CA1100867A - Method of inhibiting agricultural and horticultural crop viruses - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to a method of inhibiting the agricultural and horticultural crop viruses by spraying either on the leaves or into the soil, the diluted extracted solution of the nutrient medium and tissue-medium obtained after culturing the mycelia of such edible fungi as Lentinus edodes which belong to the basidio mycetes either in a solid culture medium or in a liquid medium containing the component of vegetable fibers.

Description

11()(~67 BACKGRoUND OF TIIE INVENTION
This invention relates to an inhibitory method designed to inhibit the agricultural and horiticultural crop viruses or plant viruses with the use of the extract solution extracted from the nutrient medium and tissue-medium made available after culturing the mycelia of the basidio mycetes which offer such edible fungi as Lentinus edodes in a culture medium containing the component of vegetable fibers.
The fundamental difference among the bacteria, mycoplasma, rickettsia, gramidia, and virus is found in their sizes Their sizes decrease in the order described above, and there is a discontinuation between the virus and the rest.
However small and simple the virus may be, the virus is a single-cell microorganism and multiplies by the binary fission. In this sense it cannot be said that the virus is a microorganism in the true sense.

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-ll()U 867 At present the kinds of typical animal viruses in-cluding vertebral and non-vertebral ones number several hundreds, while more than plant viruses have been confirmed.
The tObacco mosaic virus (TMV) and the cucumber mosaic virus (CMV) are the two typical viruses which represent the confirmed plant viruses. Recently, the carcinogenic virus theory is becoming more convincing.
It is proved in an animal test that a virus which enters a normal cell forms an unknown protein in it and turns the cell into cancer. A similar process may be conceived in plant. There have been several vaccines developed for the prevention of the diseases due to animal viruses. But no effective vaccine has been developed for inhibiting the diseases due to plant viruses.
This is because the viruses are not bacteria but at the level of bacteria and the destruction of the viruses leads to the destruction of normal cells. Consequently, the damage of the agricultural crops due to the plant viruses to which there is no effective cure is said to reach 200 billion a year in this country.
An inhibitor which is applied to agricultural and horticultural crops must be free from contamination, easy to apply, and inexpensive. Since the extract which is used in an embodiment of this invention is extracted from the mycelia of edible fungi, the extract is harmless not only to plants but also to human being and animals and features a simple and inexpensive mass production and extreme economics.

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By the way, there are recent reports on the tests on anti-plant virus effect with the use of an extract solution which was obtained by boiling slices of the fruit body of Lentinus edodes in distilled water and then filtering with gauze. They are "Inhibitory effect of aqueous extract of the fruit bodies of basidio mycetes on plant virus" by Keiichi Tomaru and Akira Udagawa (Shokubutsu Boeke, Vol.
29, No, 1 (1975)) and "Inhibitory effect of aqueous extract of the fruit bodies of Lentinus edodes on plant virus" by Yasushi Takagi and Yukio Sugimura (Proc. Kansai Pl. Prot.
Soc., 19~ 16 (1977)).
In the above two reports the infection-preventive effect was observed only when the plant was vaccinated with a mixture of equal amounts of the aqueous fruit body extract solution and a virus solution or by means of the so-called mixed vaccination, and it was found that the infection-preventive effect decreased sharply with increasing time. It was confirmed that a method of vaccinating the back of a leaf first with the aqueous extract solution by means of a paintbrush and then vaccinating the surface of the same leaf in 24 hours or the so-called backside leaf application was ineffective at all. Therefore, the above-mentioned fact indicates that the aqueous fruit body extract solution hardly penetrate the tissue of the plant directly and instead acts directly on the virus particles.
By the way, the inventors of this invention have com-pleted many inventions in relation to the methods of extrac-ting the pharmaceutically effective component contained in .~. , .
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the cultures of edible mycelia of the basidlo mycetes after many years of research on them, while they have come to know, by chance, the contribution of the aqueous mycelia extract solution to the growth of plant, and so the aqueous mycelia extract solution was analyzed. As the results of the analysis the aqueous mycelia extract solution was found to contain active substances of cytokinin system in addition to germanium in the form of organic compounds, and these substances were confirmed to contribute to the growth of plant. Inlater experiments the aqueous mycelia extract solution was confirmed to not only contribute to the growth of plant but also have an anti-virus effect. As a result the inventors of this invention thought in their first step that the germanium in the form of organic compounds and active substances of cytokinin system had some effect on the anti-virus mechanism and carried out experiments on the anti-virus effect together with the extraction of the above-mentioned substances.
The results of these experiments showed no particular dependence between the above-mentioned substances and the anti-virus effect and indicated that the anti-virus effect did not result from any one particular component in the extract solution extracted from the cultures of the mycelia but did result from several components which are contained in the extract solution and are organically bonded with one another.

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1101~67 It is an object of this invention to offer an inhibi-tory method of agricultural and horticultural crop viruses which is free from contamination, easy to apply, and inexpensive in applications to plant virus inhibition.
It is a further object of this invention to offer an inhi-bitory method of agricultural and horticultural crop viruses which has an excellent infection-preventive effect.
It is a still further object of this invention to offer an inhibitory method of agricultural and horticultural crop viruses which warrants the normal growth of plant by inhi-biting plasmolysis of the plant tissue and promoting the deplasmolysis and thus has an excellent anti-virus effect.
SUMMARY OF THE INVENTION
According to the invention, a method for inhibiting agricultural and horticultural crop viruses comprises spraying the surface of the leaves of said crop or spraying the soil surrounding said crop with a virus inhibiting effective amount of a diluted aqueous extract obtained by culturing the mycelia of edible fungi of the basidiomycetes family in a liquid nutrient medium containing fibrous components of plant tissue.

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BRIEF DESCRIPTION OF THE DRAWINGS
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Figs. l(A) and l(B) are photos in place of drawings showing the effect given to the deplasmolysis wherein Fig. l(A) is a microphotograph showing the cells of the tissue immersed in purified water with Fig. 1(~) showing the cells of the tissue immersed in the aqueous mycelia extract solution.
Figs. 2(A) and 2(B) are photos in place of drawings showing the effect given to the inhibition of the plasmolysis wherein Fig. 2(A) is a photomicrograph showing the cells of the tissue immersed in purified water with Fig. 2(B) showing the cells of the tissue immersed in the aqueous mycelia extract solution.

- 5a -1~0~ 7 Fig. 3(A) is an electron photomicrograph showing purified TMV.
Eig. 3(s) is an electron photomicrograph showing purified TMV
when it mixed in aqueous solution of the F. D. powder of mycelia extract solution.

DETAILED DESCRIPTION OF THE INVENTION
The edible fungi of the basidio mycetes which are used in this invention include shiitake (lentinus edodes), hiratake (pleurotus ostreatus), enokidake (flammulina velutipes sing), nameko (ploliata nameko), kawaratake (coriolus versicolor), and sarunokoshikake (rigiooporus ulmarius).
Among them, however, the extract solution from the culture of the mycelia of shiitake (lentinus edodes) showed the highest activity and had an excellent anti-virus effect. The above-mentioned anti-virus effect of the extract solution extracted from the culture of the mycelia of shiitake may have a closer relation with the components of the medium where shiitake is grown.
The fibrous component of high molecular carbohydrate which is the main component of the medium is absorbed by edible fungi to be lower molecular polysaccharide, or inorganic substances such as magnesium, phosphorous, copper, and iron, and these com-ponents are combined with the mycelia organically. The anti-virus effect may be ascribable to such combination. For this reason a medium composed chiefly of bagasse containing large quantities f fibrous components, but a liquid medium is applied in this invention being superior to the solid medium in the points of handling and productivity.

. ., '` ll!D~67 In this latter case 20 to 50% of components of vegetable tissues is added to the G. P. Y. medium which is commonly used.
Since the above-mentioned bagasse conventionally finds no definite use and is disposed of mainly by incineration, it is readily available at low cost. As components of vegetable tissues: saw dust, straw, husk of rice, and peanut epidermis may be added in addition to the above-mentioned bagasse.
Or the concentrated boiled solution of these materials may be used in a suitable amount, or high molecular carbohydrate such as L-cellulose, pentsan, hemicellulose and lignin which were comprised in the fibrous component as main ingredient, or inorganic substances such as magnesium,phosphorous, copper and iron may be added. In this invention the mycelia are cultured by the usual method in the above-mentioned nutrient medium, the effective components as anti-virus are extracted from the cultures of the mycelia. Extraction by heat, extrac-tion by alkali, extraction by acid, extraction by solvent, extraction by homogenizer may be adopted as extraction method.
Extraction being separated mycelia culture and filtrate culture, 3 liters of purified water was added to 100 grams of the mycelia culture. The solution heated and extracted was mixed in the filtrate culture containing metabolites and substances of the mycelia culture which was separated at first, was offered to obtain raw solution.
As is seen from the above, the mycelia culture and the filtrate culture were mixed once more in order to mix effective substances being contained both in the mycelia culture and in the filtrate culture.

The results of the analyses of these mycelia extract solutions showed the presence of nucleic acid-derived substances including mainly RNA, sugar-alcohols including inositol and munnitol, 18 kinds of amino acids, 18 kinds of organic acids including succinic acid and citric acid, vitamins, phosphorus, copper, magnesium, iron, organic germanium, active substances of cytoknin system and other unknown components. The inventors of this invention conducted several experiments to isolate the anti-virus component contained in these extract solutions but each isolated component did not show much effect, indicating that study on the effective component by isolation had little significance.
In this invention the anti-virus effect is found only in the aqueous extract solution extracted from the culture of mycelia. The inventors of this invention estimated from the results of many experiments that the mechanism of the anti-virus effect was ascribable to the following.
It is already reported by Fujio Suzuke et al that an inter-feron derived substance of RNA which has a double bond is present in the extract solution extracted from the mycelia of shiitake. The inventors of this invention tested the effect of the scale cell of onion on plasmolysis and deplas-molysis by the following means using an extract solution extracted from the culture of the mycelia of shiitake.
NOTE:
l. Object of experiment; To study the effect of the mycelia extract solution on the deplasmolysis.

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Extract solution used; Extract solution of mycelia of shiitake.
Material; Concave epidermis of scales of onion.
Method of treatment; The epidermis was immersed in 0.5M
sucrose solution for 15 minutes and then in the extract solution for one minute.
Purified water was used as the control, and the condition was observed by means of a microscope at a 150-fold magnification.
Results; As is evident from the comparison between Figs.
l(A) and l(B), the deplasmolysis was promoted in the sections treated with the extract solution.

2. Object of experiment: To study the effect of the mycelia extract solution on the plasmolysis.
The epidermis of onion was immersed in the extract solution for 30 minutes and then in 0.SM sucrose solution to observe the change in protoplasm with time by means of a microscope at a 150-fold magnification.
Purified water was used as the control.
Results; As is known from the table given below and Figs.
2(A) and 2(B), treatment with the extract solution evidently inhibited the plasmolysis.

~ I c ¦ d e 1 'I - I - -_ 5 ¦ + ¦ _ ++ h 10 I ++ I -_ 15! ++ I +
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- `` 11()(~67 a -- Tlme (in minute) e -- Extent of plasmolysis b -- Section f -- None c -- Control g -- Slightly seen d -- Extract solution h -- Clearly seen It was confirmed from the above experiments that the extract solution had properties to promote the deplasmolysis and cause the plasmolysis with difficulty. Also, the results indicated that the semipermeability of the cell walls of the scales of onion was not damaged and that there was an increase in the concentration of the protoplasm, suggesting that something was synthesized within the cells of the onion. It is thought that such inorganic components in the nutrient medium, solid or liquid, as magnesium, phosphorus, copper, iron which are contained in the fib~rs of plant were organically combined with the components present in the mycelia within the cells of a plant to form a substance of a kind similar to interferon, but this is not evident at this point. At any rate, however, it is evident from the above experiments that the treatment with the extract solution caused a change in the cell itself.
The extract solution has, in addition to the anti-virus effect, an effect to promote the growth of plant.
Particularly, the extract solution has an outstanding effect to promote the rooting and the growth of root. It has another effect to accelerate the biosynthesis in the plant, such as increasing the chlorophyll. It has also another effect to normalize the metabolism of water and make the photosynthesis proceed smoothly. This and other effects help the plant perform its inherent functions since the plant has, to some extent, Al `~

resistance to withstand external minus factors if the plant is grown in a sufficiently normal manner.
It was known from the results of the experiments that the cold reslstance, hot weather resistance, poor growth due to the defect of the sunshine, etc. were improved.
In testing agricultural and horticultural viruses, the growth of the crops which are used as the control becomes a point of great importance, and depending upon their growth conditions (soil, fertilizer, light, humidity, temperature), the results of the tests are greatly influenced. And, varied environments affect the extent to which the crops are subjected to sickness. In other words, this means that depending upon environments the plant body has sufficient resistance against virus. That is to say, to the plant body its normal growth of plant, as described earlier, through its inhibitory action on plasmolysis and deplasmolysis, and the anti-virus effect is supposed to derive from this inhibitory effect.
The infection-preventive effect of the extract solution does not lower with time. It is confirmed, on the contrary, that the infection-preventive effect increases with time and that the infection-preventive effect can be shown to be effective either by the so-called backside leaf application or by the soil-spraying. This indicates that the extract solution directly penetrates the plant tissue.
In this respect the extract solution is essentially different from any fruit body extract solution.
The mycelia extract solution of th s invention is effec-tive against agricultural and horticultural viruses and may be so , I

when applied to the human being. E`urther, the mycelia extract solution seems to be effective to the prevention of the death of cultured pink salmon due to the destruction of their pancreas resulting from the infection with a pathogenic fungus or a virus, for which there is no preventive method at all, at present but trying merely to avoid the infection by mixing the mycelia extract solution with the feedstock to the cultured pink salmon.
The mycelia extract solution is applied by diluting it with water or by diluting freeze-dried powder of the mycelia extract solution with water and then spraying the diluted solution into the soil or on the surface of leaves.
Some examples of the invention are given in the following.
(Example l) 30 to 40% (weight ratio) of ground baggase was added to G. P. Y. nutrient medium being composed of 50 grams of glucose, 2 grams of peptone, and 3 grams of yeast extract.
The liquid nutrient medium thus obtained was sterilized in the usual manner, where the cultured solid seed structure of shiitake was planted, was then placed in an air-conditioned culture chamber at a temperature of 18 to 20C to start the culture of the mycelia. It hecame sufficiently prevalent with the mycelia in lO days, then moved to treatment chamber in order to apply temperature change treatment.
It will take much time for nutrient that in case solid nutrient medium containing baggase, yeast bran, and other nutrient sources is used. Liquid nutrient medium was selected for this reason.

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It was held at a high temperature of 32 to 34C for a period of 24 to 48 hours in treatment chamber, and subjected to a low temperature treatment of 5 to 8C for a period of 5 to 7 hours.
Thereafter, it was moved to a growth chamber and left there.
At the same time, mycelia nutrient medium and filtrate nutrient medium came to be separated, and water was added at a rate of 6 liters per 100 grams of the mycelia to a tank where mycelia was discharged into. It being left for one hour at the tempera-ture of 40 to 50C, the mixture was mixed under agitation for 10 4 to 5 hours at a temperature of 60 to 130C.
By this agitation, the effective components of the mycelia of shiitake can be extracted, and thus the suspension was obtained by the mixing the extract solution and filtrate solution.
The suspension was filled in a filter bag made of flannel, and filtered under pressure. The filtrate was filtered with a membrane filter and sterilized to obtain an aqueous extract solution containing the essence which was the products of the metabolism of the mycelia of shiitake. The aqueous extract solu-tion was freeze-dried to obtain powder at a rate of about 20 7 grams per liter. The 7-gram powder was diluted with 100-fold distilled water, and the solution was offered as a raw solution.
In a similar manner to the above example, the mycelia of enokidake, hiratake, and kawaratake were cultured, and an extract solution of each kind was obtained and offered in the following experiments.
(Experiment 1) Test to confirm the infection-preventive rate of the aqueous D

mycelia extract solution against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV).
tl) Testing method Viruses used:
A tobacco mosaic virus system (TMV - OM) (Purified sample (0.25 - 0.5 ug/ml) ) A cucumber mosaic virus (CMV - Y) (Juice of infected tobacco leaves (25 - 50 fold diluted)) Both viruses were suspended separately in a 0.lM
phosphoric acid buffer solution having a PH 7Ø
Method of inoculation and examination:
Each extract solution was mixed with each virus suspension in equal amounts. As to Nicotiana glutinosa, tobacco (bright yellow), and kidney bean the infection-preventive rate was calculated by the half leaf method which counts the number of spots using the following formula. As to cowpea the infection-preventive rate was calculated by the pairing leaf method which counts the number of spots using the following formula.
Percent of the infection-preventive rate = ( (number of spots in the control section - number of spots in the treatment section / number of spots in the control section ) ) X 100 Extract solutions used:
Extract raw solutions each of the mycelium of shiitake, .
-' 11~ 67 enokidake, hiratake, and kawaratake were used.
The non-water content was 0.7 - 0.8%. In the experiments each solution was diluted with 500-fold distilled water and used.

* results TMV
_ ,_ _ _ ___ N-glutionsa kidney bean .
. treat- control I.P.rate treat- control I.P.rate ment ment shiitake 82053 99.956 315 82.2 ~nokidake 26¦ . 3024 99.167 283 76.3 ..., _ ~
hiratake 132556 99.567 302 77.8 aratake 12¦ 2265 99.556 271 79.3 N-glutinosa : Total of ten half leaves kidney bean : Total of ten half leaves ,~ ; ' CMV

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tobacco (bright yellow) ¦asparagus bean cowpea ¦treat- jcortrol treat- ~ control L,P.rate ment ment I

shiitake 239 ¦ 863 72.3 21 76 72.4 .
enokidake 214 724 70.4 13 48 73.0 .
hiratake 282 766 63.2 - 22 54 59.3 .
kawaratake 341 913 62.7 1 16 42 62.0 .

kobacco: Total of twelve half leaves asparagus bean cowpea: Total of ten half leaves The above results showed that each mycelia extract solution had a high infection-preventive rate against TMV
had a preventive effect against agricultural and horticultural crop vlruses.
(Experiment 2) Since it was confirmed in Experiment 1 that each extract solution had an infection-preventive effect against agricul-~ tural and horticultural crop viruses, a test to confirmthe preventive effect against the occurrence of a tobacco mosaic disease was conducted in a field.

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* Site for the test:
Asahi-mura, Kashima-gun, Ibaragi-ken * Variety:
MC of the second yellow kind * Outline of cultivation:
The setting was done on April 2, 1976. The large-scale mulching was done on May 1, 1976. The start of budding was seen on June 5, 1976.

* Scale of the test:
Four fields where the mosaic disease is seen.
10 acres for each treatment section, 10 acres for the control section, and number of tobacco plants : 2020/10 acres.

* Mycelia extract solution used:
Shiitake mycelia extract solution * Method of treatment:
The roots were immersed in a 500-fold diluted shiitake mycelia extract solution at the time of the setting, and the same diluted solution was sprayed on the surface of the leaves at a rate of 150 liters/10 acres at the time of mulching.

* Determination:

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0~867 The cases wherein all the leaves suffered from slight disease or heavier disease were regarded as rate of sick stabs, and the infection-preventive rate was calculated by the following formula.
Percent of the infection-preventive rate = ( (number of sick stabs in the control section - number of sick stabs in the treatment section)/ number of sick stabs in the control section) X 100 * Results:

(A) (B) (C) 1 415 1423 70.8 2 152 861 82.4

3 35 426 91.8

4 203 1153 82.4 (A) ----- Number of sick stabs in the treatment section (B) ----- Number of sick stabs in the control section (C) ----- Infection-preventive rate In each of the four fields tested, the infection-preventive effect was evidently confirmed. The average infection-preventive rate was some 80% strong.

~lV~867 (Experiment 3) The same extract solution as in Experiment 2 was given to Yasumichi ~ishi, Laboratory of plant pathology, vegetable and ornamental crops research station, Ministry of Agriculture and Yasuo Komuro, 2rd research division, Laboratory of therap-eutics, Ministry of Agriculture which conducted an entrusted test, The following results were obtained.

(Entrusted test on anti-virus effect) ~ ~B C ~ D E

b272 246 320 618 650 516 377 605 521 0%
c323 332 160 36 164 109! 99 15 39 69.0%

16 32 19 61 69 401 26 17 46 92,1%

21 35 13 37 21 16¦ 19 18 31 ¦ 94.8%

a.-------- Section, b. -------- Control section c.-------- Shiitake mycelia extract solution, 10-fold d.-------- Shiitake mycelia extract solution, raw solution e.-------- Shiitake mycelia solution, sprayed twice A.-------- Repetition~ B. -------- 1st time C.-------- 2nd time, D. -------- 3rd time E.-------- Infection-preventive rate ~ 19 --111)~867 July 29, 1977 9 : 00 - regent sprayed 12 : 00 - TMV 5000-fold 0.001 Mpp buffer planted by coating Plant used: N. glutinose, 3 pieces/section, 3 - 4 leaves inoculated F ~ I __ f G H I ¦ J K
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g 893 638 734 649 379`288 525 574 615 532 308 512 0%
_ . .__ h 38 24 19 ¦ 12 13 9 2 38 15 12 9 7 97.0~
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i 1 1 4 25 18 36 20 46 9 2 0 7 97.5%
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f. -------- Section g. -------- Control section h. -------- Shiitake mycelia extract solution, 10-fold i. -------- Shiitake mycelia extract solution, raw solution F. -------- Repetition G. -------- 4th time H. -------- 5th time I. -------- 6th time J. -------- 7th time .K. -------- Infection-preventive rate September 18, 1977 14 : 30 regent sprayed " 19, 1977 15 : 30 TMV 500-fold O.OlMpp buffer inoculated by coating Plant used: N. glutinose, 3 leaves each, 4 stabs inoculated D

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111)(~867 ] L M
¦ 9 90%

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j. --------Section, k. -------- Control section 1. --------Shiitake mycelia extract solution, 10-fold m. --------Shiitake mycelia extract solution, raw solution L. --------Number of stabs M. --------Number of sick stabs N. --------Rate of sick stabs Viruses used: CMV
Plant used: cucumber Treatment: October 1, 2, 29, 31, 1977 sprayed 4 times Inoculation: Seed of juice was inolculated Examination: November 17, 197i Directed by: Yasumichi Nishi, Laboratory of plant pathology, vegetable and ornamental crops research station, Ministry of ~griculture . I . ~
n O P Q
o 11 5 45%
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` ` 11()~67 (Note) The shiita]ce mycelia extract solution used indicates the solution prepared by freeze-drying of the extract solution and diluting the freeze-dried powder with lOO fold water.

(1) The raw solution indicates a 100-fold diluted solution (2) The 10-fold indicates 1000-fold dilution.

The results of the entrusted experiment showed surprisingly good infection-preventive values by comparison with conven-tional values and showed that the infection-preventive effect was not affe_ted at all as the time passed, on the contrary, increased with time.

(Experiment 4) Further experiment was carried out to see anti-virus effect.

* Directed by: Treatment laboratory, Plant virus Institute, Ministry of Agriculture * Viruses used: Purified TMV (150 mg/ml) * object: To test anti-virus effect of aqueous solu-tion of the extract solution in concentra-tion 1~ and virus used.
20 * Results: Fig. 3(A) is 40,000 magnification photo-micrograph of purified TMV
Fig. 3(B) is 40,000 magnification photo-micrograph showing the anti-virus effect when said TMV mixed in the extract solution.

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110~ 7 It is observed that virus surrounded by their membrances came to be closed and at least cohered. At the same time, inactivity of viruses took place of activity and infection-preventive and multiplication-preventive were thus made.

(Experiment 5) An anti-virus effect was tested by coating the extract solution to the backside of the leaves. The anti-virus effect against tobacco mosaic virus (T~lV) was confirmed with the use of the mycelia extract solution of the basidio mycetes.

(1) Testing method * Site for the test:
Soil fertilizer laboratory, Chiba agricultural experiment station * Viruses used:
TMV common variety Purified sample (0.25 to 0.5 ug/ml) Suspended in a O.lM phosphorus acid buffer solution at PH 7Ø

* Extract solutions used:
Extract solutions each of shiitake, enokidake, hiratake and kawaratake were used. The non-water contents of three mycelia solutions are 0.7 to 0.8%.

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(Example 2) The mycelia of shiitake was cultured in the liquid nutrient medium as given in Example 1. 3 liters of purified water was added to 100 grams of this cultured mycelia.
The extract solution thus obtained in the same manner as Example 1, was diluted 100 times by purified water and was used as raw solution applying no freeze-drying manner.
The same result was obtained as in said example.
For the use of the extract solution, the condition of freeze-drying powder is of great advantage to storage and conveyance.
- (Example 3) 20 grams of powdered yeast extract was added to 1 liter of water, moreover, boiled and concentrated solution was added.
(10 liters of purified water was added to 1 kilogram of bagasse containing 18% of moisture, and boiled down to 5 liters) Cultured solid seed structure of kawaratake was planted in the liquid nutrient, and was then placed in an air-conditioned culture chamber to start the culture of the mycelia. In the same manner as in Example 1, separating cultured mycelia and filtrate solution, wherein 3 liters of purified water was added to 100 grams of the cultured mycelia and thus extract solution was obtained.
100-fold extract solution diluted by purified water was used as raw solution. The same result as the said example was obtained.

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Claims (11)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A method for inhibiting agricultural and horticultural crop viruses comprising spraying the surface of the leaves of said crop or spraying the soil surrounding said crop with a virus inhibiting effective amount of a diluted aqueous extract obtained by culturing the mycelia of edible fungi of the basidiomycetes family in a liquid nutrient medium containing fibrous components of plant tissue.

2. A method according to claim 1 wherein the fungi are selected from the group consisting of shiitake, hiratake, enokidake, nameko, kawaratake and sarunokoshikake.

3. A method according to claim 2 wherein the fungus is shiitake.

4. A method according to claim 1 wherein the extract is freeze-dried before dilution.

5. A method according to claim 1 wherein the fibrous com-ponents of plant tissue comprise at least one material selected from the group consisting of saw dust, bagasse, rice husks and peanut epidermis.

6. A method according to claim 5 wherein the fibrous com-ponents of plant tissue comprise bagasse.

7. A method according to claim 1 wherein the liquid nutrient medium comprises yeast, bagasse and water.

8. A method according to claim 6 wherein the liquid nutrient medium contains from about 30% to about 40% by weight bagasse.

9. A method for inhibiting agricultural and horticultural crop viruses comprising spraying the surface of the leaves of said crop or spraying the soil surrounding said crop with a virus inhibiting effective amount of a diluted aqueous extract obtained by culturing the mycelia of edible fungi of shiitake in a liquid nutrient medium containing fibrous components of plant tissue, said fibrous components comprising bagasse.

10. A method according to claim 9 wherein the liquid nutrient medium contains from about 30% to about 40% by weight bagasse.

11. A method for inhibiting agricultural and horticultural crop viruses comprising spraying the surface of the leaves of said crop or spraying the soil surrounding said crop with a virus inhibiting effective amount of a diluted aqueous extract obtained by culturing the mycelia of edible fungi of shiitake, in a liquid nutrient medium composed of 50 gms. of glucose, 2 gms. of peptone, and 3 gms. of yeast extract per litre of water, and 30 to 40% by weight of ground bagasse, and after culturing, the material is filtered, the mycelia are diluted with water and maintained at a temperature of 40 to 50°C for one hour and then agitated at 60 to 130°C for 4 to 5 hours, and an aqueous sterilized extract is prepared.