Pesticide for spider mites belonging to family tetranychidae

Abstract

Disclosed is a pesticide for spider mites belonging to the family Tetranychidae, the pesticide comprising any of the following (a) to (c): (a) a double-stranded RNA that has an RNAi effect on syntaxin genes and / or the syntaxin12 gene; (b) an antisense nucleic acid for a transcript of a syntaxin gene and / or the syntaxin 12 gene, or a portion thereof; and (c) a nucleic acid construct that generates (a) or (b) in a cell. Also disclosed is a method for controlling spider mites belonging to the family Tetranychidae, the method comprising a step for applying any of the following (a) to (c): (a) a double-stranded RNA that has an RNAi effect on syntaxin genes and / or the syntaxin12 gene; (b) an antisense nucleic acid for a transcript of a syntaxin gene and / or the syntaxin 12 gene, or a portion thereof; and (c) a nucleic acid construct that generates (a) or (b) in a cell.

Description

Control agent for spider mites belonging to the family Tetranychidae

[0001] The present invention relates to an agent for controlling spider mites belonging to the Tetranychidae family and a method for controlling spider mites belonging to the Tetranychidae family.The present invention also relates to a nucleic acid and a transgenic plant transformed with the nucleic acid.

[0002] Until now, chemical pesticides containing organic or inorganic compounds as active ingredients have been used to control pests. Chemical pesticides target proteins, and may be effective in other organisms if the protein structure is the same.

[0003] Biological pesticides are a technology that can replace chemical pesticides. Among biological pesticides, there are RNAi pesticides that utilize RNA interference (RNAi) as a new next-generation pesticide. RNAi is a phenomenon that suppresses gene expression by degrading mRNA that has a sequence that matches 15 to 20 or more consecutive bases with the administered double-stranded RNA (dsRNA). RNAi pesticides are characterized by targeting base sequences (mRNA) and acting only on the targeted organism. Therefore, they are considered to be highly safe for the environment and the human body. Furthermore, even if resistance develops due to mutations in the target gene, it can be addressed by changing the sequence of the dsRNA.

[0004] It is known that in some invertebrates, RNAi is induced by dsRNA ingested mainly orally from the external environment. In this way, by ingesting dsRNA from the external environment, it is not necessary to introduce a dsRNA expression construct into cells, and dsRNA can be used without genetic modification without producing GM crops. Two-spotted spider mites (Tetranychus urticae) are a global agricultural pest and have also been reported to be resistant to chemical pesticides. It has been reported that RNAi was induced by orally ingesting dsRNA in such two-spotted spider mites (Non-Patent Document 1).

[0005] Control methods using RNAi pesticides have been reported in, for example, Patent Documents 1 to 3.

[0006] Patent Document 1 reports methods and compositions using silencing elements that, when ingested by pests such as Coleoptera plant pests or Chrysomelidae plant pests, reduce expression of a target sequence in the pest.

[0007] Patent Document 2 reports nucleic acid molecules and methods of use thereof for controlling pests through RNA interference-mediated inhibition of target coding sequences and transcribed non-coding sequences in pests, including Coleoptera and / or Hemiptera pests.

[0008] US Patent No. 5,999,949 reports compositions and methods that utilize double-stranded ribonucleic acid (dsRNA) to control insects, including whiteflies.

[0009] International Publication No. WO 2011 / 025860 International Publication No. WO 2018 / 175382 International Publication No. WO 2020 / 047053

[0010] Journal of Pesticide Science, Japan 46(2), 92-99(2021)

[0011] The present invention aims to provide an agent for controlling spider mites belonging to the Tetranychidae family, a method for controlling spider mites belonging to the Tetranychidae family, a nucleic acid effective for controlling spider mites belonging to the Tetranychidae family, and a transgenic plant transformed with the nucleic acid.

[0012] As a result of extensive research to achieve the above-mentioned object, the present inventors have discovered that by using syntaxin and syntaxin 12 as new target genes for RNAi pesticides, excellent acaricidal activity and oviposition suppression activity against two-spotted spider mites can be obtained.

[0013] Furthermore, the RNAi pesticide was found to have excellent acaricidal and oviposition-suppressing activity against multiple spider mites, such as the Kanzawa spider mite and the citrus red mite, but did not have acaricidal or oviposition-suppressing activity against the natural enemy, Phytoseiulus californicus.

[0014] The present invention was completed based on these findings and through further investigation, and provides the following agent for controlling spider mites belonging to the Tetranychidae family, method for controlling spider mites belonging to the Tetranychidae family, nucleic acid, and transgenic plant.

[0015] Item 1. A control agent for spider mites belonging to the Tetranychidae family, comprising any one of the following (a) to (c): (a) a double-stranded RNA having an RNAi effect on the syntaxin gene and / or the syntaxin 12 gene, (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or the syntaxin 12 gene or a part thereof, or (c) a nucleic acid construct that produces (a) or (b) intracellularly. Item 2. The control agent according to Item 1, wherein the spider mite belonging to the Tetranychidae family is at least one species selected from the group consisting of the genera Tetranychidae, Tetranychidae, and Tetranychidae. Item 3. The control agent according to Item 1, wherein the spider mite belonging to the Tetranychidae family is at least one species selected from the group consisting of Tetranychidae urticae, Tetranychidae Kanzawai, Tetranychidae three-toed spider mite, citrus red mite, red spider mite, and mango red mite. Item 4. Item 5. The control agent according to any one of Items 1 to 3, wherein the double-stranded RNA has a length of from 15 bp to the full length of the mRNA of syntaxin or syntaxin 12. Item 5. A method for controlling spider mites belonging to the Tetranychidae family, comprising the step of applying any of the following (a) to (c): (a) a double-stranded RNA having an RNAi effect on the syntaxin gene and / or the syntaxin 12 gene, (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or the syntaxin 12 gene or a part thereof, or (c) a nucleic acid construct that produces (a) or (b) intracellularly. Item 6. The control method according to Item 5, wherein the spider mites belonging to the Tetranychidae family are at least one species selected from the group consisting of the genera Tetranychidae, Tetranychidae, and Tetranychidae. Item 7. Item 5. The control method according to Item 5, wherein the spider mite belonging to the Tetranychidae family is at least one species selected from the group consisting of twospotted spider mite, Kanzawa spider mite, three-toed spider mite, citrus red mite, European red mite, and mango red spider mite. Item 8. The control method according to any one of Items 5 to 7, wherein the double-stranded RNA has a length of from 15 bp to the full length of the mRNA of syntaxin or syntaxin 12. Item 9. The control method according to any one of Items 5 to 8, wherein the application method is spraying, soil drench, or coating on plants, or infiltration on spider mites, or use of bait containing any of (a) to (c).Item 10. A method for controlling spider mites belonging to the Tetranychidae family, comprising using a transgenic plant transformed with a nucleic acid construct that produces intracellularly (a) double-stranded RNA that has an RNAi effect on the syntaxin gene and / or syntaxin 12 gene, or (b) an antisense nucleic acid against a transcript of the syntaxin gene and / or syntaxin 12 gene, or a portion thereof. Item 11. A nucleic acid set forth in any of (A) to (C) below: (A) a double-stranded RNA that has an RNAi effect on the syntaxin gene, the syntaxin 12 gene having a nucleotide sequence that is 75% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1, or the syntaxin 12 gene having a nucleotide sequence that is 75% or more identical to the nucleotide sequence set forth in SEQ ID NO: 2, (B) an antisense nucleic acid against a transcript of the syntaxin gene, the syntaxin 12 gene having a nucleotide sequence that is 75% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1, or a portion thereof, of the syntaxin 12 gene, the syntaxin 12 gene having a nucleotide sequence that is 75% or more identical to the nucleotide sequence set forth in SEQ ID NO: 2, or (C) a nucleic acid construct that produces (A) or (B) intracellularly. Item 12. The nucleic acid according to Item 11, wherein the double-stranded RNA has a length ranging from 15 bp to the full length of the mRNA of syntaxin or syntaxin 12. Item 13. A transgenic plant transformed by (C) according to Item 11.

[0016] According to the present invention, by targeting and suppressing the expression of syntaxin and syntaxin 12 mRNA, a compound having acaricidal activity and oviposition-suppressing activity against spider mites belonging to the Tetranychidae family is obtained, which is useful for controlling spider mites belonging to the Tetranychidae family. Furthermore, while the present invention has a control effect against multiple types of spider mites, it does not have a control effect against natural enemies, making it highly safe.

[0017] 1 is a diagram showing a list of PCR primers designed to obtain template DNA for dsRNA synthesis. FIG. 2 is a diagram showing a list of PCR primers designed to obtain template DNA for dsRNA synthesis. FIG. 3 is a diagram showing a list of PCR primers designed to obtain template DNA for dsRNA synthesis. FIG. 4 is a diagram showing a list of PCR primers designed to obtain template DNA for dsRNA synthesis. FIG. 5 is a schematic diagram showing the corresponding site of each dsRNA to syntaxin of the two-spotted spider mite used in the examples. FIG. 6 is a schematic diagram showing the corresponding site of each dsRNA to syntaxin 12 of the two-spotted spider mite used in the examples. FIG. 7 is a diagram showing the results of administering dsRNA solutions against 3Bc, vATP, syntaxin, and syntaxin 12 to two-spotted spider mites by the mesh method. FIG. 8 is a graph showing the survival rate of two-spotted spider mites over time (days 0 and 6). A solution (Water) containing no dsRNA was used as a control. "3Bc" is a sequence that has RNAi activity in Botrytis cinerea, but does not exhibit RNAi activity in two-spotted spider mites. Values ​​are means ± SD, n = 6. This figure shows the results of administering dsRNA solutions of various chain lengths for syntaxin and syntaxin 12 (syntaxin F0 to F12, syntaxin 12 F0 to F6, F8 to F13, F15) to twospotted spider mites using the mesh method. Graph showing survival rates of twospotted spider mites (days 0 and 6). 3Bc solution was used as a control. Values ​​are means, n = 5. This figure shows the results of administering dsRNA solutions of various chain lengths for syntaxin and syntaxin 12 (syntaxin F0 to F12, syntaxin 12 F0 to F6, F8 to F13, F15) to twospotted spider mites using the mesh method. Graphs showing the number of eggs laid per adult female twospotted spider mites (over 6 days) show the results of administering dsRNA solutions of various chain lengths for syntaxin and syntaxin 12 (syntaxin F0 to F12, syntaxin 12 F0 to F6, F8 to F13, F15) to twospotted spider mites using the mesh method. Both the top and bottom graphs show the number of eggs laid per adult female twospotted spider mites (over 6 days). 3Bc was used as a control. Values ​​are mean ± SD, n = 5. This figure shows the results of administering dsRNA solutions of various concentrations (40, 160 ppm) of syntaxin and syntaxin 12 derived from citrus red mites to twospotted spider mites using the mesh method. Graph showing the survival rate of twospotted spider mites (days 0, 6, and 9). 3Bc was used as a control. Values ​​are mean ± SD, n = 6. This figure shows the results of administering dsRNA (F0) solutions of various concentrations (40, 160 ppm) of syntaxin and syntaxin 12 derived from citrus red mites to twospotted spider mites using the mesh method (for 9 days).Both the top and bottom graphs show the number of eggs laid per adult female twospotted spider mite (3Bc was used as control). Values ​​are mean ± SD, n = 6. This figure shows the results of administering a solution of dsRNA (F0) against syntaxin and syntaxin 12 derived from twospotted spider mites to citrus red mites using the mesh method. Top: Graph showing the survival rate of citrus red mites (days 0, 3, and 6). Bottom: Graph showing the number of eggs laid per adult female citrus red mites (6 days). 3Bc was used as control. Values ​​are mean ± SD, n = 6. This figure shows the results of administering a solution of dsRNA (F3) against syntaxin and syntaxin 12 derived from Kanzawa spider mites (Tk) and twospotted spider mites (Tu) to Kanzawa spider mites using the mesh method. Top: Graph showing survival rate of Kanzawa spider mites (days 0, 3, and 6). Bottom: Graph showing the number of eggs laid per adult female Kanzawa spider mites (6 days). 3Bc was used as a control. Values ​​are mean ± SD, n = 6. Figure shows the results of administering a solution of dsRNA (F3) against syntaxin and syntaxin 12 derived from Kanzawa spider mites (Tk) and twospotted spider mites (Tu) to six twospotted spider mites using the mesh method. Top: Graph showing survival rate of twospotted spider mites (days 0 and 3). Bottom: Graph showing the number of eggs laid per adult female twospotted spider mites (3 days). 3Bc was used as a control. Values ​​are mean ± SD, n = 6. Figure shows the results of administering a solution of dsRNA against syntaxin and syntaxin 12 fragment (F0) derived from twospotted spider mites to six Phytoseiulus californicus mites using the mesh method. Top: Graph showing survival rate of Phytoseiulus californiae (days 0 and 4); Bottom: Graph showing number of eggs laid per adult female Phytoseiulus californiae (4 days); 3Bc was used as control. Values ​​are mean ± SD, n = 8. Figure 1 shows the results of administering a solution of syntaxin derived from twospotted spider mites (Tu) and mango spider mites (Oc) and dsRNA (F3) against syntaxin 12 to mango spider mites by the mesh method. Top: Graph showing survival rate of mango spider mites (days 0 and 10); Bottom: Graph showing number of eggs laid per adult female Phytoseiulus californiae (10 days); Water was used as control. Values ​​are mean ± SD, n = 5 for Oc, n = 3 for Tu.

[0018] Hereinafter, embodiments of the present invention will be described in detail.

[0019] In the present invention, unless otherwise specified, the term "gene" includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof. Furthermore, in the present invention, unless otherwise specified, the term "gene" refers to a regulatory region, a coding region, exons, and introns without distinction.

[0020] In the present invention, the terms "nucleic acid," "nucleotide," and "polynucleotide" are synonymous and include both DNA and RNA, and may be double-stranded or single-stranded.

[0021] The agent for controlling spider mites belonging to the Tetranychidae family of the present invention (hereinafter, sometimes referred to as "the control agent of the present invention") is characterized by containing any one of the following (a) to (c): (a) double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene, (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or syntaxin 12 gene or a part thereof, or (c) a nucleic acid construct that produces (a) or (b) in a cell.

[0022] The method for controlling spider mites belonging to the Tetranychidae family of the present invention (hereinafter, sometimes referred to as the "control method of the present invention") is characterized by comprising the step of applying any of the following (a) to (c): (a) double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene, (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or syntaxin 12 gene or a part thereof, or (c) a nucleic acid construct that produces (a) or (b) in a cell.

[0023] The control agent and control method of the present invention are applicable to spider mites belonging to the Tetranychidae family, and are particularly used for pests (agricultural pests) that harm crops (including horticultural crops and stored crops). Examples of spider mites belonging to the Tetranychidae family include spider mites belonging to the genus Tetranychus, spider mites belonging to the genus Panonychus, and spider mites belonging to the genus Oligonychus. Specific examples of spider mites belonging to the genus Tetranychus include Tetranychus urticae, Tetranychus kanzawai, Tetranychus evansi, and Tetranychus ludeni. Specific examples of spider mites belonging to the genus Panonychus include the citrus red mite (Panonychus citri) and the apple red mite (Panonychus ulmi). Specific examples of spider mites belonging to the genus Oligonychus include Oligonychus biharensis, Oligonychus coffeae, Oligonychus hondoensis, Oligonychus afrasiaticus, Oligonychus gossypii, Oligonychus indicus, Oligonychus mangiferus, Oligonychus mcgregori, Oligonychus pratensis, Oligonychus sacchari, Oligonychus thelytokus, and the like. In particular, the control agent and control method of the present invention are suitably used against two-spotted spider mites, Kanzawa spider mites, three-toed spider mites, citrus red mites, European red mites, mango red mites, and the like.

[0024] In the present invention, "control" means creating a state in which the spider mites cause no or little harm, and includes killing the spider mites, inhibiting the reproduction or growth of the spider mites, rendering the spider mites harmless or less harmful, and inhibiting diseases transmitted by the spider mites.

[0025] The control agent and control method of the present invention may target one type of spider mite belonging to the Tetranychidae family, or may target two or more types of spider mites.

[0026] Syntaxins are a family of membrane-bound Q-SNARE proteins involved in exocytosis. Syntaxins typically have three major domains: a C-terminal transmembrane region, a central SNARE domain, and an N-terminal regulatory domain. The present invention targets the syntaxin gene of spider mites belonging to the Tetranychidae family. Multiple syntaxin isoforms exist, and the present invention particularly targets the syntaxin gene and syntaxin 12 gene. The nucleotide sequences of syntaxin genes are registered on the NCBI website under RefSeq Accession No. XM_015934672 (Tetranychidae) (SEQ ID NO: 3, CDS is SEQ ID NO: 1) and XM_053356926 (Citrus red mite) (SEQ ID NO: 4), among others. The nucleotide sequences of the syntaxin 12 gene have been registered on the NCBI website under RefSeq Accession No. XM_015934443 (Tetranychus urticae) (SEQ ID NO: 5, CDS is SEQ ID NO: 2) and XM_053356930 (Panthera citri) (SEQ ID NO: 6). The nucleotide sequence of the syntaxin gene of Kanzawa spider mite is set forth in SEQ ID NO: 7, and the nucleotide sequence of the syntaxin 12 gene is set forth in SEQ ID NO: 8. The nucleotide sequence (partial sequence) of the syntaxin gene of mango spider mite is set forth in SEQ ID NO: 9, and the nucleotide sequence (partial sequence) of the syntaxin 12 gene is set forth in SEQ ID NO: 10.

[0027] The syntaxin gene and syntaxin 12 gene of the present invention include degenerates and mutants thereof, even if they do not have the nucleotide sequences registered in the databases described above. Examples of mutants include syntaxin genes and syntaxin 12 genes derived from other types of spider mites. Examples of mutants include genes consisting of nucleotide sequences that share 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the nucleotide sequences registered in the databases described above.

[0028] The identity of the nucleotide sequences can be calculated using analytical tools that are commercially available or available via telecommunications lines (Internet). The identity (%) of the nucleotide sequences can be determined using a program commonly used in the art (e.g., BLAST, FASTA, etc.) with the default settings.

[0029] By using the above (a) to (c) to knock down the expression of the syntaxin gene and syntaxin 12 gene in spider mites, a miticidal effect and oviposition suppression effect against spider mites can be obtained, making it possible to control spider mites belonging to the Tetranychidae family. These methods do not involve genetic modification of plants, and therefore do not have a genetic effect. The above (a) to (c) are explained below.

[0030] (Double-stranded RNA having RNAi effect) By using double-stranded RNA having RNAi effect on the syntaxin gene and / or syntaxin 12 gene, the expression of the syntaxin gene and / or syntaxin 12 gene can be inhibited or suppressed.

[0031] RNAi refers to the phenomenon in which double-stranded RNA consisting of sense RNA with a sequence identical to the mRNA sequence of a target gene and antisense RNA with a sequence complementary to that of the sense RNA is introduced into cells, thereby inducing destruction of the mRNA of the target gene and inhibiting translation into protein, thereby inhibiting expression of the target gene. Although the details of the RNAi mechanism are still unclear, it is believed that the main mechanism is that an enzyme called DICER (a member of the RNase III nuclease family) comes into contact with double-stranded RNA and degrades the double-stranded RNA into small fragments called small interfering RNA (siRNA). The double-stranded RNA having the RNAi effect in the present invention also includes such siRNA.

[0032] In addition, the double-stranded RNA having the RNAi effect in the present invention also includes molecules in which one end of the double-stranded RNA is closed, such as hpRNA or shRNA with a hairpin structure. That is, the above-mentioned RNA also includes molecules that can form a double-stranded RNA structure within the molecule. In addition, dumbbell-shaped RNA, which is a molecule in which both ends of the double-stranded RNA are closed, is also included in the double-stranded RNA having the RNAi effect in the present invention.

[0033] The RNA used for RNAi in the present invention does not need to be completely identical to the syntaxin gene and syntaxin 12 gene or a partial region of said genes, but it is preferable that they have complete identity. As will be shown in the Examples below, even when double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene of another genus and species belonging to the Tetranychidae family is used, it is possible to control spider mites belonging to the Tetranychidae family.

[0034] The double-stranded RNA having the RNAi effect of the present invention is typically double-stranded RNA consisting of a sense RNA having a sequence identical to a contiguous arbitrary RNA region in the mRNA of the syntaxin gene and syntaxin 12 gene, and an antisense RNA having a sequence complementary to the sense RNA. When targeting the genes of pests such as insects, long double-stranded RNA of several hundred nucleotides or more is more effective. The length of the "contiguous arbitrary RNA region" is typically from 15 bp to the full length of the mRNA of syntaxin or syntaxin 12 (1150 bp and 1360 bp, respectively). Examples of the length of the "contiguous arbitrary RNA region" include 16 bp or more, 17 bp or more, 18 bp or more, 19 bp or more, 20 bp or more, 21 bp or more, 30 bp or more, 40 bp or more, and 50 bp or more. The length of the "any contiguous RNA region" can be, for example, 1100 bp or less, 1000 bp or less, 900 bp or less, 800 bp or less, 700 bp or less, or 600 bp or less. Even long RNAs that do not have the RNAi effect in their original length can be degraded into siRNAs with the RNAi effect in cells, so the length of the double-stranded RNA in the present invention is not particularly limited. Furthermore, long double-stranded RNAs corresponding to full-length or nearly full-length regions of the mRNAs of the syntaxin gene and syntaxin 12 gene can be degraded in advance, for example, with DICER, and the degradation products can be used as the double-stranded RNA of the present invention. These degradation products can include double-stranded RNA molecules (siRNAs) with the RNAi effect.

[0035] In addition, since double-stranded RNA having an overhang of several bases at its end is generally known to have a high RNAi effect, the double-stranded RNA of the present invention may have an overhang of several bases at its end. The length of the bases forming this overhang is not particularly limited, but preferably is a two-base overhang. In the present invention, for example, double-stranded RNA having an overhang such as TT (thymine x 2) or UU (uracil x 2) can be used. The double-stranded RNA of the present invention also includes molecules in which the bases forming the overhang are DNA.

[0036] Furthermore, double-stranded RNA containing natural nucleotides is susceptible to degradation by ribonucleases. To overcome this drawback, in the present invention, 2'-O-methylated double-stranded RNA may be synthesized by methylating the 2'-OH groups of uridine and cytidine in the double-stranded RNA, or phosphorothioation may be performed.

[0037] The "double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene" of the present invention can be prepared based on information on the nucleotide sequences of the syntaxin gene and syntaxin 12 gene that are targets of the double-stranded RNA. For example, based on the nucleotide sequences set forth in SEQ ID NOs: 1 to 8, any continuous RNA region of mRNA, which is a transcription product of the sequence, is selected, and double-stranded RNA corresponding to this region is prepared.

[0038] Specific examples of the "double-stranded RNA having RNAi effect" in the present invention include the double-stranded RNAs (SEQ ID NOS: 9 to 39) described in the Examples.

[0039] Furthermore, in the double-stranded RNA of the present invention, all nucleotides do not need to be ribonucleotides (RNA). That is, in the present invention, one or more ribonucleotides constituting the double-stranded RNA can be replaced with the corresponding deoxyribonucleotides.

[0040] In the present invention, the nucleic acid constituting the double-stranded RNA can be a nucleic acid analog such as LNA (Locked Nucleic Acid), which is resistant to nucleases and therefore allows the RNAi effect to last for a longer period of time.

[0041] As for double-stranded RNA having an RNAi effect, two or more types of double-stranded RNA can be used for one target gene.

[0042] The double-stranded RNA of the present invention can be synthesized chemically or in vitro or in vivo using DNA encoding the double-stranded RNA of the present invention. The method using DNA encoding the double-stranded RNA is effective for preparing relatively long double-stranded RNA. Double-stranded RNA with RNAi effect can be designed by selecting an appropriate target sequence using a known program or algorithm.

[0043] (Antisense Nucleic Acid) By using an antisense nucleic acid against a transcription product of the syntaxin gene and / or syntaxin 12 gene or a part thereof, the expression of the syntaxin gene and / or syntaxin 12 gene can be inhibited or suppressed.

[0044] In one embodiment of the present invention, designing an antisense sequence complementary to the untranslated region near the 5' end of the mRNA of the syntaxin gene or syntaxin 12 gene can be effective in inhibiting gene translation. Sequences complementary to the coding region or the 3' untranslated region can also be used. Thus, nucleic acids containing antisense sequences to sequences in not only the translated regions of the syntaxin gene or syntaxin 12 gene but also the untranslated regions can be included in the antisense nucleic acids of the present invention.

[0045] The sequence of the antisense nucleic acid is preferably complementary to the target gene or a portion thereof, but does not need to be completely complementary as long as it can effectively suppress gene expression. It is sufficient for the sequence to have at least 90% identity, more preferably at least 95% identity, with the transcription product of the target gene. Generally, the longer the antisense nucleic acid, the more mismatches there are, allowing it to form a stable duplex with the target gene. To effectively suppress target gene expression using an antisense nucleic acid, the length of the antisense nucleic acid is preferably 15 bases or more. Furthermore, to avoid nonspecific effects, it is preferable to use multiple antisense nucleic acids complementary to different sequences of the target gene. The antisense nucleic acid may be DNA, RNA, or a chimeric mixture or derivative thereof. Antisense nucleic acid derivatives can be modified in the base moiety, sugar moiety, phosphate backbone moiety, etc. to improve stability.

[0046] The antisense nucleic acid of the present invention can be synthesized by a conventional method, for example, by using a commercially available nucleic acid synthesizer.

[0047] (Nucleic Acid Construct) The double-stranded RNA having the RNAi effect of the present invention may utilize a nucleic acid construct capable of expressing the double-stranded RNA in cells. Such a nucleic acid construct capable of expressing double-stranded RNA is usually a nucleic acid construct having a structure in which DNA encoding one strand of the double-stranded RNA and DNA encoding the other strand of the double-stranded RNA are linked to a promoter so that they can be expressed independently or sequentially. The nucleic acid construct can be easily produced by known genetic engineering techniques. Examples of the nucleic acid construct include expression vectors obtained by inserting DNA encoding the RNA of the present invention into a known expression vector.

[0048] The antisense nucleic acid of the present invention may also utilize a nucleic acid construct capable of expressing the antisense nucleic acid in cells. The nucleic acid construct can be easily produced by known genetic engineering techniques. Examples of the nucleic acid construct include an expression vector obtained by inserting DNA encoding the antisense nucleic acid of the present invention into a known expression vector.

[0049] In order to control spider mites belonging to the Tetranychidae family, it is necessary to incorporate the control agent of the present invention into the body of the spider mite. The method for incorporating the control agent of the present invention into the body of the spider mite is not particularly limited. For example, the control agent of the present invention can be attached to a plant that the spider mites feed on by spraying or applying it, or the control agent of the present invention can be infiltrated into the body of a plant that the spider mites feed on by spraying or applying it, and the spider mites can then ingest and incorporate the control agent. The control agent of the present invention can also be directly sprayed or applied to the spider mites, allowing it to infiltrate and be incorporated into the spider mites. Alternatively, bait containing any of the above (a) to (c) can be placed at the site of spider mite emergence or invasion route, whereby the spider mites ingest the bait, and thereby any of the above (a) to (c) is incorporated into the body of the spider mite.

[0050] Alternatively, the plant to be damaged may be a transgenic plant transformed with a nucleic acid construct that produces intracellularly (a) double-stranded RNA that has an RNAi effect on the syntaxin gene and / or syntaxin 12 gene, or (b) an antisense nucleic acid against a transcription product or a part thereof of the syntaxin gene and / or syntaxin 12 gene. By using such a transgenic plant, when spider mites eat the transgenic plant, (a) or (b) is taken up into the body of the spider mites.

[0051] The control agent and control method of the present invention may also use a nucleic acid described in any of the following (A) to (C): (A) a double-stranded RNA that has an RNAi effect on a syntaxin gene consisting of a base sequence having 75% or more identity to the base sequence set forth in SEQ ID NO: 1 or a syntaxin 12 gene consisting of a base sequence having 75% or more identity to the base sequence set forth in SEQ ID NO: 2, (B) an antisense nucleic acid against a transcription product or a part thereof of a syntaxin gene consisting of a base sequence having 75% or more identity to the base sequence set forth in SEQ ID NO: 1 or a syntaxin 12 gene consisting of a base sequence having 75% or more identity to the base sequence set forth in SEQ ID NO: 2, or (C) a nucleic acid construct that produces (A) or (B) in a cell.

[0052] Examples of the identity value include 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more.

[0053] The length of the double-stranded RNA (A) is typically from 15 bp to the full length of the mRNA of syntaxin or syntaxin 12 (1150 bp or 1360 bp, respectively). Examples of the length of the double-stranded RNA include 16 bp or more, 17 bp or more, 18 bp or more, 19 bp or more, 20 bp or more, 21 bp or more, 30 bp or more, 40 bp or more, and 50 bp or more. Examples of the length of the double-stranded RNA include 1100 bp or less, 1000 bp or less, 900 bp or less, 800 bp or less, 700 bp or less, and 600 bp or less.

[0054] When the above (a) to (c) are used as active ingredients in the pesticide of the present invention, they can be formulated together with pesticide adjuvants in various forms, such as emulsifiable concentrates, suspensions, dusts, granules, wettable powders, water-soluble powders, solutions, flowables, water-dispersible granules, aerosols, pastes, trace dusts, fumigants, and microcapsules, as in the case of conventional pesticide formulations. The blending ratio of the above (a) to (c) in the pesticide of the present invention is usually 0.1 to 500,000 ppm, preferably 1 to 100,000 ppm, and more preferably 1 to 1,000 ppm (when used diluted, the final concentration is shown). When actually using these formulations, they can be used as is, or diluted to the desired concentration with a diluent such as water. The blending ratio of the above (a) to (c) in the pesticide of the present invention can be appropriately changed depending on the form of the formulation, the purpose of application, the time, location, and pest occurrence status.

[0055] Examples of agricultural chemical adjuvants include carriers, emulsifiers, suspending agents, dispersing agents, spreading agents, penetrating agents, wetting agents, thickeners, stabilizers, pH adjusters, colorants, and fragrances.

[0056] If necessary, it can be mixed or used in combination with other agricultural chemicals such as insecticides, acaricides, nematicides, fungicides, antivirals, attractants, herbicides, plant growth regulators, etc., and in this case, even more excellent effects may be obtained.

[0057] The application amount of the control agent of the present invention is usually 0.000015 to 150,000 g, preferably 0.0015 to 1,500 g per hectare, as the amounts (a) to (c) above. However, in special cases, the amount may deviate from this range.

[0058] The control agent of the present invention can be applied by any commonly used application method, such as spraying (spraying, spraying, misting, atomizing, granulation, application to water surface, etc.), soil application (mixing, irrigation, etc.), surface application (painting, dusting, covering, etc.), immersion poison bait, etc. Application can also be performed by the so-called ultra-low volume spraying method. This method makes it possible to incorporate 100% of the above (a) to (c).

[0059] The plants targeted by the control agent and control method of the present invention are not particularly limited as long as the effects of the present invention can be obtained, and various plants can be targeted. Specific examples of such plants include the following: leafy vegetables (e.g., Chinese cabbage, spinach, cabbage, lettuce, komatsuna, bok choy, crown chrysanthemum, celery, leeks, onions, broccoli, cauliflower, asparagus, mulukhiyah), root vegetables (e.g., carrot, radish, burdock, sweet potato, taro, Chinese yam, taro, lotus root, turnip, cassava), fruit vegetables (e.g., tomato, cherry tomato, eggplant, bell pepper, bottle gourd, cucumber, zucchini, bitter melon, watermelon, strawberry, pumpkin, melon, corn, sweet corn), etc.; fruit trees (e.g., apple, pear, grape, persimmon, banana, coffee tree), soybean, edamame, beans such as adzuki beans, broad beans, peanuts, and kidney beans; tea; tobacco; cotton; wheat such as barley, wheat, oats, and rye; rice; flowers such as pansies, marigolds, salvia, petunias, periwinkles, chrysanthemums, carnations, roses, gentians, perennial baby's breath, gerberas, statice, lisianthus, alstroemeria, lilies, tulips, cyclamen, gladioli, freesia, primula, dendrobium, begonias, cymbidium, poinsettias, phalaenopsis, violas, daisies, scoparia, and calibrachoa; succulents such as cacti; trees such as shrubs, conifers, broad-leaved trees, and deciduous trees; grasses, etc.

[0060] (Transgenic Plant) The transgenic plant of the present invention is characterized in that it is transformed with a nucleic acid construct that produces intracellularly (a) double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene, or (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or syntaxin 12 gene or a part thereof. The transgenic plant of the present invention may also be a transgenic plant transformed by the above-mentioned (C).

[0061] By using such a transgenic plant, when spider mites eat the transgenic plant, the above (a) or (b) is taken up into the body of the spider mites, making it possible to control spider mites belonging to the Tetranychidae family.

[0062] The nucleic acid construct is introduced into a plant in a form in which the double-stranded RNA and antisense nucleic acid encoded therein are expressed. The expression form typically refers to a state in which transcription of RNA derived from the nucleic acid construct is carried out. Examples of such nucleic acid constructs include expression vectors obtained by inserting DNA encoding the double-stranded RNA or antisense nucleic acid into a known expression vector. To achieve this, the nucleic acid construct typically contains a promoter and terminator effective for expression, as well as other transcription / translation regulatory sequences, and is introduced into the plant. "Introduction into a plant" refers to the introduction of the nucleic acid construct into the plant as genetic material. Examples of promoters include the ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the actin promoter, and the promoter of the nopaline synthase gene. Examples of terminators include the CaMV 35S RNA gene terminator, the nopaline synthase gene terminator, and the octopine synthase gene terminator. The expression vector may further contain a selection marker gene such as an ampicillin resistance gene, a neomycin resistance gene, or a hygromycin resistance gene.

[0063] Methods for introducing nucleic acid constructs into plants include those using nucleic acid transfer reagents, particle guns (gene guns), electroporation, or Agrobacterium or viruses. These methods can be performed using known methods. The duration of the effect of the introduced genetic material can vary from transient to permanent, inherited to progeny; either method can be used. Transgenic plants include the "T1 generation" produced by transformation, the "T2 generation" derived from the seeds of the plant, and subsequent progeny plants (such as the T3 generation).

[0064] Plants to be transformed are not particularly limited and may be agricultural crops, ornamental plants, or the like, and may be either dicotyledonous or monocotyledonous plants. Specific examples of such plants include: leafy vegetables (e.g., Chinese cabbage, spinach, cabbage, lettuce, komatsuna (Japanese mustard spinach), bok choy, crown chrysanthemum, celery, leek, onion, broccoli, cauliflower, asparagus, mulukhiyah), root vegetables (e.g., carrot, radish, burdock, sweet potato, taro, Chinese yam, taro, lotus root, turnip, cassava), fruit vegetables (e.g., tomato, cherry tomato, eggplant, bell pepper, bottle gourd, cucumber, zucchini, bitter melon, watermelon, strawberry, pumpkin, melon, corn, sweet corn), and other vegetables; fruit trees (e.g., apple, pear, grape, persimmon, banana, coffee tree), soybean, edamame, beans such as adzuki beans, broad beans, peanuts, and kidney beans; tea; tobacco; cotton; wheat such as barley, wheat, oats, and rye; rice; flowers such as pansies, marigolds, salvia, petunias, periwinkles, chrysanthemums, carnations, roses, gentians, perennial baby's breath, gerberas, statice, lisianthus, alstroemeria, lilies, tulips, cyclamen, gladioli, freesia, primula, dendrobium, begonias, cymbidium, poinsettias, phalaenopsis, violas, daisies, scoparia, and calibrachoa; succulents such as cacti; trees such as shrubs, conifers, broad-leaved trees, and deciduous trees; grasses, etc.

[0065] The control agent of the present invention has acaricidal activity and oviposition-inhibitory activity against spider mites belonging to the Tetranychidae family by targeting and suppressing the expression of syntaxin and syntaxin 12 mRNA, and is useful for controlling spider mites belonging to the Tetranychidae family, making it highly useful as an RNAi pesticide. Furthermore, while the present invention has a control effect against multiple types of spider mites, it does not have a control effect against natural enemies, making it highly safe.

[0066] The natural enemies include organisms that prey on crop pests, such as Phytoseiulus californicus, Phytoseiulus swirskii, Phytoseiulus cucumeri, Phytoseiulus persimilis, Phytoseiulus kousukei, Phytoseiulus longicornis, Phytoseiulus nisellago, Phytoseiulus oryzae, Phytoseiulus nigricans ... deuteranthes, Phytoseiulus limonica, and Phytoseiulus monteri (Typhlodromips montdorensis), phytoseiid mites such as Andasonic Phytoseiulus, Amblyseiulus orbiculatus and Phytoseiulus hairi, mites of the Takaramitidae family such as the wall-attached Takaramitidae, mites of the Longnaria family, thrips such as Mallotus japonica, Anthothrips punctatus, Dioscorea orbiculatus, Chinese thrips and Hadenia thrips, tobacco stink bug, Orius strigiformes, Orius strigiformes and Examples include stink bugs such as the shiny stink bug, coleoptera such as the small-veined ladybug, the seven-spotted ladybug, the small red-spotted ladybug, the red-spotted ladybug and the two-spotted ladybug, beetles such as the small brown ladybug, the seven-spotted ladybug, the small red-spotted ladybug, the red-spotted ladybug and the two-spotted ladybug, bees such as the brown-veined gnat, the greenhouse gnat, the gifu gnat, the green leafminer gnat, the isaea gnat, the desert gnat and the coleman gnat, and flies such as the food gnat and the black-flowered house fly.

[0067] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present invention encompasses all arbitrary combinations of the constituent elements described in this specification.

[0068] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present invention may be combined in any way to specify the subject matter encompassed by the present invention, i.e., the present invention encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0069] The present invention will be described in detail below with reference to examples, but these examples are intended to explain specific embodiments of the present invention and are not intended to limit or restrict the scope of the invention disclosed in this application. It should be understood that various embodiments based on the concept of the present specification are possible in the present invention.

[0070] General biochemistry and molecular biology experimental methods, such as protein purification and electrophoresis, DNA cleavage and ligation, bacterial transformation, gene sequencing, and PCR, were basically performed in accordance with the instructions provided with the commercially available reagents and machinery used in each procedure, as well as in laboratory manuals (e.g., "Molecular Cloning: A Laboratory Manual (Third Edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press)"). PCR reactions were performed using a GeneAmp™ PCR system 9700 (Applied Biosystems). Unless otherwise specified, the instrument was operated according to the standard operating procedures described in the instructions provided with the instrument. All examples, unless otherwise described in detail, were or can be performed using standard techniques, which are well known and routine to those skilled in the art.

[0071] <Materials and Methods> 1. Obtaining cDNA of Syntaxin and Syntaxin 12 Homologous Genes (1) The test insects used were the two-spotted spider mite (scientific name: Tetranychus urticae, English name: Two-spotted spider mite), which was provided by the Akita Agricultural Experiment Station and serially reared at the Central Research Institute of Ishihara Sangyo Kaisha, Ltd.; the Kanzawa spider mite (scientific name: Tetranychus kanzawai), which was naturally reared at the Central Research Institute of Ishihara Sangyo Kaisha, Ltd.; the citrus red mite (scientific name: Panonychus citri), which was naturally reared at the Central Research Institute of Ishihara Sangyo Kaisha, Ltd.; and the mango spider mite (scientific name: Oligonychus coffeee), which was provided by the JA in Nanjo City, Okinawa Prefecture, and serially reared at the Central Research Institute of Ishihara Sangyo Kaisha, Ltd.

[0072] (2) Preparation of cDNA Total RNA was extracted from insects including all stages from first instar larvae to adults for two-spotted spider mites (Tu), all stages from first instar larvae to adults for Kanzawa spider mites (Tk), all stages from first instar larvae to adults for citrus red mites (Pc), and all stages from first instar larvae to adults for mango red mites (Oc) by the guanidine hydrochloride method using RNeasy Plant Mini Kit (QIAGEN). Using this RNA as a template, PrimeScript ＴＭ Single-stranded cDNA was synthesized according to the kit protocol of the RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Bio Inc.).

[0073] 2. RNAi method (1) Amplification of templates for dsRNA synthesis For syntaxin (SEQ ID NO: 3) and syntaxin 12 (SEQ ID NO: 5) of the two-spotted spider mite, the full-length genes obtained from RT-PCR products were used as templates. For the citrus red mite, the Kanzawa spider mite, and the mango red spider mite, the RT-PCR products were used as templates.

[0074] To obtain a PCR product (dsRNA synthesis template DNA) used as a template for dsRNA synthesis, a PCR primer containing the promoter sequence of T7 RNA polymerase was designed (Figure 1). KOD ONE (Toyobo Co., Ltd.) was used as the PCR polymerase. The PCR product obtained in the above reaction was subjected to agarose electrophoresis, and after confirming that the desired PCR product was obtained, it was purified using Wizard SV Gel and PCR Clean-Up System (Promega).

[0075] (2) Synthesis of dsRNA Using 1 μg of the above template DNA, RNA was synthesized according to the ScriptMAX™ Thermo T7 Transcription Kit (Toyobo Co., Ltd.) and dissolved in an appropriate amount of nuclease-free ultrapure water. A small amount of this dsRNA was used to measure the concentration using a NanoDrop 2000 (Thermo Fisher Scientific) and confirm the synthesis by agarose electrophoresis. The dsRNA for which synthesis was confirmed was stored at -80°C. The site of each synthesized dsRNA is shown in Figures 2 and 3. The sequence of each dsRNA is indicated by the following sequence number.

[0076]

[0077] (3) Administration of dsRNA-TuSyntaxin and TuSyntaxin 12 (Two-spotted Spider Mite, Mesh Method) 20 μl of dsRNA solution (40 ng / μl) was dropped onto a micromesh (mesh opening: 100 μm) with liquid impregnated into pores resembling leaf cells, and a moisture-proof thin film (Parafilm) was placed over the mesh to create a feeding device. After the device was completed, adult female two-spotted Spider Mite were released into the device and allowed to suck the dsRNA solution for 24 hours. After sucking, treated mite individuals were transplanted onto 2 cm diameter leaf discs of common bean leaves, and the number of surviving individuals and the number of eggs laid were observed over time.

[0078] (4) Confirmation of chain length-dependent efficacy TuSyntaxin Test sample: dsRNA (TuSyntaxin (F0 to F12), TuSyntaxin 12 (F0 to F6, F8 to F13, F15) derived from two-spotted spider mites) (40 ng / μl 20 μl) Test method: The dsRNA was administered using the mesh method described in (3).

[0079] (5) Confirmation of the efficacy of PSyntaxin and PSyntaxin 12 derived from citrus red mites against other pest mites (two-spotted spider mites, mesh method) Test sample: dsRNA (PSyntaxin (F0), PSyntaxin 12 (F0)) (40 ng / μl or 160 ng / μl 20 μl) Test subject: two-spotted spider mites Test method: dsRNA was administered using the mesh method described in (3).

[0080] (6) Confirmation of the efficacy of TuSyntaxin and TuSyntaxin 12 derived from two-spotted spider mites against other pest mites (citrus red mite, mesh method) Test sample: dsRNA (TuSyntaxin (F0), TuSyntaxin 12 (F0)) (40 ng / μl 20 μl) Test subject: citrus red mite Test method: dsRNA was administered using the mesh method described in (3).

[0081] (7) Confirmation of the efficacy of TuSyntaxin and TuSyntaxin 12 derived from two-spotted spider mites and TkSyntaxin and TkSyntaxin 12 derived from Kanzawa spider mites against other pest mites (Kanzawa spider mites, mesh method) Test sample: dsRNA (TuSyntaxin (F3), TuSyntaxin 12 (F3), TkSyntaxin (F3), TkSyntaxin 12 (F3)) (40 ng / μl 20 μl) Test subject: Kanzawa spider mites Test method: dsRNA was administered using the mesh method described in (3).

[0082] (8) Confirmation of the efficacy of TuSyntaxin and TuSyntaxin 12 derived from two-spotted spider mites and TkSyntaxin and TkSyntaxin 12 derived from Kanzawa spider mites against other pest mites (two-spotted spider mites, mesh method) Test sample: dsRNA (TuSyntaxin (F3), TuSyntaxin 12 (F3), TkSyntaxin (F3), TkSyntaxin 12 (F3)) (40 ng / μl 20 μl) Test subject: two-spotted spider mites Test method: dsRNA was administered using the mesh method described in (3).

[0083] (9) Confirmation of the efficacy of TuSyntaxin and TuSyntaxin 12 against natural enemies (Phytoseiulus californicus, mesh method) Test sample: dsRNA (TuSyntaxin (F0), TuSyntaxin 12 (F0)) (40 ng / μl 20 μl) Test subject: Phytoseiulus californicus Test method: The dsRNA was administered using the mesh method described in (3).

[0084] (10) Confirmation of the efficacy of TuSyntaxin derived from twospotted spider mites and OcSyntaxin and OcSyntaxin 12 derived from mango spider mites against other pest mites (mango spider mites, mesh method) Test sample: dsRNA (TuSyntaxin (F3), OcSyntaxin (F3), OcSyntaxin 12 (F3)) (40 ng / μl 20 μl) Test subject: mango spider mites Test method: dsRNA was administered using the mesh method described in (3).

[0085] <Results> Abbreviations may be used in figures showing test results. For example, PS12-F0-160 means that the F0 fragment of syntaxin 12 derived from the citrus red mite was applied at 160 ppm.

[0086] (1) Administration of dsRNA-TuSyntaxin and TuSyntaxin12 (Two-spotted Spider Mite, Mesh Method) The results are shown in Figure 4. TuSyntaxin F3 and TuSyntaxin12 F3 showed higher acaricidal activity than dsRNA against v-ATPase, which has been reported to have acaricidal activity against spider mites. Here, the concentrations of dsRNA against v-ATPase, TuSyntaxin, and TuSyntaxin12 were lower than those used in the literature (PLoS ONE 12(7):e0180654), and the dsRNA of the present invention showed activity even at low concentrations.

[0087] (2) Confirmation of chain length-dependent acaricidal effect The results for TuSyntaxin and TuSyntaxin12 TuSyntaxin-F0 to F12 and TuSyntaxin12-F0 to F6, F8 to F13, and F15 are shown in Figure 5. As the chain length became shorter, the acaricidal activity tended to decrease. Chain length (TuSyntaxin) F6 1150bp>F12 806bp>F0 689bp>F7 626bp>F8 604bp>F3 497bp>F1 465bp>F2 415bp>F4 268bp>F5 226bp>F10 148bp>F11 108bp>F9 80bp Chain length (TuSyntaxin12) F9 1360bp>F15 828bp>F4 621bp>F5 600bp>F0 577bp>F10 512bp>F3 426bp>F2 415bp>F1 407bp>F6 221bp>F8 195 bp > F11 120 bp > F13 112 bp > F12 106 bp TuSyntaxin-F0, F3, F6, F8 and TuSyntaxin12-F0, F1, F2, F3, F5, F10 had particularly high acaricidal activity.

[0088] (3) Confirmation of chain length-dependent spawning inhibitory effect. The results for TuSyntaxin and TuSyntaxin12 TuSyntaxin-F0 to F12 and TuSyntaxin12-F0 to F6, F8 to F13, and F15 are shown in Figure 6. As the chain length decreased, the spawning inhibitory activity tended to decrease. Furthermore, TuSyntaxin-F3, F6, and F8 and TuSyntaxin12-F3 and F5 had particularly high spawning inhibitory activity.

[0089] (4) Confirmation of efficacy of PSyntaxin and PSyntaxin 12 derived from citrus red mite against other pest mites (two-spotted spider mite, mesh method) The results are shown in Figures 7 and 8. PSyntaxin and PSyntaxin 12 also showed miticidal and oviposition-inhibitory effects against two-spotted spider mites.

[0090] (5) Confirmation of the efficacy of TuSyntaxin and TuSyntaxin 12 against other pest mites (citrus red mite, mesh method) The results are shown in Figure 9. TuSyntaxin and TuSyntaxin 12 also showed efficacy against citrus red mite. From this, it can be seen that dsRNA against syntaxin and syntaxin 12 exhibits miticidal and oviposition suppression effects not only in the Tetranychus genus but also in the Panonychus genus. Therefore, it can be said that Syntaxin is effective against a wide range of pest mites.

[0091] (6) Confirmation of the efficacy of TuSyntaxin and TuSyntaxin 12 derived from two-spotted spider mites, and TkSyntaxin and TkSyntaxin 12 derived from Kanzawa spider mites against other pest mites (Kanzawa spider mites, mesh method) The results are shown in Figure 10. TuSyntaxin, TuSyntaxin 12, TkSyntaxin, and TkSyntaxin 12 also showed miticidal and oviposition-inhibiting effects against Kanzawa spider mites.

[0092] (7) Confirmation of efficacy of TuSyntaxin and TuSyntaxin 12 derived from Tetranychus urticae and TkSyntaxin and TkSyntaxin 12 derived from Tetranychus kanzawai against two-spotted spider mites (two-spotted spider mites, mesh method) The results are shown in Figure 11. TuSyntaxin, TuSyntaxin 12, TkSyntaxin and TkSyntaxin 12 also showed miticidal and oviposition-inhibiting effects against two-spotted spider mites.

[0093] (8) Confirmation of efficacy of TuSyntaxin and TuSyntaxin 12 against natural enemies (Phytoseiulus californiae, mesh method) The results are shown in Figure 12. TuSyntaxin and TuSyntaxin 12 did not have acaricidal or oviposition-inhibitory activity against Phytoseiulus californiae.

[0094] (9) Confirmation of efficacy of TuSyntaxin derived from twospotted spider mites, and OcSyntaxin and OcSyntaxin 12 derived from twospotted spider mites against mango spider mites (mango spider mites, mesh method) The results are shown in Figure 13. TuSyntaxin, OcSyntaxin, and OcSyntaxin 12 also showed miticidal and oviposition-inhibiting effects against mango spider mites.

[0095] This application is based on patent application No. 2024-151902 filed in Japan (filing date: September 4, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A control agent for spider mites belonging to the Tetranychidae family, comprising any one of the following (a) to (c): (a) double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene; (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or syntaxin 12 gene or a part thereof; (c) a nucleic acid construct that produces (a) or (b) within a cell.

2. The control agent according to claim 1, wherein the spider mites belonging to the Tetranychidae family are at least one species of spider mites selected from the group consisting of the genera Tetranychidae, Tetranychidae and Tetranychidae.

3. The control agent according to claim 1, wherein the spider mite belonging to the family Tetranychidae is at least one species selected from the group consisting of two-spotted spider mite, Kanzawa spider mite, three-toed spider mite, citrus red mite, European red mite, and mango red mite.

4. A control agent described in any one of claims 1 to 3, wherein the double-stranded RNA has a length ranging from 15 bp to the full length of syntaxin or syntaxin 12 mRNA.

5. A method for controlling spider mites belonging to the Tetranychidae family, comprising the step of applying any of the following (a) to (c): (a) double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene; (b) an antisense nucleic acid against a transcription product of the syntaxin gene and / or syntaxin 12 gene or a part thereof; or (c) a nucleic acid construct that produces (a) or (b) within a cell.

6. The control method according to claim 5, wherein the spider mites belonging to the family Tetranychidae are at least one species of spider mites selected from the group consisting of the genera Tetranychidae, Tetranychidae and Tetranychidae.

7. The control method according to claim 5, wherein the spider mite belonging to the family Tetranychidae is at least one species selected from the group consisting of two-spotted spider mite, Kanzawa spider mite, three-toed spider mite, citrus red mite, European red mite, and mango red mite.

8. The control method described in claim 5, wherein the double-stranded RNA has a length ranging from 15 bp to the full length of the syntaxin or syntaxin 12 mRNA.

9. The control method according to any one of claims 5 to 8, wherein the application method is spraying, soil drench or coating on plants, or infiltration or use of bait containing any of (a) to (c) on spider mites.

10. A method for controlling spider mites belonging to the Tetranychidae family, characterized by using a transgenic plant transformed with a nucleic acid construct that produces intracellularly (a) double-stranded RNA having an RNAi effect on the syntaxin gene and / or syntaxin 12 gene, or (b) an antisense nucleic acid against a transcription product or a portion thereof of the syntaxin gene and / or syntaxin 12 gene.

11. A nucleic acid selected from any of the following (A) to (C): (A) a double-stranded RNA having an RNAi effect on the syntaxin gene, which consists of a base sequence having 75% or more identity with the base sequence set forth in SEQ ID NO: 1, or the syntaxin 12 gene, which consists of a base sequence having 75% or more identity with the base sequence set forth in SEQ ID NO: 2; (B) an antisense nucleic acid against a transcription product or a part thereof of the syntaxin gene, which consists of a base sequence having 75% or more identity with the base sequence set forth in SEQ ID NO: 1, or the syntaxin 12 gene, which consists of a base sequence having 75% or more identity with the base sequence set forth in SEQ ID NO: 2; (C) a nucleic acid construct that produces (A) or (B) in a cell.

12. The nucleic acid of claim 11, wherein the double-stranded RNA is from 15 bp to the full length of the syntaxin or syntaxin 12 mRNA.

13. A transgenic plant transformed by (C) according to claim 11.

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