JP2013158314A - Filamentous fungus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microbe utilizable as a microbial formulation that has excellent control effects on many disease injuries occurring during a raising seedling period of rice plant and many disease injuries occurring in fruits and vegetables, has high safety to men and beasts and a low environmental load.SOLUTION: This filamentous fungus is Penicillium pinophilum RD50109 strain (NITE P1032) belonging to the genus Penicillium.

Description

  The present invention relates to a novel microorganism having an antagonism against many diseases occurring during the seedling raising period of rice and pathogens causing many diseases occurring in fruit trees and vegetables.

  In the cultivation of rice, the period from germination to young seedling, which is the seedling raising period, is likely to be violated by various diseases, and controlling these diseases is one of the important tasks in raising healthy seedlings. In addition, fruit trees and vegetables are susceptible to various diseases throughout the cultivation period, and controlling these diseases is one of the important tasks. Usually, control with chemically synthesized pesticides is performed.

  Chemically synthesized pesticides used to control diseases during the rice seedling season that have been used in the past include triflumizole for rice seedling disease, rice blast disease, and sesame leaf blight that are seed-borne diseases caused by filamentous fungi. Drugs containing DMI agents such as ipconazole, prochloraz, etc. are mainly used. Oxolinic acid etc. are used for rice seed blight bacterial diseases, seedling bacterial diseases, and brown stripe diseases, which are seed-borne diseases caused by bacteria. Contained drugs are mainly used. For rice blight, a soil-borne disease, drugs containing hydroxyisoxazole, benomyl and the like are mainly used. In addition, various chemically synthesized pesticides have been used for controlling diseases of fruit trees and vegetables, including drugs containing DMI agents, methoxyacrylate agents and antibiotics.

  However, in recent years, pathogenic bacteria having reduced sensitivity have emerged against the above-mentioned chemically synthesized pesticides, which is a problem. In addition, due to concerns about food safety and environmental impact, there is a need to reduce the amount of chemical synthetic pesticide used and the number of times it is used. There is a need for the development of technologies and materials that can help reduce this. Specific examples of these technologies and materials include natural products, highly safe substances that can be used as foods and food additives, and agricultural chemicals using living organisms.

  As a disease control technique using living organisms, control using a microbial preparation is known. For example, Patent Document 1 describes a microbial preparation for controlling rice seedling diseases containing Talaromyces spp., And Patent Document 2 describes a microbial preparation for controlling rice seedling diseases containing Pseudomonas spp.

JP 2007-31294 A JP 2002-17343 A

  The present invention has been made from the above viewpoint, and has an antagonistic action against many pathogens of many diseases occurring in the seedling raising period of rice, many pathogens of diseases causing fruit trees and vegetables, It is an object of the present invention to provide a novel microorganism that can be used for controlling these diseases by exerting antagonism with persistence.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that various strains of fungi that cause new diseases of the fungi belonging to the genus Penicillium cause disease in rice seedling raising time, fruit trees, vegetables, etc. On the other hand, it has been found that it has a lasting antagonism, and the present invention has been completed.

  Means for solving the above-mentioned problems are as follows.

(1) A filamentous fungus belonging to the genus Penicillium, Penicillium pinophilum, strain RD50109 (NITE P1032).
(2) Rice blast fungus (Gibberella fujikuroi), Rice blast fungus (Pyricularia oryzae), Rice sesame leaf blight fungus (Cochliobolus miyabeanus), Rice blast fungus (Pseudomonas glumae), Rice seedling blight fungus (Pseudomonas plantarii) , And rice seed-borne pathogens including rice brown streak fungus (Pseudomonas avenae), and rice seedling blight fungus {Fusarium spp., Pythium graminicola, Rhizopus spp. spp.) and at least one of Trichoderma viride. }, The filamentous fungus according to the above (1), which has an antagonistic action against soil-borne pathogens and can be used to control diseases that occur during the rice seedling raising period caused by at least one of these pathogens.
(3) Pear red blight fungus (Gymnosporangium asiaticum), pear black spot fungus (Alternaria kikuchiana), apple spotted leaf blight fungus (Alternaria mali), peach perforated fungus fungus (Xanthomonas campestris pv. Pruni), grape black fungus (Elsinoe ampelina), causing fruit tree disease including citrus scab (Xanthomonas campestris pv. Citri), grapes, oysters, citrus gray scab (Botrytis cinerea), and plum scab (Pseudomonas syringae pv. Morsprunorum) The filamentous fungus according to (1) above, which has an antagonistic action against pathogenic bacteria and can be used for controlling diseases caused by at least one of these pathogenic bacteria.
(4) Cucumber powdery mildew (Sphaerotheca fuliginea), cucumber spotted bacterial fungus (Xanthomonas campestris pv. Lachrymans) tomato leaf mold (Passalora fulva), tomato subtilis fungus (Psedocercospora fuligena), tomato wilt fungus (porumium s. lycopersici), Xanthomonas campestris pv. Ralstonia solanacearum, wheat powdery mildew (Erysiphe graminis), various vegetable gray mold (Botrytis cinerea), various vegetable sclerotia (Sclerotinia sclerotiorum), various vegetable soft rot fungi (Erwinia carotovora) and various bacterial pathogens {Rhizoctonia solani culture type IIIA) and Psium Comprising at least one of (Pythium spp.). }, The filamentous fungus according to the above (1), which has an antagonism against pathogenic bacteria that cause diseases of vegetables, and can be used to control diseases caused by at least one of these pathogenic bacteria.

  According to the present invention, it has an antagonistic action against many pathogenic fungi that occur during the seedling season of rice, and many pathogenic fungi that occur in fruit trees and vegetables, and control of the diseases caused by these pathogenic fungi. An effective filamentous fungus can be provided. In addition, the microorganism preparation containing the filamentous fungus is safe and has little influence on the environment, and has no or very few restrictions on the number of times of use compared to chemically synthesized pesticides.

Hereinafter, the present invention will be described in detail.
The present invention relates to Penicillium pinophyllum belonging to the genus Penicillium, which is a novel microorganism having an antagonistic action against pathogenic bacteria causing disease during the seedling raising period of rice and pathogenic bacteria causing disease during the growing season of fruits and vegetables. penicillium pinophilum) and RD50109 strain (NITE P1032).

(Definition of plant growth stage)
In the present invention, “seedling” refers to a young plant body before planting rice. “Raised seedling” means growing a seedling, and “nurturing time” indicates a cultivation time before planting a seedling. The seedlings at the seedling raising time may indicate the growth time of the seedlings by the number of leaves the seedling has, such as the first leaf stage, the second leaf stage, and the third leaf stage. In general, seedlings at the time of raising seedlings refer to seedlings up to about the fourth leaf stage including incompletely developed leaves, which is a suitable transplanting period in transplantation cultivation of rice.

(About antagonism)
In the present invention, “antagonism” refers to either or both of an effect of reducing the number of cells and an effect of suppressing proliferation of pathogenic bacteria that cause various diseases. This is also called “antibacterial activity”.

(Strain of the present invention)
The filamentous fungus of the present invention is a novel microorganism having the above-described antagonistic action, and more specifically, “penicillium pinophilum / RD50109 strain (NITE P1032)”.

  As a result of BLAST (Altschul et al., 1997) homology search against Apollon DB-FU, which is a DNA database for identifying microorganisms, the ITS-5.8rDNA nucleotide sequence of the filamentous fungus of the present invention is penicillium pinophilum, a kind of Ascomycota It showed a homology of 99.4% with the nucleotide sequence of NRRL6420 strain (Accession No. GQ221867). As a result of homology search against international nucleotide sequence databases such as Genbank / DDBJ / EMBL, the ITS-5.8SrDNA nucleotide sequence of the filamentous fungus of the present invention shows a homology of at least 99.5% with multiple nucleotide sequences of penicillium pinophilum. It was. In the phylogenetic tree created based on the high-order base sequence obtained by homology search against Apollon DB-FU and the international base sequence database, the filamentous fungus of the present invention is the same strain as the reference strain MUCL38548 (GU396556) of penicillium pinophilum Formed a tree. Therefore, from the results of ITS-5.8rDNA nucleotide sequence analysis, the filamentous fungus of the present invention is considered to belong to penicillium pinophilum.

  As a result of observing colony properties and morphology after culturing at 25 ° C. for 7 days in a PDA (potato dextrose agar) medium, the filamentous fungi of the present invention formed ocher to yellow-green, velvety colonies, resulting in a bicyclic penicillus 1-cell fialo-type conidia were formed.

  In addition, as a result of colony properties and morphology observation after culturing at 25 ° C. for 7 days in an MDA (malt extract / dextrose agar) medium, the filamentous fungus of the present invention has the same ocher to yellow-green color as when cultured in a PDA medium. A velvety colony was formed, and a 1-cell fiaro-type conidia was formed in a bicyclic penicillus.

From these results, it was considered that the filamentous fungus of the present invention is similar to the characteristics of penicillium pinophilum (Pitt, 2000) whose assignment is estimated from ITS-5.8 rDNA nucleotide sequence analysis.
From the above results of ITS-5.8rDNA base sequence analysis and simple morphology observation, the filamentous fungus of the present invention is presumed to be penicillium pinophilum.

  The filamentous fungus of the present invention was isolated from domestic soil and presumed to belong to penicillium pinophilum, but as described later, diseases occurring in the seedling raising season of rice and diseases occurring in fruit trees and vegetables On the other hand, it can be clearly distinguished from conventionally known strains in that it has a very excellent control effect.

  Penicillium pinophilum strain RD50109 was newly isolated from domestic soil and was deposited as NITE P1032 on January 13, 2011 at the National Institute of Technology and Evaluation Patent Microorganism Depositary. ing.

  When the filamentous fungus of the present invention is used as a microbial preparation, it is preferable to use a culture obtained by culturing a microbial cell in a medium in which the microbial cell can grow, as in the case of using a microorganism for a normal microbial pesticide. More preferably, a culture containing the filamentous fungus in which spores are sufficiently formed by culture is used.

  The cultivation of the filamentous fungus of the present invention can be carried out in the same manner as the usual filamentous fungus cultivation method, and can be carried out, for example, by ordinary liquid culture or solid culture, but to increase the collection efficiency of spores. It is preferable to perform solid culture. Specifically, in the liquid culture, for example, a microbial culture of the filamentous fungus can be obtained by culturing at 15 ° C. to 35 ° C. for 3 to 20 days using a medium such as a potato dextrose medium or a Sabouraud medium. In solid culture, cereals such as rice, wheat, corn, and soybeans, solid components derived from cereals such as bran and soy bean, and solid carriers such as clay minerals containing nutrient sources are used as needed. A medium containing a source or the like can be used. Similarly to liquid culture, a cell culture can be obtained by culturing at 15 ° C. to 35 ° C. for 3 to 20 days.

  About the culture | cultivation conditions of the filamentous fungus of this invention, it is preferable to carry out on aerobic conditions by methods, such as aeration, stirring, and shaking, and culture | cultivation temperature has preferable 15 to 35 degreeC. The culture period is preferably 3 to 20 days. In addition, as for the fungal body of the filamentous fungus of this invention, it is desirable that it is a spore from a viewpoint of the preservability as an agrochemical formulation for disease control. Therefore, in order to spore the filamentous fungus, if necessary, it is desirable to adjust the conditions such as the composition of the medium, the pH of the medium, the culture temperature, the culture humidity, and the oxygen concentration so as to match the spore formation conditions. .

  The thus obtained filamentous fungus culture of the present invention can be used as it is, but after pulverizing or breaking the culture as necessary, or from the culture by centrifuging, etc. It can be used for a microbial preparation after separating the cells or drying the cells mainly composed of culture and spores.

The microorganism preparation using the filamentous fungus of the present invention is usually a culture containing 1 × 10 ⁶ to 1 × 10 ¹² cfu (colony forming units) per 1 g of the filamentous fungus of the present invention or a pulverized product of the culture. Is blended into various types of preparations suitable for the purpose and usage.

  The dosage form of the microbial preparation using the filamentous fungus of the present invention is not particularly limited, and is the same dosage form as that of a normal microbial pesticide, for example, a wettable powder, a flowable agent, an emulsion, a powder, a granule, a soil mixture, and the like. be able to.

  The production of the microbial preparation containing the filamentous fungus of the present invention can be carried out in the same manner as a normal microbial pesticide. For example, the microbial preparation can be produced by blending the filamentous fungus of the present invention with a carrier. . As the carrier, inorganic or organic materials that are usually used for microbial pesticides can be used as the main material. Specifically, as the solid carrier, red ball soil, calcined red ball soil, Kanuma soil, black soil , Acid clay, talc, clay, carillon clay, pyroferrite clay, bentonite, montmorillonite, diatomaceous earth, synthetic hydrous silicon oxide, vermiculite, perlite, zeolite, attapulgite, Otani stone, anthracite, coal ash, lime, salt, carbonate , Inorganic materials such as sulfate, nitrate, urea, rice husk, bran, wheat flour, corn cobs, peanut husk, peat moss, pulp, straw, bacus, wood flour, rice bran, rice bran powder, oil cake, starch, fish cake, bone meal , Dry manure, crab, shrimp, krill fine powder, charcoal, kun charcoal, bark charcoal, rice husk charcoal, grass charcoal, active , Can be used ash, fossil shells, D- sorbitol, lactose, maltitose, glucosamine, the organic materials such as oligosaccharides. As the liquid carrier, water, vegetable oil, animal oil, mineral oil, synthetic water-soluble polymer and the like can be used. These components can be used alone or as a mixture of two or more.

  In addition, microbial preparations include natural polysaccharides such as casein, gelatin, gum arabic, alginic acid, celluloses, carboxymethylcellulose, xanthan gum, chitins, chitosans, polyvinyl alcohols, polyacrylic acids, bentonite, etc. as adjuvants. For the purpose of viscosity, fixation, dispersion, etc., it can be contained as required.

  In addition, the microbial preparation can contain dihydric alcohols such as ethylene glycol and propylene glycol as needed for the purpose of preventing freezing.

  Microbial preparations can contain anionic, cationic, and amphoteric surfactants as needed for the purposes of dispersion stability, aggregation prevention, emulsification, and the like.

  The microorganism preparation containing the filamentous fungus of the present invention obtained as described above can be applied to rice seeds, seedlings, seedling soil, seedling culture media, fruit trees, vegetables and other crops. It is appropriately selected depending on the use form and disease. Specifically, seed soaking treatment, seed dressing treatment, seed application treatment, seed spraying treatment, seed spraying treatment, soil spraying treatment, soil spraying treatment, soil admixing treatment, soil irrigation treatment, plant base application, ground part liquid spraying , Methods such as above-ground solid dispersion.

  In addition, when applying the microorganism preparation containing the filamentous fungus of the present invention, at least one of fungicides, insecticides, nematicides, acaricides, herbicides, plant growth regulators, fertilizers, soil improvement materials, etc. It is also possible to apply these in a mixed manner or alternately without mixing, or simultaneously.

The application amount of the microbial preparation containing the filamentous fungus of the present invention cannot be defined unconditionally depending on the type of disease or the like. For example, in the case of seed immersing treatment, the microbial preparation is 10 to 1000 times (mass) as a seed immersing solution. It is preferable to dilute with water or the like, and the bacterial cell concentration is preferably in the range of usually 1 × 10 ³ to 1 × 10 ¹⁰ cfu per ml of immersion liquid.

In the case of seed dressing treatment of the microbial preparation containing the filamentous fungus of the present invention, it is preferable to apply 1 to 20 mass% of the microbial preparation with respect to the seed mass, and the bacterial cell concentration is usually 1 per 1 g of the seed mass. It is preferably in the range of × 10 ³ to 1 × 10 ¹⁰ cfu.

When applying the microbial preparation containing the filamentous fungus of the present invention to soil spraying or soil irrigation, it is preferable to apply 50 to 1000 ml per nursery box (area of about 1800 cm ² ) usually used for seedling transplanting, The bacterial cell concentration is preferably in the range of usually 1 × 10 ³ to 1 × 10 ¹⁰ cfu per 1 ml of sprayed or irrigated liquid.

When the microorganism preparation containing the filamentous fungus of the present invention is mixed with soil, it is preferably applied in an amount of 0.1 to 100 g per nursery box (area of about 1800 cm ² ) usually used for seedling transplanting. The body concentration is preferably in the range of 1 × 10 ² to 1 × 10 ⁹ cfu per ml of soil.

When the microbial preparation containing the filamentous fungus of the present invention is sprayed on the foliage, the amount of sprayed liquid is preferably adjusted between 100 ml to 300 L per 10 ares according to the type of plant, and the bacterial cell concentration is The range is preferably 1 × 10 ³ to 1 × 10 ¹⁰ cfu per 1 ml of spray solution.

  The number of times of use of the microorganism preparation containing the filamentous fungus of the present invention is not particularly limited. For example, 1 to 1 can be used between the seed sowing time and the rice seedling raising time or between the plant growing time and the harvesting time. By using it five times, it is possible to control the disease.

  The microbial preparation containing the filamentous fungus of the present invention obtained as described above can be used, for example, to control diseases that occur at the time of rice seedling raising and diseases that occur in fruit trees and vegetables.

  The pathogenic bacteria that cause diseases at the seedling raising season include rice blast fungus (Gibberella fujikuroi), rice blast fungus (Pyricularia oryzae), rice sesame leaf blight fungus (Cochliobolus miyabeanus), rice blast fungus (Pseudomonas glumae), rice Infectious pathogens of rice seeds such as Pseudomonas plantarii and Pseudomonas avenae, Fusarium spp., Pythium (Pythium) graminicola), Rhizopus spp., Trichoderma viride and the like. } And other soil infectious pathogens.

  The pathogens that cause fruit tree diseases include pear scab (Gymnosporangium asiaticum), pear black spot fungus (Alternaria kikuchiana), apple spotted leaf rot fungus (Alternaria mali), and peach perforated fungus (Xanthomonas campestris pv. Pruni) , Grape black fungus (Elsinoe ampelina), citrus scab (Xanthomonas campestris pv. Citri), grape, oyster, citrus gray scab (Botrytis cinerea), and plum scab (Pseudomonas syringae pv. Morsprunorum) Is mentioned.

  The pathogens that cause vegetable diseases include Sphaerotheca fuliginea, Xanthomonas campestris pv. Lachrymans, Tomato leaf fungus (Passalora fulva), Tomato subtilis fungus (Psedocercospora fuligena), Tomato Fungus (Fusarium oxysporum f.sp.lycopersici), Pepper spot bacterial fungus (Xanthomonas campestris pv.vesicatoria), Strawberry anthracnose fungus (Colletotrichum fragariae), Brown rot fungus (Pseudomonas syringae pv. solani culture type IV), solanaceae vegetables Ralstonia solanacearum, wheat powdery mildew (Erysiphe graminis), various vegetables, gray mold (Botrytis cinerea), various vegetables, sclerotia (Sclerotinia sclerotiorum) , Soft rot fungus of various vegetables (Erwinia carotovora), and pathogenic fungus of various bacterial diseases {Rhizoctoni genus (Rhizoctoni a solani culture type IIIA) and Pythium spp. } Etc. are mentioned.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited only to these examples.

<Example 1 Rice Bone Seedling Control Test>
(1) Preparation of Infected Seeds Rice (variety: Koshihikari) seeds were harvested from paddy rice with frequent shoot seedling diseases in Honda and used as seeds for shoot seedling infection.

(2) Preparation of the drug Penicillium pinophilum / RD50109 strain was inoculated into a bran medium (mixed at a volume ratio of bran: distilled water = 2: 1 and then autoclaved at 120 ° C. for 20 minutes). The cells were cultured in a room at 25 ° C. for 14 days. After completion of the culture, distilled water containing 0.1% (volume) of surfactant tween20 was added to the medium in an equal volume (volume), stirred vigorously, and filtered through gauze (single) to remove the medium residue. A stock solution (about 1 × 10 ¹¹ cfu per ml) of a spore suspension of the strain was obtained. This was diluted with distilled water containing 0.1% (volume) of the surfactant tween20 to obtain a spore suspension containing 1 × 10 ⁷ cfu of spore per ml and used as a drug. .

(3) Chemical treatment Rice seedlings were seeded with a bath ratio of cocoon: distilled water = 1: 2 (volume ratio) and allowed to stand at 15 ° C. for 7 days. The water was changed on the third day. After completion of the soaking, the above chemical was used and the bath ratio was soaked to be 籾: drug = 1: 2 (volume ratio), and the chemical treatment was performed by germination treatment at 30 ° C. for 24 hours. . At the same time, the soot after completion of soaking was soaked so that the bath ratio was soot: distilled water = 1: 2 (volume ratio), and sprouting was performed at 30 ° C. for 24 hours, which was set as an untreated section.

(4) Growth of test plant After the sprouting treatment, the seed pods were air-dried and sown in a 16.5 cm x 11 cm plastic pot filled with paddy rice seedling culture soil. Two repetitions were made for both the drug-treated group and the non-treated group. After sowing, the seedling was covered with a paddy rice seedling culture medium, and then cultivated and managed in a glass greenhouse according to a regular method.

(5) Control effect investigation Two weeks after sowing, the seedlings expressing extreme symptom symptoms caused by stupid seedling disease were used as diseased seedlings. The diseased seedling rate was calculated based on (Formula 1). Based on the diseased seedling rate, the control value of the chemical treatment area was calculated based on the following (formula 2), and evaluated as the control effect.

<Formula 1> Disease seedling rate (%) = number of diseased seedlings / total number of surveyed seedlings × 100
<Formula 2> Control value = {(Non-treatment district diseased seedling rate-Drug treatment zone diseased seedling rate) / Non-treatment zone diseased seedling rate} × 100

(6) Test results The survey results are shown in Table 1. As is apparent from Table 1, the treatment value of Penicillium pinophilum / RD50109 strain was 100, indicating a high control effect against rice seedling diseases.

Example 2 Rice Seedling Bacterial Disease Control Test
(1) Preparation of Infected Seeds Rice (variety: Koshihikari) seeds were put into a suspension containing a rice seedling bacteriomycetes and forcibly inoculated into the seeds under reduced pressure. After air drying, this seed pod was used as a seedling infected with rice seedlings.

(2) Chemical Treatment Rice seedling-infected bacterial disease-infected seeds were soaked so that the bath ratio was 籾: distilled water = 1: 2 (volume ratio), and allowed to stand at 15 ° C. for 7 days. The water was changed on the third day. After completion of the soaking, the medicine prepared in the same manner as above was used, and the bath ratio was 籾: drug = 1: 2 (volume ratio), and the sprouting treatment was performed at 30 ° C. for 24 hours. Was treated. At the same time, the soot after completion of soaking was soaked so that the bath ratio was soot: distilled water = 1: 2 (volume ratio), and sprouting was performed at 30 ° C. for 24 hours, which was set as an untreated section.

(3) Growth of test plant After the above sprouting treatment, the seed pods were air-dried and sown in a 9 cm × 9 cm plastic pot filled with paddy rice seedling culture soil. Two repetitions were made for both the drug-treated group and the non-treated group. After sowing, the seedling was covered with a paddy rice seedling culture medium, and then cultivated and managed in a glass greenhouse according to a regular method.

(4) Pest control effect investigation Two weeks after sowing, the seedlings with the disease symptoms of seedling bacterial disease such as rot, wilting, and whitening of the seedlings are used as diseased seedlings, and the number of diseased seedlings in two treatments in each treatment area Using the average value, the diseased seedling rate was calculated based on the above (Formula 1). Based on the above-mentioned diseased seedling rate, the control value of the chemical treatment section was calculated based on the above (Formula 2), and evaluated as the control effect.

(5) Test results The survey results are shown in Table 2. As is clear from Table 2, the control value of Penicillium pinophilum / RD50109 strain was 100, indicating a high effect against rice seedling blight.

<Example 3 rice brown streak disease effect test result>
(1) Preparation of infected seeds Rice (variety: Koshihikari) seeds were put into a suspension containing rice brown streak fungus and forcibly inoculated into the seeds under reduced pressure. After air drying, this seed pod was used as a rice brown streak infected seed.

(2) Chemical treatment Rice brown stripe disease infected seeds were soaked so that the bath ratio would be cocoon: distilled water = 1: 2 (volume ratio), and allowed to stand at 15 ° C. for 7 days. The water was changed on the third day. After completion of the soaking, the medicine prepared in the same manner as above was used, and the bath ratio was 籾: drug = 1: 2 (volume ratio), and the sprouting treatment was performed at 30 ° C. for 24 hours. Was treated. At the same time, the soot after completion of soaking was soaked so that the bath ratio was soot: distilled water = 1: 2 (volume ratio), and sprouting was performed at 30 ° C. for 24 hours, which was set as an untreated section.

(3) Growth of test plant After the above sprouting treatment, the seed pods were air-dried and sown in a 9 cm × 9 cm plastic pot filled with paddy rice seedling culture soil. Two repetitions were made for both the drug-treated group and the non-treated group. After sowing, the seedling was covered with a paddy rice seedling culture medium, and then cultivated and managed in a glass greenhouse according to a regular method.

(4) Investigation of control effect Two weeks after sowing, seedlings that develop brown streak symptoms such as curvature, poor growth, and death with brown water-immersed streaks appear on the seedlings. Based on the above (Equation 1), the diseased seedling rate was calculated using the average value of the number of diseased seedlings of 2 repetitions. Based on the above-mentioned diseased seedling rate, the control value of the chemical treatment section was calculated based on the above (Formula 2), and evaluated as the control effect.

(5) Test results The survey results are shown in Table 3. As is apparent from Table 3, the treatment with penicillium pinophilum and RD50109 strain showed a control value of 80, showing a high effect on rice brown stripe disease.

<Example 4 Rice Bacterial Bacterial Disease Effect Test>
(1) Preparation of Infected Seeds Rice (variety: Koshihikari) seeds were put into a suspension containing rice blast fungus and were forcibly inoculated into the seeds under reduced pressure. After air-drying, this seed pod was used as a rice wilt bacterial seed.

(2) Chemical Treatment Rice seedlings infected with rice blast blight were soaked so that the bath ratio was 籾: distilled water = 1: 2 (volume ratio) and allowed to stand at 15 ° C. for 7 days. The water was changed on the third day. After completion of the soaking, the medicine prepared in the same manner as above was used, and the bath ratio was 籾: drug = 1: 2 (volume ratio), and the sprouting treatment was performed at 30 ° C. for 24 hours. Was treated. At the same time, the soot after completion of soaking was soaked so that the bath ratio was soot: distilled water = 1: 2 (volume ratio), and sprouting was performed at 30 ° C. for 24 hours, which was set as an untreated section.

(3) Growth of test plant After the above sprouting treatment, the seed pods were air-dried and sown in a 9 cm × 9 cm plastic pot filled with paddy rice seedling culture soil. Two repetitions were made for both the drug-treated group and the non-treated group. After sowing, the seedling was covered with a paddy rice seedling culture medium, and then cultivated and managed in a glass greenhouse according to a regular method.

(4) Pest control effect investigation Two weeks after sowing, the average number of diseased seedlings of two repeated treatments in each treatment area was treated with the seedlings with the symptoms of bacterial wilt disease such as spoilage, wilting and whitening of the seedlings. Based on the above (Formula 1), the diseased seedling rate was calculated using the value. Based on the above-mentioned diseased seedling rate, the control value of the chemical treatment section was calculated based on the above (Formula 2), and evaluated as the control effect.

(5) Test results The survey results are shown in Table 4. As is apparent from Table 4, the treatment value of Penicillium pinophilum / RD50109 strain was control value = 93, showing a high effect on rice blast blight.

<Example 5-Antibacterial activity test against pathogenic filamentous fungi that can be cultured in an artificial medium>
Among plant pathogenic filamentous fungi, those that can be cultured in an artificial medium can be confirmed for antibacterial activity by an anti-culture method. It can be inferred that microbial strains with antibacterial activity can be used for disease control. Hereinafter, the test results of the microorganism strain of the present invention by the anti-culture method will be described.

(1) Test method A high-pressure, high-temperature sterilized PDA medium was poured into a sterilized plastic petri dish and solidified, and used as a basic medium. Penicillium pinophilum / RD50109 strain was precultured in the basic medium as a test bacterium, and after confirming that the diameter of the flora grew to about 2 cm, each pathogen was separated from the periphery of the flora by about 1 cm. Transplanted to different positions.
The pathogenic fungi evaluated were the pathogens of rice seedling blight (Fusarium spp., Pythium graminicola, Rhizopus spp., Trichoderma viride), Rice sesame leaf blight fungus (Cochliobolus miyabeanus), rice blast fungus (Pyricularia oryzae), grape, oyster, citrus, vegetable gray mold fungus (Botrytis cinerea), vegetable sclerotia fungus (Sclerotinia sclerotiorum), apple leaf spot Alternaria mali, tomato leaf mold (Passalora fulva), tomato subtilis fungus (Psedocercospora fuligena), tomato wilt fungus (Fusarium oxysporum f.sp.lycopersici), strawberry anthracnose fungus (Colletotrichum fragariae), potato black wilt (Rhizoctonia solani culture type IV), a pathogen of bacterial wilt of vegetables {Rhizoctonia solani culture type IIIA, Pythium spp.}

(2) Evaluation method of antibacterial activity By transplanting each pathogenic bacterium and comparing the degree of growth of the pathogenic bacterium after 7 to 10 days with an untreated group (a group in which only the pathogenic bacterium is cultured), macroscopically visually according to the following criteria: evaluated.

+: The diameter of the flora of pathogenic bacteria is about 10% or less of the untreated area (has strong activity)
±: The diameter of the bacterial flora of the pathogen is about 10% to 30% of the untreated area (has weak activity)
-: The diameter of the flora of pathogenic bacteria is about 30% or more of the untreated area (no activity or almost no activity)

(3) Test results The survey results are shown in Table 5. As is apparent from Table 5, the Penicillium pinophilum RD50109 strain was shown to have a strong antibacterial activity against any of the above pathogenic bacteria. Therefore, it is inferred that Penicillium pinophilum / RD50109 strain can be used for controlling diseases caused by these diseases.

<Example 6-Antibacterial activity test against pathogenic bacteria that can be cultured in an artificial medium>
Since phytopathogenic bacteria can be cultured in an artificial medium, antibacterial activity can be confirmed by an anti-culturing method. It can be inferred that microbial strains with antibacterial activity can be used for disease control. Hereinafter, the test results of the microorganism strain of the present invention by the anti-culture method will be described.

(1) Test method A high-pressure and high-temperature sterilized PDA medium (1% by weight of peptone added) was poured into a sterilized plastic petri dish and solidified, and used as a basic medium. Penicillium pinophilum / RD50109 strain was precultured in the basic medium as a test bacterium, and after confirming that the diameter of the flora grew to about 2 cm, each pathogenic bacterium was about 1 cm from the periphery of the flora. The streak was transplanted at a remote location. The pathogenic bacteria evaluated were peach perforated bacteria (Xanthomonas campestris pv. Pruni), citrus scab (Xanthomonas campestris pv. Citri), plum scab (Pseudomonas syringae pv. Morsprunorum), cucumber spot bacterial bacterium (Pseudomonas syringae) lachrymans), Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. ).
(2) Evaluation method of antibacterial activity By transplanting each pathogenic bacterium and comparing the degree of growth of the pathogenic bacterium after 7 to 10 days with the untreated group (the group in which only the pathogenic bacterium is cultured), it is visually observed according to the following criteria Macroscopically evaluated.

+: The degree of growth of colonies of pathogenic bacteria is about 10% or less of the untreated group (has strong activity)
±: The degree of growth of colonies of pathogenic bacteria is about 10% to 30% of the untreated group (has weak activity)
-: About 30% or more of the colony of pathogenic bacteria in the untreated area (no activity or almost no activity)
(3) Test results The survey results are shown in Table 6. As is apparent from Table 6, Penicillium pinophilum RD50109 strain was shown to have strong antibacterial activity against any of the above pathogenic bacteria. Therefore, it is inferred that Penicillium pinophilum / RD50109 strain can be used for controlling diseases caused by these diseases.

<Example 7: Pear Red Star Disease Control Test>
(1) Test method The test was carried out in a field (open field) where an 18-year-old pear tree (variety: Hosui) where red star disease naturally occurs was planted. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 5.0 × 10 ⁷ cfu of spore per ml, and this was used as a reagent. As a control drug, thionoc flowable (active ingredient thiuram = 40%) was diluted 500 times with tap water and used.
Three trees were used per ward, and 3 liters per tree were sprayed three times at intervals of 4 days using a power-type sprayer. 1 round of spraying was set to be before about 10 days from the first generation of gymnosporangium.

(2) Survey of control effect Survey was conducted 4 days and 15 days after the final spraying. For each 100 leaves (total 300 leaves / district), the number of lesions per leaf was investigated, the disease severity was evaluated based on the following disease index, and the disease severity and control value were calculated from the following equations. .

Disease index 0: No disease 1: 1 to 3 lesions 2: 4 to 10 lesions 3: 11 to 15 lesions 4: 16 or more lesions onset = Σ Number x index) x 100 / (number of surveys x 4)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results The survey results are shown in Table 7. As is clear from Table 7, the drug-treated section of penicillium pinophilum / RD50109 strain has a control value of 98.7, which is more effective than thionoch flowable of the control drug. It was revealed that it has a high control effect.

<Example 8-Grape black scab control test>
(1) Test method The test was carried out using a pot (diameter: 25 cm) in which a 5-year-old tree (variety: Kyoho) of grapes in which black pepper naturally occurs was planted. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 1.0 × 10 ⁷ cfu of spores per ml, and this was used as a reagent. As a control drug, Dimandaisen Flowable (active ingredient Manzeb = 20%) was diluted 800 times with tap water and used.
Three trees were used per ward, and 3 liters per tree were sprayed four times at intervals of 7 days using a back-up power sprayer. The first spraying was set to be about 3-4 weeks before the first occurrence of black depression.

(2) Investigation of control effect An investigation was conducted 16 days after the final spraying. For each 100 leaves (total 300 leaves / district), the number of lesions per leaf was investigated, the disease severity was evaluated based on the following disease index, and the disease severity and control value were calculated from the following equations. .

Disease index 0: No disease 1: 1 to 10 lesions 3: 11 to 30 lesions 4: 31 or more lesions onset = Σ (number of leaves according to disease severity x index) x 100 / (investigation) Number x 4)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results Table 8 shows the survey results. As is clear from Table 8, the drug-treated area of penicillium pinophilum and RD50109 strain had a control value of 65.4, which was slightly inferior to the control Zymandene flowable, but against grape black depression It was revealed that it has a controlling effect.

<Example 9: Tomato bacterial wilt control test>
(1) Test method It implemented using the tomato (variety: House Momotaro) of about 15 cm of plant height. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 1.0 × 10 ⁷ cfu of spores per ml, and this was used as a reagent. As a control drug, mycoshield (active ingredient oxytetracycline quaternary ammonium salt = 31.5%) was diluted 500 times with tap water and used.
The drug-treated area used 11 shares per ward, and the untreated area used 15 shares. In the drug-treated section of penicillium pinophilum • RD50109 strain, the tomato roots washed with water after removing the soil were immersed in the drug for 24 hours at 25 ° C. In the control drug-treated section, the drug was immersed in the drug for 10 minutes at 25 ° C. After the chemical treatment, each tomato seedling was transplanted into a No. 6 pot containing red soil.
A cell suspension of bacterial wilt (Ralstonia solanacearum) was prepared so as to be 1.0 × 10 ⁷ cfu or more per ml, and this was inoculated by irrigating 50 ml in each pot. After inoculation, it was put in a 20 ° C. incubator and cultivated.

(2) Control Effect Investigation Seven days after the inoculation, the ground part was cut and the degree of browning of the conduit part and the level of wilting of the above-ground part were observed and evaluated with the naked eye according to the following criteria.

0: No or almost no symptoms 1: Conduit browning and wilting degree 30% or less of uninoculated area 2: Condense browning and wilting degree 30% or more of non-inoculated area Number of leaves by degree of disease x index) x 100 / (number of surveys x 2)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results Table 9 shows the survey results. As is clear from Table 9, the drug-treated section of Penicillium pinophilum / RD50109 strain has a control value of 68, showing an effect superior to that of the control drug, Mycoshield, against bacterial wilt of solanaceous vegetables. On the other hand, it became clear that it has a control effect.

<Example 10: Cucumber powdery mildew control test>
(1) Test method It implemented using what cultivated the cucumber (variety: Sharp 301) in which about 15 to 20 leaves of main leaves were cultivated in a Wagner pot of 1 / 5,000 are. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 2.0 × 10 ⁶ cfu of spores per ml, and this was used as a reagent. As control drugs, Botokiller wettable powder (active ingredient Bacillus subtilis spore = 1.0 × 10 ¹¹ cfu / g) diluted 1000 times and Berkut flowable (active ingredient iminoctadine albecylate = 30.0%) 2000 Double dilution was used.
The drug-treated area used 2 shares per area, and the non-treated area used 3 shares. In all the treatment sections, a sufficient amount of the drug dripped or dropped using a sprayer was sprayed twice at 7-day intervals before the onset of powdery mildew. During the test period, the plants were cultivated in a glass house at 15 ° C to 30 ° C. Disease onset was a naturally occurring condition.

(2) Control effect investigation Seven days after the second spraying, the degree of symptom appearing on the upper leaves from the lower to fifth leaves was observed and evaluated with the naked eye according to the following criteria.

0: No or almost no symptom 1: The area of the symptom is about 10% or less of the entire leaf surface 2: The area of the symptom is about 10% to 30% of the entire leaf surface 3: The area of the symptom is the leaf surface About 30% or more of the total disease severity = Σ (number of leaves by degree of disease x index) x 100 / (number of leaves surveyed x 3)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results Table 10 shows the survey results. As is apparent from Table 10, the drug-treated section of Penicillium pinophilum / RD50109 strain has a control value = 66, which is inferior to the chemical synthesis agent Berkut flowable (control value = 100), but microorganisms It showed an effect superior to that of the botulizer wettable powder (control value = 44), and has a control effect against cucumber powdery mildew.

<Example 11: Strawberry anthracnose control test>
(1) Test method It carried out using the strawberry (variety: Toyooka) which the main leaf developed 10 leaves (double leaf) or more and grew sufficiently.
In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 2.0 × 10 ⁶ cfu of spores per ml, and this was used as a reagent. As a control agent, a quinone dough flowable (active ingredient 8-hydroxyquinoline copper = 35.0%) diluted 500 times was used.
A sufficient amount of the drug was sprayed on one strain per ward using a sprayer. After air-drying, the leaves were cut into 3 leaves (double leaves), placed on a vat with moistened kitchen paper, and the leaves were scratched with a needle. The periphery of the flora of strawberry anthracnose fungus (Colletotrichum fragariae) previously cultured on PDA medium was punched with a 6 mm diameter cork borer, and this section was inoculated by placing it on a wound on a leaf. The vat was sealed so as not to dry, and cultured in a room at 25 ° C. for 4 days.

(2) Control Effect Survey Four days after the inoculation, the degree of leaf symptom was observed and evaluated with the naked eye according to the following criteria.

0: No or almost no symptom 1: Progress of symptom is less than 2 mm from the periphery of the slice of the inoculation source 2: Progress of symptom is about 2 mm or more from the periphery of the slice of the inoculation source

Disease severity = Σ (number of leaves according to disease severity x index) x 100 / (number of surveys x 2)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results Table 11 shows the survey results. As is clear from Table 11, the drug-treated section of Penicillium pinophilum strain RD50109 has a control value of 94, showing an effect superior to that of the control drug, quinone do flowable, and having a control effect on this disease. It became clear.

<Example 12: Tomato leaf mold control test>
(1) Test method It implemented using the tomato (variety: House Momotaro) of about 15 cm of plant height. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 1.0 × 10 ⁷ cfu of spores per ml, and this was used as a reagent. As a control drug, Berkut flowable (active ingredient iminoctadine albecylate = 30.0%) was diluted 2000 times with tap water and used.
A sufficient amount of the drug was sprayed on 5 strains per ward using a sprayer and air-dried. Distilled water was poured into a flora of tomato leaf mold (passalora fulva) previously cultured on PDA medium, and the surface was rubbed with a brush to obtain a spore suspension. A spore suspension of a pathogenic bacterium prepared to 1.0 × 10 ⁶ cfu / ml was spray-inoculated using a sprayer, then left in a room at 25 ° C. maintained at a humidity of 90% or more and cultivated for 14 days.

(2) Investigation of control effect 14 days after the inoculation, the degree of disease symptoms appearing on the upper leaves (leaves developed at the time of inoculation) from the lower third to the third leaves was visually observed and evaluated according to the following criteria. Newly developed leaves after inoculation were not considered.

0: No or almost no symptom 1: The area of the symptom is about 10% or less of the entire leaf surface 2: The area of the symptom is about 10% to 30% of the entire leaf surface 3: The area of the symptom is the leaf surface About 30% or more of the total disease severity = Σ (number of leaves by degree of disease x index) x 100 / (number of leaves surveyed x 3)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results Table 12 shows the survey results. As is clear from Table 12, the drug-treated section of Penicillium pinophilum / RD50109 strain has a control value of 68, showing the same effect as the control drug Berkut flowable, and having a control effect against this disease. It became clear.

<Test Example 13: wheat powdery mildew control test>
(1) Test method It implemented using the wheat (variety: Norin 61) by which it sown in the field and the leaf fully developed. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 0.5 × 10 ⁷ cfu of spore per ml, and this was used as a reagent. As a control drug, cumulus (active ingredient: sulfur = 79.2%) was diluted 500 times with tap water and used.
A sufficient amount of the drug was sprayed twice with a sprayer at 6-day intervals on wheat development leaves in the range of 1.5 m per ward in a cocoon with a width of about 1 m. Three repetitions were made per ward. Disease onset was a naturally occurring condition.

(2) Control effect investigation 14 days after the second spraying, the disease area occupied on the foliage for each wheat strain was indexed objectively to calculate the disease severity. Ten strains (× 3 repetitions) were investigated per ward.

0: No or almost no symptom 1: The area of the symptom is about 10% or less of the entire leaf surface 2: The area of the symptom is about 10% to 30% of the entire leaf surface 3: The area of the symptom is the leaf surface About 30% or more of the total disease severity = Σ (number of leaves by degree of disease x index) x 100 / (number of leaves surveyed x 3)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results Table 13 shows the survey results. As is apparent from Table 13, the drug-treated section of Penicillium pinophilum / RD50109 strain has a control value of 81, which is slightly inferior to the control drug cumulus but has a sufficient effect, and has a control effect against this disease. It became clear to have.

Example 14 Pear Black Spot Control Test
(1) Test method The test was carried out in a field (open field) where an 18-year-old pear tree (variety: nova) that naturally developed black spot disease was planted. In the same manner as in Example 1, a spore suspension of Penicillium pinophilum RD50109 strain was prepared to contain 1.0 × 10 ⁷ cfu of spores per ml, and this was used as a reagent. As a control drug, a score wettable powder (active ingredient difenoconazole = 10%) was diluted 4000 times with tap water and used.
Three trees were used per ward, and 3 liters of medicine per tree was sprayed four times at 7-day intervals using a back-up power sprayer. The first spraying was set to be about 3 to 4 weeks before the first occurrence of black spot disease. Disease onset was a naturally occurring condition.

(2) Control Effect Survey The survey was conducted 16 days after the final spraying. For each 100 leaves (total 300 leaves / district), the number of lesions per leaf was investigated, the disease severity was evaluated based on the following disease index, and the disease severity and control value were calculated from the following equations. .

Disease index 0: No disease 1: 1 to 3 lesions 2: 4 to 7 lesions 5: 8 or more lesions onset = Σ (number of leaves according to disease severity x index) x 100 / (survey Number x 5)
Control value = {(Non-treatment district disease severity-Drug treatment zone disease severity) / Non-treatment zone disease severity} × 100

(3) Test results The results of the investigation are shown in Table 14. As is clear from Table 14, the drug-treated section of Penicillium pinophilum strain RD50109 has a control value of 90.4, which is almost the same as the control score wettable powder, and the black spots of pears It became clear that it had a control effect against the disease.

Claims (4)

  A filamentous fungus belonging to the genus Penicillium, Penicillium pinophilum, RD50109 strain (NITE P1032).   Rice blast fungus (Gibberella fujikuroi), rice blast fungus (Pyricularia oryzae), rice sesame leaf blight fungus (Cochliobolus miyabeanus), rice blast fungus fungus (Pseudomonas glumae), rice seedling blight fungus (Pseudomonas plantarii), and rice Infectious pathogens of rice seeds including Pseudomonas avenae, and pathogens of rice seedling blight (Fusarium spp.), Pythium graminicola, Rhizopus spp. And at least one of Trichoderma viride. The filamentous fungus according to claim 1, which has an antagonism against soil-borne pathogens containing the lysate and can be used to control diseases that occur during rice seedling raising caused by at least one of these pathogens.   Pear red blight fungus (Gymnosporangium asiaticum), pear black spot fungus (Alternaria kikuchiana), apple spotted leaf fungus (Alternaria mali), peach perforated fungus fungus (Xanthomonas campestris pv. Pruni), grape black fungus (Elsinoe ampelina) ), Pathogens causing fruit tree diseases including citrus scabs (Xanthomonas campestris pv. Citri), grapes, oysters, citrus gray scabs (Botrytis cinerea), and plum scabs (Pseudomonas syringae pv. Morsprunorum) The filamentous fungus according to claim 1, which has an antagonistic action and can be used for controlling diseases caused by at least one of these pathogenic bacteria.   Cucumber powdery mildew (Sphaerotheca fuliginea), cucumber spotted bacterial fungus (Xanthomonas campestris pv. Lachrymans) Of Xanthomonas campestris pv. Vesicatoria, Colletotrichum fragariae, Pseudomonas syringae pv. Theae, potato black rot (Rhizoctonia solani culture type IV), solanaceous vegetables Ralstonia solanacearum, wheat powdery mildew (Erysiphe graminis), various vegetable gray molds (Botrytis cinerea), various vegetable sclerotia (Sclerotinia sclerotiorum), various vegetables soft rot (Erwinia carotovora) ), And various bacterial pathogens (Rhizoctonia solani culture type IIIA) and Pythium (Pythi) um spp.). } The filamentous fungus according to claim 1, which has an antagonism against pathogenic bacteria that cause diseases of vegetables including the same, and can be used for controlling diseases caused by at least one of these pathogenic bacteria.