KR101079491B1 - Biological germicide for preventing fruit tree disease - Google Patents

Abstract

The present invention relates to a fungicide for fruit tree disease control using antagonistic microorganisms, wherein the microbial fungicide of the present invention is isolated by the present inventors as an antagonist against fruit tree disease ( Bacillus subtilis KB-401, KFCC 11388P). ) Is similar to conventional chemical fungicides by using tea or turmeric extract as a bactericidal plant extract and castor oil, clove bud, thyme and the like as a bactericidal plant essential oil. However, it does not cause resistance and is harmless to the human body, and provides an eco-friendly fungicide with a bactericidal power comparable to a conventional chemical pesticide.

Melanose, Citrus Diaporthe citri, Bacillus subtilis, Melaleuca alternifolia, Castor oil

Description

BIOLOGICAL GERMICIDE FOR PREVENTING FRUIT TREE DISEASE}

The present invention relates to a fungicide for fruit tree disease control using antagonistic microorganisms, and more specifically, to an orchard disease such as citrus black spot disease, using antagonistic microorganisms and bactericidal / antibacterial plant extracts, bactericidal and antimicrobial plant essential oils to prevent natural pollution. It relates to a biological fungicide.

Black spot disease (Melanose) is the most damaging disease for citrus farmers in Jeju Island. In severe areas, more than half of the total number of pesticide sprayings is done to control black spot disease. In particular, the southeastern part of Jeju Island, including Seogwipo, is more affected by black spots than other areas. In addition, black spots are the cause of resin stems or stem rot, a major storage disease, in which the entire stem or tree suddenly dies.

The host ranges from citrus plants, and it occurs in all citrus fruits, but there are some differences in the onset depending on the type. Although it is widely distributed in the citrus region, the incidence is relatively low in areas where there is little rainfall during the growing period of citrus fruits, and in southeastern Jeju Island where rainfall is high.

Black spots pathogens ascus spores in fungi belonging to the three ascus bacterium is a conidia three Diaporthe citri FA Wolf is Phomopsis citri Fawcett. Pyramids that form conidia, conidia, mainly occur on dead branches, especially recently dead branches, black oval or round, and the size is 200-450 μm. Bottle spores form two forms, alpha (α) spores and beta (β) spores. Alpha spores are colorless and monofoam, 5 ~ 9 × 2 ~ 4㎛ in size, which directly invade host plants, while beta spores are colorless, It is a silk-shaped thread, and its size is 20 ~ 30 × 0.7 ~ 1.5㎛ and it doesn't germinate so it doesn't directly invade host plants.

The despoilers erupt from the crustacean in the form of sticky yellow mucus in moist conditions and in the form of tendrils under relatively dry conditions, and spores from these are known to be very resistant to drying. Asympias occurs on the branch almost finished with crustacean formation. It is black and has a long beak shape. The tip is tapered and its length is 200 ~ 800㎛. Ascima is a long club, containing 8 capspores, each of which is colorless, 2, and two gulutulae, 11 ~ 15.5 × 3 ~ 5㎛. The optimum temperature for mycelial growth is 24 ℃, and germination starts in 4 hours at 20 ~ 28 ℃, but requires 10 hours at 15 ℃.

In the occurrence of black spot disease, pathogens overwinter in the form of sickle or asymptomatic, and infectious agents are diseased spores and vesicles produced from them. Asymptomatic spores are scattered in the air and can propagate pathogens over long distances by wind. Morphos are produced from the awakenings from dry branches on trees or dead branches left in orchards and scattered with raindrops. In the case of fruit, the disease can develop from the time of fall to 5 months after the fall, and the degree of disease varies according to the amount of dead branches remaining in the water, the size of fruit (growing stage), and the wetting period due to rainfall or dew.

Pathogens settle on dead or pruned branches and form spores in two to three months, becoming infectious agents, and spore production continues for about a year. Pathogens that invade greenery are often alive without dying, and after the greenery dies (not sure if it dies by pathogens), pathogens become infectious agents. When spores invade fruits or leaves, host plants secrete antimicrobial substances as a defense against pathogens, killing invading pathogens, and reacting with them to form black spots.

Spores need to be wetted for more than 8 hours at 24 ~ 28 ℃ and more than 12 hours at 20 ℃. The incubation period is 1 ~ 2 days at 25 ℃ and 7 days at 10 ℃. Therefore, even if there is not much rain in the afternoon when the rain and dry in the state at a certain night temperature is high enough to cause the disease. If the disease condition is met, the disease will occur until early October, but the disease is rare since it is resistant after fruit hypertrophy except in special cases.

Symptoms of black spot disease appear only about one week after the invasion of pathogens. They develop on leaves, branches, and fruit, and vary widely depending on the time of infection, the condition of the fruit, and the climatic conditions at the time of infection. There are three types, lump form and platen form. Epidermal cells of plants invaded by pathogens necrosis up to six layers of cells from the invasion site and are embedded with hard, dark red rubbery material, which is a representative sunspot. The affected area before the inflorescence is slightly recessed, and in some cases the depression is maintained until the harvest season, and the area remains blue even when pigmented and then gradually progresses. The size of the sunspots varies so much that during the infall phase, the affected lesions are relatively large in size, and in the more mature state, the lesions form relatively small lesions, and the shape of the sunspots becomes a distinct small bump, and the fruit's hypertrophy is almost over. Infected tissues are reddish in lesion and relatively unspotted. In addition, when the pathogen concentration is high, it becomes a dark red scab or swell-like shape.

In the case of leaves, the leaves start to be infected before the leaves are hardened in early and mid-May, and they are loomed with typical black spots. In the case of stems, the young buds or greenery have been infected since the beginning of May and around mid-May.

As a method of controlling the black spot pattern disease, first, the dead branches and pruning branches are removed. Since black spot disease is transmitted by spores formed on dead branches, if there are many infectious agents, that is, dead branches, no amount of pesticides can be used to control them. Thoroughly remove dead branches left on the tree during harvesting or pruning, and pruning branches can be burned or crushed to eliminate disease sources rather than stacked around orchards.

Drug control varies depending on the region, but the drug is sprayed four to five times every 15 to 20 days or between 200 and 250 mm of accumulated rainfall between mid-May and late-May to late-August. Drugs that have been announced include Mankoji (Daisen M-45), Dich (Dellan Hydrating), Mechiram (Sunup Granular Hydrating), and Propy (Anthracol Hydrating) (Research Institute for Horticultural Diseases, Rural Development Administration, pp. 297 ~ 301). ). However, there are currently no eco-friendly drugs that can be used in pesticide-free citrus farmers.

Recently, a number of biological control methods using microorganisms and various extracts have been studied. Since these methods do not cause problems such as environmental pollution, resistance induction and residual toxicity, they are considered to be very desirable control methods because they can be used in non-pesticide citrus farmers. .

Biomaterials that can control phytopathogens include antagonistic microorganisms, plant extracts, and antimicrobial oils. Antagonist microorganisms include Bacillus subtilis, Bacillus pumilus, Bacillus Licheniformis, Paenibacillus polymyxa, Ampelomyces quisqualis, Streptomyce genus, and many researches have been conducted at home and abroad. There is no microbial pesticides. Plant extracts are known to have a bactericidal effect on plant pathogens such as oleander, purslane, widower, and turmeric, but they are not commercialized as effective products. In addition, antimicrobial oils include thyme oil, clove bud oil, rose geranium oil, sassafras oil, castor oil, etc., and are known to have bactericidal effects, but they also do not show satisfactory control value for single use.

Biopesticides that are developed using the components of these plants have been widely commercialized as insecticides, but fungicides are relatively weak and do not vary widely. In addition, there is a lack of formulation technology capable of properly exerting an effect on the formulation of plant extracts and other mixed ingredients, the effect is weak compared to chemical pesticides or synthetic fungicides. In addition, in order to improve farm household income and secure stable profitability, it is necessary to develop technology to produce crops in an environmentally friendly manner.

In tangerines, pests such as black spots, lancets, and ulcers, and mites, aphids, worms, and whiskers are causing great damage. Among them, eco-friendly control agents for pest control have been developed and sold in 7 ~ 8 companies in Korea, the effect is known to be very excellent. The damage of citrus disease is about 80% of black spot disease, about 5% of window disease, about 10% of ulcer disease, and about 5% of other diseases. Therefore, development of biopesticides for black spot disease control is urgently needed to produce no pesticide citrus. Do. Therefore, in the present invention, an environmentally friendly fungicide capable of controlling the black spot pattern disease, which is the most damaging in the pesticide-free citrus cultivation, to reduce the high-rise of farmers who want to grow pesticide-free citrus.

An object of the present invention is to solve the problems described above, by providing a powerful antagonist that has a control effect on fruit trees, particularly citrus black spot pattern disease, to solve the problems caused by conventional chemical pesticides, there is no induction of resistance, harmless to the human body In addition, to provide an environmentally friendly microbial fungicide.

Hereinafter, the present invention will be described in detail.

The present invention provides a microbial fungicide for fruit disease control using a Bacillus subtilis KB-401 (KFCC 11388P) isolated by the present inventors as a bacteriostatic antagonist as a bactericidal active ingredient. In the present invention, 'antibacterial' may be used Bacillus subtilis, in particular, Bacillus subtilis KB-401 ( Bacillus subtilis KB-401, KFCC 11388P) separated and deposited by the present inventors the most effective control ability see.

An object of the present invention is to provide an environmentally friendly fungicide against diseases occurring in fruit trees, but is not specifically limited to the types of fruit trees, but through experiments, preferably citrus black spots ( Diaporthe citri ), pear black pattern Bottle (Alternaria kikuchiana), pear black star (Venturia nashicola), apple black star (Venturia inaequalis), gray mold (Botrytis cinerea), tomato leaf fungus (Cladosporium fulvum), pepper anthrax (Colletotrichum coccodes) (Fusarium oxysporum), potato sage (Fusarium solani), pepper blight (Phytophthora capsici), melon blight (Phytophthora melonis), tomato sclerotinia minor, Fusarium moniliforme, grass anthrax (Colletotrichum cauda) It can be applied variously to Pythium sp., Grass brown spread disease (Rhizoctonia solani), but most preferably Citrus black spot disease ( Diap) orthe citri ).

In addition, the microbial fungicide for controlling the fruit disease of the present invention may include a bactericidal plant extract in addition to the Bacillus subtilis KB-401, KFCC 11388P as an antagonist against the disease, preferably tea or It is preferable to further include any one or more plant extracts selected from the group consisting of turmeric, garlic, brood, purslane, sesame, cedar, pasqueflower as sterilizing active ingredients, and among these, tea or turmeric extracts to enhance the sterilizing power. More preferred.

In addition, the microbial fungicide for controlling fruit orchard diseases according to the present invention is bactericidal vegetable oil in addition to Bacillus subtilis KB-401 (KFCC 11388P) as an antagonist against diseases, castor oil (Castor oil), cloves Bud (Clove bud), thyme (Thyme) may further include a vegetable essential oil extracted from one or more selected as a sterilizing active ingredient.

The microbial fungicide for controlling fruit disease of the present invention is weighted when it contains both Bacillus subtilis KB-401 and KFCC 11388P as antagonists and tea tree extract as a bactericidal plant extract and castor oil as a bactericidal plant essential oil. It can provide an excellent fruit disease control effect.

The microbial fungicide of the present invention may further include an adhesion promoter for vaporizing the surface adhesion of the microorganism, a sunscreen agent for reducing the ultraviolet destruction of the microorganism, or a moisturizer for maintaining the humidity of the microorganism.

In the present invention, the term 'polysaccharide' is a material extracted from a plant, and may be liquid, concentrated, mixed or solid, or powdered, and may be used as a concept including a crude extract, and further purified crude extract. It is a concept to include. In addition, 'polysaccharide' may be preferably used to refer to the extract extracted from the 'Tea tree ( Melaleuca alternifolia )' separated by the present inventors.

Vegetable essential oil in the present invention includes a vegetable oil, the oil is an essential oil extracted from the plant is used in the concept including the liquid phase, concentrate or mixed liquid, and particularly in the present invention preferably includes 'castor oil'.

In the present invention, the 'efficacy enhancer' includes an 'adhesive enhancer' to enhance the adhesion of the active ingredient, an 'ultraviolet ray blocker' to protect the antagonistic spores from ultraviolet rays, and a 'humidant' to maintain and moisturize the antagonistic spores. It is a concept.

In the present invention, "%" of the blending ratio means "volume%" unless otherwise stated.

The active ingredient in the present invention, gilhanggyun, "" biocidal plant extract (anti-microbial polysaccharide), 'vegetable oil (antibacterial oil), as an interior effect to the concept of the form to be mixed minutes three kinds of efficacy are black spots pathogen (Diaporthe citri) To evaluate the effect on the packaging, the control effect was measured in citrus cultivated farmers.

The present invention can be used to cultivate antagonists against citrus black spot disease by mixing tea extracts (polysaccharides), castor extracts (castor oil) using a antagonist culture medium and lower alcohol or methanol of C1 ~ C4 at a constant ratio, respectively. The effective ratio of is not limited in the mixing ratio, but for optimum sterilization effect, it is more preferable that they are mixed in equal proportions.

The extraction of the plant is preferably extracted with a lower alcohol of C1 ~ C4, more preferably with methanol. The extraction temperature is not limited but is about 50 ° C., and the extraction time is not limited but preferably about 24 hours or more.

The application of the fungicide is preferably applied to citrus black spot pattern disease, but as long as it provides a fungicide for the disease of the fruit tree, it is not limited thereto and may be applied to the fungal disease of other fruit trees.

In addition, the present invention provides acaricides further comprising a potentiator to increase the sterilization efficiency of the three active ingredients.

The additive is not particularly limited, but as the adhesion promoter, carboxy methyl cellulose (Carboxy Methyl Cellulose, CMC), gelatin (Gelatin) is preferred, and gelatin is more preferred. 2-Hydroxy-4-octyloxybenzophenone and 2--2-hydroxy-5-methylphenylbenzotriazole (2- (2-Hydroxy-5-methylphenyl) benzotriazole as sunscreen ), More preferably 2-hydroxy-4-octyloxybenzophenone (2-Hydroxy-4-octyloxybenzophenone). As a moisturizing agent, sodium hyaluronate and sodium 2-pyrrolidone-5-carboxylate are preferable, of which sodium 2-pyrrolidone-5-carboxylate More preferred is (Sodium 2-pyrrolidone-5-carboxylate).

In addition, the present invention provides a fungicide further comprises a physical property modifier and stabilizer to stabilize the three active ingredients.

The physical property modifier is not particularly limited, but polyoxyethylene monooleate, polyoxyethylene dodecyl mono ether, polyethylene glycol-400, ethoxylated castor oil (Castor oil, ethoxylated) is preferred.

The stabilizer is sodium benzoate, potassium sorbate, sodium propionate, and more preferably, potassium sorbate.

The fungicide is used in combination with three active ingredients, an effect enhancer, a physical property modifier and a stabilizer. Bacillus subtilis KB-401 in the active ingredient is preferably 1 × 10 ⁹ cfu / ml or more for all the fungicides. It is preferable that the antimicrobial oil is about 80 to 86%, the potentiator is preferably 0.5 to 3.5%, the water modifier is 2 to 10%, and the stabilizer may be a small amount.

The content of the active ingredient of the present invention is preferably applied differently depending on the formulation of the fungicide, the spraying method. The formulation of the disinfectant for the black spot disease in the present invention is in the liquid phase and the spraying method can be dilute in water sufficiently to treat the foliage.

As described above, the bactericide using Bacillus subtilis KB-401 (KFCC 11388P), tea extract and castor oil of the present invention has the best control effect against citrus black spot disease, Compared with chemical pesticides, it has more than 90% control effect compared with chemical pesticides, and it is practically useful as an eco-friendly agricultural material for controlling citrus black spots.

Hereinafter, the microbial fungicide for controlling fruit disease of the present invention will be described in more detail with reference to the following examples. This embodiment only describes a preferred embodiment of the present invention, the technical spirit of the present invention is not limited thereto.

Example 1 Selection of Citrus Black Spot Pattern Antagonists

Bacillus subtilis KB-401 was used as an antagonist of citrus black spot disease used in the present invention.

The present inventors separated a total of 254 microorganisms from domestic soil, and evaluated the antimicrobial activity against Diaporthe citri , safety evaluation on citrus trees, artificial culture evaluation, and durability formation evaluation in indoor and pot. Finally, one of the most effective bacteria was selected and named Bacillus subtilis KB-401 (KFCC 11388P), and as of April 24, 2007 of Culture Collections) was deposited with the accession number of KFCC 11388P.

The main bacteriological characteristics of Bacillus subtilis KB-401 are as follows.

[Characteristics in NA Medium]

Color Light pale yellow Flora circle Balance total surface Convex Balance total size 1.0-3.0 mm

[Physiological characteristics]

Gram reaction positivity Resistant Spore Formation U Cell morphology Simplified Cell activity U Oxygen composition Aerobic Anaerobic growth Impossible Catalase production U VP test positivity Oxidation glucose U Xylose U Mannitol U Arabinose U Glucose rotor gas generation U Hydrolysis Casein positivity gelatin positivity starch positivity Citrate Availability U Propionate Availability radish Tyrosine resolution radish Nitrate reducing ability U Indole formation ability radish Salinity composition radish Growth at 7% salinity U Urea Hydrolysis radish Growth by temperature lowest 15 ℃ optimal 30 ℃ Best 40 ℃

[Antibacterial Spectrum]

Disease name (pathogen) Antimicrobial activity Pear Black Pattern Disease ( Alternaria kikuchiana ) + Citrus black spot disease ( Diaporthe citri ) +++ Pear Black Star Pattern ( Venturia nashicola ) + Apple Black Star Pattern ( Venturia inaequalis ) + Gray Mold Disease ( Botrytis cinerea ) ++ Tomato leaf fungal disease ( Cladosporium fulvum ) + Pepper Anthrax ( Colletotrichum coccodes ) ++ Tomato withering ( Fusarium oxysporum ) ++ Fusarium solani ++ Pepper Plague ( Phytophthora capsici ) ++ Melon plague ( Phytophthora melonis ) + Tomato Sclerotinia minor + Fusarium moniliforme ++ Turf anthrax ( Colletotrichum caudatum ) ++ Turfium leaf blight ( Pythium sp. ) + Grass brown spread disease ( Rhizoctonia solani ) +++

      ※ Antibacterial activity degree (㎜) 0 ~ 10: +, 10 ~ 20: ++, 20 or more: +++

     Bacillus subtilis KB-401, an antagonistic bacterium of the present invention, has an activity of remarkably inhibiting mycelial growth and spore germination of citrus black spot pattern pathogens, thereby controlling citrus black spot patterns without adversely affecting crops or the environment.

Example 2: Preparation of antagonist culture medium of antagonist Bacillus subtilis KB-401

The antagonistic Bacillus subtilis KB-401 was cultured using M-2 medium (Table 1) to confirm the packaging production. The culture was incubated for 48 hours at 30 ℃, 250 L fermenter (Fermenter) at 150 rpm, 1.0 ~ 1.5 vol / vol / min, pH 6.0 ~ 7.0 for 48 hours, it was confirmed by the microscope (x400) that all cells were changed to spores.

M-2 badge composition Badge composition Content (/ ℓ) Glucose 20g Soybean flour 25 g KH ₂ PO ₄ 1 g K ₂ HPO ₄ 1 g CaCO ₃ 1 g MgSO _{4 7} H ₂ O 0.3 g CuSO ₄ · 5H ₂ O 0.01 g MnSO ₄ 0.02 g FeSO ₄ 7H ₂ O 0.02 g ZnSO ₄ · 7H ₂ O 0.02 g

Experimental Example 1: Evaluation of antifungal activity of Bacillus subtilis KB-401

In order to evaluate the antifungal activity of Bacillus subtilis KB-401 cultured above, antifungal activity against Citrus black spotted pathogen ( Diaporthe citri ) was evaluated along with other antagonists . That is, the filtrate was prepared by passing the antagonistic culture medium through a 0.45 μm filtration membrane, and 40 μl / disc was dropped according to the paper disc method, and the antifungal activity was shown in Table 2 below.

Evaluation Results of Antifungal Activity of Antagonist Culture Solution Target pathogen Growth inhibitory activity of pathogens of antagonists (growth inhibiting ring diameter; mm) KB-401 KB-291 KB-423 KB-525 Citrus Black Spot Pattern
( Diaporthe citri ) 21.8 11.4 7.6 10.3

Example 3 Preparation of Crude Extracts from 48 Plants

Through various literatures, 48 kinds of plants shown in Table 3 below, which are expected to have bactericidal effects against various phytopathogens, were selected, and then, each of them was pulverized finely in a blender, 200g each, put in a glass bottle, and then mixed with 1000ml of methanol at about 50 ° C. It was left for 24 hours. After 24 hours, the mixture was filtered and the extract solution was concentrated using a rotary vacuum concentrator (EYELA N-1000, Japan). The concentrate was further mixed with methanol to prepare a crude methanol extract.

Botanical name Scientific name Botanical name Scientific name Rhododendron Caulophyllum thalictroicdes Cucumber Sanguisorba officinalis Country Chrysanthemum morifolium Gentian Gentiana scabra Michaelmas daisy Aster tataricus Turmeric Curcuma longa Hazel Psoralea corylifolia Honeysuckle Lonicera japonica Poker Plagiorhegma dubium Ginkgo Ginkgo biloba ground cherry Physalis franchetii Yin Kalopanax pictus Sticky spatula Drosera rotundifolia cranesbill Geranium nepalense Soothe Allium grayi Jangguchae Melandrium firmum Fowl of chicken Commelina communis Scoop Sasamorpha purpurascens rhubarb Rheum undulatum Jovan Cephalonopsis segetus Macaw Xanthium strumarium violet Viola mandshurica garlic Allium sativum Rat vine Aristolochia contorta knotgrass Polygonum aviculare Jimo Anemarrhena asphodeloides Spicy Wasabi Wasabia japonica Plantain Plantago asiatica Macmundong Liriope platyphylla tea Melaleuca alternifolia elecampane Inula helenium A bullshit Rumex crispus Windproof Saposhnikovia divaricata Cypress Thuja orientalis ringworm Dictamnus dasycarpus Nasturtium Eclipta prostata Loquat Eryobotrya japonica pasqueflower Pulsatilla koreana Cedar Artemisia capillaris figwort Scrophularia oldhami Japanese tree Zanthoxylum piperitum Oleander Nerium oleander Sounder Rumex crispus Brother-in-law Schizonepeta tenuifolia Pine tree Picrasma ailanthoides Widower Chloranthus japonicus purslane Portulaca oleracea Round ginseng Humulus japonicus

Experimental Example 2: Evaluation of antifungal activity against crude extract

In order to evaluate the antifungal activity of the 48 crude preparations prepared above, antifungal activity against citrus fruit black spots ( Diaporthe citri ) was evaluated. The evaluation method was to drop the crude extract in 1000ppm / disc, 500ppm / disc, 100ppm / disc concentration according to the paper disc method to evaluate the antifungal activity and the results are shown in Table 4 below.

Evaluation result of antifungal activity of crude extracts (growth-lowering ring diameter; mm) Crude extract Treatment Concentration (ppm) Crude extract Treatment Concentration (ppm) 1000 500 100 1000 500 100 Rhododendron 10.3 9.5 9.0 Cucumber 9.7 9.2 9.0 Country 9.5 9.3 - Gentian 9.5 9.1 9.0 Michaelmas daisy 10.0 9.0 Turmeric 19.2 16.5 11.3 Hazel 9.7 9.3 9.0 Honeysuckle 10.2 9.5 9.3 Poker 11.0 10.2 9.4 Ginkgo 12.4 10.8 10.0 ground cherry 10.6 9.7 9.1 Yin 10.2 9.5 9.0 Sticky spatula 10.9 10.2 9.0 cranesbill 9.8 9.3 9.1 Soothe 9.5 9.1 - Jangguchae 9.3 - - Fowl of chicken 9.3 9.0 - Scoop 10.0 9.2 9.0 rhubarb 9.8 9.4 9.0 Jovan 9.5 9.0 - Macaw 10.8 9.7 9.0 violet 9.6 9.0 - garlic 13.6 11.3 10.8 Rat vine 9.3 9.0 - knotgrass 9.0 - - Jimo 9.5 9.1 - Spicy Wasabi 9.5 9.0 - Plantain 9.5 9.0 - Macmundong 9.7 9.2 tea 21.7 17.4 12.8 elecampane 9.3 - - A bullshit 15.2 13.0 11.2 Windproof 9.7 9.1 9.0 Cypress 15.9 13.7 10.6 ringworm 9.4 9.0 9.0 Nasturtium 11.8 10.5 9.8 Loquat 10.1 9.6 9.1 pasqueflower 13.6 10.7 9.4 Cedar 12.2 10.9 9.2 figwort 10.5 9.5 9.1 Japanese tree 12.8 11.0 9.8 Oleander 10.7 9.7 9.0 Sounder 17.4 14.2 11.0 Brother-in-law 10.3 9.0 - Pine tree 11.3 10.1 9.4 Widower 9.6 9.1 9.0 purslane 17.6 14.7 11.5 Round ginseng 10.2 9.3 9.0

Example 4 Preparation of Plant Essential Oil (Antibacterial Oil) from 11 Plants

Plant essential oils were prepared by steam distillation from the plants of Table 5, which are known to have bactericidal effects on various phytopathogens through various literatures.

Botanical Plant name Clove bud Eugenia caryophyllata Castor oil Ricinus communis L. Aniseed Pmpinella anisum Thyme Thymus vulgaris Eucalyptus leaf Eucalyptus globulus Patchouli leaf Pogostemon cablin Rosemary leaf Rosmarinus officinalis Rose geranium Pelargonium rosium Petitgrain Citrus aurantium amara Cinnamon bark Cinnamomum cassia Sassafras Sassafras albidum

Experimental Example 3: Evaluation of antifungal activity on plant essential oils

In order to evaluate the antifungal activity of 11 plant essential oil prepared in the above were evaluated for antifungal activity against the citrus black spot patterns pathogen (Diaporthe citri). The evaluation method was to drop the anti-fungal activity of each plant essential oil in 1000ppm / disc, 500ppm / disc, 100ppm / disc in accordance with the paper disc method and the results are shown in Table 6 below.

Evaluation results of antifungal activity of plant essential oils (growth-lowering ring diameter; mm) Botanical Treatment Concentration (ppm) 1000 500 100 Clove bud 19.3 16.5 12.4 Castor oil 20.9 17.5 13.1 Aniseed 14.6 12.3 10.2 Thyme 18.7 16.4 12.8 Eucalyptus leaf 13.9 11.7 10.6 Patchouli leaf 10.8 10.3 9.5 Rosemary leaf 11.2 9.9 9.3 Rose geranium 13.6 11.0 9.8 Petitgrain 14.2 11.8 10.4 Cinnamon bark 14.0 11.6 10.5 Sassafras 15.7 13.0 10.6

Experimental Example 4: Evaluation of antifungal activity by mixing three active ingredients

In order to more effectively enhance the bactericidal effect of the three selected active ingredients (Bacillus subtilis KB-401, tea tree extract, castor oil), the respective effective ingredients are used alone and the three effective ingredients have a certain ratio. The antifungal activity against citrus black spot pattern pathogens was evaluated when used as a mixture. Antagonist culture medium was cultured Bacillus subtilis KB-401 and added to the spore density of 1 × 10 ⁹ cfu / ㎖ or more, tea extract and castor oil was mixed in the same ratio.

Comparison of Antifungal Activity by Mixing Three Active Ingredients Active ingredient Treatment Concentration (ppm) 1000 500 100 Antagonist Culture 21.8 17.5 12.6 Tea Tree Extract 21.7 17.4 12.8 Castor Oil 20.9 17.5 13.1 Antagonist culture medium + tea extract 23.2 19.1 13.8 Antagonist culture medium + castor oil 22.9 18.3 14.2 Tea Tree Extract + Castor Oil 22.7 18.5 14.0 Antagonist Culture + Tea Extract + Castor Oil 25.4 20.2 16.5

Example 5 Preparation of Fungicides Further Containing Efficacy Enhancers

In order to enhance the bactericidal effect of the three active ingredients selected in Table 7, an adhesion enhancer, a sunscreen, and a moisturizer were added.

Adhesion enhancers were made to ensure the proper density of microorganisms at desired sites using viscous materials such as Avicel, CMC, Xanthan Gum, Rosin, Glycerol, and Gelatin. Dilutions were prepared for each concentration, and then mixed with three active ingredients, and the physical properties and stability of the product were compared. Gelatin 0.5% showed excellent physical properties and stability.

Sunscreens can effectively block effective microorganisms from UV rays by using various types of sunscreens used in cosmetics and industrial applications. Candidates include Octyl p-methoxycinnamate, 2- (2-Hydroxy-5-methylphenyl) benzotriazole, 2,4-Dihydroxybenzophenone, Bis (2,2,6,6-tetramethyl-4-piperidinyl) sebacate, Phenyl salicylate, 2 -Hydroxy-4-octyloxybenzophenone was used. Each sunscreen agent was treated by concentration and mixed with antagonistic spore solution. After comparing the UV blocking effect, it was found that the UV protection effect was superior to 2-Hydroxy-4-octyloxybenzophenone more than 2.5% and it was also effective for germination of antagonistic spores.

Moisturizers use various non-toxic moisturizers used in cosmetics to maintain proper humidity in the target area and allow spores of effective microorganisms to germinate. Candidates include hyaluronic acid, sodium hyaluronate, sodium 2-pyrrolidone-5-carboxylate, and glyceryl dimaltodextrin (Glyceryl). dimaltodextrin) and potassium cellulose succinate. Each moisturizer was treated by concentration and mixed with the antagonistic spore solution, and the moisturizing ability was confirmed. As a result, spore germination effect and sodium bipyrrolidone-5-carboxylate were 0.5% or higher. Moisturizing ability was found to be excellent.

Example 6: Preparation of acaricides further comprising physical property modifiers and stabilizers (selection of additives for formulation)

In order to formulate three active ingredients and potentiators of the present invention, the stability and preservation stability of the active ingredients are required.

Therefore, various nonionic surfactants were mixed in the mixture of the potentiator selected from Example 5 and the three active ingredients as concentration modifiers and stabilizers according to concentrations to observe physical property changes and to select formulation additives.

As a result, 2% polyoxyethylene monooleate as a physical property modifier, 3% polyoxyethylene dodecyl mono ether, 3% polyethylene glycol-400, ethoxylate caster When more than 2% of oil (Castor oil, ethoxylated) was mixed, there was no precipitation, layer separation, etc. of the active ingredient.

As a stabilizer, sodium benzoate, potassium sorbate, and sodium propionate were treated by concentration, and when 0.5% of potassium sorbate was mixed, the physical properties were not changed. It appeared to show stable physical properties.

Example 7 Preparation of Fungicides According to Final Formulation

Using the three active ingredients and potentiators, physical properties, and stabilizers selected from the above to prepare a fungicide according to the final formulation. Bacillus subtilis Kb-401 as an active ingredient was added to 1 × 10 ⁹ cfu / ㎖, tea extract of antimicrobial polysaccharides 40%, and castor oil of antimicrobial oil 40%. Adhesion enhancer Gelatin 0.5%, sunscreen 2-Hydroxy-4-octyloxybenzophenone 2.5%, moisturizer 0.5% sodium 2-pyrrolidone-5-carboxylate Mixed. Physical property modifiers are 2% polyoxyethylene monooleate, 3% polyoxyethylene dodecyl mono ether, 3% polyethylene glycol-400, ethoxylated castor oil ( Castor oil, ethoxylated) 2% was mixed, and stabilizer was prepared by mixing 0.5% potassium sorbate (Potassium sorbate).

Test of bactericidal effect in the packaging of the bactericide of the present invention

Experimental Example 5: Pavement test of Gangjeong-dong, Seogwipo-si

The fungicides prepared in Example 7 were tested for bactericidal effects against citrus black spots in Gangjeong-dong, Seogwipo-si, Jeju-do. The bactericidal effect assay in packaging was four times with leaf dilutions at 500-day and 1000-fold dilutions at 14-day intervals, and compared with dithianon hydrate (dithianon, 1000-fold) as a control. Pavement management was conducted according to the farming general seedling method, and the test plot was repeated for 2 weeks and 3 repetitions, and the survey method examined the incidence of the entire section per test plot.

Test drug Foot disease degree (%) valence
(DMRT) Control
(%) 1 repetition 2 repetitions 3 repetitions Average Example 7 (500 times) 3.7 5.2 3.9 4.3 b 84.5 Example 7 (1000x) 4.9 6.6 6.4 6.0 b 78.4 Dithianon Hydration (1000x) 1.7 2.3 3.2 2.4 b 91.4 No treatment 23.8 29.5 30.2 27.8 a -

CV (%) ----------------------------------------- 18.8%

As shown in Table 8, the control effect of 84.5% was shown at 500 times of Example 7, and the control effect of 78.4% was shown at 1000 times. Compared with the dithianon hydrate, the control drug, it was 500 to 6.9% weaker, but it showed a practical control effect as an eco-friendly agricultural material.

Experimental Example 6: Pavement Test of Sinrye-ri, Namwon-eup, Seogwipo-si

The fungicides prepared in Example 7 were tested for bactericidal effects on citrus black spots in the package of Sinrye-ri, Namwon-eup, Seogwipo-si, Jeju-do. The bactericidal effect assay in packaging was four times with leaf dilutions at 500-day and 1000-fold dilutions at 14-day intervals, and compared with dithianon hydrate (dithianon, 1000-fold) as a control. Pavement management was conducted according to the farming general seedling method, and the test plot was repeated for 2 weeks and 3 repetitions, and the survey method examined the incidence of the entire section per test plot.

Test drug Foot disease degree (%) valence
(DMRT) Control
(%) 1 repetition 2 repetitions 3 repetitions Average Example 7 (500 times) 4.5 3.5 3.1 3.7 b 84.8 Example 7 (1000x) 6.4 4.8 4.2 5.1 b 79.1 Dithianon Hydration (1000x) 2.3 1.2 1.3 1.6 b 93.4 No treatment 26.1 19.6 27.5 24.4 a -

CV (%) ----------------------------------------- 25.6%

As shown in Table 9, the control effect of 84.8% was shown at 500 times of Example 7, and the control effect of 79.1% was shown at 1000 times. Compared with dithianon hydration, a control drug, it was 500 to 8.6% weaker, but it showed practical control effect as an eco-friendly agricultural material.

Experimental Example 7: Namwon-ri, Namwon-eup, Seogwipo City

The fungicides prepared in Example 7 were tested for bactericidal effects on citrus black spots in Namwon-ri, Namwon-eup, Seogwipo-si, Jeju-do. The bactericidal effect assay in packaging was four times with leaf dilutions at 500-day and 1000-fold dilutions at 14-day intervals, and compared with dithianon hydrate (dithianon, 1000-fold) as a control. Pavement management was conducted according to the farming general seedling method, and the test plot was repeated for 2 weeks and 3 repetitions, and the survey method examined the incidence of the entire section per test plot.

Test drug Foot disease degree (%) valence
(DMRT) Control
(%) 1 repetition 2 repetitions 3 repetitions Average Example 7 (500 times) 3.1 4.5 4.0 3.9 b 85.5 Example 7 (1000x) 5.3 5.7 6.2 5.7 b 78.8 Dithianon Hydration (1000x) 1.4 1.8 2.5 1.9 b 92.9 No treatment 20.8 29.2 30.7 26.9 a -

CV (%) ----------------------------------------- 28.3%

As shown in Table 10, the control effect was 85.5% at 500 times of Example 7, and 78.8% at 1000 times. Compared with the dithianon hydrate, the control drug, it was shown to be 500 ~ 7.4% weaker, but it showed practical control effect as an eco-friendly agricultural material.

1 is a graph showing the sporulation rate with time of the Bacillus subtilis KB-401 of the present invention.

Claims (9)

In microbial fungicide using fruit tree disease antagonists, A microbial fungicide for controlling Citrus citri , which is a bactericidal active ingredient using Bacillus subtilis KB-401, KFCC 11388P. delete delete The microorganism fungicide of claim 1, further comprising a tea tree extract or turmeric extract as an antiseptic active ingredient. The microorganism fungicide of claim 1, further comprising, as an antiseptic active ingredient, any one or more plant extracts selected from the group consisting of garlic, broccoli, purslane, sesame, cypress, and pasqueflower. According to claim 1, Castor oil (Castor oil), Clove bud (Clove bud), a microorganism for controlling fruit orchard disease further comprising a vegetable essential oil extracted from at least one selected from thyme as a sterilizing active ingredient disinfectant. The method of claim 4, further comprising a vegetable essential oil extracted from at least one selected from castor oil (Castor oil), clove bud (Clove bud), time (Thyme) as an antiseptic active ingredient Microbial fungicides. delete delete

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