JP6655910B2 - Method of controlling plant virus disease transmission - Google Patents

Description

  The present invention relates to a method for controlling the transmission of plant virus diseases.

Plant diseases caused by plant viruses, fungi, and the like are a major threat to crop production, and account for 10% of the world's food production loss. Above all, since there is no means for directly treating plant virus diseases, control measures centered on the use of virus-resistant varieties and the extermination of agents for vector-borne organisms have been taken.
However, these control means cannot sufficiently suppress the occurrence of plant virus disease.

For example, tomato yellow leaf curl virus (hereinafter sometimes referred to as “TYLCV”) is transmitted by tobacco whitefly and poses a major threat to tomato production, and its control has been largely dependent on pesticide application. Was. This is one of the reasons, and biotypes with low drug sensitivity are dominant and their distribution is expanding, posing a further threat.
Therefore, as a control means of TYLCV, a combination with a control method other than the application of the chemical has been adopted. Examples of the control method other than the chemical spraying include concrete control of insect insect nets, physical control of UV-cutting materials, cultivation of resistant varieties, and cultivation control such as adjustment of cropping time.
Such a situation is common when there are insects that transmit plant viruses such as aphids and thrips, and the threat of plant virus infection and onset is still large for TYLCV.

Patent Literature 1 discloses that tomato yellow leaf curl virus (non-insectophile TYLCV) that is not transmitted by vector is not transmitted from disease-resistant tomatoes to TYLCV-sensitive tomatoes. The disease-resistant tomatoes pre-inoculated with the medium TYLCV do not infect the disease-resistant tomatoes, and naturally, the disease-resistant tomatoes pre-inoculated with the non-media-type TYLCV are further transferred to other TYLCV-sensitive tomatoes. No transmission is described.
In addition, as a method for controlling a virus with an attenuated vaccine (also referred to as an attenuated virus) using a virus with reduced pathogenicity, cucumber mosaic virus (hereinafter, sometimes referred to as “CMV”) or zucchini yellow mosaic virus (hereinafter, referred to as “CMV”). , "ZYMV" in some cases) has been put to practical use using an attenuated virus (Non-Patent Documents 1 and 2). However, in some cases, despite the same type of virus, a mixed infection occurs due to insufficient interference effect (Non-Patent Documents 3 and 4).

International Publication No. 2012/105696

Phytopathology, 1993, 83, p. 405-410 Plant Disease, 1997, 81 (7), p. 733-738 Japanese Society of Plant Pathology, 1996, 62 (3), p. 332 Japanese Society of Plant Pathology, 1998, 64 (4), p. 428

  However, it is possible that such cases can occur with attenuated viruses. If this occurs in the field, plant virus disease may spread to neighboring fields that have not been attenuated.

  The problem to be solved by the present invention is to provide a new control method for preventing transmission of plant virus diseases.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, by inoculating a plant virus in advance with a plant virus, even if other plant viruses that later infected the plant multiply, The present inventors have found that they have an action of suppressing the transmission of other plant viruses, and have completed the present invention.

That is, the present invention is as follows.
(1)
A method of reducing the transmission of a secondary virus that has infected the plant, including the primary virus, by vaccinating the plant with the primary virus.
(2)
The method according to (1), wherein the secondary virus is insect-borne.
(3)
The method according to (1) or (2), wherein the primary virus is tomato yellow leaf curl virus (TYLCV).
(4)
The method according to (1) or (2), wherein the primary virus is zucchini yellow mosaic virus (ZYMV).
(5)
A plant comprising the primary virus according to any one of (1) to (4).
(6)
The plant of (5), further comprising the secondary virus according to any one of (1) to (4).
(7)
The plant according to (5) or (6), which is derived from callus or the like.
(8)
A plant obtained by vegetatively propagating the plant according to (7).

  ADVANTAGE OF THE INVENTION According to this invention, the new control method which prevents transmission of a plant virus disease can be provided.

A part of the results of the secondary virus infection analysis at 5 weeks after inoculation of the secondary virus to the primary virus-treated tomato and to the uninoculated tomato (hereinafter referred to as “challenge inoculation”) is shown. In the primary virus-inoculated group, it can be confirmed that the detection band of the secondary virus is thinner than that in the control group (primary virus-inoculated group). P indicates a secondary virus positive control. The results of primary virus and secondary virus extraction from pumpkin leaves and virus detection using the detection primers are shown. a and b show the results of amplification with the primary virus (ZY-02) detection primer, and c and d show the results of amplification with the secondary virus (4S) detection primer. Lanes 1-9 of a and c show the primary and secondary virus inoculated strains, and lanes 10-12 of a and c show the inoculated strain of primary virus only. Lanes 13-21 of b and d show the strain inoculated with the secondary virus only, and lanes 22-24 of b and d show the virus-free strain. In a and c, and b and d, the same lane means that they are derived from the same strain of pumpkin.

  Hereinafter, embodiments for carrying out the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist.

  The present invention is a method for reducing the transmission of a secondary virus that has infected the plant, including the primary virus, by vaccinating the plant with the primary virus.

In the present invention, the primary virus means a virus having an interference effect of suppressing infection of a virus of the same species in a plant containing the primary virus.
That is, a virus has an interference effect of suppressing infection, which means that even if another virus is transmitted to a plant infected with the primary virus because the primary virus has been inoculated (vaccinated) in advance, the transmitted virus Does not infect plants.
The primary virus in the present invention not only exhibits an interference effect with respect to the same kind of virus, but also has an effect of reducing secondary transmission with respect to a secondary virus having an incomplete interference effect.
By immunizing a plant with a primary virus, for example, even if a vector insect poisoning the same virus later comes into contact with a plant pre-inoculated with the primary virus, the strongness of the vector insect is maintained. The poisonous insect-borne virus has an interference effect that does not infect plants inoculated with the primary virus before, and the method of the present invention provides that the secondary virus infects plants inoculated with the primary virus in advance, It differs in that it is a method of suppressing secondary transmission to other plants thereafter.

Examples of the primary virus and the secondary virus in the present invention include viruses belonging to Geminiviridae, Potyviridae, Bromoviridae, and Closteroviridae.
Among them, Arch Virol 2008, 153, p. Geminiviridae, Begomovirus, and Virus Taxonomy 2005, p.783-821. 819-842, viruses classified as the genus Potyviridae (Potyviridae), and the like, are preferred.
Examples of the virus include, for example, tomato yellow leaf curl virus (TYLCV), bean golden mosaic virus (BGMV), East African cassava mosaic virus (EACMV), pepper golden mosaic virus (PGMV), and tobacco cigar Japanese virus (TLCJV) of the Geminiviridae family. ), Tomato leaf curl China virus (ToLCCCV), tomato yellow leaf curl China virus (TYLCCNV), and sweet potato cigar Japanese virus (SPLCJV), zucchini yellow spot mosaic virus (ZYMV) of the potyviridae family, potato Y virus (PVY), beet Mosaic virus (BtMV), clover leaf vein yellowing virus (CLYVV), ume ring print virus (PPV), potato A virus (PVA) And tobacco plaque virus (TVMV), cucumber mosaic virus (CMV) of the bromoviridae family, tomato aspami virus (TAV), and peanut dwarf virus (PAV), citrus tristeza virus (CTV) of the closteroviridae family, And tobacco mosaic virus (TMV).
Among them, tomato yellow leaf curl virus (TYLCV), East African Cassava mosaic virus (EACMV), zucchini yellow mosaic virus (ZYMV), cucumber mosaic virus (CMV), citrus tristeza virus (CTV), and tobacco mosaic virus (TMV) And the like are more preferred viruses, more preferably tomato yellow leaf curl virus (TYLCV) and zucchini yellow mosaic virus (ZYMV).

As the primary virus, a virus known as a virus having an interference effect can be used.
As the virus having an interference effect, specifically, as tomato yellow leaf curl virus (TYLCV), 17G strain described in WO2012 / 105696, and as zucchini yellow mosaic virus (ZYMV), ZY- 02 strain, TMV-L11A strain, CMV-KO2 strain, CTV-HM55 strain and the like.

The primary virus may be an attenuated virus as a virus having an interference effect.
In the present invention, the attenuated virus is a virus that does not cause a plant-derived symptom or causes only a minor symptom in a plant.
In the present invention, as the primary virus, an attenuated virus having an interference effect can be used. Further, it is preferable that the primary virus is non-insectophile that is not transmitted by a vector.

In the present invention, a plant to be vaccinated with a primary virus means a plant that is a host of the primary virus.
That is, the virus is not particularly limited as long as it is a virus that is infected by the primary virus. Examples of the virus include plants such as Solanaceae, Cucurbitaceae, Legume, and Rutaceae.
Examples of solanaceous plants include, for example, tobacco, tomato, eggplant, bell pepper, pepper, and the like, and examples of cucumber plants include, for example, cucumber, melon, pumpkin, zucchini, and the like, and legumes include, for example, broad beans. , Kidney beans, soybeans, and the like, and the Citrus family plants include citrus fruits, such as Satsuma Mandarin, Yuzu, and Hassaku.

In the present invention, vaccination means infecting a plant with a primary virus. Prior infection of the plant with the primary virus can take place at any stage of the growth of the plant and can be achieved by grafting. In order to efficiently carry out grafting and subsequent infection with the primary virus, it is preferable to carry out the transplantation on about 2 to 6 true leaves.
In addition, the primary virus can be inoculated into a plant by, for example, rubbing a seedling of a plant with a swab coated with celite as an abrasive using the infected leaves of the primary virus as an inoculum. .
The primary virus is preferably maintained by grafting or cutting propagation. As a plant infected with the primary virus, aside buds can be used, provided that the size of the side buds is two or more true leaves. Thus, it can be confirmed that the virus is sufficiently contained.

  "Infecting" a plant virus means not only when the plant virus has not been confirmed in the plant, but also when the virus manifests itself in the plant. Includes cases where it can be confirmed by genetic analysis etc.

In the present invention, the secondary virus is, for example, a virus of the same species as the primary virus, but a virus of a different strain.
When a virus susceptible to the interference of the primary virus is transmitted to a plant infected with the primary virus, the transmitted virus does not infect the plant, but the secondary virus in the present invention is a primary virus, for example, A heterologous virus that infects plants.

In the present invention, reducing the transmission means that the plant infected with the primary virus is infected with the secondary virus, but the transmission of the secondary virus from the infected plant is reduced. .

The decrease in transmission can be confirmed, for example, by the following method.
Infect a plant vaccinated with the primary virus with the secondary virus, and confirm whether the plant transmits the secondary virus to a virus-free plant (secondary transmission rate 1).
At the same time, plants that have not been vaccinated with the primary virus are infected with the secondary virus, and it is confirmed whether the plant can transmit the secondary virus to virus-free plants (secondary transmission rate 2).
Here, by comparing the secondary transmission rates of the two, the secondary transmission rate from plants vaccinated with the secondary virus is higher than the secondary transmission rate from plants not vaccinated with the secondary virus. If the value is low, it can be confirmed that the transmission of the secondary virus has decreased.
The reduction of the secondary virus transmission rate is, for example, as a secondary transmission rate 1 / secondary transmission rate 2 × 100, preferably 50% or less, preferably 20% or less, more preferably 10% or less. preferable. In the present invention, the secondary transmission rate of the secondary virus is substantially larger than zero.

In the present invention, the transmission of the secondary virus is not particularly limited, but is transmitted by vectors, transmission by food grafting, egg transmission, contact transmission, seed transmission, soil transmission, and infected leaf ground juice. Transmission by mechanical inoculation, etc., but in order to confirm that the transmission of the secondary virus is reduced, transmission by a vector is preferable, and in this case, the secondary virus is an insect-borne virus. It is. In the present invention, it is preferred that the primary virus is a non-insectotropic virus and the secondary virus is a combination of insect-borne viruses.
In the present invention, by inoculating the primary virus in advance, even if the secondary virus infected later proliferates, it has the effect of suppressing subsequent transmission by vector insects. This means that in crop fields where a large number of vector insects are present, it reduces the uptake of secondary viruses by vector vectors, makes plant viruses more difficult to spread, and prevents the spread of viral diseases. Leads to. Therefore, according to the present invention, it is possible to provide a method for overcoming the limitations of the existing interference effects and preventing the spread of plant virus.
As shown in the following Examples, the present inventors have discovered that a plurality of plant virus species and plant species have an effect of suppressing secondary transmission of a secondary virus. According to the present invention, a new method for controlling plant viruses is provided.

The present invention also provides a plant infected with the primary virus and containing the primary virus. Also provided are plants infected with the primary and secondary viruses.
Examples of the plant include, but are not particularly limited to, leaves, stems, roots, flowers, seeds, fruits, seedlings, cut flowers, bulbs, scales, cell clumps, callus, and protoplasts obtained from the plant. Can be
The plant may be a plant derived from callus or the like, or may be a plant vegetatively propagated from a plant derived from callus or the like.
As the plant infected with the primary virus, a plant resistant to the primary virus species can be used. Examples of disease-resistant plants include plants having a resistance gene that can be infected with a primary virus but suppress the symptoms of a viral disease caused by the primary virus.
The disease-resistant plant may be a wild-type strain that has naturally acquired the resistance gene, a plant into which the resistance gene has been introduced by crossing, or a plant into which the resistance gene has been introduced by genetic recombination technology. Good.

  The plant body may be a plant in a form after grafting, and is not particularly limited, but can obtain a plant seedling. Also, for obtaining a grafted seedling, a scion, a rootstock, an intermediate tree, and the like. Is mentioned.

  Seedlings grown from seeds are called "self-rooted seedlings", while seedlings obtained by grafting another plant or another varieties resistant to diseases to rootstocks are called "grafted seedlings". The plant may be a self-rooted seedling or a grafted seedling.

  Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1]
As test tomatoes in the primary virus inoculation zone, tomato seedlings (variety: Oyasu Yoshinobu, Nantes seedlings) were infected with TYLCV as the primary virus, and the whole plant was infected. A test tomato infected with TYLCV was placed in an acrylic cage. As the TYLCV as the primary virus, the 17G strain described in WO2012 / 105696 was used.
The full-length DNA sequence of the 17G strain was inserted into the binary vector for Agrobacterium pCAMBIA2300 (CAMBIA) and received as a plasmid (pCAM17G1) at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology on November 2, 2010. The accession number FERM-AP22037 was assigned, and the accession number FERM P-22037 was assigned on December 3, 2010. The plasmid was used to infect a plant body by the agroin filtration method, and it was confirmed that the virus was expressed and the non-insect medium was maintained. In addition, the plasmid and the 17G strain are preserved and maintained by the present applicant, and the applicant guarantees the sale according to the provisions of Article 27-3 of the Japanese Patent Law Enforcement Regulations.
The 17G strain is a plant virus having a DNA represented by the nucleotide sequence of SEQ ID NO: 1 as described in WO2012 / 105696, and is a tomato yellow leaf curl virus that is not transmitted by vector insects. It is a plant virus that can be said to be a representative strain of (non-insectoid TYLCV). Non-insectoid TYLCV can be suitably used as the primary virus in the present invention.
In addition, as the non-insect medium of TYLCV, it is thought that three amino acids present in the CP region of TYLCV are involved in insect mediated properties, and the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 encoding the CP region is It is believed that TYLCV, having the represented DNA, has the property of being non-insectophile. TYLCV having a peptide represented by the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 also has a property of being non-insectophile.
In addition, if TYLCV having the property of non-insect medium can be provided, an amino acid sequence in which one or several amino acids of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 have been deleted, added, and / or substituted. TYLCV with the indicated peptides can also be used as the primary virus. Furthermore, if TYLCV having the property of non-insect medium can be provided, for example, 80%, preferably 85%, more preferably 90%, even more preferably 95% of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. TYLCV having a peptide represented by an amino acid sequence having a homology of 100%, and even more preferably 98%, can also be used as the primary virus.
In the present invention, TYLCV having arginine (80), phenylalanine (147), and asparagine (171) as amino acids of the coat protein (CP) can also be used as a primary virus as a non-insect-borne virus. Furthermore, each of the three amino acids does not have to be exactly at positions 80, 147, and 171 in the CP region. When the amino acids are aligned by a method known to those skilled in the art, the corresponding amino acids are arginine and phenylalanine, respectively. A virus having an amino acid sequence of asparagine can also be used as the primary virus. Arginine (80), phenylalanine (147), and asparagine (171) may be substituted with structurally similar amino acids as long as each of them can maintain the non-insectophile function in the virus. Arginine (80), phenylalanine (147), and asparagine (171) are represented as R80, F147, and N171 when represented by one letter. In the present invention, TYLCV having three amino acids R80, F147, and N171 in the coat protein is a virus showing non-insectitis, and may be used as a primary virus unless the function of the coat protein is lost. it can. In the present invention, non-insectivorous TYLCV having arginine (80), phenylalanine (147), and asparagine (171) as amino acids in the coat protein of the virus are Q80R, Y147F, and K171N in the CP protein of insectivorous TYLCV. Can be a virus having R80, F147, and N171 as a mutation corresponding to, and even if not derived from a parasite TYLCV having strictly Q80, Y147, and K171, The corresponding amino acids only need to have arginine, phenylalanine and asparagine, respectively.
Virus-free tomato seedlings (variety: Yoshinori Oyasu, Nantes seedlings) were used as test tomatoes in the control plot (primary virus-inoculated plot).
Next, in order to perform challenge inoculation, virus-free tobacco whitefly was released on tomato seedlings (variety: Yoshinori Oyasu, Nantes seedlings) infected with TYLCV as a secondary virus for 5 days to obtain secondary virus poisoning insects. Was. As TYLCV as a secondary virus, Israel mild strain 233G strain was used.
The secondary virus poisoning insects were released into the acrylic cage in which the test tomatoes prepared above were placed so that the number of the test tomatoes was 10 or more per test tomato. The tobacco whiteflies used in the challenge inoculation had a poisoning insect rate of 100%, confirming that the virus was sufficiently poisoned.
After challenge inoculation, test tomatoes were grown in an isolated screen room.
In order to confirm the infection of the secondary virus, the upper leaves of the test tomatoes 5 weeks after the challenge inoculation were collected and the viral DNA was extracted. The method for extracting viral DNA is as follows.
Tomato leaves were triturated with a buffer (100 mM Tris HCl pH 8.0, 50 mM EDTA, 500 mM NaCl, 0.001% 2-Mercaptoethanol), treated with 10% SDS, and treated at 65 ° C. for 10 minutes. Next, 5M potassium acetate was added and centrifuged. The resulting supernatant was treated with phenol / chloroform (1: 1), then precipitated with 2-propanol, and the DNA was concentrated and recovered. After extracting the viral DNA, using the extracted DNA as a template, the secondary virus detection primers TYmdv1 (5′-ATTGACCAAGAATTTTTACACTTATCCC-3 ′: SEQ ID NO: 6), TYunic1 (5′-AAGTGGRTCCCACATAATTGCAAGA-3 ′: SEQ ID NO: 7) and GoTaq Amplification of viral DNA was attempted by PCR using (registered trademark) Green Master Mix (promega). The results are shown in FIG.
The number of secondary virus detection strains in the control plot (in FIG. 1, no primary virus inoculation plot) was 18/20 (number of test tomato strains), whereas in the primary virus inoculation plot, 16/18 (number of test tomato strains). There was no significant difference in the number of infected strains of secondary virus. However, in the primary virus inoculated group, the band of the secondary virus electrophoresis detected by PCR was clearly thinner and thinner than that in the control group.

The amount of extracted viral DNA can be estimated to some extent by the density of the band of the amplified product electrophoresed after PCR. At 10 weeks after challenge inoculation, viral DNA was extracted, amplified by PCR, and the amplified product was compared by electrophoretic migration band intensity. The secondary virus band in the primary virus-inoculated tomato strain showed that the primary virus The band tended to be lighter than in the case without inoculation. Among the tomato strains that were PCR-positive, the percentage of the band with a faint band was 87.5% in the tomato inoculated with the primary virus, and 22.2% in the tomato without inoculated primary virus (Table 1).
This suggests that the inoculation of the primary virus may suppress the growth of the subsequently transmitted secondary virus.
In addition, from the results of the 5th and 10th weeks after challenge vaccination, tomatoes inoculated with the primary virus cannot prevent the infection of the secondary virus, but must keep suppressing the virus growth after the secondary virus infection. Was suggested.

Table 1. Difference of band concentration by secondary virus PCR in TYLCV (10th week of inoculation)

Twelve weeks after inoculation of the secondary virus, the tomato strain with a dark electrophoretic band after PCR and a thin tomato strain were separated, and these tomato strains were sufficiently inoculated to white tobacco whitefly as an infectious source. Infected. Specifically, the experiment was performed in the following procedure.
Infectious tomato strains were individually caged one strain at a time and inoculated with virus-free tobacco whitefly for 5 days. 10 tomato seedlings (variety: House Momotaro, Takii seedlings) were newly prepared as test tomatoes for the secondary transmission test per tomato strain of the transmission source, and 10 or more were released per seedling for 5 days. The inoculated juice was sucked.
As a result, 75% of the secondary transmission was observed in the section where the band was dark in the control section, and 20% was observed in the section where the band was light. On the other hand, in the primary virus inoculated plot, the secondary transmission was 30% in the plot with the darker band, but the secondary transmission did not occur in the plot with the lighter band.
The difference in the secondary virus transmission rate between the case where the virus was primarily inoculated and the case where the virus was not inoculated was calculated by multiplying the density of the PCR detection band by the transmission rate. Comparing the total value of the transmission rates with and without the primary virus inoculation, in the control group not inoculated with the primary virus, the overall secondary transmission rate was 62.7% (secondary virus detection band). Is 58.3% when the virus concentration is high and 4.4% when the virus concentration is low, whereas 3.8% (3.8% when the secondary virus detection band is dark) in the primary virus inoculation group. (Sum of 0% of cases) (Table 2).
Therefore, when the primary virus is inoculated, the growth of the secondary virus is suppressed, and it is expected that the transmission rate of the secondary virus will be about 1/16 compared to the case where the primary virus is not inoculated.
From the above results, even if the presence of the primary virus does not prevent the infection of the secondary virus, the effect of suppressing the growth and lowering the secondary transmission rate, that is, the effect of the secondary virus from tomatoes inoculated with the primary virus, It was presumed that secondary transmission of the secondary virus was unlikely to occur.

Table 2. Secondary transmission rates of secondary virus from tomatoes challenged with primary virus and uninoculated tomatoes with secondary virus

[Example 2]
Twenty-four virus-free pumpkin seedlings (variety: Ebisu, Takii seedlings) are prepared, and half of them are ZYMV as primary virus (see strain ZY-02, Journal of General Plant Pathology, 2006, 72, p. 52-56). It was arranged in an inoculated section and a non-inoculated section (control section).
Ten days after inoculation of the primary virus, ZYMV (4S strain, see Japanese Society of Plant Pathology, 2006, 72, p. 40) was inoculated as a secondary virus.
On the 8th day after challenge inoculation, the fifth leaf of the true leaf was sampled, and mixed infection analysis was performed using discriminating primers for the primary virus and the secondary virus.
As a primary virus detection primer (detecting ZY-02), Z5-457F (5′-CGCTCGTATGAGCATGATG-3 ′: SEQ ID NO: 8) and Z5-793R (5′-TTCCTCATGCCGCGACTGACACC-3 ′: SEQ ID NO: 9) were used. As secondary virus detection primers (detecting 4S), Oki4S-386F (5′-GCGGACTTAGAGGCCTTGTT-3 ′: SEQ ID NO: 10) and Oki4S-1087R (5′-TATCGCCGGCAAATTTTGAGG-3 ′: SEQ ID NO: 11) were used. RT-PCR was performed using PrimeScript II High Fidelity One Step RT-PCR Kit (manufactured by TaKaRa).
Primary virus was detected in all inoculated pumpkins and not in uninoculated pumpkins (FIGS. 2a, b). The secondary virus was detected 100% in the control group not inoculated with the primary virus, but was detected in 67% in the group inoculated with the primary virus (FIGS. 2c and 2d).

Therefore, pumpkin seedlings inoculated with the secondary virus were used as the transmission source, cotton aphids were poisoned, and virus-free pumpkins were secondarily transmitted. Specifically, the experiment was performed in the following procedure.
Virus-free cotton aphid 100 which was fasted for 3 hours using nine pumpkin seedlings inoculated with both the primary virus and the secondary virus used in the analysis and nine pumpkin seedlings infected with the secondary virus in the control plot as a poisoning source More than one head was released to each pumpkin and was poisoned for 20 minutes. Sixteen virus-free test pumpkins (variety: Ebisu, Takii seedlings) for the secondary transmission test were prepared for each poison source, and five poultry insects were inoculated per strain. The inoculation time was 2 hours. After the inoculation, the insecticide was sprayed to remove the insect pest. Table 3 shows the results.
Of 9 pumpkin seedlings inoculated with both the primary virus (ZY-02 strain) and the secondary virus (4S), 6 were pumpkin seedlings that could be confirmed to be infected with 4S, and the remaining 3 were 4S uninfected. Was. Among the pumpkin seedlings confirmed to be infected with 4S, only three of the pumpkin seedlings showed secondary transmission three weeks after inoculation, and the secondary transmission rate of the secondary virus was 6 to 13%. There was no secondary transmission from the three poisoning sources for which no 4S infection could be confirmed. As a result, both the primary virus and the secondary virus were inoculated, and when both were infected, the total insect transmission rate (secondary transmission rate) was 5% (5/96).
On the other hand, of 9 pumpkin seedlings in the control plot (only inoculated with the secondary virus), two were used as poisoning agents for secondary transmission tests. As a result of performing infection analysis by RT-PCR three weeks after the inoculation, the secondary transmission rates were 38% (6/16) and 56% (9/16) (47% overall: 15/32).
From the above, even in the combination of pumpkin and ZYMV, when a secondary virus is transmitted to a plant vaccinated with the primary virus, the secondary virus transmission rate (secondary transmission) is suppressed as compared with the control plot. It has been found.

Table 3. Secondary transmission rates of secondary virus from pumpkins inoculated with primary virus and challenged with non-inoculated pumpkin with secondary virus

SEQ ID NO: 1 shows the entire nucleotide sequence of 17G strain.
SEQ ID NO: 2 shows the entire nucleotide sequence of the 17G strain CP region.
SEQ ID NO: 3 shows the entire nucleotide sequence of the CP region of a non-insectin chimeric clone obtained from the TYLCV-Isr (ISR10-1) strain.
SEQ ID NO: 4 shows the amino acid sequence of the CP region of 17G strain.
SEQ ID NO: 5 shows the amino acid sequence of the CP region of a non-enzootic chimeric clone obtained from TYLCV-Isr (ISR10-1) strain.
SEQ ID NO: 6 shows the nucleotide sequence of TYmdv1 of the secondary virus detection primer.
SEQ ID NO: 7 shows the nucleotide sequence of TYunic1 of the secondary virus detection primer.
SEQ ID NO: 8 shows the base sequence of Z5-457F of the primary virus detection primer (detecting ZY-02).
SEQ ID NO: 9 shows the base sequence of Z5-793R of the primary virus detection primer (detecting ZY-02).
SEQ ID NO: 10 shows the base sequence of Oki4S-386F of the secondary virus detection primer (detects 4S).
SEQ ID NO: 11 shows the base sequence of Oki4S-1087R of the secondary virus detection primer (detects 4S).

Claims (2)

By inoculating a plant with a non-insectotropic primary virus, reducing the transmission of the insect- borne secondary virus that has infected the plant, including the non- insectative primary virus ,
The method wherein the non-insectophil primary virus is a Zucchini yellow mosaic virus (ZYMV) ZY-02 strain and the secondary virus is a Zucchini yellow mosaic virus (ZYMV) . 2. The method according to claim 1, wherein the secondary virus is a 4S strain.