KR101478408B1 - Biological control product of vegetable powdery mildew by microorganism and natural materials - Google Patents

Abstract

Fruit and Vegetable Powdery mildew which is harmful to cucumber, pumpkin, melon and the like by the optimal combination of antagonistic microorganisms and natural materials which have the function of inhibiting the growth of powdery mildew ( Sphaerotheca fusca ) control effect, and at the same time has an immediate effect and a sustainability. The present invention is Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens ) KB-MJK 601 (accession number: KCTC 11748BP) and eucalyptol oil as effective ingredients for preventing powdery mildew.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for controlling powdery mildew disease caused by microorganisms and natural materials,

More particularly, the present invention relates to a complex crop protection agent for controlling powdery mildew of foaming and crops using specific microbial strains and natural material-derived materials.

The present invention was derived from research carried out as part of the R & D joint research project / national policy development project.

[Project number: PJ008976, Title: Development of an eco-friendly antiseptic for mildew disease and nematode disease using microbial and natural material-derived substances]

Powdery mildew is an active parasitic organism, and because of the entanglement of mold mycelium on young plant tissue, large and small white or grayish white lesions appear like flour spreading. Pathogens ryeojyeo Oh, about 20 species and Erie Príncipe (Erysiphe) in micro Spa as (Microsphaera) in grapes spa called (Podosphaera), A pillar Sancti California (Phillactinia), A ungki Cronulla (Uncinula), A Spy It is caused by a special variant of fungal species belonging to the genus Sphaerotheca .

These pathogens become the primary infectious agents by wintering in the remnants of the diseased plants in the form of phloem or mycelium, and in the cultivation of the plants, the fungus grows continuously with air transmission. In addition, it is common in areas where temperature difference between night and day is severe or in areas where ventilation is not good, and the annual plant growth and the decrease in yield due to powdery mildew are more severe than other diseases and cause enormous damage to farming. This powdery mildew is a disease that is difficult to eradicate by the chemical method because it always exists in the plantation site.

It is known that chemical crops are mainly used in plantation sites including fruit and vegetables, and in fact, chemical pesticide control is carried out about 10 times or so in a small plant. According to the recent well-being trend, consumers are demanding high-quality safe agricultural products, and the opening of agricultural imports by FTA is expanding, and as sustainable agriculture is needed for farmers, eco-friendly agriculture is becoming urgent. In addition, the international trend of agriculture is also going toward eco-friendly agriculture.

Microbial antagonists are known to biologically control fungi of powdery mildew. Antagonistic microorganisms can prevent disease caused by antibiotic production, pathogenic bacteria, habitat competition, nutrition ingredient competition, etc. Is a functional microorganism. Antagonistic microorganisms are biologically active materials that can control plant diseases in an environmentally friendly manner by inhibiting plant pathogens very efficiently and continuously. However, they have no short-lived effect, . As antimicrobial antagonistic microorganism being used to date Agrobacterium (Agrobacterium) into Bacillus (Bacillus), A penny Bacillus (Paenibacillus) genus Enterobacter (Enterobacter) genus Pseudomonas (Pseudomonas) genus Streptomyces (Streptomyces ), Ampelomyces , and Trichoderma microorganisms. There have been many studies in domestic and foreign countries. However, there are very few commercialized products.

In addition to these antagonistic microorganisms, antimicrobial plant extracts derived from natural products, antimicrobial oil components, and the like are biomaterials capable of controlling powdery mildew. As antimicrobial plants, it is recognized that phytopathogenic fungi, ganghwam, Saururus chinensis, and Artemisia spp. Are effective against plant pathogenic fungi. However, this also shows no satisfactory control effect. The antimicrobial oil includes citronella oil, jojoba oil, canola oil and the like, and it has a certain degree of antibacterial activity, but the effect is too low to be used as a disease control agent . Other useful ingredients known to have other antimicrobial activity are also the same situation.

On the other hand, in Japanese Patent Application Laid-Open No. 2008-0045346 and Japanese Patent Publication No. 1139794, there is disclosed a microorganism preparation which contains a strain of Bacillus subtilis M27 (accession number: KACC 91208P) as an effective ingredient, Microbial agents have been disclosed, but the effect of controlling the powdery mildew of fruits and vegetables has not been mentioned.

In addition, in Patent Publication No. 1079491, a new strain Bacillus subtilis Subtilis KB-401 was introduced as a microbial disinfectant mixed with botanical extracts to produce Diaporthe citri ), but it does not mention the control effect of powdery mildew on fruits and vegetables.

Further, in Unexamined Patent Publication No. 2012-0094647 No. Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens ) KB-MJK 601 (accession number: KCTC 11748BP), various biological pathogens that inhibit the growth of various plant pathogenic bacteria have been disclosed. Especially, the effect of controlling the powdery mildew of cucumber, It is not mentioned specifically.

In addition, Japanese Patent Publication No. 1098304 discloses a microbial fungicide for controlling cucumber powdery mildew, which comprises Bacillus subtilis KB-401, Pseudomonas sylvestris extract and canola oil as a sterilizing effective ingredient, and has a relatively excellent control effect However, the development of a microorganism preparation having a better control effect than that in consideration of the harm caused by powdery mildew has been desired.

Accordingly, the present invention relates to a method for producing a powdery mildew ( Sphaerotheca) which is harmful to cucumber, amber, melon and the like by optimally combining an antagonistic microorganism having a function of inhibiting the growth of fruit and vegetable powdery mildew and a naturally- fusca ), which has the most effective control effect, but also has both rapid efficacy and persistence.

Also, it is intended to provide an environmentally friendly crop protection agent having various action mechanisms that can be practically applied in a farmhouse by optimally blending antagonistic microorganisms and naturally occurring substances together with additives such as fixing agent, moisturizing agent, and physical property stabilizer.

In order to solve the above-mentioned problems, the present invention relates to a composition for controlling powdery mildew diseases on foams and crops containing Bacillus amyloliquefaciens KB-MJK 601 (accession number: KCTC 11748BP) and eucalyptus oil as active ingredients Provide crop protection.

In addition, the powdery mildew on the above-mentioned foams and crops is caused by the presence of cucumber powdery mildew ( Sphaerotheca The present invention provides a complex crop protection agent for controlling powdery mildew of foaming and crops.

Furthermore, the Bacillus amyl Lowry Quebec Patience (Bacillus Amyloliquefaciens KB-MJK 601 (Accession No .: KCTC 11748BP) is contained in an amount of 2 to 50% by volume as a culture medium at a concentration of 10 ⁵ to 10 ¹⁰ cfu / ml, and the eucalyptus oil is contained in an amount of 50 to 98% And a complex crop protection agent for controlling powdery mildew of a plant and a plant.

Also, the present invention provides a complex crop protection agent for controlling powdery mildew of a foaming and a crop, wherein the tea plant extract (teacandy) further comprises 10 to 30 parts of skin to 100 parts of the skin of the complex crop protection agent.

Also, a polyalkylene oxide modified heptamethyltrisiloxane (polyalkyleneoxide modified heptamethyltrisiloxane) is added to the skin of 100 parts of the composite crop protecting agent in an amount of 5 to 10 parts, and the composite crop protection agent for preventing powdery mildew .

Also, it is preferable that polyoxyethylene dodecyl mono ether or methyloleate is further contained in 3 to 10 parts skin to 100 parts skin of the complex crop protection agent. Provides a complex crop protection agent for control.

According to the present invention, it is possible to provide a complex crop protection agent of various mechanisms exhibiting a strong controlling effect of not less than 80%, preferably not less than 85%, against a powdery mildew of a persimmon crop, particularly by combining a specific antimicrobial microorganism and an antimicrobial natural- .

In addition, the present invention can provide an environment-friendly crop protection agent that can be applied at an optimum level in combination with a fixing agent, a moisturizer, a dispersant, and an emulsifier so that the same level of efficacy as chemical pesticides can be exhibited.

FIG. 1 is a photograph showing electrodeposition and dispersibility test results according to the kind and content of the dispersant added in the preparation of the green complex crop protection agent of the present invention,
2 is a graph showing the microbial stability over time of the combination preparation according to the present invention,
FIG. 3 is a photograph showing the result of carrying out the pavement proof test on cucumber and pumpkin of the complex crop protection agent according to the present invention.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Accordingly, it is to be understood that the constituent features of the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the inventive concepts of the present invention, so that various equivalents, And the like.

The inventors of the present invention have found that it is not easy to implement a preparation having excellent performance in the control of powdery mildew by using a microorganism or a plant extract as a single component or a combination thereof, and as a result, they have surprisingly found that a specific antimicrobial microorganism and an antimicrobial natural- The present inventors have found that it is possible to have the best effect of controlling powdery mildew by complementing the disadvantages of each component when they are combined optimally.

Accordingly, the present invention relates to a method for producing a microorganism, which comprises the steps of: (1) cultivating a microorganism in a microorganism, such as Bacillus amyloliquefaciens KB-MJK 601 strain (accession number: KCTC 11748BP; available from the Korea Research Institute of Bioscience and Biotechnology); and eucalyptol A complex crop protection agent of various mechanisms having a powdery mildew control effect is disclosed.

Each component content of the crop protection compound according to the invention the Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens KB-MJK 601 is a culture medium having a concentration of 10 ⁵ to 10 ¹⁰ cfu / ml and may be contained in an amount of 10 to 30% by volume as a culture medium having a concentration of 2 to 50% by volume, preferably 10 ⁷ to 10 ¹⁰ cfu / ml, The eucalyptus oil may contain 50 to 98% by volume, preferably 70 to 90% by volume.

Hereinafter, the process for deriving the environmentally friendly complex crop protection agent according to the present invention will be described in detail.

Microbial and natural products Compound condition  optimization

(1) Antimicrobial microorganism assay

In the environment-friendly composite crop protection according to the invention in order to select the optimal antibacterial microorganism Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens ) KB-MJK 601 strain was cultured in various culture media to produce cucumber powdery mildew ( Sphaerotheca fusca ), and the results are shown in Table 1 below.

As shown in Table 1, the Bacillus amyloliquefaciens KB-MJK 601 strain showed the highest inhibitory effect of 81% in LB medium, and the MY (M) and MY (CM) (CM: culture media) containing spores rather than metabolites (M: metabolites). As described above, in the case of the LB medium, the MY (CM) medium, which has a 71% control effect, was selected and tested for effect because the test medium is a test medium having a high control effect but a low industrial utilization.

(2) Antimicrobial properties Test of natural products

Optimum Antimicrobial Activity in Protecting Environmentally Friendly Composite Crops According to the Present Invention In order to select naturally occurring substances, eucalyptol oil and clove oil were sphaerotheca fusca ) and the results are shown in Table 2 below.

Referring to Table 2, it can be seen that the eucalyptol oil-controlling effect of cucumber powdery mildew was superior to other plant extracts, and the best effect was obtained at 500-1000 ppm concentration.

(3) Confirm synergy effect of active ingredient

To check for powdery mildew control synergy according to the effective ingredient of the present invention in combination, Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens ) KB-MJK 601 strain and eucalyptus oil (cucumber powdery mildew). The synergistic effect of controlling the mildew disease according to the above combination was compared with the effect on each single component. In addition, Bacillus subtilis ( Bacillus subtilis) Subtilis KB-401 strain and eucalyptol oil were compared and the results are shown in Table 3 below.

Referring to Table 3, when the Bacillus amyloliquefaciens KB-MJK 601 strain and eucalyptol oil were treated in combination as compared with the case where they were treated alone, the control effect against cucumber powdery mildew was found to be better than that of Bacillus amyloliquefaciens KB- It can be seen that it is excellent.

Meanwhile, Bacillus subtilis subtilis) KB-401 For the strain when only treatment Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens ) KB-MJK 601 strain, but the effect of Bacillus subtilis subtilis) KB-401 strain, and when a combination treatment of the eucalyptus oil (eucalyptol) Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens ) KB-MJK 601 strain and eucalyptus oil (eucalyptol) combination treatment, the control effect of the Bacillus amyloliquefaciens KB-MJK 601 strain and eucalyptus oil (eucalyptus oil) ), The synergistic effect on the control of cucumber powdery mildew is very excellent.

(4) Test for possibility of adding active ingredients

The possibility of incorporation of Bacillus amyloliquefaciens KB-MJK 601 strain and eucalyptol oil, which showed excellent antibacterial activity as an active ingredient in the aforementioned antimicrobial microorganism test and antimicrobial natural product assay, was tested. In this test 2.4 × 10 ⁹ cfu / ㎖ concentration of Bacillus amyl Lowry Quebec Patience (Bacillus amyloliquefaciens KB-MJK 601 strain and eucalyptol oil (eucalyptol, 50%) were mixed at a ratio of 1: 4, and the stability of the compound (log of colonies / ml) Respectively.

Referring to Table 4, when the selected antimicrobial microorganisms and eucalyptus oil (eucalyptol, 50%) were mixed, the density of the microorganisms was hardly changed for 4 months. Therefore, the effective ingredient composition of the crop protection agent which can exert excellent control performance by various action mechanisms for the final fruit and vegetable powdery mildew is Bacillus amyloliquefaciens KB-MJK 601 strain of 2.4 × 10 ⁹ cfu / ml concentration and eucalyptus oil (eucalyptol, 50%) was determined as a composite of 1: 4 by volume and used as a sample for formulation.

Formulation according to effective concentration

(1) Improvement of stickiness by biodegradable adhesives

(60%), paraffin oil (65%) and tea tree extract (teacandy, < RTI ID = 0.0 > 50%) was examined by the following method, and the results are shown in Table 5 below.

[Experimental Method]

Each fixing agent was added to the mixture according to the above mixture sample in an amount of 3 to 30 parts per 100 parts of the above mixture sample, and the composition was diluted 1,000 times with respect to the leaf of the plant. Firstly, the appearance of the diluted solution on the leaf of the plant was observed, and the appearance of Bacillus amyloliquefaciens KB-MJK 601 existing in 1 g of the plant leaf of each fixing agent treatment was observed under a microscope. The results of observations for each treatment were compared with each other and it was judged as excellent (⊚), good (◯), normal (△), and bad (x).

It is most preferable to select tea extract (teacandy, 50%) as a fixing agent in the formulation of the crop protection agent according to the present invention, and the content thereof is preferably 10 to 30 parts by weight per 100 parts of the active ingredient combination It can be confirmed that it is preferable to include it.

(2) Electrodepositable diffusion agent selected

The surface of the hyphae and crops of most molds including mildew disease are composed of the phospholipid layer and the cuticle layer, and the drug is not easily applied. To overcome this, the nonionic surfactant which is easy to disperse and diffuse is selected by the following method in order to make the active ingredient according to the present invention more easily contact with the powdery mildew and the surface of the crop. The results are shown in FIG. 1, Table 7 shows the results. Fig. 1 is a photograph showing the results of electrodeposition and dispersibility test according to the type (a) and the content (b) of the dispersant added in the formulation of the environmentally friendly complex crop protection agent of the present invention. In FIG. 1, PMF means polyalkyleneoxide modified heptamethyltrisiloxane, PTE means polyoxyethylene tridecyl ether, and OA means oleic acid.

[Experimental Method]

A representative surfactant used in the agricultural field was first selected on the basis of the concentration of 0.05% for the selection of the surfactant, and the selected surfactant was added to 100 parts of the above-mentioned active ingredient combination sample to prepare a spore solution containing 1 to 10 parts skin The pathogenic fungi Rhizoctonia solani) was determined by contact between (wetting) and diffusibility (determine the degree of dispersion), and the relatively good compared to the observed results (+++), medium (++), poor (+) was inoculated to the subculture group .

Referring to Table 6 and FIG. 1, it is most preferable to select polyalkylene oxide modified heptamethyltrisiloxane as the electrodepositable dispersant in the formulation of the crop protection agent according to the present invention .

Also, referring to Table 7 and FIG. 1, it can be confirmed that 5 to 10 parts of skin is preferably included in 100 parts of the active ingredient combination.

(3) Selection of emulsifier

In order to select the optimum emulsifier for formulation of the eco-complex crop protection agent according to the present invention, polyoxyethylene dodecyl mono ether, polyoxyethylene monooleate, methyloleate, The polyoxyethylene sorbitan monooleate was examined for its emulsifiability and properties by the following method, and the results are shown in Table 8 below.

[Experimental Method]

Each emulsifier was visually observed for emulsification while diluting 500 times of the spore solution containing 1 to 10 parts of skin to 100 parts of the above-mentioned mixture sample in the mixture according to the above compound sample. Here, the observation results of the respective treatments were compared with each other to determine excellent (?), Good (?), Normal (?), And poor (x).

Referring to Table 8, it is preferable to select polyoxyethylene dodecyl mono ether or methyloleate as an emulsifier in the formulation of the crop protection agent according to the present invention, It can be confirmed that it is preferable to include 3 to 10 parts of skin to 100 parts of skin.

(4) Test for efficacy and safety of compound crop protection agent

Based on the preparation studies based on the optimum combination preparation described above, each of the selected additives was formulated into the combination described in Table 9 below.

The composite formulation formulated in Table 9 was tested for changes in pH, layer separation, precipitation, and microbial density at 4, 25, and 54 ° C in the following manner, And Fig. Fig. 2 is a graph showing the microbial stability over time of the combination preparation according to the present invention. Fig.

[Experimental Method]

a. pH

And measured using a hydrogen ion concentration meter (pH meter). The measurement range is from -9.000 to +23.000 and accuracy is ± 0.01 pH after calibration. The instrument is calibrated after calibration. When the result is stable, the pH meter Were used.

b. Layer separation

The prepared products were observed by temperature.

c. Precipitation

The prepared products were observed by temperature.

d. Microbial density

Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) in samples stored at 4 < 0 > C, 25 & Amyloliquefaciens KB-MJK 601 was prepared by spreading 1 ml of the prepared preparation on a tryptone soytone agar (TSA) plate and incubating at 30 ° C for 24 hours. The number of colonies formed on the medium was measured at the initial, Week, for 12 weeks.

Referring to Table 10, it was confirmed that pH, layer separation and precipitation of the complex crop protection agent prepared according to the present invention were observed for not less than 4 weeks, showing no specific property change.

2, the effective microorganism Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) in the complex crop protection agent prepared according to the present invention amyloliquefaciens ) KB-MJK 601 showed good stability due to the maintenance of microbial durability over a period of 12 weeks after preparation.

Meanwhile, in order to confirm the field applicability of the complex crop protection agent according to the present invention, the composite preparation blended with the composition shown in Table 9 was diluted 500-fold and 250-fold to perform a package demonstration test for cucumber and pumpkin, As shown in Table 11, Table 12 and FIG. 3, it was confirmed that the field applicability was also excellent. The pavement test was carried out at Geumgok-ri, Jungnam-myeon, Hwaseong-si, Gyeonggi-do.

The preferred embodiments of the present invention have been described in detail with reference to the drawings. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing detailed description, and all changes or modifications derived from the meaning, range, and equivalence of the claims are included in the scope of the present invention Should be interpreted.

Claims (6)

delete delete As a complex crop protection agent for preventing powdery mildew against foaming and crops containing Bacillus amyloliquefaciens KB-MJK 601 (accession number: KCTC 11748BP) and eucalyptol oil as active ingredients,
Powdery mildew of the foliage and crops is a powdered cucumber powdery mildew caused by Sphaerotheca fusca ,
The Bacillus amyloliquefaciens KB-MJK 601 (Accession No .: KCTC 11748BP) is contained in a concentration of 10 ⁷ to 10 ¹⁰ cfu / ml in a concentration of 10 to 30% by volume and the eucalyptus oil is contained in an amount of 500 to 1,000 by weight, based on the total weight of the mixture, of 70 to 90% by volume. delete delete The method of claim 3,
The complex crop protection agent for controlling powdery mildew disease comprises 10 to 30 parts of a teacandy extract, 5 to 10 parts of polyalkyleneoxide modified heptamethyltrisiloxane, 100 parts of poly (ethylene oxide) modified heptamethyltrisiloxane, A polyoxyethylene dodecyl monoether or methyloleate in an amount of 3 to 10 parts by weight based on the total weight of the composition.

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