JP5455114B2 - Plant disease control agent - Google Patents

Description

  The present invention relates to a phytopathogenic fungus Verticillium lecanii having a pest control effect, a strain having a high plant disease inhibitory effect even after inactivation regardless of the life or death of the fungus, and a plant containing the strain The present invention relates to a disease control agent and a plant disease control method using the same.

  Current pest control of plants is mainly done with chemical insecticides and chemical fungicides using chemical substances. Many of these chemical pesticides have excellent effects and have contributed greatly to the current agricultural production. On the other hand, environmental pollution by chemical pesticides, safety to humans and animals, and a decrease in efficacy due to the emergence of resistant pests and resistant bacteria have also become apparent.

  Against this background, efforts for integrated pest management technology (IPM) that does not rely solely on chemical pesticides have become active. One approach is to control plant pests using microorganisms that have a lower environmental impact than chemical pesticides and that specifically act only on the target pests.

  As pest control methods using microorganisms, methods using entomopathogenic fungi such as Beauveria, Metarhizium, Nomuraea, Verticillium, etc. It has been known. On the other hand, disease control methods using microorganisms include methods using filamentous fungi such as Fusarium, Trichoderma, Bacillus, Pseudomonas, Erwinia, and Erwinia. A method using bacteria such as genus bacteria is known.

  Among these, it is known that Verticillium lecani has different characteristics such as pathogenicity against pests and pest species that can be controlled by fungal strains. Among the strains possessed by the inventors, the strains having high pathogenicity to pests include MG-VL-18 and MG-VL-45, and among these, MG-VL-18 has particularly high pathogenicity to whiteflies. MG-VL-45 is known to show high pathogenicity particularly against aphids (for example, Non-Patent Document 1).

  In addition, Verticillium lecani is actually registered and marketed as an agrochemical. As microbial pesticides containing such Verticillium lecani as active ingredients, there are “Batalek (registered trademark)” and “Mycotal (registered trademark)”. Batalek has a high pathogenicity against aphids, and mycotal is a whitefly. It is a strain that has a strong pathogenicity against the species. Some strains of Verticillium regani are known to be pathogenic to insects such as scale insects and thrips, and mites. Furthermore, it is known that there are strains that parasitize cyst nematodes, root-knot nematodes, phytopathogenic filamentous fungi such as rust fungi and powdery mildews, and have a resistance action (for example, Non-Patent Document 2). ).

  In addition, a plant characterized by containing a conventional Verticillium lecani strain as a pest parasite in terms of pathogenicity against pests on the surface of plants such as leaves. Body surface-fixing microbial pesticides (for example, Patent Document 1) and pest control microbial pesticides characterized by containing, as effective bacteria, a strain of Verticillium regani having the ability of pest control and plant disease control (for example, Patent Document 2) has been proposed.

  In general, the insecticidal effect of Verticillium lecani is that the spores come into contact with the pests, the spores germinate and infest the pests, and then the mycelium grows and fills in the pests, causing the pests to die. It is. Since infestation takes place immediately after germination of the spores, the insects are quickly infected after the bacteria are treated, and the bacteria grow within the insects and exhibit an insecticidal effect. On the other hand, for the manifestation of the plant disease suppressing effect, it is necessary that the cells remain on the plant for a relatively long time. However, Verticillium lecani loses its activity within a relatively short period of time and is killed under environmental conditions unsuitable for survival such as high temperatures and direct sunlight. And it was not known about the presence of the strain which has a plant disease inhibitory effect among dead vicinalium rekani even if it became dead cells.

JP 2003-335612 A JP 2006-169115 A

Journal of Japanese Society of Applied Animal Entomology, Volume 36, p239-245 Agriculture of the month September 2001, p72-77

  The object of the present invention is to suppress plant disease even if it becomes a dead cell in Verticillium lecani that loses its activity within a relatively short time under environmental conditions unsuitable for survival such as high temperature and direct sunlight. It is providing the plant disease control agent which consists of a strain which has an effect, and the plant disease control method using the said plant disease control agent.

  As a result of intensive studies on the entomopathogenic fungus Verticillium lecani strain that is known to exhibit a high control effect against pests in order to solve the above problems, the present inventors have determined that Verticillium lecani The MG-VL-45 strain (NITE P-493) and the Verticillium recani MG-VL-101 strain (NITE P-494) are effective against a wide range of plant diseases even in dead cells that have lost activity. The inventors have found that generation can be suppressed and completed the present invention.

That is, the present invention relates to (1) Verticillium lecani MG-VL-45 strain (NITE P-493) or MG-VL-101 strain (NITE P-494 ) in which the dead spores after heat treatment have the ability to suppress plant diseases. viable cells of) relates viable cells filtrate, dead cells, the plant disease controlling agent characterized by containing at least one of dead cells filtrate, and culture filtrate.

The present invention also relates to ( 2 ) Verticillium regani MG-VL-45 strain (NITE P-493) or MG-VL-101 strain (NITE P-494 ), in which dead spores after heat treatment have the ability to suppress plant diseases. ) Live cell bodies, live cell body filtrates, dead cell bodies, dead cell body filtrates, and culture filtrates are used as plant disease control agents, or ( 3 ) dead spores after heat treatment are plant diseases. Viable cell, viable cell filtrate, dead cell, dead cell of Verticillium lecani MG-VL-45 strain (NITE P-493) or MG-VL-101 strain (NITE P-494) having inhibitory ability The present invention relates to a method of using at least one of a body filtrate and a culture filtrate for the preparation of a plant disease control agent.

The present invention relates to (4) above (1) Symbol placement of plant disease inhibitor, and method for controlling plant diseases, which comprises using at least one of the soil portion including aerial plant parts and plant rhizosphere, ( 5 ) It relates to the Verticillium regani MG-VL-101 strain (NITE P-494) in which the dead spores after the heat treatment have the ability to suppress plant diseases.

  Verticillium regani, which is used in the plant disease control agent of the present invention and whose dead spores after heat treatment have the ability to suppress plant disease, has a control effect on a wide range of plant pests and plant diseases, and is applied to plants. In this case, the live spores attached to the pests quickly infect the pests and eventually die, while the live spores that did not adhere to the pests adhere to the plant body and show a disease suppressing effect. Become. However, the live bacteria of this Verticillium regani cannot survive for a long time on the plant body due to environmental conditions, but it will die, but this fungus is the same as or better than the live cell body even with dead cells. Therefore, even if it is sprayed under an environment condition unsuitable for survival such as daylight in a high temperature atmosphere or direct sunlight, it exhibits a plant disease suppressing activity even if it is killed in a relatively short time. This eliminates restrictions on spraying time zones and environmental conditions in the facility, such as conventional microbial pesticides. In addition, the plant disease control agent of the present invention can control both pests and diseases at the same time, and also has a disease control effect other than the treated part such as the part where the spray liquid is not directly applied, thus reducing agricultural chemicals, labor saving, cost reduction Can be achieved.

As the plant disease control agent of the present invention, the dead spore after the heat treatment has the ability to suppress plant disease, the viable cell body, viable cell filtrate, dead cell body, dead cell filtrate, and culture filtrate. As long as it contains at least one of the above, it is not particularly limited, and Verticillium regani, in which dead spores after heat treatment have a plant disease suppressing ability, can be screened as follows, for example. First, stocks and isolates of Verticillium lecani were left to stand at 27 ° C. for 7 days on a Sabouraud agar medium or potato dextrose agar medium (PDA) containing yeast extract, and then sterilized water was added to produce conidia ( Conidia) is scraped off, the mycelium is filtered through gauze and suspended in sterile water to produce a conidia spore suspension (living spore suspension) adjusted to a concentration of 1 × 10 ⁶ spores per ml. . Next, a dead spore suspension is prepared by heat sterilizing the living spore suspension, for example, at 60 ° C. for 2 hours or at 80 ° C. for 10 minutes. Then, when this dead spore suspension is sprayed on the foliar surface, when the plant diseases such as powdery mildew are suppressed statistically significantly (P <0.05) as compared with the case of no treatment, such a vertici It is determined that the heat-treated dead spores have the ability to suppress plant diseases.

  The present invention also includes at least one of viable cell bodies, viable cell filtrates, dead cell cells, dead cell filtrates, and culture filtrates of Verticillium regani whose dead spores after heat treatment have a plant disease suppressing ability. Also included are a method for use as a plant disease control agent and a method for use in the preparation of a plant disease control agent.

  Examples of the viable cells of Verticillium lecani include viable mycelium and viable spores (conidia, blast spore) of Verticillium lecani, and the live spore suspension can be preferably exemplified. it can. Examples of the viable cell filtrate of Verticillium lecani include a liquid obtained by removing the live spores from the live spore suspension by filtration and centrifugation. Examples of dead cells of Verticillium lecani include dead mycelium and dead spores after heat sterilization, and preferred examples include the heat sterilized dead spore suspension. Examples of the dead cell filtrate of Verticillium lecani include a liquid obtained by removing the dead spores from the dead spore suspension by filtration and centrifugation of the dead spore suspension. Examples of the culture filtrate of Verticillium lecani include a culture filtrate in a Sabouraud liquid medium containing yeast extract and a potato dextrose liquid medium (PDB). In addition, since live spores are required to control insect damage, in the case of microbial pesticides that require both insect damage control effects and plant disease control (suppression) effects, a culture solution containing live spores and A suspension of live spores and a powder containing suspension containing live spores are preferably used, and among these, a powder containing suspension containing live spores excellent in preservability is particularly preferably used. In the case of a plant disease control agent for the purpose of plant disease control only for the purpose of controlling and controlling plant diseases, in addition to the culture solution of Verticillium regani, whose dead spores after heat treatment have the ability to suppress plant diseases, culture Filtrate, viable cell filtrate, dead cell (eg, dead spore suspension), and dead cell filtrate can be advantageously used.

  Specific examples of Verticillium regani having a plant disease-suppressing ability after the above-described heat treatment include Verticillium regani MG-VL-45 (NITE P-493) and Verticillium regani. Examples thereof include MG-VL-101 strain (NITE P-494) and these mutant strains in which dead spores after heat treatment have the ability to suppress plant diseases. The Verticillium regani MG-VL-45 strain is a strain isolated from cotton aphids in an open field field in Natori City, Miyagi Prefecture. The strain was deposited with the number of NITE P-493 at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation on February 21, 2008. In addition, Verticillium regani MG-VL-101 is a novel strain isolated from the potato aphid in an open field in Furukawa City, Miyagi Prefecture. The strain was deposited with the NITE P-494 number at the Patent Microorganism Depositary Center, National Institute of Technology and Evaluation, February 21, 2008. Although the MG-VL-45 strain is a conserved strain, the newly isolated MG-VL-101 strain has a sharp end (from the thick base to the tip) in the middle and tip of the colorless conidia pattern. The phialide (thinly and gradually thinning) diverges in two to three rings, and a spherical spore mass is formed at each tip of the phialide. The conidia is colorless, elliptical to cylindrical, rounded at both ends, and is a single cell. As a result, it was identified as a novel strain of Verticillium lecani.

  In the plant disease suppressant of the present invention, the dead spore after the heat treatment is a viable cell, live cell filtrate, dead cell, dead cell filtrate, culture filtrate, etc. Each can be used alone or in combination, but can also be diluted with an inert liquid or solid carrier and used as a preparation with surfactants and other adjuvants added as necessary. Specific examples of preparations include dosage forms such as granules, powders, wettable powders, suspension preparations, and emulsions. Examples of the carrier include hydrotalcite, kaolinite, montmorillonite, talc, clay, diatomaceous earth, bentonite, white carbon, kaolin, vermiculite, calcium carbonate, potassium chloride, sodium sulfate, calcium sulfate, ammonium sulfate, starch, sucrose, glucose And liquid carriers such as water, isopropyl alcohol, xylene, cyclohexanone, methylnaphthalene, and alkylene glycol. Examples of the surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkyl ester, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl phenyl ester, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, lignin. Examples thereof include sulfonates.

  As an applicable disease of a plant disease inhibitor containing at least one of viable cell, live cell filtrate, dead cell, dead cell filtrate, and culture filtrate of Verticillium lecani of the present invention, The disease is not particularly limited as long as the disease shows a bactericidal effect, and examples thereof include viral diseases, bacterial diseases, and fungal diseases. Specifically, tomato powdery mildew (Oidium lycopersici) ), Eggplant powdery mildew (Erysiphe polygoni), cucumber powdery mildew (Sphaerotheca cucurbitae), strawberry powdery mildew (Sphaerotheca humuli), tomato leaf mold (Fulvia fulva), eggplant subtilis (Mycovellosiella nattrassii) It can be expected to be effective for controlling.

  The plant disease control agent containing viable cells of Verticillium lecani according to the present invention can also be used as a pest control agent when using live bacteria (live spores). Examples of applicable pests include whiteflies, aphids, thrips and the like. Specifically, examples of whiteflies include white spotted whitefly, tobacco whitefly, silver leaf whitefly, citrus whitefly, marrat whitefly, grape Examples of whitefly, camellia whitefly, blue whitefly, Japanese whitefly, linden whitefly, citrus whitefly, azalea whitebird, yamako whitefly, azalea whitefly, strawberry whitefly, etc. The aphid can be exemplified, and examples of the thrips include citrus thrips, southern thrips, chano thrips, and thrips. It is possible.

  The method for controlling plant diseases of the present invention is not particularly limited as long as it is a method using the plant disease control agent of the present invention in at least one of the plant ground part and the soil part containing the plant rhizosphere, As in the case of using ordinary chemical pesticides, it can be appropriately selected depending on the types of pests and plant diseases, the types of applied plants and the like. For example, the plant disease control agent of the present invention may be directly applied or sprayed on plants, or may be mixed, sprayed or irrigated, etc. on soil containing plant rhizosphere (plant cultivation soil). For example, when treating the cultivated soil of the plant using the plant disease control agent of the present invention, the plant may be planted after treating the soil with the plant disease control agent of the present invention, or the plant was planted in the soil. Later, the soil may be treated with the plant disease control agent of the present invention.

When treating at least one of the above-ground part of the plant and the soil part including the plant rhizosphere using the plant disease inhibitor of the present invention, the culture solution or the culture filtrate may be used without diluting. You may dilute and use with the quantity of water etc. When using live spores or dead spores as an active ingredient of the plant disease suppressant of the present invention, the content varies depending on the treatment method, the types of pests and / or plant diseases, the types of applied plants, etc. Although it cannot be defined, for example, when spraying an agent containing live spores or dead spores to the plant above-ground part, the number of spores per 1 mL of the treatment agent is 1 × 10 ³ to 1 × 10 ¹² , preferably The number of spores can be 1 × 10 ⁴ to 1 × 10 ⁹ , and when irrigating the soil, the number of spores is 1 × 10 ² to 1 × 10 ¹¹ , preferably 1 × 10 ⁴ to 1 × 10 ^9. It can be a range. Moreover, the amount of application and the number of times of application can be appropriately selected depending on the type of plant disease, the type of applied plant, the degree of disease, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, the technical scope of this invention is not limited to these illustrations.

[Preparation of spores]
Verticillium regani MG-VL-45 strain and MG-VL-101 strain were each cultivated at 25 ° C. for 7 days in a Sabouraud plate medium containing yeast extract, and then sterilized by adding about 10 ml of sterilized water. The conidia were scraped off with a brush and the mycelium was filtered with gauze and suspended in sterile water to prepare a conidia spore suspension having a spore count of 1 × 10 ⁶ / ml. The live spore suspension was then heat sterilized at 60 ° C. for 2 hours to produce a dead spore suspension with a spore count of 1 × 10 ⁶ / ml.

[Cutting effect test for powdery mildew of cucumber]
Live cells (live spores) and dead cells (dead spores) of Verticillium lecani MG-VL-45 strain, and live cells (live spores) and dead cells (dead) of MG-VL-101 strain Spores) were each adjusted to a concentration of 1 × 10 ⁶ spores per ml and sprayed once on a cucumber at the trilobal stage. Seven days after the application to the first to third leaves, the number of powdery mildew diseased leaves and the number of lesions were investigated, and the rate of diseased leaves and the number of lesions per leaf were calculated. Moreover, the investigation of the 4th leaf which was not developed at the time of spraying was similarly conducted 19 days after the spraying, and the diseased leaf rate and the number of lesions per leaf were calculated. Further, the control value was also calculated by the following formula. In addition, each test was carried out 10 to 12 repetitions with 1 strain per repetition. The results are shown in Table 1. As shown in Table 1, the Verticillium recani MG-VL-45 and MG-VL-101 strains in the present invention have a controlling effect on cucumber powdery mildew in both live and dead cells, The control effect was shown also in the part which those spray liquids do not apply directly.

Control value = {1− (number of lesions per leaf in test area / number of lesions per leaf in untreated area)} × 100

[Testing effect of strawberry on powdery mildew]
The dead cells (dead spores) of the MG-VL-45 strain were adjusted to a concentration of 2 × 10 ⁵ spores per ml, and sprayed four times at intervals of about 10 days on the cultivated strawberries. Six days after the third spraying and 7 days after the fourth spraying, the top 4 compound leaves (12 leaflets) were examined according to the number of powdery mildew diseased leaflets and the criteria in Table 2, and the disease severity and control value were calculated. Moreover, the calculation of the disease severity was obtained by the following formula. The test was carried out 3 times with 20 strains per replicate. The results are shown in Table 3. As shown in Table 3, the dead cells of Verticillium lecani MG-VL-45 strain in the present invention showed a control effect on strawberry powdery mildew.

Disease severity = {Σ (number of diseased leaflets by incidence × attack index) × 100 / (number of leaflets surveyed × 4)}
According to the formula.

[Testing effect on strawberry yellowing]
MG-VL-45 strain dead spores were adjusted to a concentration of 1 × 10 ⁶ spores per ml and sprayed on pot-planted strawberries three times at intervals of about 10 days. Two days after the second spraying, artificial strawberry inoculation with Fusarium oxysporum was performed. After 19 days from the inoculation of the pathogenic bacteria, a survey was conducted according to the criteria shown in Table 4, and the severity of the disease was calculated. One strain / one repetition was carried out and repeated 8 times. The results are shown in Table 5. As shown in Table 5, the dead cells of Verticillium lecani MG-VL-45 strain in the present invention can be applied to the above-ground part of strawberry and can suppress the yellowing disease which is a soil infectious disease. It was.

[Tomato leaf mold, powdery mildew control effect test]
The spore of MG-VL-45 strain was adjusted to a concentration of 2 × 10 ⁶ and 2 × 10 ⁵ spores per ml, and sprayed twice at 7-day intervals on tomatoes grown in the facility. Seven days after the second spraying, the leaflets of leaf mold and powdery mildew were investigated, and the leaflet incidence and control value were calculated. The control value here was determined by the following formula. The test was carried out 3 times with 8-10 strains / repetition. The results are shown in Table 6. As shown in Table 6, the viable cells of Verticillium lecani MG-VL-45 strain in the present invention showed a controlling effect on tomato leaf mold and powdery mildew.

Control value = {1− (rate of diseased leaflets in treated area / rate of diseased leaflets in untreated area)} × 100
The control value was also calculated by the following formula.

[Test of control effect of tomato powdery mildew on irrigation treatment]
The spore of the MG-VL-45 strain was adjusted to a concentration of 2 × 10 ⁵ spores per ml, and was poured into a pot-planted tomato four times at intervals of about 7 days. The investigation was conducted according to the criteria shown in Table 7 for all the 6 to 8 compound leaflets, and the disease severity was calculated. The disease severity was calculated by the following formula. The test was carried out 6 to 9 times with 1 strain / repetition. The results are shown in Table 8. As shown in Table 8, powdery mildew, a terrestrial disease, could be suppressed by irrigation treatment into soil.

Disease severity = {Σ (number of diseased leaflets by incidence × attack index) × 100 / (number of leaflets surveyed × 5)}

[Control effect test of culture filtrate against cucumber powdery mildew]
Suspensions in which live cells (live spores) and dead cells (dead spores) of MG-VL-45 strain were adjusted to a concentration of 2 × 10 ⁷ spores per ml, respectively, and 0.45 μm The viable cell filtrate and the dead cell filtrate filtered through a sterilizing filter were sprayed three times at intervals of 5 to 6 days on the cucumber at the 1-leaf stage. The severity of powdery mildew was examined according to the criteria shown in Table 9 at intervals of 2 to 6 days from 7 days after the first application (immediately before the second application). The degree of disease occurrence was determined by Equation 7. The test was performed at 1 strain / repetition and 9 to 10 repetitions. The results are shown in Table 10. Dispersion of dead cell suspension and dead cell filtrate showed extremely high disease control effect against powdery mildew. Dispersion of viable cell filtrate also showed a high disease suppression effect. Although the viable cell suspension had a disease-inhibiting effect, its persistence was low. The effect showed a tendency to increase by filtration and killed bacteria treatment.

Disease severity = {Σ (number of diseased leaves by degree × morbidity index) × 100 / (number of leaves surveyed × 4)}
According to the formula.

[Preparation of wettable powder]
8 parts by weight of a spore count of 1 × 10 ⁶ / ml of the prepared Verticillium lecani G-VL-101 strain, diatomaceous earth 40 parts by weight, talc 40 parts by weight, bentonite 10 parts by weight poly 1 part by weight of oxyethylene fatty acid ester and 1 part by weight of polyoxyethylene alkyl ether are mixed and dried, and then pulverized to obtain a wettable powder.

[Preparation of granules]
6 parts by weight of a spore suspension of 1 × 10 ⁶ / ml of the prepared Verticillium regani G-VL-101 strain, 1 part by weight of sodium salt of lauryl alcohol sulfate, sodium lignin sulfonate After 1 part by weight, 2 parts by weight of carboxymethylcellulose, 40 parts by weight of diatomaceous earth, and 50 parts by weight of clay are uniformly mixed and pulverized, and the mixture is processed into granules of 14 to 32 mesh using an extrusion granulator, Dry into granules.

  According to the present invention, there is no restriction on the spraying time zone and environmental conditions in the facility like conventional microbial pesticides, and both pests and diseases can be controlled at the same time. It is possible to provide a plant disease control agent that can be expected to have a disease suppressing effect other than the portion.

Claims (5)

Viable cells and viable bacteria of Verticillium lecani MG-VL-45 strain (NITE P-493) or MG-VL-101 strain (NITE P-494) in which dead spores after heat treatment have the ability to suppress plant diseases A plant disease control agent comprising at least one of a body filtrate, dead cell, dead cell filtrate, and culture filtrate. Viable cells and viable bacteria of Verticillium lecani MG-VL-45 strain (NITE P-493) or MG-VL-101 strain (NITE P-494) in which dead spores after heat treatment have the ability to suppress plant diseases A method of using at least one of a body filtrate, dead cell, dead cell filtrate, and culture filtrate as a plant disease control agent. Viable cells and viable bacteria of Verticillium lecani MG-VL-45 strain (NITE P-493) or MG-VL-101 strain (NITE P-494) in which dead spores after heat treatment have the ability to suppress plant diseases A method of using at least one of a body filtrate, dead cell, dead cell filtrate, and culture filtrate for the preparation of a plant disease control agent. The plant disease inhibitor of claim 1 Symbol placement, method for controlling plant diseases, which comprises using at least one of the soil portion including aerial plant parts and plant rhizosphere. Verticillium lecani MG-VL-101 strain (NITE P-494) in which dead spores after heat treatment have plant disease suppression ability.