JP2011051925A - Method for controlling plant disease caused by nematode - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling plant diseases caused by nematode using biological control techniques. <P>SOLUTION: The method for controlling plant diseases caused by nematode includes inoculating nonpathogenic filamentous bacteria to stems and leaves of plants. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for controlling plant diseases caused by nematodes. More particularly, the present invention relates to a method for controlling plant diseases caused by nematodes, particularly root-knot nematodes, by inoculating plant stems and leaves with non-pathogenic filamentous fungi.

  Nematode is considered one of the most harmful pests because it has a wide host range and causes a significant loss in yield and death from a wide range of crops ranging from open-ended root vegetables to institutional fruit vegetables, and it is difficult to eradicate them when invaded. The nationwide soil screening project carried out for 10 years since 1959 revealed that harmful nematodes live at a high density in 53% of the 6,400 ha screening area. With the complete abolition of methyl bromide for soil disinfecting applications excluding essential uses since fiscal 2005, nematode damage that had been controlled unconsciously until now has become apparent and expanding. On the other hand, introduction of environmental conservation type organic technology and organic farming technology is strongly demanded in crop prevention.

  The mainstream of conventional nematode control measures is chemically synthesized pesticides. Dedicated and general-purpose chemically synthesized pesticides are used as nematocides. These are broadly divided into granules and fumigants, and the former is an organic phosphorus phosphiazate and the latter is a DD. Physical control measures include solar soil sterilization that seals the house and heats the cultivated soil, reduced soil sterilization that mixes organic matter in the house soil and heats it in a sealed manner, and hot water that heats the soil directly by supplying hot water from a movable boiler. There are soil disinfection and long-term flooding treatment of arable soil. Cultivating control techniques include the cultivation of nematode-resistant plants and nematode resistant varieties that, when cultivated, reduce the nematode density in the soil. Marigold's “African Thor” and wild oat's “Hey Oats” are typical nematode counter plants. Examples of registered biopesticides include budding bacteria, Pastria wettable powder (Pasturia penetrans), and filamentous fungi Nemahumann (monacrosporium fimatopagum).

Organophosphate-based chemically synthesized pesticides have become a social problem in crop residues. Effects of soil fumigants such as chlorpicrin and DD agents on human health, destruction and disturbance of soil microbiota, groundwater Causes environmental impact such as pollution. Although chemically synthesized pesticides and physical control technologies generally show dramatic control effects, nematodes remain deep in the soil, so nematode density recovery after cultivation is rapid and on the contrary promotes nematode damage (induction) Multiple). Countering plant use, which is cultivated control, has problems such as crop cultivation interruption, weeding, and the occurrence of other pests. In general, resistant varieties are often not marketable, and some vegetables do not have nematode resistant varieties (for example, Cucurbitaceae vegetables). In addition, nematode lines (resistance-breaking lines) that parasitize resistant varieties emerge due to the continuous use of resistant varieties, and the resistance of nematodes to resistant varieties has already been overcome in tomatoes. As for biological materials, Pastria wettable powder suppresses nematodes by a single mechanism of egg-laying suppression and nemahumanton predatory, but the cost is extremely high and the dramatic control effect is poor. The fungus (Fusarium, Paecilomyces, Fimatopagam, Trichoderma, etc.) fungus or spores mixed with root-knot nematode contaminated soil before planting the root-knot (insects) Methods for reducing the number of gills and the soil density of the second root-knot nematode larvae are also known (eg Paecilomyces lilacinus , strain PL 251. BioAct (R) WG (http://www.prophyta.de/englisch /pdf/BioAct_Product_Information.pdf), and non-patent documents 1 to 4). However, when applying these methods in the field, a large amount of fungus is required, and the nematode control effect is often smaller in the field than in the pot test.

  The nematode control means described above are aimed at direct killing or inactivation of nematodes regardless of chemical, physical, or biological methods, and nematodes or plant pathogens parasitic on nematodes (mainly pathogenic filamentous fungi) It is not aimed at strengthening the defense response of plants against.

  The inventor's research group has reported a biological control technique by strengthening the defense reaction against parasites on the host plant side, which is another important control factor (Patent Document 1). The main point is that by controlling the root-knot nematode by treating a solanaceous plant with a non-pathogenic Fusarium fungus, the synergistic action of using a combination of the non-pathogenic Fusarium fungus and an attenuated strain of the Tobamovirus genus It was to control root-knot nematodes.

JP 2009-155229 A

BARRON, G. L., The nematode-destroying fungi.Canadian Biological Publications, Guelph, 140pp, 1977 DRECHSLER, C., Mycologia 46, 762-782, 1954 Hallmann J, Sikora RA, J Plant Dis Protect 101: 475-481, 1994 Sharon, E., Bar-Eyal M., Chet, I., Herrera-Estrella, A., Kleifeld, O., Spiegel, Y., Phytopathology 91: 687-693, 2001

  An object of the present invention is to provide a method for controlling plant diseases caused by nematodes using biological control techniques.

  The present inventor conducted further diligent studies on the nematode control technology using a non-pathogenic filamentous fungus that was previously reported. It has been found that growth and plant diseases can be suppressed. In addition, the combination of attenuated plant virus and non-pathogenic filamentous foliar inoculation and the application of attenuated plant virus causes the plant to co-infection with these two species, and the growth of nematode and plant diseases are more significantly suppressed. I also found. The present invention has been completed based on these findings.

That is, the outline of the present invention is as follows.
(1) A method for controlling plant diseases caused by nematodes, characterized by inoculating non-pathogenic filamentous fungi on plant foliage.
In the above method, it is preferable to apply the attenuated plant virus to the plant before inoculation of the non-pathogenic filamentous fungus into the foliage of the plant.
In the above method, it is preferable to use a non-pathogenic Fusarium bacterium as the non-pathogenic filamentous fungus. Non-pathogenic filamentous fungi can also be inoculated, for example, in the form of a flora disc or suspension.
Further, in the above method, the nematode is, for example, a root-knot nematode.
(2) A foliar treatment agent for controlling plant diseases caused by nematodes, containing a non-pathogenic filamentous fungus as an active ingredient.
In the foliar treatment agent, the non-pathogenic filamentous fungus is preferably a non-pathogenic Fusarium fungus.

  According to the present invention, plant diseases caused by nematodes can be biologically controlled. Stem and leaves inoculation with non-pathogenic filamentous fungi, and inoculation with attenuated plant viruses and non-pathogenic filamentous fungi can exhibit a nematode control effect comparable to an organophosphorus agent that is a chemical control means. Unlike the conventional chemical control methods, the method and the foliar treatment agent of the present invention have a slight environmental impact and impact on human livestock or other crops, so without disturbing the harmony of the ecosystem, In addition, plant diseases caused by nematodes can be controlled stably and continuously without adversely affecting beneficial microorganisms living in the soil. In addition, the present invention can be widely applied to plants having nematodes parasitic and having inherent viral diseases, Fusarium diseases, and the like.

Figure 2 shows nematode damage in tomato plants inoculated with the attenuated tomato mosaic virus and stalked with a non-pathogenic Fusarium fungus. A is a root-knot class value, B is a root-kump index, and C is a nematode larva density, indicating nematode damage. Shows nematode damage in tomato plants inoculated with stems of non-pathogenic Fusarium fungus at various times. A indicates the root nose class value, and B indicates the number of root nodules (goals) of the root-knot nematode. The nematode damage (the number of nematode invading to tomato roots) in a tomato plant inoculated with a non-pathogenic Fusarium fungus is shown. A shows a case where a non-pathogenic Fusarium bacterium is inoculated as a PDA disc, and B shows a case where the non-pathogenic Fusarium bacterium is inoculated as a suspension. The results of a field test comparing the inoculation of non-pathogenic Fusarium stalks with soil mixing are shown. A is a nematode larva density, and B is a root-knot class value, showing the nematode damage of each treated plant.

  The method for controlling plant diseases caused by nematodes according to the present invention is characterized by inoculating plant stems and leaves with non-pathogenic filamentous fungi, thereby reducing the damage caused by nematode growth and parasitism and / or associated with nematodes. Plant diseases caused by other parasites (filamentous fungi, bacteria, viruses, etc.) can be reduced.

  Nematode is a pest that parasitizes plants and takes nutrients from the protoplasts of plant cells. For example, about 70 species (Meloidogyne arenaria, M. incognita, M. hapla, M. javanica, etc.) have been confirmed in the taxonomy of root-knot nematode belonging to the genus Meloidogyne. Parasitic. The whole species has been reported to affect over 2000 species of plants, among which are important crops such as tomato, pepper, cucumber, potato, sweet potato, eggplant, carrot, burdock, spinach, chard, garlic, Includes leeks, ginger, peas, kidney beans, cowpeas and the like.

  Nematodes not only harm plants alone, but also cause soil pathogens and complex infections. When nematode is infected, there are no obvious signs in the ground that can help diagnose the infestation early in the infection, but in the basement, bumps (gall or knot) begin to form. The size of the bumps varies depending on the variety, but in many cases it is about 1 to 2 mm and is difficult to confirm with the naked eye. On the other hand, eggs are laid on the surface and roots of the bumps, and the mass of the eggs can be confirmed with the naked eye. Roots infected with nematodes parasitize nematodes at various stages of development. Nematode infection reduces crop yield or loses market value such as parasitic tuberous roots.

  The nematode to be controlled in the present invention is not particularly limited as long as it is a harmful plant parasitic nematode, and is not limited. Meloidogyne nematodes such as root-knot nematodes (Meloidogyne arenaria), Ditylelenchus nematodes such as Ditylelenchus destructor, Ditylelenchus dipsaci, and Pratylenchus penis (trans) Examples include Pratylenchus genus such as Negrass nematode (Pratylenchus cffeae) and Chromeges nematode (Pratylenchus vulnus). In the present invention, it is possible to effectively control nematodes parasitic on the roots, particularly root-knot nematodes.

  Nematodes are also involved in infection with filamentous fungi, bacteria, and viruses. For example, fungi belonging to the genus Fusarium (Fusariumoxysporum f.sp. lycopersici, Fusarium oxysporum f.sp. cucumerinum), Rhizoctonia genus (Rhizoctonia solani), Pythium genus (Pythium cucurbitacearum, etc.), Pseudomonas Plant diseases such as wilt disease, bacterial wilt disease, blight disease, vine split disease, hip fracture disease, and clubroot disease occur in plants. Therefore, “control of plant diseases caused by nematodes” means both of controlling the attack and proliferation of nematodes and / or controlling plant diseases caused by infection with the above-mentioned filamentous fungi, bacteria, viruses and the like.

  The non-pathogenic filamentous fungus used in the method of the present invention is not particularly limited as long as it is a filamentous fungus that is not pathogenic to plants. For example, a filamentous fungus belonging to the genus Fusarium, Trichoderma, etc. (Siddiqui IA and Shaukat, SS (2003) Letters in Applied Microbiology 2003, 37, 109-114; Diedhiou PM, Hallmann J., Oerke EC, Dehne HW (2003) Mycorrhiza (2003) 13: 199-204) . Of these, non-pathogenic Fusarium bacteria are preferable, non-pathogenic Fusarium oxysporum is more preferable, and Fusarium oxysporum F13 is particularly preferable. Fusarium oxysporum F13 was separated from the soil in Shimane University and was filed by the applicant on July 20, 1995, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 Tsukuba, Tsukuba, Ibaraki) No. 6) is deposited under the accession number FERM P-1557 (International Deposit Number FERM BP-5825) (Japanese Patent Laid-Open No. 9-67218). Moreover, although not non-pathogenic filamentous fungi, for example, non-pathogenic bacteria belonging to the genus Pseudomonas can be used in the same manner as non-pathogenic filamentous fungi (for example, Ali NI et al. (2002) Soil Biology & Biocemistry 34: 1051-1058).

  The culture of non-pathogenic filamentous fungi used in the present invention can be carried out by using a general method for culturing microorganisms known in the art, such as potato dextrose agar medium, potato sucrose agar medium, tour peck Solid culture using solid plate culture medium or slant culture medium such as agar culture medium, solid component derived from plants such as bran, solid culture using porous material impregnated with sugar or nitrogen source, potato / dextrose liquid culture medium, potato -Liquid culture using various liquid culture media such as sucrose liquid culture media and tourpec liquid culture media are all possible. The culture method can be shake culture, jar fermenter culture, stationary culture, or the like. For example, the culture conditions for culturing Fusarium oxysporum F13 are preferably aerobic conditions and cultured at a temperature of 15 to 35 ° C. and a pH of 4 to 8 for 5 to 8 days.

  The non-pathogenic filamentous fungus can be used in a culture of a filamentous fungus, a form in which the culture is crushed or shredded with a medium, or a form in which the filamentous fungus is separated from the medium. In addition, filamentous fungi were shaken and cultured in potato dextrose (PD) broth or sucrose-added potato broth liquid medium (PS) broth known in the art, and then the mycelium fragments were removed by filtration through double gauze. Sprouting cells (such as small conidia) can be used.

  Therefore, the foliar treatment agent of the present invention can contain any non-pathogenic filamentous fungus in the above-mentioned form. In addition, the foliar treatment agent contains a non-pathogenic filamentous fungus as an active ingredient, which contains a carrier, a sticking agent, a dispersing agent, an auxiliary agent, and the like, a fungus disc (for example, PDA disc), a paper file disc, a suspension, It can be in the form of a wettable powder, solution, emulsion, powder, granule or the like.

  Examples of the carrier include water-soluble polymer compounds (polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, etc.), water, vegetable oil, liquid animal oil and other liquid carriers; and talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, Examples thereof include solid carriers such as vermiculite and silica sand. Examples of the fixing agent include casein, gelatin, gum arabic, and alginic acid. Examples of the dispersant include alcohol sulfate esters and polyoxyethylene glycol ether. Examples of the auxiliary agent include carboxymethyl cellulose, starch, and lactose.

  In the production of the foliar treatment agent of the present invention, the carrier, the sticking agent, the dispersing agent and the auxiliary agent can be used alone or in appropriate combination depending on the purpose.

For example, when preparing a PDA disk as a flora disk, a small conidia of a non-pathogenic filamentous fungus is applied to a 1.0% agar medium (PDA medium) mixed with a potato dextrose liquid medium, and the whole surface of the PDA medium is applied. A PDA disk having an appropriate size (for example, a diameter of about 0.5 cm) can be prepared from a well-organized flora using a cork borer or the like. Also when preparing the suspension for example, prepared by shaking culture of conidia small amount of non-pathogenic fungi, by diluting to conidia small content contained ¹⁰⁵ degree per 1 ml, the suspension can do. In addition, when preparing a paper file disc, a liquid containing small conidia is dropped on a paper file disk of 5 mm or less in the straight shape and dried, or a liquid containing small conidia is dropped on a filter paper disk of 5 mm or less in the straight shape. It can be prepared by air drying.

The content of the non-pathogenic filamentous fungus in the foliar treatment agent of the present invention is not particularly limited. For example, the range is 1 × 10 ⁴ to 1 × 10 ⁸ cfu / g, preferably 1 × 10 ⁶ to 1 × 10 ⁷ cfu / g. The range of g. Moreover, what is necessary is just to prepare a foliage treating agent so that it may become the range of said density | concentration at the time of inoculation, and a non-pathogenic filamentous fungus can be contained by the density | concentration of several times-several thousand times of the said range at the time of formulation. The non-pathogenic filamentous fungus may be a mycelium or a spore, and the units of cfu / g and cfu / ml may be read as spore / g and spore / ml.

  Inoculation with non-pathogenic filamentous fungi is carried out in the field before the plant is infected with nematode, at the same time as or after the infection, preferably after the plant is infected with nematode, for example, plants grown in soil that has exterminated nematode in the field. After planting (more preferably 1 to 3 days after planting), it is performed on the foliage of the plant. In the present invention, the “stems and leaves” means any part of the stems and leaves in the above-ground part of the plant.

The method for inoculating the non-pathogenic filamentous fungi can be performed by any method known in the art. The inoculation of foliage is, for example, by immersing a needle in a suspension of a non-pathogenic filamentous fungus and damaging the foliage of the plant with the needle, or damaging the foliage of the plant, where the flora of non-pathogenic fungal fungi is present. This can be done by attaching a disc. For example, when inoculating a paper file disc containing non-pathogenic filamentous fungi, the paper file disc may be rubbed against the stem and fixed with a belt-like parafilm. The fungus can be introduced into the plant body by piercing with a needle after fixing to the stem. The inoculation amount of the non-pathogenic filamentous fungus is not particularly limited as long as the fungus can grow, but is preferably in the range of 10 ³ to 10 ⁶ cfu / strain, more preferably in the range of 10 ⁴ to 10 ⁵ cfu / strain. It is.

  In the method for controlling plant diseases caused by nematodes according to the present invention, when an attenuated plant virus is applied to a plant before inoculation of a non-pathogenic filamentous fungus into the foliage of the plant, the control effect is further enhanced. Two types of microorganisms (attenuated plant viruses and non-pathogenic filamentous fungi) work together to strongly inhibit nematode infestation and growth, possibly through multiple mechanisms such as strengthening plant defense mechanisms.

  The attenuated plant virus used in the present invention is not particularly limited as long as it is a microorganism that is not pathogenic to plants, and examples thereof include an attenuated tobamovirus genus. In the attenuated tobamovirus genus, for example, attenuated tomato mosaic virus, red pepper mild mottle virus, cucumber mosaic virus, tobacco mosaic virus and the like are preferable (for example, Oshima, N. 1981. Control of tomato mosaic disease by attenuated virus. Japan Agric Res. Quart. 14: 222-228).

  When the non-pathogenic filamentous fungus used in the present invention is a non-pathogenic Fusarium oxysporum, it is preferable to use an attenuated strain of tomato mosaic virus and an attenuated strain of capsicum mild mottle virus as the attenuated plant virus. Of these, the tomato mosaic virus L11A237 strain is preferred. Tomato mosaic virus L11A237 strain is MAFF No. 260002 or No. It is stored as 260004 shares in the Agricultural Resource Research Institute, Genebank, and can be obtained from distribution application.

  Attenuated plant viruses (viruses that do not cause damage even if they infect plants) can be created by mutations (for example, artificial mutations) from highly toxic plant viruses (viruses that infect plants and cause damage) Can be separated from the natural world.

  The attenuated plant virus can be used, for example, in the form of a milling solution of a plant infected with the virus. Virus may be collected or concentrated by centrifuging the grinding liquid. In addition, attenuated plant virus particles or viral nucleic acids may be used alone, or mixed with a buffer solution or the like to form an attenuated plant virus solution in the form of a solution, emulsion, suspension or the like. Examples of the buffer solution include a sodium phosphate buffer solution and a potassium phosphate buffer solution. Examples of the liquid agent may include adjuvants such as trehalose and sucrose, chelating agents such as EDTA, stabilizers such as polyethylene glycol, and the like. In addition, a grinding liquid or a liquid preparation may be freeze-dried and prepared with water, a buffer solution or the like at the time of application.

  The content of the attenuated plant virus in the liquid preparation or the like is not particularly limited. For example, the virus particle concentration is preferably in the range of 0.0001 to 0.5% by weight, more preferably 0.001 to 0.01% by weight. It is a range.

  Application of the attenuated plant virus to plants can be carried out by spraying a grinding solution or solution onto plants (preferably leaves of seedlings, stems, etc.) or smearing them. At that time, an abrasive such as carborundum may be used in combination, and the above-mentioned grinding liquid or liquid agent is smeared with absorbent cotton or the like on the leaves sprayed with the abrasive. Moreover, after spraying a grinding liquid or a liquid agent etc. to a plant, you may damage the surface of a plant using the roller etc. which adhered the abrasive | polishing agent. Alternatively, the plant may be sprayed by applying pressure to the grinding liquid or liquid agent and carborundum using a sprayer or a spray gun. The application amount of the attenuated plant virus to the plant is not particularly limited as long as the virus infects the plant.

  Plants to be controlled in the present invention are plants infested with nematodes, such as solanaceae plants (tomatoes, peppers, eggplants, potatoes, peppers, tobacco, etc.), cucurbitae plants (cucumbers, cucumbers, pumpkins, etc.), convolvulaceae. Plants (such as sweet potatoes), celery family plants (carrots, celery, celery, etc.), asteraceae plants (such as burdock, garlic, etc.), red crustaceae (spinach, chard, etc.) ), Ginger family plants (such as ginger), and legumes (such as peas, kidney beans, cowpeas), but are not limited thereto.

  In the method for controlling plant diseases according to the present invention, first, seedlings are grown in soil subjected to fumigation, sterilization, etc., and an attenuated plant virus is applied before planting the seedlings. Next, non-pathogenic filamentous fungi are inoculated into the foliage of the plant to cause superinfection. Specifically, it is possible to inoculate the seedlings before planting the non-pathogenic filamentous fungus in the field (before nematode infection), or inoculate the seedlings when planting the seedling in the field, but more preferably Is inoculated with a foliage several days after planting (preferably 1-3 days after planting).

  Plants treated as described above reduce nematode, especially root-knot nematode infestation, and also reduce plant diseases due to infection with filamentous fungi, bacteria and viruses. In particular, the present invention has an advantage that a small amount of inoculation is sufficient because non-pathogenic filamentous fungi are inoculated on the foliage of the above-ground part. Therefore, fungi with low culture efficiency can be made into materials, and the production cost and use cost of the filamentous fungus material can be reduced. In addition, the inoculation of the above-ground foliage is labor-saving, which contributes to labor cost reduction. Therefore, this invention can be utilized in the scene which controls the root-knot nematode damage of a plant, especially a crop. In particular, nematode control using crop physiological changes such as induced resistance of microorganisms such as filamentous fungi can be expected to have a stable control effect.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

[Example 1]
In the pot cultivation of tomatoes, experiments were conducted on the root-knot nematode control effect when non-pathogenic Fusarium fungi were inoculated with stems and leaves in an early, simultaneous and late time series relative to the time of infection with root-knot nematodes.

(1) Nematode and microorganism strains used in the experiment (i) Nematode Sweet potato nematode Nematode: Meloid guinea incognita (derived from house-grown tomatoes in Kajigawa-mura, Kitaharabara-gun, Niigata) A line that has been maintained for generations, hereinafter referred to as the “Niigata Ganbaru Root Line”) was used. This strain is a so-called “resistance-breaking strain” in which the infectivity for tomato varieties (NR varieties: Nematode resistant variety) incorporating the nematode resistance gene (Mi gene) is equivalent to the infectivity for susceptible varieties. . This system was established by MAFF No. 108258 is registered.
(Ii) Non-pathogenic Fusarium Fusarium oxysporum F13 strain was used.

(2) Seedling and Cultivation Tomato varieties were NR varieties (nematode resistance), Momotaro (Takii). The seedling bed soil used was a mixture of 50% Kureha Horticulture soil and sandy loam soil. This floor soil was filled into a black vinyl pot (volume: 300 ml) having a diameter of 90 mm and a height of 80 mm, and 2 seeds were sown per pot. After germination, the seedlings were thinned and made into single seedlings. Eleven days after sowing, the attenuated strain of tomato mosaic virus was inoculated into tomato seedlings in half of the total number of pots. 18 days after sowing, the above seedling bed soil was used to tamper with a black vinyl pot (volume: 300 ml) having a diameter of 90 mm and a height of 80 mm. Sowing, raising seedlings, inoculation of test microorganisms, and tomato seedling management after inoculation were all carried out in the glass room of the Central Agricultural Research Center located at 3-1-1 Kannondai, Tsukuba, Ibaraki Prefecture. The seedlings were managed at 25 ° C., and were grown until the planting date 57 days after sowing.

(3) Inoculation source preparation and inoculation method (i) nematode Inoculation source sweet potato root nematode Niigata gambaru root line is inoculated with 500 individuals of second stage larvae and cultivated in a greenhouse controlled at 25-30 ° C for about 80 days It was obtained from the tomato (Strong Rice Life No. 2). That is, the soil was removed from the roots of the tomatoes by washing with water, and the egg sac formed on the roots of the exposed roots was collected with tweezers while observing under a stereomicroscope (60 ×). 100 egg sacs were transferred to a Syracuse watch glass containing 5 ml of tap water, and another watch glass was placed on top of this to prevent water transpiration. After incubation at 25 ° C. for 2 weeks, only hatched second stage larvae were collected by sucking with water using a glass Komagome pipette and prepared to contain 250 second stage larvae in 1 ml with a graduated cylinder. did. Inoculation of nematodes into tomato seedlings (six-leaf stage) is performed by drilling 4 holes with a diameter of 10 mm and a depth of 30 mm on the outside 30 mm from the root using glass tube bottles, and 1 ml of the prepared nematode suspension in each hole. It was carried out by pouring each time. 1000 nematodes were inoculated per pot.

(Ii) Non-pathogenic Fusarium fungus F13 cells of inoculation source are filled with 2 liters of Fusuma medium in a plastic bag for paper mushroom culture (Shinano pack) with circular vents made of paper. Sterilized twice, and inoculated with a sterile aqueous suspension of F13 mycelium passaged on potato-dextrose agar medium (PDA medium) in vitro and aseptically isolated in approximately two weeks. Incubated. Spores and hyphae were not purified. The bacterial density of F13 in the naturally air-dried culture was assayed using MP medium, and confirmed to be a bacterial density of 5.67 × 10 ⁷ cfu / g dry matter. The fungus was stored in a cold room at 10 ° C. until use.

For soil inoculation, immediately before inoculation, the F13 bran culture is crushed with a blender, the required amount is weighed and mixed with steam sterilized sand loam, and soil containing 1 × 10 ⁷ cfu of F13 per ml. 1600 ml was prepared. Next, the tomato was extracted from the vinyl pot of 90 mm × height 80 mm together with the root and soil (300 ml) and replaced with a 1 / 10,000 Wagner pot (volume 1000 ml) having a diameter of 120 mm × height 145 mm, and then F13 was mixed. 400 ml of soil was filled in the gap between the pots. Therefore, the inoculation density of F13 was approximately 6 × 10 ⁶ cfu / ml soil.

  Inoculation source F13 cells used for stem inoculation were obtained by shaking and culturing F13 in PD broth at 25 ° C. for about 6 days, and then filtering with double gauze to remove mycelial fragments. The mixture is centrifuged at 3000 rpm for about 15 minutes, the supernatant is removed, and sterilized distilled water is added thereto for washing, and then the mixture is centrifuged again at 3000 rpm for about 10 minutes to completely remove the supernatant. By removing it, paste-like cells (small conidia) were obtained. The paste-like cells (small conidia) were taken at the tip of an injection needle [standard of 22G × 1 1/4 (0.70 × 32 mm)] and inoculated into a plant stem. The amount of the spore when it was covered with a 22G × 1 1/4 needle was 6.6E + 05 spore ± 0.7E + 05.

(4) Test zones Seven test zones were established as shown below.
(I) F13 stem-2 section Fusarium fungus F13 small conidia is put at the tip of the injection needle and stabbed into the stem of the cotyledon position of a tomato seedling, and after 2 days, the tomato seedling is 120 mm in diameter and 145 mm in height. The plant was replanted in a pot and immediately inoculated into the tomato culture with the second larvae of the root line nematode of Niigata.

(Ii) F13 Sat-2 Ward Tomato seedlings were planted together with 400 ml of soil containing F13 culture into a Wagner pot with a diameter of 120 mm and a height of 145 mm and contacted with F13, and two days later, the root system nematode of Niigata does its best Inoculated the second stage larvae on tomato culture.

(Iii) F13 stem ± 0 section Tomato seedlings were replanted into Wagner pots with a diameter of 120 mm x height of 145 mm, and the second stage larvae of the root line nematode of Niigata Ganbaru were immediately inoculated into the tomato soil, and 2 hours later on the same day A small conidia of Fusarium fungus F13 is inoculated by taking the tip of an injection needle and piercing the stem at the cotyledon level.

(Iv) F13 stem + 2 sections Tomato seedlings were replanted into Wagner pots with a caliber of 120 mm in diameter and 145 mm in height, and the second-stage larvae of the root line nematode of Niigata Ganbatsu were immediately inoculated into the tomato culture, and Fusarium fungus F13 two days later. A small conidia is placed at the tip of an injection needle and stabbed into a cotyledonal stem.

(V) F13 soil + 2 wards The second stage larvae of Niigata gambaru root line nematode is inoculated into the tomato culture soil, and two days later, Wagner pot 120 mm in diameter and 145 mm in height together with 400 ml of soil containing Fusarium F13 culture Replanted and brought into contact with F13.

(Vi) F13 stem + 8 wards Tomato seedlings were replanted into Wagner pots with a diameter of 120 mm x height of 145 mm, and the second-stage larvae of the root line nematode of Niigata Ganbatsu were immediately inoculated into the rhizosphere soil of tomatoes, and Fusarium 8 days later A small conidia of a fungus is taken at the tip of an injection needle and stabbed into a cotyledonal stem.

(Vii) Control group Tomato seedlings were replanted into a Wagner pot with a diameter of 120 mm and a height of 145 mm together with 400 ml of sterile sandy loam (no contact with F13). Inoculated in tomato culture.
Each test group was 4 replicates. The pot was placed on a bench in a glass chamber and managed at 25 ° C to 30 ° C.

(5) Evaluation method 42 days after inoculating nematodes in the test plots of the Examples, the tomatoes in each test plot were cut off from the ground, soil was removed from the roots, and removed into polyethylene bags. The roots were carefully washed with tap water.

(I) Nematode second stage larva count 20 g of soil was weighed in triplicate from the soil removed in polyethylene bags prior to washing the tomato roots and separated by the Bellman funnel method for 72 hours. The water containing the isolated nematode was placed on a plankton counting slide, and the second stage larvae of root-knot nematode were counted while observing at 150 times using a biological microscope (Olympus BX-50). The average of 3 replicates was counted as the second stage larva density per 20 g of soil.

(Ii) 0 to 10 levels
According to the method of Zeck (Zeck, WM (1971): Pflanzenschutz-Nachichten. Bayer AG, 24, 141-144.), The degree of root nodule formation was evaluated by the following class values.
0: No humps are observed.
1: Several small root bumps can be recognized by careful observation.
Several small root bumps similar to 2: 1 can be easily identified.
3: There are many small roots, some of which are fused. The root function is almost intact.
4: There are many small roots and some large roots. Many of the roots are functioning.
5: 25% of the roots are markedly rooted and not functioning.
6: 50% of the roots are markedly rooted and not functioning.
7: 75% of the roots are markedly humped and the ability to regenerate the roots is lost.
8: There are no healthy roots and plant nutrient absorption is inhibited. The foliage is still blue.
9: The root system completely covered by the hump is decaying. The plant is dying.
10: Both plants and roots withered.

(Iii) Root Hump Index A 5-step rating of 0 to 4 was given according to the degree of root galling, and the root galling index was calculated by the following formula (1). Although the root-knot index is a subjective evaluation method, it is most widely used as an evaluation index of nematode damage.
Root kump index = ((4VI + 3III + 2II + 1I) / (4 × number of surveyed strains)) × 100 (1)
0: Number of plants in which nodules were found in 0% of the roots
I: Number of plants with knots found in 1-25% of roots
II: Number of plants with humps found in 26-50% of the roots
III: Number of plants with humps found in 51-75% of roots
IV: Number of plants with humps in 76-100% of roots (iv) Browning of vascular bundles by non-pathogenic Fusarium

35 days after inoculating nematodes in the test plots of the Examples, the tomatoes in each test plot were cut from the ground part, and a 5-stage evaluation of 0 to IV was performed according to the degree of vascular browning 5 cm above the ground part. And the morbidity index of each treatment was calculated by the following formula (2).
Disease index = ((4IV + 3III + 2II + 1I) / (4 × number of strains)) × 100 (2)
0: No vascular bundle browning
I: Number of plants with browning observed in 1/4 of the vascular bundle
II: Number of plants with browning observed in half of the vascular bundle
III: Number of plants with browning observed in 3/4 of the vascular bundle
IV: Number of plants with dead or browned vascular bundles

(V) Statistical processing When testing is possible, 5% level significant difference is detected by one-way analysis of variance when the test is possible. If a significant difference is detected in the test interval, HSD Tukey is detected. Multiple comparisons were made by the method. In the figure, the alphabets “a” and “b” indicate that there is a significant difference between the test plots marked with “a” and the test plots marked with “b”.

(6) Results FIG. 1A, B, and C show the root nodule class value, root nodule index, and second stage larva density in each test section, respectively. Fusarium soil inoculation and stalk inoculation (needle) were compared. In Fusarium small conidia stalk inoculation, the damage was mitigated by inoculation approximately 2 days later than nematode inoculation, similar to the soil inoculation of fungus bodies. The density of nematode larvae in the soil was also low when the stem was inoculated with Fusarium fungus 2 days after nematode inoculation.

  Although the browning of the conduit by Fusarium bacteria was observed in F13 stem-2 ward (index 10.0), F13 stem + 2 ward (6.3), F13 soil + 2 ward (18.8), wilt symptoms There was no onset and no harm.

[Example 2]
In the pot cultivation of tomato, an experiment was conducted on the root-knot nematode control effect in the case of inoculating the non-pathogenic Fusarium fungus at intervals of 8 hours over 2 to 64 hours from the root-knot nematode infection.

(1) Nematode used in experiment and strain of microorganism (i) Nematode In the same manner as in Example 1, the root strain of sweet potato root nematode Niigata was used.
(Ii) Non-pathogenic Fusarium Fusarium oxysporum F13 strain was used in the same manner as in Example 1.

(2) Seedling and Cultivation 13 g / 18 liters of chemical fertilizer (NPK component 14% each) and bitter lime were added to sand loam soil (containing about 50% sand) sterilized with a soil steam sterilizer. This floor soil was filled into a black round vinyl pot having a diameter of 90 mm and a height of 80 mm, and a tomato (Momotaro Haruka), a regenerated seedling, was transplanted. Raising seedlings, inoculation of test microorganisms, and cultivation management of tomatoes after inoculation were all carried out in the glass room of the Central Agricultural Research Center located in Tsukuba City, Ibaraki Prefecture.

(3) Preparation of inoculation source and inoculation method (i) Nematode Nematode was prepared in the same manner as in Example 1, and 1000 root-knot nematode larvae per pot were inoculated into the soil.
(Ii) Non-pathogenic Fusarium fungus As described in Example 1, small conidia of Fusarium fungus F13 were prepared. Inoculation of bacteria drilled deep 2mm pin leaves position stems cotyledons tomato stem, with a pipette fitted with a needle at the tip into the hole, the conidia small amount of distilled water 10 ⁵ / This was done by injecting 1 μl of the suspension containing μl. Tomato stems were inoculated 2, 8, 16, 24, 32, 40, 48, 56 and 64 hours after nematode inoculation. No control was inoculated.
Each test group was repeated 3 times. The pot was placed on a bench in a glass chamber and managed at 25 ° C to 30 ° C.

(4) Evaluation method 36 days after inoculating nematodes in each test section, the tomatoes in each test section were cut from the ground, soil was removed from the roots, and removed into polyethylene bags. The roots were carefully washed with tap water.
(I) 0 to 10-step gall class value Judgment was made according to the method of Zeck of Example 1 (Zeck, WM (1971): Pflanzenschutz-Nachichten. Bayer AG, 24, 141-144.).

(Ii) Number of root bumps Dissolve the tomato roots with a dissecting scissors, count the root bumps (goals) with a counting counter under a stereomicroscope (40 to 60 times) for each broken side root, add them up, The number of root nodules was counted.

(Iii) Statistical processing When testing is possible, the test data of the above-mentioned test section is detected by 5% level significant difference analysis by one-way analysis of variance. If a significant difference is detected in the test section, HSD Tukey is detected. Multiple comparisons were made by the method. In the figure, alphabets “a”, “b”, and “c” indicate that there is a significant difference (P <0.05) between the average values of the treatment sections when the same symbol is not given.

(5) Results FIG. 2A and FIG. 2B show the root-hump class value and the number of root-humps (goal number) in each test section. As a result of tracking the timing of inoculating Fusarium bacteria on the tomato stem every 8 hours, the root nodule class value (A) and goal (number of root nodules) (B) are inoculated with Fusarium fungus 2-24 hours after nematode inoculation. However, it was confirmed that it returned to its original state after 32 hours of inoculation and greatly decreased again after 48 hours of inoculation. Subsequent inoculation with Fusarium bacteria did not decrease.

[Example 3]
An experiment to introduce a tobamovirus attenuated strain as a microbial material that may further enhance the nematode control effect of non-pathogenic Fusarium fungi, and inoculate tomatoes grown in pots in glass chambers with two non-pathogenic microorganisms and nematodes The nematode control effect was examined.

(1) Nematode and microorganism strain used in the experiment (i) Nematode The same strain as in Example 1 was used.
(Ii) Non-pathogenic Fusarium bacterium The same non-pathogenic Fusarium bacterium strain F13 as in Example 1 was used.
(Iii) Tobamovirus attenuated strain Tomato mosaic virus (ToMV) attenuated virus strain L11A-237 was used.

(2) Seedling and cultivation Tomato varieties were tested using Momotaro (Takii), sterilized tomato seeds were immersed in 1.0% antiformin for 10 minutes and 70% ethanol for 5 minutes in this order, and sterilized distilled water This was done by washing 3 times at The sterilized seeds were immersed in sterilized distilled water in a conical beaker and allowed to germinate by shaking with a shaker for several days (sterilized distilled water was changed every 24 hours). Germinated seeds were sown in a 7.5 cm diameter black circle pot filled with sand.

(3) Preparation and inoculation method of inoculation source (i) Preparation and inoculation of virus attenuated strain Tomato mosaic virus (ToMV) attenuated strain L11A-237 of Tobamovirus was obtained from tobacco Nicotiana tabacum seedlings infected with this strain . About 0.1 g of leaves were cut from this plant and ground in 100 ml of phosphate buffer solution of 0.05 M and pH 7.0 using a mortar and pestle. Next, an abrasive was sprinkled on the cotyledons of tomatoes, and absorbent cotton soaked with the above-mentioned leaf grinding solution was lightly rubbed. As the abrasive, 400-600 mesh carborundum was used. ToMV (L11A-237) was applied to the cotyledons at the time when two tomato green leaves were developed.

(Ii) Preparation and inoculation of non-pathogenic Fusarium fungi The small conidia of the Fusarium fungus F13 line as the inoculum were the same as those used in Example 1. The small conidia were applied to a 9 cm diameter sterilized plastic petri dish containing 1.0% agar medium (PDA medium) mixed with Potato dextrose broth. After the application, a PDA disk having a diameter of 0.5 cm was prepared by a cork borer from those in which the bacterial flora reached the entire surface of the PDA medium. The shaking culture was a conidia this small amount, so that the conidia small fraction is included ¹⁰⁵ degree per 1 ml, were prepared suspension.

  When 2 days (48 hours) had passed since the inoculation of tomato mosaic virus, non-pathogenic Fusarium or nematode was inoculated. In the case of the PDA disc, the stock of the tomato was scratched with an injection needle, the PDA disc was affixed there, and the stock PDA disc was fixed by wrapping with parafilm. In the case of a suspension, the suspension was immersed in an injection needle, the stock was damaged, and the wound was covered with parafilm.

(Iii) Preparation and inoculation of nematode The inoculation source sweet potato nematode was the same strain as in Example 1, was grown in the same manner as in Example 1, and the second stage larvae were collected. Inoculation of nematodes into tomato seedlings was carried out at 0, 6, 12, 24, 36 and 48 hours before Fusarium inoculation, or 6, 12, 24, 36 and 48 hours after Fusarium inoculation. The head was inoculated into the soil.

(4) Test Zones The following 4 zones were established as test zones.
(I) + virus group When two days have passed after inoculating tomato mosaic virus on tomato seedlings, when inoculating root-knot nematodes, 6, 12, 24, 36 and 48 hours after root-knot nematode inoculation (6, 12, 24, 36 and 48)) inoculated with non-pathogenic Fusarium fungus, stem inoculated with root-knot nematode (± 0 Ward), or inoculated with non-pathogenic Fusarium fungus Inoculated root-knot nematodes after 6, 12, 24, 36 and 48 hours (-6, -12, -24, -36 and -48 wards).

(Ii) -virus group When a tomato seedling is inoculated with root-knot nematode, non-pathogenic Fusarium fungus is introduced at 6, 12, 24, 36 and 48 hours (6, 12, 24, 36 and 48) after root-knot nematode inoculation. 6, 12, 24, 36 and 48 hours after inoculation of stems, inoculation of non-pathogenic Fusarium fungi at the same time (± 0) with root-knot nematode inoculation, or inoculation of non-pathogenic Fusarium fungi -6, -12, -24, -36 and -48 wards) inoculated with root-knot nematodes. Tomato mosaic virus is not inoculated.

(Iii) Control group-The virus control group is a plant in which only tomato seedlings are inoculated with root-knot nematode, and neither Fusarium nor virus is inoculated. The control of + virus was inoculated with a tomato mosaic virus in a tomato seedling and inoculated with root-knot nematode only two days later, and was not inoculated with Fusarium.
For each test group, 5 repetitions were performed twice, for a total of 10 repetitions. The pot was allowed to stand on a bench in a glass room and cultivated while controlling the temperature at 20 to 25 ° C. Moreover, it watered to such an extent that it did not dry.

(5) Evaluation method After 2 weeks from the test (± 0 ward) inoculated with Fusarium and nematode simultaneously, the tomato roots of each test ward were carefully washed and seedlings were collected.
The collected roots were stained by the acidic glycerin method using a Bellman tube bottle, and the number of root-knot nematodes inside the roots was confirmed with a microscope.

(6) Results The numbers of nematodes entering the tomato root in each test section are shown in FIGS. 3A and 3B together with standard errors. FIG. 3A shows a case where Fusarium bacteria are inoculated as a PDA disk, and FIG. 3B shows a case where Fusarium bacteria are inoculated as a suspension. In FIG. 3, the horizontal axis indicates the time difference of Fusarium inoculation time based on root-knot nematode inoculation. If the number is negative, Fusarium bacteria are inoculated before nematodes, and a positive number indicates that Fusarium bacteria were inoculated later. Dark gray represents the case where Fusarium was inoculated alone, and light gray represents the combination with attenuated ToMV.
As a result of the experiment, the number of nematode intrusions observed every 12 hours was consistently less than the control and the smallest after 48 hours, unlike the number of goal formations in the pot test every 8 hours. As shown in FIGS. 3A and B, the combination with virus further reduced the number of invading nematodes.

[Example 4]
There is no nematode control effect by inoculating a tomato seedling with an attenuated strain of Tobamovirus genus and the condition of inoculating non-pathogenic Fusarium fungi at the timing of infecting nematode late 2 days later. It investigated in warming vinyl house cultivation.

(1) Nematode and microorganism strains used in the experiment (i) Nematode Sweet potato root nematode: Meloidogyne incognita (from the field of cultivation of tomato (variety: dancer) in Yamanashi Prefecture) (Tsukuba) was introduced into the vinyl house of the C4 special field, and then a line of tomatoes was used to establish this vinyl house soil. This strain is a so-called “resistance-breaking strain” in which the infectivity for tomato varieties (NR varieties: Nematode resistant variety) incorporating the nematode resistance gene (Mi gene) is equivalent to the infectivity for susceptible varieties. . This strain has not been deposited with any public organization, but can be obtained free of charge from Takayuki Mizubo, a pest detection and identification method research team, National Agricultural Research Center for Agricultural and Food Industry Research Organization.
(Ii) Non-pathogenic microorganism As in Example 1, Fusarium oxysporum F13 strain was used.
(Iii) Tobamovirus attenuated strain As in Example 3, the attenuated L11A237 strain of tomato mosaic virus ToMV was used.

(2) Seedling and Cultivation Tomato varieties were NR varieties (nematode resistance), Momotaro (Takii). The seedling bed soil used was a mixture of 50% Kureha Horticulture soil and sandy loam soil. This floor soil was filled in a black vinyl pot (volume: 100 ml) having a diameter of 60 mm and a height of 57 mm, and 2 seeds were sown per pot. After germination, the seedlings were thinned and made into single seedlings. Eleven days after sowing, the attenuated strain of tomato mosaic virus was inoculated into tomato seedlings in half of the total number of pots. 18 days after sowing, the above seedling bed soil was used to tamper with a black vinyl pot (volume: 300 ml) having a diameter of 90 mm and a height of 80 mm. As in Example 1, sowing, raising seedlings, inoculation of test microorganisms, and seedling management of tomatoes after inoculation were all carried out in the same manner as in Example 1. Glass of the National Agricultural Research Center located at 3-1-1 Kannondai, Tsukuba, Ibaraki Carried out in the room. The seedlings were managed at 25 ° C. and grown until the fixed planting date.

The cultivation test is a vinyl house (frontage 6.4m, depth 25.0m, height 3.0m) installed in the C4 special farm of the Central Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki ). Three ridges having a width of 1.0 m and a length of 21.6 m across a 0.8 m wide passage were provided. A 25 cm high polyethylene corrugated plate was embedded in each cage and partitioned every 2.4 m, and 27 plots of microplots were provided. The area of one test plot was 2.4 m ² . Nine test plots were selected that were randomly selected from these and repeated three test plots separated from each other.

  The plots used for the test were plowed with straw, then converted to 14g fertilizer (NPK: 14-14-14), mashed lime and molten phosphorus fertilizer for each plot 240g (equivalent to 100kg / 10a), 240g (Equivalent to 100 kg / 10a) and 48 g (equivalent to 20 kg / 10a) were sprayed and fertilized, and carefully mixed with a scissors.

  In the first sampling, an auger capable of collecting 100 ml of soil from the surface layer to a depth of 20 cm at a time immediately after plowing is used to collect four soil cores (subsamples) per plot (hereinafter referred to as sampling). ) The collected four subsamples were stored in a No. 12 polyethylene bag and mixed immediately after storage to make one sample.

Using a hand-operated irrigator (Kyoritsu), DD fumigant was irrigated at a depth of 15 cm at 2 cm (equivalent to 20 L / 10a) on a test plot assigned to conventional control and covered with a black vinyl multi-sheet. did. Eight days after irrigation, plowing was used to degas the fumigant component remaining in the soil.
The second and third (final) mining were performed in the same manner as the first mining.

  The planting of tomatoes was carried out by inserting the soil where seedlings were nurtured into a 9 cm diameter and 9 cm deep hole made with a multi-drilling machine from black polymulch covered with straw on May 20, 2008. Went by. In the test plot, 8 seedlings were planted in 2 rows with 50 cm between the strips and 60 cm between the plants. A 180 cm post was set up after planting.

  After planting, it was cultivated for 84 days until August 12, 2008. Irrigation was carried out once or twice a week with an irrigation tube (Sumisansui M30) installed in the lower center of the black polymulti covering the straw. The temperature inside the house was kept below 35 ° C. by opening and closing the side windows and forced ventilation with a large ventilation fan. During this period, sprouting, attracting to a support, and pest control were performed as appropriate.

(3) Preparation and inoculation method of inoculation source (i) Preparation and inoculation of virus attenuated strain The tobamovirus genus tomato mosaic virus attenuated strain of inoculum was prepared in the same manner as in Example 3, and the same method as in Example 3 Was used to inoculate tomato seedlings 14 days after sowing.

(Ii) Preparation and inoculation of non-pathogenic Fusarium fungus F13 strain was used as the non-pathogenic Fusarium fungus as the inoculation source. The inoculation source to be treated in the field was adjusted as follows. That is, 2 L of bran, 0.8 L of black soil in the field, 0.8 L of vermiculite, and 0.8 L of tap water are mixed, and 2 L each in two bags of the plastic pack for cultivating mushrooms described in Example 1 (Shinano pack). I put it in. Each pack was subjected to sterilization by autoclaving at 121 ° C. for 1 hour after the opening at the top of the pack was sealed with a heat sealer (sealer). Immediately after sterilization, the medium pack was transferred to a clean bench and allowed to cool. Subsequently, 10 ml of Fusarium fungus solution was injected using a sterilized 10 ml injection needle. The pores of the pack where the injection needle was opened were closed by applying vinyl tape. Approximately 2 × 10 ⁸ CFU / g wet culture was obtained after 2 weeks. 15 g per strain was treated by drilling holes of 3 cm straight 2 cm depth and about 5 cm depth at the side of the stock and introducing 5 g of each culture. Therefore, the inoculation amount per strain was 7.5 × 10 ⁹ cfu. F13 to be inoculated on the stem was cultured in the same manner as in Example 1 and adjusted in the same manner as in Example 1 to obtain paste-like small conidia (budding microbial cells). This was inoculated by picking up a standard 25G (straight needle 0.5 mm) needle and piercing the stem between the cotyledon part and the first true leaf at a right angle. The inoculum was 5 × 10 ⁵ / strain.

(4) Test plot (i) Control plot A seedling that has not been inoculated with the attenuated strain of the virus is planted, and 15 g of the F13 culture autoclaved to the strain is mixed 2 days after the planting, and further, with a standard 25 G needle. Inoculated into the stem with distilled water.

(Ii) F13 stem group A seedling inoculated with or without an attenuated strain of the virus was planted, and two days after the planting, the stem was inoculated with non-pathogenic Fusarium fungus F13.

(Iii) F13 strain Moto-ku A seedling inoculated with or without an attenuated strain of virus was planted, and a non-pathogenic Fusarium fungus F13 was mixed with this strain 2 days after planting.

(Iv) Phosthiazeto group A seedling that has been inoculated or not inoculated with an attenuated strain of the virus is planted and treated with 20 kg / 10a of a 1.5% granulose that is conventionally used for nematode control.
The test field was a site where DD fumigant 20 liter / 10a was treated in 2007 and tomatoes were grown.

(5) Evaluation Method After 84 days (August 12, 2008) after planting seedlings in the test plots of the examples, the stems and leaves of the tomatoes in each test plot are cut, and a shovel is placed deeply 20 cm from the center of the strain. A root system having a diameter of 40 cm and a depth of 25 cm was dug together with the soil. The soil was removed from the roots on the spot, and the degree of damage to the roots was determined by visual observation. In addition, using the auger, four soils at a position 20 cm outside the stock were collected and stored in a polyethylene bag.

(I) 0 to 10 levels
According to the method of Zeck (Zeck, WM (1971): Pflanzenschutz-Nachichten. Bayer AG, 24, 141-144.), The degree of root nodule formation was evaluated in 11 stages according to the following class values.
0: No humps are observed.
1: Several small root bumps can be recognized by careful observation.
Several small root bumps similar to 2: 1 can be easily identified.
3: There are many small roots, some of which are fused. The root function is almost intact.
4: There are many small roots and some large roots. Many of the roots are functioning.
5: 25% of the roots are markedly rooted and not functioning.
6: 50% of the roots are markedly rooted and not functioning.
7: 75% of the roots are markedly humped and the ability to regenerate the roots is lost.
8: There are no healthy roots and plant nutrient absorption is inhibited. The foliage is still blue.
9: The root system completely covered by the hump is decaying. The plant is dying.
10: Both plants and roots withered.
(Ii) Nematode second stage larvae The number of nematode second stage larvae per 20 g of soil was counted in the same manner as in Example 1.

(6) Results FIG. 4A and FIG. 4B show the larvae density and the hump class value in each test section, respectively. In FIG. 4A, the nematode larvae density is shown logarithmically. When the non-pathogenic Fusarium fungus was inoculated on the stem, the larval density decreased from the typical nematode phosphiazeate granule at the time of inoculation 2 days later, and it tended to be lower in the seedlings inoculated with tomato mosaic virus (FIG. 4A). ). Although the degree of damage to the roots (root hump class value) varied greatly, the hump class value of stem inoculation and tomato mosaic virus treatment had little variation, and the degree of suppression was stable (FIG. 4B).

  The present invention is widely applicable to plants having parasitic virus disease or Fusarium disease, which are parasitic on root-knot nematodes. According to the present invention, plant diseases caused by root-knot nematodes can be avoided safely and efficiently, so that the present invention is useful in the agricultural field, agrochemical field, and the like.

Claims (7)

  A method for controlling plant diseases caused by nematodes, which comprises inoculating a non-pathogenic filamentous fungus on a plant foliage.   The method of claim 1, wherein the attenuated plant virus is applied to the plant prior to inoculation of the non-pathogenic filamentous fungus into the plant foliage.   The method according to claim 1 or 2, wherein the non-pathogenic filamentous fungus is a non-pathogenic Fusarium fungus.   The method according to any one of claims 1 to 3, wherein the non-pathogenic filamentous fungus is inoculated in the form of a flora disk or a suspension.   The method according to any one of claims 1 to 4, wherein the nematode is a root-knot nematode.   A foliar treatment agent for controlling plant diseases caused by nematodes, comprising a non-pathogenic filamentous fungus as an active ingredient.   The foliar treatment agent according to claim 6, wherein the non-pathogenic filamentous fungus is a non-pathogenic Fusarium bacterium.

Images