatment, and preparing 0.3 percent Tween 80+ PVP K30 aqueous solution as a contrast agent.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 16 nematicidal Activity of azelaic acid before and after nanocrystallization

And (4) conclusion: the insecticidal activity of the azelaic acid to the pine wood nematode is greatly improved after the azelaic acid is processed by nano treatment, and the improvement range is 62.12 percent. Example 7 synergistic Effect of 2-Hydroxypentanoic acid Compound pesticide on Bursaphelenchus xylophilus poisoning Activity

Preparing a medicament:

preparing the 1.2-hydroxyvaleric acid of the group into a 10g/L aqueous solution by using water;

group 2. matrine is prepared into 0.6mg/L aqueous solution by sterilized water;

group 3, preparing the abamectin into 18mg/L aqueous solution by using sterilized water;

group 4. Compound 1 is prepared by adding 2-hydroxy valeric acid and matrine into sterilized water, wherein the final concentration of 2-hydroxy valeric acid is 10g/L, and the final concentration of matrine is 0.6 mg/L;

group 5. the compound medicament 2 is prepared by adding 2-hydroxyvaleric acid and abamectin into sterilized water, wherein the final concentration of the 2-hydroxyvaleric acid is 10g/L, and the final concentration of the abamectin is 0.6 mg/L;

group 6. water as control group;

preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

Synergistic effect of compound pesticide of table 172-hydroxyvaleric acid on poisoning activity of pine wood nematodes

Group of	Number of dead insects	Total insect number	Number of dead insects	Total insect number	Mortality of nematodes％)	Corrected mortality (%)
							1	83	123	86	135	65.5	63.8
2	67	161	53	155	38.0％	35.0
							3	65	177	50	162	33.9	30.7
4	132	158	112	143	81.0	80.1
							5	95	139	101	146	68.8	67.3
6	7	158	8	168	4.6	0

Synergistic effect of surface 182-hydroxyvaleric acid compound pesticide on poisoning activity of pine wood nematodes

And (4) conclusion: as can be seen from the table, 2-hydroxyvaleric acid, abamectin and matrine have obvious synergistic effect.

Example 8 preparation method of 2-Hydroxyvaleric acid nanometer preparation and Activity experiment on Bursaphelenchus xylophilus

1) The preparation method of the 2-hydroxyvaleric acid nano preparation comprises the following steps:

0.1g to 0.5g of Tween 80 and PVP K30 are respectively added into 100ml of deionized water and dissolved in a magnetic stirrer at 500r/min to form a water phase. Weighing 1g of 2-hydroxyvaleric acid, adding the 2-hydroxyvaleric acid into the solution, uniformly mixing, transferring the mixture into a WG-0.3L medium grinder, selecting zirconia beads with the diameter of 0.2mm as grinding media, taking the filling amount of the media as 80 percent of the cavity of the grinder, grinding the mixture for 30min at the speed of 2500r/min, equally dividing the ground nanosuspension into 4 parts, respectively placing the nanosuspension at the temperature of 52 ℃ for 0.5h, 2d, 6d and 12d, measuring and counting the particle size, the polydispersity and the decomposition rate of the 2-hydroxyvaleric acid of the nanosuspension, wherein detailed data are shown in the following table.

TABLE 19 indexes of nanosuspension

After being ground by the wet medium, the 2-hydroxyvaleric acid has average particle size smaller than 500nm and polydispersity PDI smaller than 0.25 when being stored at 25 ℃ for 12 days, which shows that the solution particles not only reach the nano level, but also the 2-hydroxyvaleric acid has narrow distribution in nano suspension and better system uniformity. In the wet medium grinding process, due to the fact that a certain decomposition rate exists in the effective components due to repeated collision of the grinding medium, the decomposition rate of the 2-hydroxyvaleric acid in the nanosuspension is less than 5% through high performance liquid chromatography quantitative analysis, the GB/T19136-one 2003 'determination method for pesticide thermal storage stability' provides that the pesticide passes a thermal storage chemical stability test, and the medicine is qualified when the decomposition rate of the effective components is less than 5%. In conclusion, the average particle size of the active ingredients of the 2-hydroxyvaleric acid nanosuspension prepared by the medium grinding method, the dispersibility of the active ingredients in the system, the stability of the active ingredients and the like meet the requirements, so that the qualified 2-hydroxyvaleric acid nanosuspension can be prepared by the method.

2) The poisoning activity of the 2-hydroxyvaleric acid nanocrystallized preparation on the pine wood nematodes is determined:

preparing a medicament: taking the prepared 2-hydroxyvaleric acid nanometer suspension with the effective component concentration of 10g/L, preparing 2-hydroxyvaleric acid solution with the concentration of 10g/L and without nanometer treatment, and preparing 0.3 percent of Tween 80+ PVP K30 aqueous solution as a contrast agent.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 202-Hydroxyvaleric acid Pre-and post-Nanocrastericidal Activity

And (4) conclusion: after the 2-hydroxyvaleric acid is subjected to nanocrystallization, the insecticidal activity of the 2-hydroxyvaleric acid on pine wood nematodes is greatly improved, and the improvement range is 39.6%. Example 9 synergistic Effect of 3-Hydroxymyristic acid Compound pesticide on Bursaphelenchus xylophilus poisoning Activity

Preparing a medicament:

group 1.3-Hydroxymyristic acid was prepared as a 10g/L solution in a DMSO test system (4% DMSO + 15% Tween 80);

group 2. matrine is prepared into 0.6mg/L aqueous solution by sterilized water;

group 3, preparing the abamectin into 18mg/L aqueous solution by using sterilized water;

group 4. Compound 1 is DMSO test system (4% DMSO + 15% Tween 80) with 3-hydroxy myristic acid and matrine added, wherein the final concentration of 3-hydroxy myristic acid is 10g/L, and the final concentration of matrine is 0.6 mg/L;

group 5. the compound preparation 2 is prepared by adding 3-hydroxymyristic acid and abamectin into a DMSO test system (4% DMSO + 15% Tween 80), wherein the final concentration of the 3-hydroxymyristic acid is 10g/L, and the final concentration of the abamectin is 0.6 mg/L;

group 6 DMSO assay system (4% DMSO + 15% tween 80) as control;

preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

TABLE 213-synergistic effect of hydroxymyristic acid compound pesticide on poisoning activity of pine wood nematodes

Group of	Number of dead insects	Total insect number	Number of dead insects	Total insect number	Nematode mortality (%)	Corrected mortality (%)
							1	71	127	72	123	57.2	55.1
2	67	161	53	155	38.0％	35.0
							3	65	177	50	162	33.9	30.7
4	123	160	110	139	77.9	76.8
							5	89	129	105	149	69.8	68.3
6	7	158	8	168	4.6	0

TABLE 223 synergistic effect of hydroxymyristic acid complex pesticides on pine wood nematode poisoning activity

And (4) conclusion: as can be seen from the table, 3-hydroxymyristic acid has obvious synergistic effect with abamectin and matrine.

Example 10 preparation method of 3-Hydroxymyristic acid nanometer preparation and Activity experiment on Bursaphelenchus xylophilus

1) The preparation method of the 3-hydroxymyristic acid nano preparation comprises the following steps:

0.1g to 0.5g of Tween 80 and PVP K30 are respectively added into 100ml of deionized water and dissolved in a magnetic stirrer at 500r/min to form a water phase. Weighing 1g of 3-hydroxymyristic acid, adding the solution into the solution, uniformly mixing, transferring the solution into a WG-0.3L medium grinder, selecting zirconia beads with the diameter of 0.2mm as grinding media, filling the medium into the cavity of the grinder, grinding the mixture for 30min at the speed of 2500r/min, equally dividing the ground nanosuspension into 4 parts, placing the nanosuspension at the temperature of 52 ℃ for 0.5h, 2d, 6d and 12d respectively, measuring and counting the particle size, the polydispersity and the 3-hydroxymyristic acid decomposition rate of the nanosuspension, wherein detailed data are shown in the following table.

TABLE 23 nanometer suspension index table

Standing at 52 deg.C	Average particle diameter (d.nm)	Polydispersity	Decomposition rate of 3-hydroxymyristic acid
				0.5h	349.2±36.02	0.05±0.04	1.35％
2d	353.4±35.64	0.05±0.05	1.41％
				6d	357.5±37.69	0.10±0.06	2.21％
12d	362.1±39.71	0.15±0.11	2.89％

After being ground by the wet medium, the 3-hydroxymyristic acid has the average particle size of less than 500nm and the polydispersity PDI of less than 0.25 when being stored for 12 days at 25 ℃, which indicates that the solution particles not only reach the nano-grade, but also the 3-hydroxymyristic acid is narrow in distribution in the nano-suspension and has better system uniformity. In the wet medium grinding process, due to the fact that a certain decomposition rate exists in the effective components due to repeated collision of the grinding medium, the decomposition rates of the 3-hydroxymyristic acid in the nanometer suspension are less than 5% through high performance liquid chromatography quantitative analysis, and the pesticide is qualified when the decomposition rate of the effective components of the drug is less than 5% through a thermal storage chemical stability test according to GB/T19136-2003 'determination method for thermal storage stability of pesticide'. In conclusion, the 3-hydroxymyristic acid nanosuspension prepared by the medium grinding method meets the requirements on the average particle size of the active ingredient, the dispersibility of the active ingredient in the system, the stability of the active ingredient and the like, so that the qualified 3-hydroxymyristic acid nanosuspension can be prepared by the method.

2) The poisoning activity of the obtained 3-hydroxymyristic acid nano preparation on the pine wood nematodes is determined:

preparing a medicament: taking the prepared 3-hydroxymyristic acid nano suspension with the effective component concentration of 10g/L, preparing a 5g/L non-nanocrystallized 3-hydroxymyristic acid solution, and preparing a 0.3% Tween 80+ PVP K30 aqueous solution as a control medicament.

Preparation of nematode suspension: suspending the pine wood nematodes on the corn kernel culture medium in a water phase by using a proper amount of sterile water, separating by a Bellman funnel method to obtain a live pine wood nematode suspension, centrifuging the nematode suspension for 2min at 2000r/min, removing supernatant, retaining precipitate, adding a proper amount of sterile water, performing microscopic examination to observe the age and activity of the nematodes and count the number of the nematodes, and finally quantifying the nematode suspension to 1000 nematodes/mL by using the sterile water, wherein most nematodes are in the J3 stage, the few are in the J2 stage and the J4 stage, and the nematode is reserved at 4 ℃.

Medicament treatment: taking 0.5mL of the prepared nematode suspension, centrifuging for 2min at 2000r/min, removing supernatant, adding 0.5mL of different reagent to be tested into the precipitate, blowing uniformly by using a pipette, covering the centrifuge tube, culturing in a climatic chamber with the temperature of 25 ℃, the relative humidity of 60% and no light, observing and recording the total number and the dead number of the nematodes under a 40-time Olympus CX33 optical microscope after 24 h.

epi243-Hydroxymyristic acid Pre-and post-Nanoclasic nematicidal Activity

And (4) conclusion: after the nanocrystallization treatment is carried out on the 3-hydroxymyristic acid, the insecticidal activity of the nanocrystallization treatment on the pine wood nematodes is improved, and the improvement range is 26.6%.