JP2008143864A - Anti-plant virus agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new anti-plant virus agent. <P>SOLUTION: The anti-plant virus agent contains 80% ammonium sulfate salting-out extract of Tricholoma matsutake. The agent is obtained by culturing Tricholoma matsutake fungus to form mycelium, crushing the mycelium to provide a supernatant, adding ammonium sulfate to the supernatant so as to become 80% saturation to carry out salting-out of the supernatant and recovering the resultant precipitation. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an anti-plant virus agent and use thereof.

  In terms of the classification and proportion of plant pathogens, about 80% are fungi, about 10% are bacteria, and the remaining 10% are viruses, viroids, phytoplasmas, nematodes, and the like. Plant viruses account for only a small percentage of pathogens. However, plant viral diseases cause serious damage to important crops such as shells, vegetables and fruit trees. Viruses that have invaded plant cells grow entirely depending on the metabolism of the cells, so in the case of antiviral agents, it is not easy to find a specific metabolic pathway targeted for attack.

  When a plant virus enters a plant cell by a wound or a vector insect, part of the coat protein is broken, and the genome nucleic acid inside the particle is uncovered. After that, replication of genomic RNA and discovery of various proteins are performed using the metabolic pathway of the host plant, and it spreads throughout the plant through cell-to-cell and long-distance transfer. Inhibits virus growth and at the same time causes phytotoxicity to plants (Kunou et al., 1998). Since plant viruses completely depend on the host plant for various metabolic processes, it is difficult to specifically inhibit only the growth of the virus while keeping the plant metabolic pathway normal. For this reason, plant viral diseases are regarded as difficult to control diseases (Frankki et al, 1979).

  Among plant diseases, it is extremely difficult to develop a control agent for viral diseases. There are two types of antiviral agents: growth inhibitors and infection inhibitors. The former includes blasticidin S (BcS) and ribosome inactivating proteins (RIPs), but unfortunately there are also phytotoxicity is there. The latter includes skim milk and sodium alginate as preventive agents, but has no effect of inhibiting virus growth after infection. Therefore, a practical antiviral agent has not been developed so far.

  Recently, there was a report that antiviral protein was extracted from maitake. Maitake mushrooms were ground and extracted by ammonium sulfate salting out. When the extract was further purified by gel filtration, Grifola frondosa antiviral protein (GFAP) was obtained. GFAP was a TMV infection inhibitory protein, containing Cys and found to be heat stable (Chen et al., 2004).

Atsushi Hirai (1979). Viruses and plants. Hitoshi Kuno, Yuki Shiraishi, So Takahashi, Shinji Tsukumu, Shigeru Kajiyama (1998). Introduction to new plant pathology. Yokendo. M. Fujiwara, te al. (2001), J. Gen. Plant Pathology.67: 159-163. R. I. B. Francki, te al. (1979). CMI / AAB Description of Plant Virus July 1979 No, 213. N. Chen, et al., (2004)., Prog. Biochem. Biophys. (2004): 31 (3) 283-286. Z. C. Chen, et al. (1991)., Plant Pathology 40: 612-620. T. Hirai, et al ,. (1966)., Phytopathology 56: 1236-1239. K. Tomaru and et al., (1975), Ann. Phytopath. Soc. Japan 41: 155-161.

  Therefore, the present invention seeks to provide a practical anti-plant virus agent.

  In order to search for practical antiviral substances, this study used mushroom mycelium extracts as candidate substances, and tried to select them and analyze the mechanism of action. As a result of selecting a candidate substance exhibiting antiviral activity using Cucumber mosaic virus (CMV) and cowpea, matsutake 80% ammonium sulfate salting out extract showed a high infection inhibitory effect. CMV, one of the plant viruses, belongs to the genus Cucumovirus of the Bromoviridae family and takes a particle form with a diameter of 28-30 nm. CMV infects more than 191 plants in 40 families and is one of the broadest host range of plant viruses (Frankki et al, 1979).

Therefore, the present invention cultivates matsutake fungi to form mycelia, crush the mycelia to obtain a supernatant, subject the supernatant to salting out with 80% saturation, and recover from the resulting precipitate. An anti-plant virus agent comprising an 80% ammonium sulfate salting out extract of matsutake is provided.
The present invention also provides a method for protecting a plant from virus infection, characterized in that the above-mentioned 80% ammonium sulfate salting out extract of matsutake is applied to the plant.

The present invention further relates to a method for producing the 80% ammonium sulfate salting out extract of the matsutake,
(1) cultivating matsutake fungi to form mycelia,
(2) The supernatant is obtained by crushing the mycelium,
(3) subjecting the supernatant to salting out to 80% saturation; and (4) recovering from the precipitate resulting from the salting out.
A method comprising the above is provided. Preferably, the recovery step is performed by dissolving the precipitate in a buffer solution and dialyzing the resulting solution against the buffer solution to remove ammonium sulfate.

I. Active ingredient 80% ammonium sulfate salting out extract of matsutake The 80% ammonium sulfate salting out extract of matsutake can be prepared as follows. (1) cultivating matsutake fungi to form mycelia; (2) crushing the mycelium to obtain a supernatant; (3) subjecting the supernatant to salting out to 80% saturation; and (4) the salt described above. It is recovered from the precipitate produced by the analysis.

  The mycelium of Tricholoma matsutake can be cultured in any medium in which the mycelium can grow. A specific example is Mr. Hamada's medium described later. The culture is performed by surface culture using a solid medium (for example, an agar medium) for storage and growth of the mycelium strain. As the medium for producing the active ingredient, a liquid medium is preferable, and for example, the following Mr. Hamada liquid medium can be raised. Culture must be performed under aerobic conditions, and surface static culture on a liquid medium is preferable. Moreover, it can also culture | cultivate in a liquid medium on aerobic conditions by usual means, such as shaking, aeration, and stirring.

When cultivating mycelia of matsutake by liquid surface culture, the antiviral active ingredient of the present invention accumulates mainly in the fungus body. For this reason, in order to obtain an 80% ammonium sulfate salting out extract of matsutake containing active ingredients, first, the mycelium is recovered from the culture solution. As a method for collecting mycelia, conventional methods can be used for collecting mycelia (mycelium) from a liquid medium, such as filtration and centrifugation.
Next, the collected mycelium is crushed. This method can also be carried out using a mixer or other conventional cell disruption means.

  Next, the cell end debris is removed from the crushed cell body to obtain a supernatant, that is, a liquid from which insoluble matter such as the cell crumb is removed from the cell crushed material. In this case as well, conventional solid-liquid separation means such as filtration and centrifugation can be used.

  Next, salting out is performed by adding ammonium sulfate to this supernatant to saturate the ammonium sulfate concentration to 80%, thereby obtaining a weekly precipitate. Next, the precipitate is dissolved in water or a suitable buffer, and ammonium sulfate is removed by dialysis against water or a suitable buffer. In this case, the pH of the buffer solution is 6-7. As a result, the “80% ammonium sulfate salting out extract of matsutake” of the present invention is obtained. This extract can be stored as it is at a low temperature, for example, frozen, or it can be stored as a solid by means of lyophilization or the like.

  The 80% ammonium sulfate salting out extract of the matsutake of the present invention loses almost no antiviral activity even when heated at 100 ° C. for 10 minutes. Therefore, the main body of antiviral activity is expected to be a low molecular weight substance. Furthermore, it is also confirmed by gel filtration that the active ingredient is a low molecular weight substance.

Next, the present invention will be described more specifically with reference to examples.
II. Materials and methods Preparation of 80% ammonium sulfate salted out extract of matsutake mushroom mycelia on Hamada plate medium (tap water 1000 mL; glucose 20 g; shrimp powder 5 g; pH = 5.1 to 5.2; agar 20 g) at 30 ° C. for 30 days The mycelium was grown by culturing. This mycelium is inoculated into 20 mL of Mr. Hamada's liquid medium (1000 mL of tap water; glucose 20 g; shrimp powder 5 g; pH = 5.1 to 5.2) in a 200 mL Erlenmeyer flask at 24 ° C. for 60 days. After static culture, the mycelium was grown on the surface of the liquid medium.

  The culture broth was filtered through a Buchner funnel (No. 2 filter paper; manufactured by Advantec) to collect matsutake mycelia, suspended in 20 mM Tris-HCl (pH 7.0) buffer, and pulverized using a mixer. The crushed cells were centrifuged (12,000 rpm, 20 minutes), and the supernatant was collected. The supernatant was salted out by adding ammonium sulfate to 80% saturation. After salting out, the precipitate is collected by centrifugation (12,000 rpm, 20 minutes), dissolved in 20 mM Tris-HCl (pH = 7.0) buffer, and this solution is buffered with 20 mM Tris-HCl (pH = 7.0). The resulting solution was dialyzed to obtain an “80% ammonium sulfate salting out extract of matsutake”.

The heat resistance of the 80% ammonium sulfate salting out extract of matsutake was examined. In the non-heated "Matsutake 80% ammonium sulfate salting out extract" and the "Matsutake 80% ammonium sulfate salting out extract" heated at 100 ° C for 10 minutes, the virus infection inhibition rate in the assay system using cowpea Examined. As a result, the virus infection inhibition rate of 90% or more (92%) was maintained even when heated. From this, it was suggested that the active ingredient in “80% ammonium sulfate salting out extract of matsutake” is a heat-stable low molecular weight substance.
Furthermore, gel filtration using Sephadex G-75 also confirmed that the inhibitor was a low-molecular substance.

2. Test plant
Cowpea (Vigna sesquipedalis cv. Kurodane sanjaku)
The seedlings sown in a flat pot filled with vermiculite and placed in a greenhouse were germinated by transplanting to a cell tray. In the experiment, primary leaves that were sufficiently developed were transplanted into a 9 cm diameter plastic pot.
Tobacco (Nicotiana tabacum cx. Xanthi nc)
The seedlings sown in a flat pot filled with vermiculite and placed in a greenhouse were germinated by transplanting to a cell tray. Two to three weeks later, one individual plant was planted in a pot, and one in the 7-8 leaf stage was used for the experiment.

3. Test virus
CMV pepo system
It was isolated from a peppo pumpkin that showed mosaic symptoms in 1973 by Ozaki et al. It is a highly virulent isolate that does not contain satellite RNA. It belongs to Lagunaria group, subgroup I.

4). CMV purification method
Tobacco-inoculated leaves on day 7 after CMV inoculation were frozen at −80 ° C. and ground with a mixer. To 5 g of frozen infected leaves, 100 ml of extraction buffer [0.5 M citric acid, 0.01 M ethylenediamine-4-acetic acid-2-sodium (EDTA); pH 6.5, 0.1% sodium thioglycolate] was added and stirred. This was allowed to stand for 5 minutes under ice cooling, and then centrifuged at 10,000 rpm for 15 minutes to obtain a supernatant. To this supernatant, 8% (w / v) polyethylene glycol 6000 (PEG) was added, completely thawed at 4 ° C., and centrifuged at 10,000 rpm for 15 minutes to obtain a virus precipitate. This precipitate was suspended in BE buffer (0.005M boric acid; pH 9.0, 0.01M EDTA), and centrifuged at 10,000 rpm for 10 minutes to remove impurities. This operation was performed 2 or 3 times to obtain a supernatant.

1% Triton X was added to the supernatant, and centrifugation was performed at 40,000 rpm for 60 minutes to obtain a virus precipitate. The precipitate was suspended in BE buffer and centrifuged at 10,000 rpm for 10 minutes to remove contaminants. This operation was performed 2 or 3 times to obtain a supernatant. The supernatant was centrifuged at 42,000 rpm for 90 minutes, and the resulting virus precipitate was suspended in 0.005 M borate buffer (pH 9.0). The suspension was centrifuged at 10,000 rpm for 10 minutes to remove impurities. This operation was performed 2 or 3 times to obtain a supernatant.
The virus suspension obtained by the above operation was quantified with a spectrophotometer (Ultrospec 3000; Amersham Pharmacia Biotech) and stored frozen at -20 ° C. The CMV concentration was set to 1 mg / ml with E ₂₆₀ = 5.

5. Particle inoculation The purified virus solution was diluted with 0.1 M phosphate buffer (pH 7.0), suspended by adding 3% carborundum, rubbed with a cotton swab, and washed with tap water.

6). The CMV and cowpea, a local lesion host, were used for the anti-leaf test using local lesion hosts. Of the two primary cowpea leaves, the active substance was applied to one, and the active substance solvent (20 mM Tris-Cl 0.1% Triton X) was applied to the other. After the leaves were dried for about 15 minutes, CMV was inoculated, and the number of necrotic lesions that appeared after 2 to 3 days was compared to examine antiviral properties. here,
Infection inhibition rate (%) = (1-candidate substance treatment district local lesion count / control district local lesion count) × 100
It was.

7). DAS-ELISA method Anti-CMV CP IgG was diluted 1/1000 with carbonate buffer [64 mM Na ₂ CO ₃ , 136 mM NaHCO ₃ , 0.8% NaN ₃ : pH 9.6], and 100 μl was dispensed on an ELISA plate. Thereafter, the plate was allowed to stand at 37 ° C. for 2 to 3 hours, or at 4 ° C. for 1 CP overnight, and then the wells were washed 3 times with PBST. The infected leaves were ground with 10 times the amount of carbonate buffer, and 100 μl of the ground liquid was dispensed into each well. Allowed to stand at 37 ° C. for 2-3 hours or at 4 ° C. overnight. Thereafter, the wells were washed 4 times with PBST, and the conjugate (AP-labeled anti-rabbit antibody) was diluted 1/1000 with PBST and dispensed in 100 μl aliquots. After allowing to stand at 37 ° C. for 2 to 3 hours, the wells were washed 3 times with PBST. The alkaline phosphatase substrate was dissolved in 10% diethanolamine solution, dispensed in 100 μl aliquots, and after color development, the absorbance was measured with a microplate reader (autoreader; Sanko Junyaku).

8). RNA extraction from purified virus 800 μl of glycine buffer [100 mM glycine, 100 mM NaCl, 10 mM EDTA: pH 9.5] was added to the sample and transferred to a 1.5 ml microtube. Add 70 μl 10% SDS, 70 μl 4% bentonite and 500 μl phenol / chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1), and centrifuge at 12,000 rpm for 4 minutes. The supernatant was transferred to another microtube. An additional 500 μl of phenol / chloroform was added and centrifuged at 12,000 rpm for 4 minutes. 45 μl of 3 M sodium acetate solution and 1 ml of ethanol were added to 450 μl of the supernatant, and the mixture was allowed to stand at −80 ° C. for 15 minutes, followed by centrifugation at 15,000 rpm for 15 minutes. The supernatant was removed, and ethanol remaining on the inner wall was completely removed with a micropipette, followed by drying under reduced pressure for 15 minutes in a desiccator. The obtained precipitate was melted in DEPC-treated water and the concentration was measured with a spectrophotometer. The CMV RNA concentration was 1 mg / ml with E ₂₆₀ = 40.

9. RNA inoculation RNA extracted from virus particles was diluted with 0.1 M phosphate buffer (pH 7.0) / bentonite and inoculated into test plants in the same manner as particle inoculation. The RNA concentration was adjusted to 0.4 μg / ml for cowpea and 2 μg / ml for tobacco.

Ten. Tissue blotting The nitrocellulose membrane and 4 filter papers were shaken in advance with 0.2 M CaCl ₂ for 15 minutes. The main veins of the sample leaves were removed and washed with distilled water. After cellulite was sprayed on the back side of the leaves, the epidermal tissue was removed by polishing with a toothbrush. Thereafter, the sample leaves were gently washed with 0.2 M CaCl ₂ . Two filter papers were placed on the gel dryer, and a nitrocellulose membrane, sample leaves, and two filter papers were stacked in this order. After sufficiently containing a transfer buffer, they were heated in a vacuum state at 50 ° C. for 15 minutes for transfer.

  After the transfer, the nitrocellulose membrane was immersed in TTBS (1 × TBS, pH 7.5 (25 mM Tris, 137 mM NaCl, 25 mM KCl), 0.05% Tween 20) and shaken for 10 minutes. After shaking, TTBS was removed, immersed in a blocking solution and shaken for 30 minutes. The blocking solution was removed and the nitrocellulose membrane was lightly washed with TTBS. Thereafter, the nitrocellulose membrane was immersed in CMV IgG (1 mg / ml) diluted 6000 times with TTBS, and shaken for 90 minutes. After completion of the reaction, the nitrocellulose membrane was washed with TTBS for 10 minutes with shaking.

This washing operation was repeated on the second floor, and then the nitrocellulose membrane was soaked in alkaline phosphatase-labeled anti-rabbit IgG-goat IgG diluted 10,000 times with TTBS and shaken for 90 minutes. After completion of the reaction, the nitrocellulose membrane was washed with TTBS for 10 minutes with shaking. After repeating this washing operation twice, the nitrocellulose membrane was rinsed lightly with distilled water. The nitrocellulose membrane was immersed in AP 9.5 (pH 7.4, 100 mM Tris, 100 mM NaCl, 50 mM MgCl ₂ ) for 5 minutes, and then immersed in BCIP / NBT for color development in the dark.

III. Result 1. Screening for Candidate Substances For the evaluation of anti-plant virus activity, CMV and Dwarf cowpea, a locally infected host of CMV, were used.
As a result, the number of local lesions was not observed in the cowpeas treated with the extract obtained from 80% ammonium sulfate salting out of matsutake mycelia, so it was considered to have a high infection inhibitory effect.

2. Antiviral properties of matsutake 80% ammonium sulfate salted out extract Next, effective concentration of matsutake 80% ammonium sulfate salted out extract, presence or absence of preventive or therapeutic effect against CMV inoculation, cowpea and CMV systemic infected host which are CMV local infection hosts The growth inhibitory activity in tobacco was investigated.

(1) Antiviral effect of matsutake 80% ammonium sulfate salting out extract (i) Antiviral property of matsutake 80% ammonium sulfate salting out extract in locally infected host (a) Effective concentration of matsutake 80% ammonium sulfate salting out extract The effective concentration of matsutake 80% ammonium sulfate salting-out extract was investigated by the counter-leaf method in cowpea. Matsutake 80% ammonium sulfate salting out extract was applied at three concentrations of 100 μg / ml, 10 μg / ml and 1 μg / m. As a result, 100 μg / ml showed an infection inhibition rate of 80% or more, and concentrations of 10 μg / ml and 1 μg / ml showed an infection inhibition rate of 40% or less (FIG. 1).
Matsutake 80% ammonium sulfate precipitation was mixed with CMV and inoculated at three concentrations of 100 μg / ml, 10 μg / ml, and 1 μg / ml. As a result, a concentration of 100 μg / ml showed a 96% infection inhibition rate. Concentrations of 10 μg / ml and 1 μg / ml showed an infection inhibition rate of 40% or less (FIG. 2).

(B) Effective time of treatment with matsutake 80% ammonium sulfate salting out extract When the matsutake 80% ammonium sulfate salting out extract was applied and CMV was inoculated immediately after drying, an infection inhibition rate of 80% or more was shown. Here, before and after application of matsutake 80% ammonium sulfate salting-out extract, time was taken, and then CMV was inoculated to investigate the persistence of the effect of matsutake 80% ammonium sulfate salting-out extract.
A matsutake 80% ammonium sulfate salting-out extract with a concentration of 100 μg / ml was applied to one of the primary leaves of cowpea with a cotton swab and a solvent was applied to the other. Particles were inoculated with 10 μg / ml CMV after 6, 12, and 24 hours. As a result, an infection inhibition rate of 84% was exhibited 6 hours after treatment with matsutake 80% ammonium sulfate salting out extract. At 12 and 24 hours after treatment, the infection inhibition rate was 40% or less (FIG. 3). Therefore, it was found that the matsutake 80% ammonium sulfate salting-out extract maintained the infection-inhibiting effect on the leaf surface for 6 hours.

  Subsequently, after treatment with CMV, the possibility of disease treatment of matsutake 80% ammonium sulfate salting-out extract was examined by treating matsutake 80% ammonium sulfate salting-out extract at a concentration of 100 μg / ml. The primary leaves of cowpea were inoculated with CMV, and after 6 to 12 hours, matsutake 80% ammonium sulfate salting out extract was applied. As a result, the infection inhibition rate was 40% or less at 6, 12 hours after CMV inoculation (FIG. 4). Therefore, the application of matsutake 80% ammonium sulfate salting-out extract after CMV inoculation did not suppress the formation of lesions, and there was no disease treatment effect.

(Ii) Antiviral activity of matsutake 80% ammonium sulfate salting out extract in systemically infected host The CMV growth inhibitory activity of matsutake 80% ammonium sulfate salting out extract was investigated using tobacco, a CMV systemic infected host.
(A) Amount of CMV CP accumulated in the tobacco leaf surface application of matsutake 80% ammonium sulfate salting out extract 30 minutes after applying 100% matsutake 80% ammonium sulfate salting out extract to tobacco and completely drying it The particles were inoculated at 20 μg / ml. The control group was coated with a solvent and inoculated with CMV particles 30 minutes after complete drying. Every two days, the upper two leaves from the inoculated leaves were sampled, and the CMV CP accumulation amount was measured by the DAS-ELISA method.

  As a result, it was observed that the amount of CMV CP accumulated in the matsutake 80% ammonium sulfate salting-out treatment group after inoculation was clearly lower than that in the control group. In the control plot, leaf malformation and clear veins were observed in the upper leaves 5 days after inoculation, and mosaic symptoms were confirmed 12 days later, but in the matsutake 80% ammonium sulfate salting-out extract treatment plot, symptoms were observed. The discovery was delayed and light yellowing was observed on the leaves only after 8 days and mosaic symptoms were confirmed after 15 days. The symptom of the matsutake 80% ammonium sulfate salting-out extract treated tobacco continued to progress and was no different from the control group (FIGS. 4, 5 and 6).

(B) Accumulated CMV CP in Tobacco Leaf Surface Mixing Process of Matsutake 80% Ammonium Sulfate Salting Extract A mixed solution of Matsutake 80% ammonium sulfate salting out extract and CMV at a concentration of 100 μg / ml was applied to tobacco. A solvent and CMV mixture was applied to the control group. Every two days, two upper leaves from the inoculated leaves were sampled, and CMV CP accumulation was measured by the DAS-ELISA method.
As a result, it was observed that the accumulated amount of CMV CP in the matsutake 80% ammonium sulfate salting-out treatment group after inoculation was clearly lower than that in the control group. In the control group, leaf deformity and vein transparency were observed in the upper leaves 6 days after inoculation, and mosaic symptoms were confirmed 11 days later, but in the matsutake 80% ammonium sulfate salting-out extract treatment group, disease symptoms were observed. The discovery was delayed and light yellowing was observed on the leaves only after 9 days, and mosaic symptoms were confirmed after 14 days. Symptoms of matsutake 80% ammonium sulfate salted-out extract treated tobacco continued to progress, and were no different from the control group (FIGS. 7, 8 and 9).

(C) Virus distribution of tobacco leaves by tissue blotting To a tobacco, a mixed solution of matsutake 80% ammonium sulfate salting-out extract and CMV having a concentration of 100 μg / ml was applied. A solvent and CMV mixture was applied to the control group. Every 2 days and 4 days, the inoculated leaves were sampled, and the virus distribution of tobacco leaves was examined by tissue blotting.
Tobacco was coated with matsutake 80% ammonium sulfate salting-out extract at a concentration of 100 μg / ml, and after 30 minutes of complete drying, CMV particles were inoculated at 20 μg / ml. The control group was coated with a solvent and inoculated with CMV particles 30 minutes after complete drying. Every 2 days and 4 days, the inoculated leaves were sampled, and the virus distribution of tobacco leaves was examined by tissue blotting.

As a result, on the second day, the mixed treatment was slightly distributed on the edge of the leaves. In the control group, it was distributed in large quantities around the leaf veins. On the 4th day, both the treated and control plots had the virus fully spread to the leaves (Figure 10).
In the applied treatment section, it was slightly distributed around the main veins of the leaves on the second day. In the control group, it was distributed in large quantities around the leaf veins. On the 4th day, both the treatment group and the control group had the virus fully expanded to the leaves (FIGS. 11 and 12).

3. Antiviral action mechanism of matsutake 80% ammonium sulfate salting out extract Next, we tried to elucidate the antiviral action mechanism of matsutake 80% ammonium sulfate precipitation.
(1) Presence or absence of induction of resistance to cowpea Induction of resistance to cowpea by applying backside treatment with 100% matsutake 80% ammonium sulfate salting out extract on the backside of the leaf and inoculating the surface with virus. The sex was investigated. As a result, the virus infection inhibition rate was 40% or less, and no induction of resistance was observed.

(2) The presence or absence of extract migration in cowpea Applying 100% matsutake 80% ammonium sulfate salting out extract to one leaf of cowpea, inoculating the virus on the other leaf, inhibiting the extract The presence or absence of migration was investigated. As a result, the infection inhibition rate was 30% or less, and no permeability was observed.

(3) Mixed inoculation of matsutake 80% ammonium sulfate salted out extract and CMV To investigate whether matsutake 80% ammonium sulfate salted out extract acts on CMV particles or CMV RNA, matsutake 80% ammonium sulfate salted out extract The mixture was inoculated with CMV RNA. A mixed solution of matsutake 80% ammonium sulfate salting-out extract and CMV RNA was prepared and inoculated in cowpea. The concentration of Matsutake 80% ammonium sulfate salting-out extract in the mixed solution was 20%.
As a result, several tens of local lesions appeared on the leaves of the control group. There were no local lesions on the leaves of the treated area, and the infection inhibition rate was 100% (FIG. 13). Therefore, it was considered that matsutake 80% ammonium sulfate salting out extract inhibits viral RNA.

III. DISCUSSION In the present invention, using mushroom-derived candidate substances as materials, selection of substances having antiviral activity was performed, and the anti-plant virus activity of the resulting matsutake 80% ammonium sulfate salting-out extract and analysis of its action mechanism Tried.
When the matsutake 80% ammonium sulfate salting-out extract was further purified, the antiviral activity of each molecular weight protein was low and the resistance to CMV was almost lost. It is considered that matsutake 80% ammonium sulfate salting out extract itself has antiviral activity. The cause of further purification is thought to be due to the antiviral activity of the matsutake 80% ammonium sulfate salting out extract due to multiple antiviral substances.

  The matsutake 80% ammonium sulfate salting-out extract suppressed the formation of lesion spots in cowpea by application treatment. By combining matsutake 80% ammonium sulfate salting-out extract and CMV particles, cowpea lesions were also suppressed. Even when application of cowpea with matsutake 80% ammonium sulfate salting out extract 6 hours before CMV inoculation, high infection inhibition rate was shown, but treatment with matsutake 80% ammonium sulfate salting out extract after inoculation lost the disease treatment effect . When matsutake 80% ammonium sulfate salting-out extract was applied to tobacco at a concentration of 100 μg / ml, symptom delay and CMV CP accumulation were reduced.

  When a mixture of matsutake 80% ammonium sulfate salting out extract and CMV particles was applied to tobacco at a concentration of 100 μg / ml, symptom delay and a decrease in CMV CP accumulation were also observed. Tobacco leaves treated with the application treatment and mixed treatment were analyzed by tissue blotting, and it was observed that leaf growth in CMV tobacco was suppressed up to 2 days. Furthermore, when the cowpea was inoculated with a mixed solution of matsutake 80% ammonium sulfate salting-out extract and CMV RNA, lesions were not completely observed. Based on the above, matsutake 80% ammonium sulfate salting out extract has infection inhibitory activity and growth inhibitory activity, not only acting directly on CMV particles but also showing inhibitory effect, also acting on CMV RNA and inhibiting It is considered to show an effect.

  Figalene is thought to penetrate into the leaves and act on the virus or induce resistance (Fujiwara et al, 2001). However, in cowpea, treatment with matsutake 80% ammonium sulfate salting-out extract after CMV inoculation has no therapeutic effect. In the cowpea, the backside treatment with matsutake 80% ammonium sulfate salted-out extract and CMV showed the same number of lesions as the control. In addition, when a migration survey was conducted by applying matsutake 80% ammonium sulfate salting-out extract to cowpea primary leaves and inoculating CMV on the other leaves, the number of lesions was the same as in the control group. Based on the above, matsutake 80% ammonium sulfate salting-out extract does not induce resistance, does not migrate from the leaves of the plant to the other leaves, is distributed in the applied leaves and inhibits the invasion of CMV particles It is also thought to inhibit the growth of CMV particles and CMV RNA.

  Alginate has a high anti-TMV effect. Moreover, it penetrates into the leaf tissue and has no migration. In addition, alginate has the property of aggregating TMV in vitro (Tomaru et al., 1965). The matsutake 80% ammonium sulfate salting-out extract also penetrates the leaves of the plant in the same way and inhibits the virus infected inside. Moreover, it is thought that it penetrates into the leaf tissue first and inhibits the entry of the virus.

  Blasticidin S (BcS) has been reported to be effective in suppressing tobacco mosaic virus (Tobacco mosaic virus: TMV) that infects tobacco leaves (Hirai et al.). The inhibitory effect is very high after inoculation and 12 hours after inoculation. Blasticidin S (BcS) inhibits amino C binding in the cytoplasm, chloroplasts and mitochondria of tobacco leaves 6-24 hours after infection. Both normal and TMV proteins are suppressed. The matsutake 80% ammonium sulfate salted out extract showed a high infection inhibition rate when mixed with CMV RNA, so it is considered to have a mechanism of action that inhibits viral RNA.

In addition, Pokeweed antiviral protein: PAP has been reported to penetrate from the lower leaves to the upper leaves through cell-to-cell transition and have a virus inhibitory effect (Chen et al.). The matsutake 80% ammonium sulfate salting-out extract is considered to have no migratory properties based on the results of the migratory investigation.
When used as an antiviral agent, it is important to show antiviral activity without showing phytotoxicity (Hirai, 1972). The matsutake 80% ammonium sulfate salted-out extract, which was the subject of this experiment, was considered promising as an anti-plant virus because it showed antiviral effects without showing any phytotoxicity.

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FIG. 1 is a graph showing the antiviral effect of matsutake 80% ammonium sulfate salting out extract against CMV. FIG. 2 is a graph showing the antiviral effect of matsutake 80% ammonium sulfate salting out extract against CMV. FIG. 3 is a graph showing the virus inhibition effective time of the matsutake 80% ammonium sulfate salting out extract. FIG. 4 is a photograph showing the delaying effect of viral symptom in matsutake 80% ammonium sulfate salting-out extract application treatment (virus inoculation after foliar application). FIG. 5 is a graph showing the CMV CP accumulation inhibitory effect (in the inoculated leaves) when tobacco leaf surface treatment (virus inoculation after leaf surface application) with matsutake 80% ammonium sulfate salting out extract is performed. FIG. 6 is a graph showing the CMV CP accumulation inhibitory effect (in the upper leaves) when tobacco leaf surface treatment with matsutake 80% ammonium sulfate salting out extract (virus inoculation after leaf surface application) is performed. FIG. 7 is a photograph showing a delayed effect of virus symptoms when a mixture of matsutake 80% ammonium sulfate salting-out extract and virus is applied to leaves. FIG. 8 is a graph showing the CMV CP accumulation inhibitory effect (in the inoculated leaves) when a mixture of matsutake 80% ammonium sulfate salting-out extract and virus is applied to the leaves. FIG. 9 is a graph showing the CMV CP accumulation suppression effect (in the upper leaves) when a mixture of matsutake 80% ammonium sulfate salting-out extract and virus is applied to the leaves. FIG. 10 is a photograph showing the leaf surface distribution of virus when a mixture of matsutake 80% ammonium sulfate salting-out extract and virus is applied to tobacco. FIG. 11 is a photograph showing the leaf distribution of virus when a mixture of matsutake 80% ammonium sulfate salting-out extract and virus is applied to tobacco with leaves on the surface (after 2 days). FIG. 12 is a photograph showing the leaf distribution of virus when a mixture of matsutake 80% ammonium sulfate salting-out extract and virus is applied to tobacco with leaves (after 4 days). FIG. 10 is a photograph showing the leaf distribution of a virus when a mixture of matsutake 80% ammonium sulfate salting-out extract and viral RNA is applied to tobacco.

Claims (4)

  Matsutake fungi are cultured to form mycelia, and the mycelia are crushed to obtain a supernatant, which is subjected to salting out with 80% saturation, and recovered from the resulting precipitate, 80% ammonium sulfate of Matsutake An anti-plant viral agent comprising an analysis extract.   A method for protecting a plant from viral infection, comprising applying the 80% ammonium sulfate salting-out extract of matsutake according to claim 1 to the plant. In the manufacturing method of the 80% ammonium sulfate salting out extract of the matsutake of Claim 1,
(1) cultivating matsutake fungi to form mycelia,
(2) The supernatant is obtained by crushing the mycelium,
(3) subjecting the supernatant to salting out to 80% saturation; and (4) recovering from the precipitate resulting from the salting out.
A method comprising that.   The method according to claim 3, wherein the recovery step is performed by dissolving the precipitate in a buffer solution and dialyzing the resulting solution against the buffer solution to remove ammonium sulfate.