Nematode killing composition and method for controlling nematodes

Abstract

The present invention belongs to the field of pesticides, and specifically relates to a nematicidal composition and a method for controlling nematodes. The present invention finds that by compounding triflupyridam and cyclobromofenamide, the effect of nematode control can be significantly improved, especially with high activity against pine wood nematodes, providing a new option for pine wood nematode control. At the same time, the nematicidal composition of the present invention exhibits a significant synergistic effect, which is not only conducive to reducing environmental load, but also can avoid resistance caused by long-term and extensive use of existing agents, which is conducive to the control of nematode resistance.

Description

Technical Field

[0001] The invention belongs to the field of pesticides, and in particular relates to a nematicide composition and a method for controlling nematodes. Background Art

[0002] Pine wilt disease is a global forest disease that causes devastating mortality in pine species caused by the pine wood nematode (Bursaphelenchus xyluphilus). Pine wood nematodes are extremely destructive and difficult to control parasitic nematodes on pine trees. They are mainly spread in nature by vector insects, which is one of the important factors leading to the rapid and large-scale spread of pine wilt disease, making the prevention and control of pine wilt disease even more difficult.

[0003] At present, the prevention and control methods of pine wood nematode disease include plant quarantine, diseased tree control, biological control and chemical control, and chemical control has become one of the most commonly used control methods due to its convenience and quick effect. The main agents for the control of pine wood nematodes are avermectin and emamectin benzoate, and there are relatively few agents to choose from, especially those with other mechanisms of action.

[0004] Cyclobutrifluram is a SDHI fungicide / nematicide developed by Syngenta. As a fungicide, it has excellent control effects on fungal diseases caused by Fusarium. As a nematicide, it can control root-knot nematodes, pine wood nematodes, stem nematodes, etc. As a nematicide, the mechanism of action of cyclobutrifluram as a nematicide is to inhibit the activity of succinate dehydrogenase, destroy the ATP production in the mitochondria of plant pathogenic nematodes, and control a wide variety of nematodes such as root-knot nematodes.

[0005] However, there is still room for further improvement in its control effect on nematodes (especially pine wood nematodes). Summary of the invention

[0006] The first object of the present invention is to provide a nematicidal composition, the active ingredients of which are triflupyridam and cycloniflavone in a weight ratio of 9:1 to 1:3.

[0007] The second object of the present invention is to provide a method for controlling nematodes, comprising: applying the nematode killing composition to the nematodes or their living environment.

[0008] The present invention finds that by compounding triflupyridam and cyclofenac, the effect of nematode control can be significantly improved, especially with high activity against pine wood nematodes, providing a new option for pine wood nematode control. At the same time, the nematicidal composition of the present invention exhibits a significant synergistic effect, which is not only conducive to reducing environmental load, but also can avoid resistance caused by long-term and extensive use of existing agents, which is conducive to nematode resistance control. DETAILED DESCRIPTION

[0009] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0010] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0011] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0012] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0013] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0014] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0015] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0016] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0017] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0018] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0019] In the present invention, the term "trifluoropyridinamide" is also known as cyclobutrifluram, cyclobutrifluram, and its English common name is Cyclobutrifluram. Trifluoropyridinamide is a mixture of N-[(1S,2S)-2-(2,4-dichlorophenyl)cyclobutyl]-2-(trifluoromethyl)pyridine-3-carboxamide and its (1R,2R)-enantiomer, the former content is ≥80%, and its molecular formula is C 17 H 13 C 12 F3N2O, relative molecular mass is 389.20, CAS registration number is 1460292-16-3. Its specific structural formula is as follows:

[0020]

[0021] In the present invention, the English common name of the term "cyclobrombenzamide" is Cyclaniliprole, which is an o-carboxamidobenzamide insecticide developed by Ishihara Sangyo Co., Ltd. of Japan. It belongs to the amide insecticide and acts on the nicotinic receptor allosteric form. Cyclanbrombenzamide has contact, stomach poison and systemic activity, and has a broad-spectrum insecticidal activity. It has a good killing effect on pests such as Lepidoptera, Coleoptera, Thysanoptera and Diptera. The CAS registration number is 1031756-98-5, the chemical name is 2',3-dibromo-4'-chloro-1-(3-chloro-2-pyridyl)-6'-{[(1RS)-1-cyclopropylethyl]carbamoyl}-1H-pyrazole-5-carboxanilide, and the molecular formula is C 21 H 17 Br2Cl2N5O2, relative molecular mass is 602.11. Its specific structural formula is as follows:

[0022]

[0023] The present invention first provides a nematicidal composition, the active ingredients of which are triflupyridam and cyclobromofenamide in a weight ratio of 9:1 to 1:3. Among them, triflupyridam is used as a nematicide, and its mechanism of action is to inhibit the activity of succinate dehydrogenase, and cyclobromofenamide acts on the nicotinic receptor allosteric form. The present invention unexpectedly found in a large number of experiments that the combination of triflupyridam and cyclobromofenamide has a significant synergistic effect on pine wood nematodes, providing a new type of nematicidal composition for the control of pine wood nematodes, and can reduce the amount of nematicides used.

[0024] In some preferred embodiments, the weight ratio of triflupyridam to cyclofenac is 6: 1 to 1: 1. Within the above weight ratio range, the synergistic effect of the two is more significant.

[0025] In some further preferred embodiments, the weight ratio of triflupyridam to cyclofenac is 6: 1 to 3: 1. Within the above weight ratio range, the synergistic effect of the two is further improved.

[0026] In some further preferred embodiments, the weight ratio of triflupyridam to cyclofenac is 4: 1 to 2: 1, more preferably 3±0.5: 1, and further preferably 3: 1. Within the above weight ratio range, the synergistic effect of the two is further improved.

[0027] In some specific embodiments, the weight ratio of triflupyridam to bromofenac can be 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1:1, 1:2, 1:3, or any value in the range of 9:1 to 1:3.

[0028] In some embodiments, the weight proportion of the active ingredient in the nematicidal composition is 1% to 40%, preferably 2% to 30%, 2% to 25%, 3% to 20%, 3% to 15%, 4% to 10%, or 5% to 10%.

[0029] In some specific embodiments, the weight proportion of the active ingredient in the nematicidal composition can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, or any value in the range of 1% to 40%.

[0030] The nematicide composition of the present invention can be prepared into a dosage form permitted by nematicides, such as being prepared into a dosage form permitted by nematicides by combining it with auxiliary components of pesticide preparations.

[0031] In some embodiments, the nematicidal composition is in the form of a soluble solution, a microemulsion, an emulsifiable concentrate, an aqueous emulsion, a suspension, a microcapsule suspension-suspension concentrate, or a wettable powder.

[0032] In some preferred embodiments, the nematicidal composition is in the form of a soluble solution, a microemulsion, or an emulsifiable concentrate.

[0033] In some embodiments, the nematicidal composition further comprises a carrier, and the carrier is selected from any one or more of water, solvents, and fillers.

[0034] In some specific embodiments, the solvent includes but is not limited to one or more of C6-C30 isoparaffin or dearomatized solvent oil, methyl laurate, diethyl maleate, tributyl phosphate and other low-toxic and odorless monoester, diester or triester solvents, diisooctyl phthalate, trans-2-hexenol, diesel, 150# solvent oil, diisopropyl diacid, dimethylformamide, N,N-dimethylacetamide, N,N-dimethylpropionamide, dimethyl phthalate, vegetable oil, engine oil and dimethyl sulfoxide.

[0035] In some specific embodiments, the filler includes, but is not limited to, one or more of kaolin, white carbon black, clay, bentonite, attapulgite, diatomaceous earth and light calcium.

[0036] In some embodiments, the nematicidal composition further comprises an adjuvant, which is selected from any one or more of a wetting agent, a dispersant, an emulsifier, a disintegrant, an antifreeze agent, a preservative, a defoamer, a thickener, an acid-base regulator, and a binder. In specific implementation, those skilled in the art can selectively add the above-mentioned adjuvants in combination with different requirements of the preparation.

[0037] In some specific embodiments, the wetting agent includes, but is not limited to, one or more of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, alkyl sulfate, naphthalene sulfonate, alkyl succinate sulfonate and alkylphenol polyoxyethylene ether formaldehyde condensate sulfonate.

[0038] In some specific embodiments, the dispersant includes but is not limited to one or more of polyether phosphates and their salts, alkylphenol polyoxyethylene ether formaldehyde condensate sulfonates, polycarboxylates, block polyethers, phenethylphenol polyoxyethylene ether phosphate triethanolamine salts, naphthalene sulfonate formaldehyde condensates, lignin sulfonates, pull-open powder, NNO, MF and IS dispersing.

[0039] In some specific embodiments, the emulsifier includes but is not limited to one or more of polyether phosphate or salt, T-60, calcium dodecylbenzene sulfonate, Ningru 33# (styrene phenol formaldehyde resin polyoxyethylene-polyoxypropylene block polyether), ethylene oxide propylene oxide block copolymer, fatty alcohol polyoxyethylene ether, castor oil polyoxyethylene ether, alkylphenol polyoxyethylene ether, alkylbenzene calcium sulfonate and block polyether.

[0040] In some specific embodiments, the disintegrant includes, but is not limited to, one or more of ammonium sulfate, sodium sulfate, glucose, corn starch, ammonium chloride, bentonite, urea, and sodium bicarbonate.

[0041] In some specific embodiments, the antifreeze agent includes, but is not limited to, one or more of ethylene glycol, propylene glycol, glycerol, sodium chloride, urea, cellosolve, sorbitol, polyvinyl pyrrolidone and calcium chloride.

[0042] In some specific embodiments, the preservative includes but is not limited to one or more of sodium benzoate, potassium sorbate, sodium dehydroacetate, sodium diacetate, casper, paraben or its sodium salt, bronopol, pentylene glycol and sodium salicylate.

[0043] In some specific embodiments, the defoaming agent includes, but is not limited to, one or more of organosilicon compounds, lower alcohols, silicone oils, phosphates, fatty acids, and fatty acid esters.

[0044] In some specific embodiments, the thickener includes but is not limited to one or more of xanthan gum, fumed SiO2, bentonite, aluminum stearate, dragon gum powder, polyvinyl alcohol, modified cellulose, magnesium aluminum silicate, gum arabic, gum, gelatin, guar gum, alginic acid, agar and tall oil.

[0045] In some specific embodiments, the acid-base regulator includes but is not limited to one or more of citric acid, oxalic acid, ammonium chloride, tartaric acid, and succinic acid.

[0046] In some specific embodiments, the binder includes, but is not limited to, one or more of polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, polyvinyl alcohol, dextrin, sodium alginate, sodium alginate, calcium lignosulfonate, and the like.

[0047] In specific implementation, the carriers and adjuvants that can be used in the nematicidal composition of the present invention include but are not limited to the components mentioned above. Those skilled in the art can combine the above embodiments with common sense to obtain more specific embodiments of the nematicidal composition of the present invention.

[0048] The present invention does not particularly limit the preparation method of the nematicidal composition. The nematicidal composition can be obtained by mixing the components involved based on common knowledge in the art.

[0049] The present invention further provides a method for controlling nematodes, comprising: applying the nematode killing composition to the nematodes or their living environment.

[0050] In some specific embodiments, the nematode is a nematode of the genus Bursaphelenchus, more preferably a pine wood nematode (Bursaphelenchus xylophilus). The nematicidal composition of the present invention has a particularly outstanding control effect on nematodes of the genus Bursaphelenchus, especially pine wood nematodes.

[0051] The living environment of nematodes mentioned in the present invention includes any environment where nematodes may exist. For example, the internal tissues of host plants, the soil and root environment of host plants, the habitat of transmission vectors, the transportation and storage environment of host plants (or related wood), etc. In some specific embodiments, the living environment includes the xylem or other internal tissues (such as phloem) of pine trees (Pinus spp.), the soil and root environment of pine trees (or pine wood), the habitat of Monochamus spp. (such as pine forests, fallen trees, etc.), the transportation and storage environment of pine trees, etc.

[0052] In some specific embodiments, the method comprises: applying the nematicidal composition to the pine tree by means of trunk injection. Infusion is a method for controlling forest pests and diseases, and is particularly suitable for controlling nematodes (such as pine wood nematodes). It injects chemical agents directly into the xylem or phloem of trees, and the agents can directly act on the site where the pests and diseases occur, and are continuously released in the tree body through the tree's transport system (ducts in the xylem), thereby effectively controlling the pests and diseases.

[0053] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0054] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0055] Test Example 1: Determination of the co-toxicity coefficient of triflupyridam and cyclobenzapamide

[0056] Test method: immersion method.

[0057] Test object: pine wood nematode.

[0058] Test agent: Weigh triflupyridam and cyclobenzapamide technical drugs, dissolve them in a small amount of acetone respectively, and then dilute them with a 0.1% Tween aqueous solution containing a surfactant to prepare a single-dose stock solution, and design 7 groups of ratios (9:1, 6:1, 3:1, 1:1, 1:3, 1:6, 1:9), and set 5 series of mass concentrations of each single dose and each group of mixed ratios in equal proportions.

[0059] Experimental treatment: Take a 96-well culture plate, add 100 μL of each concentration of drug solution to each well with 100 μL (about 100) pine wood nematode suspension and mix well. Set up 3 replicates for each treatment in the experiment, and use 0.1% Tween aqueous solution and sterile aqueous solution as blank controls. After treatment, place the culture plate in a 25℃ constant temperature box, and check the life and death of pine wood nematodes under a microscope after 24 hours. Judgment criteria: Nematodes that keep swinging or in an "S" shape, curled shape, wavy shape or spiral shape are considered alive, while those that do not move and are in a "J" or "C" shape or have a stiff body are judged to be dead.

[0060] The co-toxicity coefficient (CTC value) of the mixture was calculated using the Sun Yunpei method.

[0061] The percentage of agent A in the mixture = agent A content / (agent A content+agent B content)×100%.

[0062] Actual toxicity index (ATI) of mixture = LC of agent A 50 / LC of mixture 50 ×100.

[0063] Theoretical toxicity index (TTI) of a mixture = TI(A) × the percentage of agent A in the mixture + TI(B) × the percentage of agent B in the mixture.

[0064] Co-toxicity coefficient (CTC) = actual toxicity index (ATI) of the mixture / theoretical toxicity index (TTI) of the mixture × 100.

[0065] A co-toxicity coefficient ≥120 indicates a synergistic effect; a co-toxicity coefficient ≤80 indicates an antagonistic effect; and a co-toxicity coefficient between 80 and 120 indicates an additive effect.

[0066] Table 1 Toxicity determination of triflupyridam and cyclofenac to pine wood nematodes

[0067] Pharmacy <![CDATA[LC 50 mg / L]]> 95% Confidence Interval Virulence regression equation Trifluopyridine 1.959 1.476～2.606 Y=-0.670+2.295X Cyclofenac 41.149 32.744～51.867 Y=-3.678+2.279X

[0068] Table 2 Determination of the co-toxicity coefficient of triflupyridamole and cyclobromofenamide

[0069]

[0070] According to the results in Tables 1 and 2, the weight ratio of triflupyridam to cyclofenac is 9:1-1:3, the co-toxicity coefficient is greater than 135, and there is a significant synergistic effect; when the weight ratio of triflupyridam to cyclofenac is 6:1-1:1, the synergistic effect is more obvious, all above 180; in particular, when the weight ratio of triflupyridam to cyclofenac is 3:1, the synergistic effect is the largest, and the co-toxicity coefficient is greater than 250.

[0071] Formulation Example 1: 8% triflupyridam-cyclobenzapamide microemulsion (3:1)

[0072] 6 g of triflupyridam technical drug, 2 g of cyclobenzapamide technical drug, 2 g of fatty alcohol polyoxyethylene ether (AEO-9), 17 g of castor oil polyoxyethylene ether (EL-40), and dimethylformamide were added to make up to 100 g to prepare microemulsion preparation example 1.

[0073] Formulation Example 2: 8% triflupyridam-cyclobenzapamide emulsifiable concentrate (6:1)

[0074] Add 5 g of triflupyridam original drug, 1 g of cyclobenzapamide original drug, 3 g of calcium dodecylbenzenesulfonate, 6 g of Ningru 33#, 1 g of castor oil polyoxyethylene ether (EL-40), and N,N-dimethylacetamide to make up to 100 g to prepare emulsifiable concentrate preparation Example 2.

[0075] Comparative Example 1:

[0076] A commonly used medicine purchased on the market - 5% avermectin emulsifiable concentrate.

[0077] Comparative Example 2: 8% triflupyridam-chlorantraniliprole microemulsion (3:1)

[0078] Microemulsion Preparation Example 2 was prepared by adding 6 g of trifluralin technical drug, 2 g of chlorantraniliprole technical drug, 2 g of fatty alcohol polyoxyethylene ether (AEO-9), 17 g of castor oil polyoxyethylene ether (EL-40), and dimethylformamide to make up to 100 g.

[0079] Test Example 2: Determination of the efficacy of the preparation against pine wood nematodes indoors

[0080] Test method: immersion method.

[0081] Test object: pine wood nematode.

[0082] Test agents: Preparation Example 1, Preparation Example 2, Comparative Example 1 and Comparative Example 2.

[0083] Experimental treatment: Dilute each agent 10,000 times with sterile water; add 0.1 ml of the agent solution and 0.1 ml (about 100 pine nematodes) of the suspension to a 96-well culture plate and mix well. Set up 3 replicates for each treatment; sterile water treatment is the blank control (CK). After treatment, place the culture plate in a 25°C incubator, and check the life and death of pine nematodes under a microscope after 24 hours. Judgment criteria: Nematodes that keep swinging or in an "S" shape, curled shape, wavy shape or spiral shape are considered alive, while those that do not move and are in a "J" or "C" shape or have a stiff body are judged to be dead.

[0084] Table 3 Determination of the efficacy of preparations against pine wood nematodes indoors

[0085] Pharmacy Total number of nematodes (items) Number of deaths in 24 hours 24h protection effect Preparation Example 1 342 319 93.3％ Preparation Example 2 309 266 86.1％ Comparative Example 1 354 315 89.0％ Comparative Example 2 316 238 75.3％ CK 278 0 0

[0086] According to the results in Table 3, 8% triflupyridam. cyclofenac microemulsion (3:1 and 6:1) has excellent effect in controlling pine wood nematodes, which is equivalent to the control effect of 5% avermectin emulsifiable concentrate, the mainstream agent on the market, with little difference; and the control effect of 8% triflupyridam. cyclofenac microemulsion (3:1) on pine wood nematodes is significantly better than that of 8% triflupyridam. chlorantraniliprole microemulsion (3:1).

[0087] It can be seen that the nematicidal composition of the present invention provides a new type of agent for controlling pine wood nematodes, which has a synergistic effect, is beneficial to reducing environmental load, and can also avoid the development of nematode resistance caused by long-term use of avermectin emulsions, thereby facilitating nematode resistance management.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A nematicidal composition, wherein the active ingredients are triflupyridam and cycloniflamid in a weight ratio of 9:1 to 1:

3.

2. The nematicidal composition according to claim 1, wherein The weight ratio of triflupyridam to chlorfenapyr is 6:1 to 1:

1.

3. The nematicidal composition according to claim 2, wherein The weight ratio of triflupyridam to chlorfenapyr is 6:1 to 3:

1.

4. The nematicidal composition according to any one of claims 1 to 3, wherein The weight proportion of the active ingredient in the nematicidal composition is 1% to 40%, preferably 3% to 15%.

5. The nematicidal composition according to any one of claims 1 to 4, wherein The nematicidal composition is in the form of a soluble agent, a microemulsion, an emulsifiable concentrate, an aqueous emulsion, a suspension, a microcapsule suspension-suspension concentrate, or a wettable powder, preferably a soluble agent, a microemulsion, or an emulsifiable concentrate.

6. The nematicidal composition according to any one of claims 1 to 5, wherein The nematicidal composition further comprises a carrier, and the carrier is selected from any one or more of water, solvents and fillers.

7. The nematicidal composition according to any one of claims 1 to 6, wherein The nematicidal composition further comprises an auxiliary agent, which is selected from any one or more of a wetting agent, a dispersant, an emulsifier, a disintegrant, an antifreeze agent, a preservative, a defoaming agent, a thickener, an acid-base regulator, and a binder.

8. A method for controlling nematodes, comprising: The nematode or its living environment is treated with the nematode composition according to any one of claims 1 to 7.

9. The method for controlling nematodes according to claim 8, wherein: The nematode is a nematode of the genus Bursaphelenchus.

10. The method for controlling nematodes according to claim 9, wherein: The nematode is pine wood nematode (Bursaphelenchus xylophilus).

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