Liquid for controlling white root rot fungus of apple tree and method for using the same

Abstract

To provide an apple white root rot fungus control liquid for reducing infectivity and pathogenic power of a white root rot fungus by bringing a white root rot fungus infected with a mycovirus into contact with a mycovirus-free white root rot fungus to transfer (transmit) the mycovirus from the white root rot fungus infected with the mycovirus to the white root rot fungus.SOLUTION: The control liquid for the apple white root rot fungus is a mixed liquid comprising a culture liquid obtained by mixing and culturing the white root rot fungus carrying a mycovirus for reducing the infectivity and pathogenic power of the white root rot fungus and a bacterial group belonging to the genus 13, zinc ions, fulvic acid and water.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a control solution for white root rot fungus on apple trees, which reduces the infectivity and pathogenicity of the white root rot fungus on apple trees, thereby preventing infection of apple trees with the white root rot fungus and improving and curing the symptoms of white root rot disease, and a method for using the same. [Background technology]

[0002] The causative agent of white root rot disease of apple trees is Rosellinia necatrix, a fungus belonging to the Ascomycete fungi. White root rot fungus spreads quickly, primarily infecting and rotting the roots. For this reason, early detection of white root rot infection is difficult; by the time infection is confirmed on the above-ground parts of one apple tree, surrounding apple trees are often already infected. In order to prevent further spread of infection, infected apple trees may have to be cut down. Furthermore, the quality of apples produced by infected trees is reduced, resulting in a decrease in yield, so white root rot fungus has caused significant damage to apple tree cultivation.

[0003] Traditionally, white root rot fungus has been eradicated using chemical substances such as pesticides, but this method has limited effectiveness and has problems such as causing chemical damage and environmental pollution from the pesticides used. Meanwhile, the present inventors have discovered that waste liquid from culture medium (see, for example, Patent Document 1) discharged from an organic waste treatment system containing multiple fungal bodies is effective against white root rot fungus of apple trees, and have been investigating its application.

[0004] Here, there are mycoviruses that reduce the infectivity and pathogenicity of the white root rot fungus. In recent years, a biocontrol method has been investigated in which a mycovirus-infected, attenuated white root rot fungus is brought into contact with a mycovirus-free white root rot fungus to infect the fungus with the mycovirus, thereby reducing the infectivity and pathogenicity of the white root rot fungus.

[0005] Patent Document 2 describes a mycovirus that has a significant effect of reducing the infectivity and virulence of white root rot fungus and can be used in the biocontrol method. However, between cytoplasmically incompatible bacteria, virus transfer (transmission) from mycovirus-infected bacteria to mycovirus-free bacteria does not normally occur.

[0006] Particle transfection is a common method for transferring mycoviruses between cytoplasmically incompatible bacteria from a mycovirus-infected white root rot fungus to a mycovirus-free white root rot fungus. However, because particle transfection can only be performed in a laboratory environment, it is difficult to perform in an orchard (see, for example, Patent Document 3).

[0007] Furthermore, Patent Document 3 describes a method for transferring a mycovirus from a mycovirus-infected white root rot fungus to a mycovirus-free white root rot fungus by concurrently culturing the two fungi in a medium containing added zinc ions, thereby overcoming cytoplasmic incompatibility between the bacteria.

[0008] However, in order to transfer mycovirus using this method, a high concentration of zinc ions of 0.4 to 2.5 millimolar is required, and it has been described that this method has problems in terms of concerns about maintaining reaction conditions in an outdoor environment, environmental burden, biological toxicity, etc. (see, for example, Patent Documents 3 and 4).

[0009] There was a desire to develop a biocontrol technology that would allow direct introduction of a mycovirus into any white root rot fungus in a field environment, overcoming the cytoplasmic incompatibility reaction. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 4043473 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-255460 [Patent Document 3] Patent No. 6715021 [Patent Document 4] Japanese Patent Application Laid-Open No. 2012-110318 [Non-patent literature]

[0011] [Non-Patent Document 1] "Fraction of Fulvic Acid by Gel Filtration and Its Chelating Ability," Hidekazu Yamada, Koyo Yonebayashi, Kyosei Hattori, Shuji Morita, Abstracts of the 1972 Osaka Conference of the Japanese Society of Soil Science and Plant Nutrition (accepted July 26, 1975). https: / / core.ac.uk / download / pdf / 235429797.pdf Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention addresses the problem of providing a white root rot fungus control solution that reduces the infectivity and pathogenicity of the white root rot fungus by a biocontrol method in which a mycovirus-infected white root rot fungus is brought into contact with a mycovirus-free white root rot fungus, and the mycovirus is transferred from the mycovirus-infected white root rot fungus to the white root rot fungus. Another object of the present invention is to provide a white root rot fungus control liquid that reduces the infectivity of mycovirus-free white root rot fungus by the biocontrol method, thereby preventing infection by the white root rot fungus and treating and recovering apple trees infected with the white root rot fungus by reducing its pathogenicity, and a method for using the same. [Means for solving the problem]

[0013] The white root rot fungus control liquid of the present invention is characterized by being a mixed liquid containing a culture liquid obtained by mixed culture of white root rot fungus carrying a mycovirus that reduces the infectivity and pathogenicity of the white root rot fungus and a group of bacteria belonging to the following 13 genera, zinc ions, fulvic acid, and water. (1) Streptomyces (2) Sulfolobus (3) Saccharopolyspora (4) Nocardia (5) Pseudomonas (6) Bacillus (7)Cytophaga (8) Candida (9) Cellulomonas (10) Clostridium (11) Aspergillus (12) Penicillium (13) Rhizopus

[0014] It is preferable that the culture medium is cultured so that the total mass of the bacterial cells is in the range of 3.0 to 30.0 mass% of the total mass of the culture medium, and that the solid components are removed by filtering through a filter material with openings that allow the bacterial cells to pass through.

[0015] The white root rot fungus control solution is characterized by being a mixture of 50.0 to 150.0 liters of culture solution, 0.1 to 1.0 kg of fulvic acid, and 0.1 to 10.0 millimolar equivalents of water-soluble zinc ions, and then water is added to make up to 1000 liters.

[0016] Furthermore, the method for using the white root rot fungus control solution for apple trees of the present invention is characterized by injecting the white root rot fungus control solution for apple trees into the root zone of apple trees infected with the white root rot fungus using an underground injector.

[0017] More specifically, the method of using the white root rot fungus control solution of the present invention is preferably to inject 10 to 30 ml into each hole at intervals of 20 to 40 cm into the root zone within a radius of 40 to 100 cm from the apple tree to be treated, at a depth of 20 to 40 cm from the ground surface, using an underground injector. [Effects of the Invention]

[0018] The white root rot fungus control solution of the present invention can prevent apple trees from being infected with the white root rot fungus by infecting a mycovirus-free white root rot fungus, which has strong infectivity, with a mycovirus and reducing the infectivity of the white root rot fungus.

[0019] Furthermore, the white root rot fungus control solution of the present invention infects the white root rot fungus with a mycovirus, thereby reducing the pathogenicity of the white root rot fungus and curing white root rot, thereby restoring apple production and the quality of the apples produced.

[0020] Furthermore, the white root rot fungus control solution of the present invention treats apple trees by restoring their vigor, thereby significantly reducing the risk of having to cut down apple trees infected with white root rot fungus. DETAILED DESCRIPTION OF THE INVENTION

[0021] The white root rot fungus control solution of the present invention and its method of use will be described below. This description is for the purpose of explaining the present invention and is not intended to limit the technical scope of the present invention. The present invention can be implemented in various modifications within the scope of the present invention.

[0022] The white root rot fungus control liquid of the present invention is characterized by being a mixed liquid containing a culture liquid obtained by mixed culture of white root rot fungus carrying a mycovirus and a group of bacteria belonging to the following 13 genera, zinc ions, fulvic acid, and water.

[0023] [About the culture medium] The culture medium used in the present invention is a mixture of white root rot fungus carrying a mycovirus that has the effect of reducing the infectivity and pathogenicity of white root rot fungus and bacteria belonging to the 13 genera mentioned above, and is cultured by adding fermentable organic matter and water to the medium. The culture medium is preferably cultured so that the total mass of the bacterial cells used is 3 to 30% by mass relative to the total mass of the culture medium. If the mass of the bacterial cells is less than 3% by mass relative to the mass of the culture medium, sufficient control effect against white root rot fungus cannot be obtained when used as a white root rot fungus control liquid, while culturing so that the mass exceeds 30% by mass is often difficult and is not preferred because the activity of the fungus may decrease or change.

[0024] [About cultured bacteria] The cultured bacteria used in the present invention are preferably a mixture of white root rot fungus carrying a mycovirus that has the effect of reducing the infectivity and pathogenicity of white root rot fungus and bacteria belonging to the following 13 genera. (1) Streptomyces (2) Sulfolobus (3) Saccharopolyspora (4) Nocardia (5) Pseudomonas (6) Bacillus (7)Cytophaga (8) Candida (9) Cellulomonas (10) Clostridium (11) Aspergillus (12) Penicillium (13) Rhizopus

[0025] The white root rot fungus carrying a mycovirus was isolated from multiple strains of the fungus collected from apple orchards contaminated with the fungus, and was a strain with poor growth ability that carried an RNA virus, which was isolated in accordance with the method described in Patent Document 2.

[0026] The bacteria used for cultivation preferably include white root rot fungus carrying a mycovirus and all bacteria belonging to the bacterial group of the above 13 genera. By including all bacteria belonging to the 13 genera and white root rot fungus carrying a mycovirus, the mutual decomposition and fermentation action of the bacteria can be enhanced.

[0027] The organic matter used as the raw material for the culture medium is not particularly limited as long as it can be decomposed by fermentation under the culture conditions of the present invention. Preferred examples include, but are not limited to, waste from agricultural product processing, waste from seafood processing, and food waste.

[0028] [About zinc ions] When filamentous fungi that are cytoplasmically incompatible are co-cultured, transmission (transfer) of fungal parasitic viruses does not normally occur. However, when these fungi are co-cultured in the presence of zinc ions, the cytoplasmic incompatibility is overcome, and the virus is transferred (transmitted) from a filamentous fungus infected with a fungal parasitic virus to a filamentous fungus that is free of the fungal parasitic virus. However, in order to transfer mycoviruses using this method, a high concentration of zinc ions of about 0.4 to 2.5 millimolar is required, which poses a problem in maintaining reaction conditions in an outdoor environment (see, for example, Patent Documents 3 and 4).

[0029] In this invention, to overcome cytoplasmic incompatibility, fulvic acid, which has a strong affinity for zinc ions and aids their uptake in the body, was added to co-culture with the white root rot fungus. The growth inhibitory effect of this mixed fungus was measured, and it was found that mycovirus transfer occurred even in the presence of low concentrations of zinc ions, demonstrating that this mixed solution has a strong inhibitory effect on the white root rot fungus. In the present invention, in the biocontrol method in which a weakly virulent mycovirus-infected fungus that is cytoplasmically incompatible with a mycovirus is brought into contact with a mycovirus-free fungus in the presence of zinc ions to infect the white root rot fungus with the mycovirus, the concentration of zinc ions can be reduced by adding fulvic acid.

[0030] There are several known water-soluble zinc compounds that dissociate in an aqueous solution to give zinc ions, and any zinc salt that can be preferably used in the present invention can be used. For example, zinc chloride and zinc sulfate heptahydrate are commercially available and can be used.

[0031] [About fulvic acid] Fulvic acid is a substance that gives a yellow or orange-yellow color to the supernatant liquid that is extracted with inorganic acid after humic acid has been extracted from forest underground soil with dilute alkali and removed. When dried, it becomes an amorphous powder. The resulting powder is an amorphous acidic substance that is soluble in water and ethanol. Fulvic acid has a large molecular weight and does not have a single chemical structural formula, and its composition and molecular weight are not constant; rather, they change depending on the raw material and collection conditions. Furthermore, fulvic acid has a strong affinity for many metal ions, particularly zinc and iron ions, and assists in the uptake of zinc and iron ions into the body.

[0032] In the present invention, it is preferable to use purified fulvic acid produced by the method described in Non-Patent Document 1 as the fulvic acid, but fulvic acid in an aqueous solution can also be used by converting the fulvic acid content.

[0033] [Composition of white root rot fungus control solution] The white root rot fungus control solution of the present invention is a mixed solution obtained by mixing the above culture solution, zinc ions, fulvic acid, and water. The white root rot fungus control solution of the present invention preferably has a culture solution content of 50 to 150 L, assuming a total volume of 1000 L. If the volume of culture solution is less than 50 L, the control effect of the white root rot fungus control solution will be reduced, and adding a content exceeding 150 L is not preferred because the enhancement of the control effect of the white root rot fungus control solution will not correspond to the increase in the amount of culture solution added.

[0034] Furthermore, when the total volume of the white root rot fungus control solution of the present invention is 1,000 liters, the fulvic acid content is preferably 0.1 to 1.0 kg. By setting the content within this range, the zinc ion concentration can be reduced. If the fulvic acid content is less than 0.1 kg, the control effect of the white root rot fungus control solution will be reduced, and adding fulvic acid in an amount exceeding 1.0 kg will not increase the control effect of the white root rot fungus control solution in proportion to the increase in the amount of fulvic acid added, which is economically undesirable.

[0035] Furthermore, the zinc ion concentration is preferably 0.1 to 10.0 millimolar / white root rot fungus control solution, more preferably 0.2 to 5.0 millimolar / white root rot fungus control solution, and most preferably 0.4 to 2.5 millimolar / white root rot fungus control solution. If the zinc ion content is less than 0.1 millimolar, the control effect of the white root rot fungus control solution will be reduced, and if the zinc ion content exceeds 10.0 millimolar, environmental pollution may occur and it is economically undesirable.

[0036] The amount and method of use of the white root rot apple fungicide solution of the present invention will vary depending on the shape and symptoms of the target apple tree, the shape of the land on which it is growing, etc., but generally 10 to 30 ml can be injected into a single hole in the ground at a depth of 20 to 40 cm in the root zone, at intervals of 20 to 40 cm, within an area of ​​a radius of 40 to 100 cm from the affected tree, using, for example, an underground injector.

[0037] [Example] (1) The culture medium of the present invention preferably contains white root rot fungus carrying a mycovirus that has the effect of reducing the infectivity and pathogenicity of white root rot fungus, and a group of bacteria belonging to the following 13 genera:

[0038] [Culture solution] The 13 types of bacteria of the present invention include the following 13 genera. The attached symbols correspond to the above 13 genera. 1) Streptomycin-producing bacteria (Streptomyces griseus) Leptomyces genus) 2) Sulfolobus acidoca ldarius Sulfolobus) 3) Saccharopolyspora erythraea erythraea (Saccharopolyspora genus) 4) Nocardia asteroides Cardia genus) 5) Pseudomonas fluorescens cens Pseudomonas spp.) 6) Bacillus subtilis 7) Cytophaga hutchinsoni Cytophaga spp. 8) Candida versatillis genus) 9) Cellulomonas flavigena Cellulomonas spp.) 10) Clostridium thermocellum llum Cristridium genus) 11) Aspergillus oryzae Gillus genus) 12) Penicillium chrysogenum mn Penicillium spp. 13) Rhizopus oryzae (Rhizopus oryzae)

[0039] [Production of culture medium] All of the above 13 species of bacteria and the white root rot fungus carrying the mycovirus were cultured at 1 × 10 6 cfu / cm 3The seed cultures of the two concentrations were mixed in equal amounts and pre-cultured until the total mass of all the cells accounted for 10% by mass of the total culture solution, to produce a crude culture solution. This crude culture solution was then filtered using a filter with openings large enough to pass the cells through, to obtain the culture solution of the present invention.

[0040] [Manufacturing of white root rot fungus control solution] Water was added to 100 liters of the culture solution, 1.0 kg of fulvic acid produced according to the method described in Non-Patent Document 1, and a predetermined amount of zinc sulfate heptahydrate (see paragraph

[0042] ) to produce 1,000 liters of a white root rot control solution. In this case, the water used to produce the white root rot control solution is preferably water that does not contain an oxidizing agent.

[0041] [Spraying white root rot fungus control solution] In the present invention, the white root rot control solution prepared as described above is diluted 2 to 20 times with water, and then 10 to 30 ml of the solution is injected into each hole at intervals of 20 to 40 cm and a depth of 20 to 40 cm using an underground injector into the root zone within a range of 40 to 100 cm from the center of the apple tree to be treated.

[0042] [Test example] Comparison of the pathogenicity of white root rot fungus control liquid against apple seedlings The following white root rot fungus control solutions 1 to 8 with different compositions were prepared, and the pathogenicity of the white root rot fungus confronted with each white root rot fungus control solution was compared. Sample composition solution 1: Water was added to 100 liters of culture solution to make up to 1000 liters. Sample composition solution 2 Add 28.8 g of zinc sulfate heptahydrate to 100 liters of culture solution, then Water was added to bring the total volume to 1000 liters (zinc ion concentration 0. 1 mmol). Sample composition solution 3 Add 288g of zinc sulfate heptahydrate to 100 liters of culture solution. Water was then added to make the total volume 1000 liters (zinc ion concentration 1.0 mmol. Sample composition solution 4 Add 2876g of zinc sulfate to 100 liters of culture solution, then add water. The total volume was 1000 liters (zinc ion concentration 10.0 min Limol). Sample composition solution 5 Add 300g of fulvic acid to 100 liters of culture solution, then add water. The total volume was set at 1000 liters. Sample composition solution 6: Add 300g of fulvic acid and 2g of zinc sulfate to 100 liters of culture solution. 8.8g was added, and water was added to bring the total volume to 1000 liters ( lead ion concentration 0.1 mmol). Sample composition solution 7: Add 300g of fulvic acid and 28g of zinc sulfate to 100 liters of culture solution. Add 7.6g and then add water to bring the total volume to 1000 liters. (zinc ion concentration 1.0 mmol). Sample composition solution 8: Add 300g of fulvic acid and zinc sulfate to 100 liters of culture solution. Add 2876g and then add water to bring the total volume to 1000 liters. (zinc ion concentration 10.0 mmol).

[0043] Each of the sample composition solutions 1 to 8 was mixed with virus-free white root rot fungus on a cut apple branch and co-cultured for 14 days, and each section was used as an inoculum source for the white root rot fungus control solution. The above inoculum was placed 7 cm below the taproot of apple seedlings, buried, and cultivated in a greenhouse for 3 weeks. The number of dead seedlings in each group was then compared. The results are shown in Table 1.

[0044] [Test Results] [Table 1]

[0045] As shown in Table 1, in sample composition solution No. 3, which did not contain fulvic acid, seven apple seedlings died at a zinc ion concentration of 1.0 mmol / L, whereas in sample composition solution No. 4, which had a zinc ion concentration of 10.0 mmol / L, all apple seedlings survived. On the other hand, in the composition solutions of sample composition solutions Nos. 5 to 8 containing fulvic acid, 2 / 10 of the apple seedlings died when the zinc ion concentration was 0.1 mmol, but all of the apple seedlings survived when the concentrations were 1.0 mmol and 10.0 mmol.

Claims

1. A white root rot fungus control solution for apple trees, characterized by being a mixed solution containing a culture solution obtained by mixed culture of white root rot fungus carrying a mycovirus that reduces the infectivity and pathogenicity of the white root rot fungus and a group of bacteria belonging to the following 13 genera, zinc ions, fulvic acid, and water. (1) Streptomyces (2) Sulfolobus (3) Saccharopolyspora (4) Nocardia (5) Pseudomonas (6) Bacillus (7) Cytophaga (Cytophaga) (8) Candida (9) Cellulomonas (10) Clostridium (11) Aspergillus (12) Penicillium (13) Rhizopus

2. 2. The apple tree white root rot fungus control solution according to claim 1, characterized in that the culture solution is cultured so that the total mass of the bacterial cells of the bacterial group is in the range of 3.0 to 30.0 mass% of the total mass of the culture solution, and is filtered through a filter material with mesh openings that allow the bacterial cells of the bacterial group to pass through, thereby removing solid components.

3. The white root rot fungus control solution for apple trees according to claim 2, characterized in that the white root rot fungus control solution is a mixed solution obtained by adding 50.0 to 150.0 liters of the culture solution, 0.1 to 1.0 kg of fulvic acid, and 0.1 to 10.0 molar equivalents of water-soluble zinc ions, and further adding water to make up 1000 liters.

4. A method for using a white root rot fungus control solution for apple trees, comprising injecting the white root rot fungus control solution for apple trees according to any one of claims 1 to 3 into the root zone of an apple tree infected with the white root rot fungus using an underground injector.

5. 5. A method for using the white root rot fungus control solution for apple trees according to claim 4, characterized in that 10 to 30 ml of the control solution for apple trees according to any one of claims 1 to 3 is injected into the root zone within a radius of 40 to 100 cm from the apple tree to be treated, at intervals of 20 to 40 cm, to a depth of 20 to 40 cm from the ground surface, into each hole.

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