KR101332360B1 - Composition of dioscoreacea extract and microorganism for preventing storage disease of fruits - Google Patents

Abstract

The present invention relates to a composition for controlling citrus fungal diseases comprising a cheonsan extract and a microorganism strain or a culture solution of the microorganism strain, and more particularly, a citrus fruit having excellent antibacterial activity against penicillium pathogen, a fruit storage disease. As a composition for controlling fungal diseases, the present invention relates to a composition for controlling a phytic acid extract and a microbial strain. The Bacillus sp. Strain may be Bacillus velezensis or Bacillus amyloliquefaciens, and the strain of Burkholderia sp. It may be Burkholderia vietnamiensis.
Citrus fungal disease control composition of the present invention is more environmentally friendly than conventional organic synthetic pesticides, it can satisfy the safety of the environment and the needs of consumers.

Description

Composition of dioscoreacea extract and microorganism for preventing storage disease of fruits

The present invention relates to a composition for controlling citrus fungus diseases comprising extracts for cheonsan and microbial strains, and more specifically, mixed and treating microbial strains showing additional antibacterial activity to extracts for cheonsan having antimicrobial activity against citrus fungus-induced pathogens. The present invention relates to a novel citrus fungal disease control composition having high activity.

In fruits, diseases caused by Penicillium spp. Are not caused during the growing period of the crop, but are mainly caused by wounds in the process of harvesting, storing, and transporting the crops. 10-30%, and some underdeveloped countries make up more than 30%. In addition, if the disease occurs during the storage of fruits, such as pears, apples, citrus fruits, and grapes, which have high value added, the merchandise will be greatly degraded, resulting in economic losses.

Among citrus stockpiles, the loss caused by P. digitatum, which we commonly refer to as mixed blue mold, and by penicillium P. italicum, which causes blue mold, are lost during full storage. It accounts for more than 90% of the losses incurred. Among them, the most damaging disease during the storage of citrus fruits is green mold disease. Green fungal disease caused by P. digitatum causes millions of spores to form at the infected area and scatters, causing them to act as inoculum. Can grow at 6-37 degrees

On the other hand, P. italicum can grow at lower temperatures than P. digitatum and even at zero degrees. Among these two pathogens, P. digitatum, the penicillium app., Which causes fruit storage disease, has the strongest pathogenicity among the pathogens. Therefore, green mold disease control is very important.

The most effective control agents for the green and blue mold diseases, which are the main citrus storage rots, are organic biological pesticides such as sodium bicarbonate, Imazaryl (IMZ: imazalil), TBZ (thiabendazole), and pyrimethaneyl ( Single agents and mixtures of PYR: pyrimethanil), fludioxonil (FLUD) and sodium o-phenylphenate have been widely used, but pharmaceuticals for imazalil, thiabendazole and sodium o-phenylphenate With the development of resistant P. digitatum bacteria, the development of new control agents has been required.

In addition, the use of chemical synthetic pesticides in the control of plant diseases all over the world has been decreasing due to the Rio Convention, etc., and it is necessary to use low-toxic and eco-friendly pesticides to prevent the corruption of storage fruits due to the need for environment-friendly disease control methods. Development of the control methods used is becoming important.

 In Korea, it is reported that the drug for the control of citrus reservoirs is not activated, and water spraying 1,000 fold of thiophanate-methyl, an organic synthetic pesticide 10 days before harvesting, is effective for the control. However, this drug is also resistant to the development of resistant bacteria is very far from the situation. Therefore, there is a need for a new control method that can replace organic synthetic pesticides and solve problems such as the emergence of resistant bacteria.

As an example, jasmonic acid, amino acid, and its derivative beta-aminobutyric acid, which regulate plant growth, to improve the resistance of fruit to disease of P. digitatum Studies on physical bottle control methods such as the use of natural compounds such as and washing with UV and hot water are being conducted.

In addition, low-toxic and eco-friendly Penicillium control method has been attempted to control plant extracts and microorganisms.

For example, Patent Publication No. 10-0382164 (2003.04.26,) describes a Bacillus sp. Strain having antifungal activity isolated from soil, and is disclosed in Korean Patent Publication No. 10-2012-0030630. (2012.03.29.) Describes a novel Bacillus amylolyquiecaptions JBC36 strain that has excellent control against green and blue mold diseases of fruits.

However, the development of biopesticides derived from natural products or microorganisms is very few compared to the vast resources, and the development cost is cheaper and eco-friendly than chemical pesticides. Is required.

Patent Publication No. 10-0382164 (2003.04.26,) Publication No. 10-2012-0030630 (2012.03.29.)

In order to solve the above problems, the present invention as a composition for controlling environmentally friendly citrus fungus disease, an object of the present invention is to provide a natural pesticide by mixing a natural product extract and microbial strains. More specifically, the present invention is to provide a novel citrus fungal disease control composition having a high activity by mixing the microbial strain showing an additional antimicrobial activity to the extract for citric acid having antimicrobial activity to citrus fungal disease-inducing pathogens.

It is another object of the present invention to provide an environment-friendly composition for controlling citrus fungal diseases, which can replace organic synthetic pesticides, can be mass-produced, and has non-toxic properties.

In order to solve the above problems, the present invention provides a composition for controlling citrus fungus disease comprising a strain for antimicrobial activity, including an antimicrobial component protodioscin (protodioscin) extracts and antibacterial activity on citrus fungus.

More specifically, the present invention is an extract for cheonsan containing an antimicrobial component protodioscin (protodioscin); And strains of the genus Bacillus sp. Or strains of the genus Burkholderia sp. Showing antibacterial activity against citrus fungal diseases.

In the present invention, the Bacillus sp. Strain may be Bacillus velezensis or Bacillus amyloliquefaciens, and the strain Berkholderia sp. It may be Burkholderia vietnamiensis.

As described above, the composition for controlling the citrus fungus disease provided by the present invention has an advantage in that it is possible to control the citrus storage bottle more effectively by mixing an antimicrobial microbial strain with a heavenly extract containing protodioscin.

In addition, the present invention is capable of mass production and non-toxic properties can be environmentally friendly control, it is expected to replace the existing organic synthetic pesticides is expected to have high practical potential.

1 is a photograph showing the antifungal activity of medicinal plant extracts against Penicilliun digitatum.
Figure 2 is a photograph showing the results of confirming the mycelial growth for 2 weeks by inoculating the mycelial tip of P. digitatum in PDA medium containing the antimicrobial protodioscin 50, 100, 250, 500, 1,000 ppm concentration .

Hereinafter, with reference to the accompanying drawings will be described in detail including a preferred embodiment that can be easily implemented by those of ordinary skill in the art. In the drawings of the present invention, the sizes and dimensions of the structures are enlarged or reduced from the actual size in order to clarify the present invention, and the known structures are omitted so as to reveal the characteristic features, and the present invention is not limited to the drawings . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail to avoid obscuring the subject matter of the present invention.

The present invention for the development of a biopesticide for the control of citrus fungus disease caused by P. digitatum that has a great damage in the process of storage and transport after harvesting citrus fruits, microorganisms showing additional antimicrobial activity to the extract for cheonsan having antimicrobial activity to the storage pathogen It is to provide a new bio pesticide for the control of citrus fungus disease that can replace the existing organic medicine by mixing the strain.

The present invention is an extract for cheonsan containing an antimicrobial component protodioscin (protodioscin); And strains of the genus Bacillus sp. Or strains of the genus Burkholderia sp. Showing antibacterial activity against citrus fungal diseases.

The strain of the genus Bacillus sp. Is a representative Gram-positive bacterium that forms spores having heat resistance and dry resistance, and has many advantages in the formulation and formulation of biological agents, and in fact, microbial fungicides using B. subtilis have been developed. It is reported that B. amyloliquefaciens, which has recently been reported to be the same strain as B. velezensis, has the potential as a biological control agent against various phytopathogens. Can be used.

 In addition, Pseudomonas (Burkholderia) cepacia, P. glathei, and P. syrinae strains, which showed control against green fungi caused by citrus P. digitatum and blue fungi by P. italicum, have been suggested as excellent biocontrol agents. In the present invention, it can be used as an antibacterial component of the composition for controlling citrus fungus disease.

In the present invention, the Bacillus sp. Strain may be Bacillus velezensis or Bacillus amyloliquefaciens, and the strain Berkholderia sp. It may be, but not limited to, Burkholderia vietnamiensis, the Bacillus (Bcillus) strain or Burkholderia sp. Strains may be any strain that exhibits antimicrobial activity against citrus fungal disease. . ,

The citrus fungal disease refers to a green mold disease caused by penicillium digitatum and a blue mold disease caused by penicillium italicum, and is mainly caused by penicillium digitatum. Can be.

In addition, the composition may be used in the concentration range of the protodiosin content 50 to 2000ppm, it can be treated by varying the concentration range according to the control conditions.

The composition for controlling citrus fungus of the present invention can be used together with a dispersant or an adhesive. More specifically, the composition for controlling the citrus fungus disease may include a dispersant because it may dissolve in organic alcohols, but may not be completely mixed with water, so when sprayed with water, only the water may be sprayed or not sprayed at the correct concentration. In addition, when sprayed on the fruit may be further included a pressure-sensitive adhesive to the sprayed active ingredient to remain in the fruit to maintain the control effect.

Example

1. Identify and secure pathogens

For the study of the control of storage diseases caused by the genus Penicillium, Penicillium bacteria which were actually generated in fruit phase were isolated and identified. To separate pathogens from diseased fruit, isolate spores and mycelium from pathogens on citrus fruit surface aseptically, inoculate in PDA medium, grow 24 hours after incubation in 24 incubator, isolate potato and agar spores (PDA: Potato Dextrose Agar medium) and incubated in 24 incubator for 3 days. For the identification of the cultures, the form and color of spores growing on the medium were confirmed, and spores and mycelia were observed by microscopic examination at 400 times. As a result, it was possible to identify and isolate pure penicillium digitatum and penicillium italicum from infected citrus fruits.

 In addition to the two pathogens isolated above, five penicillium genera, as shown in Table 1, were used in the experiment from the Korea Agricultural Culture Collection Center (KACC) in order to secure the genus Penicillium strain causing storage diseases.

Predistributed Penicillium genus is a major Penicillium genus pathogen that occurs during storage of mushrooms, garlic and vegetables as well as high value-added fruits such as citrus fruits, pears, grapes and apples.

Penicillium spp . Source  ( KACC No .) Penicillium digitatum KACC41898 Penicillium italicum KACC43474 Penicillium expansum KACC40814 Penicillium solitum KACC40818 Penicillium polonicum KACC41353

2. Selection of antimicrobial active medicinal crops

In order to examine the antifungal activity against Penicillium digitatum, storage diseases in 32 medicinal plants, including Cheonsan, which are expected to contain useful antimicrobial substances, the degree of inhibition of mycelial growth was confirmed.

Each of the 32 plants was crushed and dried in a grinder, and then 100 g of 100% methanol was placed in 5 l of shaking culture for 24 to 48 hours, and the extract was filtered three times using gauze and filter paper. The filtered material was completely removed by using a rotary pressure reducer and lyophilized.

The lyophilized plant extracts were adjusted to 100-fold concentration and then placed on a paper plate, and then placed on a PDA medium coated with Penicillium digitatum, and assayed. Mycelial growth was inhibited in the extract of No. 18 in Table 2 and FIG. It could be confirmed. Table 2 shows the antifungal activity of medicinal plants at 5 mg / concentration against Penicillium digitatum.

(-: Promote growth, +: less than 25%, ++: 25 ~ 50%, +++: 50 ~ 75%)

3. Mass production of extracts

Cheonsan used in the present invention is a medicinal plant of the horse as its scientific name is Dioscorea nipponica Makino. Cheonsan used in the experiment was purchased from the commercial Chinese herbal medicine store, and such extract for mass production can be mass-produced by the following method.

1) 1kg of dried Cheonsan and 100% solvent methanol or butanol 500l in 500l reactor and shake culture for 24 to 48 hours.

2) The cultured extract is filtered through medicinal gauze five times.

3) The filtered material is removed from the solvent by using a 20l rotary depressurizer, and lyophilized in a 200l vacuum depressurizer.

4. Analysis of Active Ingredients of Chun-san Extract

Extracts of n-hexane, ethyl ether, chloroform, ethyl acetate, n-butatnol, and H2O were obtained from the extract for cheonsan by solvent fractionation, and their antimicrobial activity fractions were identified by agar diffusion method. Column chromatography was performed on the n-butatnol fractions that showed strong antimicrobial activity. The chloroform: methanol (100: 0 ~ 60: 40, V / V) is sequentially lowered through the silica gel open column (70 ~ 230 mesh, for column chromatography, Merck, Germany) to obtain the chloroform: methanol (80:20) fraction. The extracts were sequentially eluted with 100% Methanol using Sephadex LH-20 (column chromatography, Sweden), and the active fractions were assayed using the agar diffusion method.

 Columns were subjected to column chromatography on the n-butatnol fractions, and ODS Thin layer chromatography was performed on the fractions showing strong antimicrobial activity. The spots were identified using -anisaldehyde (Sigma, USA) to identify spots. Rf values were determined by assaying the activity, and 1 H- and 13 C-NMR analysis were performed. To determine the molecular weight of the purified and purified antimicrobial active material was measured by ESI-Mass, the structure was analyzed by measuring the 1H-1H COSY, HMBC spectrum, and the antimicrobial material was identified.

As a result of analyzing the active ingredient of the extract for cheonsan, it was confirmed that the active ingredient of the substance having antimicrobial activity was protodioscin.

5. Control of Citrus Green Fungal Diseases from Protodioscin Extracts

 end. Antibacterial Activity Assay on Medium

   Mycelial growth inhibitory activity against phytopathogenic bacteria of antimicrobial actives containing cheonsanyong was tested. The mycelial growth of P. digitatum was inoculated into PDA medium containing protodioscin, an antimicrobial active agent at 50, 100, 250, 500, and 1,000 ppm concentrations, and mycelial growth was confirmed for 2 weeks. A strong mycelial growth inhibition of 100% was shown (Figure 2, A: Control, B: 50 ppm, C: 100 ppm, D: 250 ppm, E: 500 ppm, F: 1,000 ppm).

Inhibition of mycelial growth of 50% at 100 ppm and more than 85% at 500 ppm of steroid saponin-based substance was observed in the medium, and more potent mycelial growth was observed in protodioscin.

I. Method to test control effect after inoculation of pathogen

One citrus unscathed (breed: early maturing) three needles with 1 mm to hurt the deep three replications, each insert 6 back, a spore suspension of P. digitatum spores 110 ⁵ / the citrus surface in a plastic container of 350 volume per Was inoculated. After 3 hours, 100, 250 and 500 ppm concentrations of 100 to 250 ppm of protodioscin were sprayed onto the citrus surface. After treatment, the incidence and the control value were investigated after storing for 23 days and 7 days in a sealed state.

^a) 0: Onset, 1: 1-5%, 2: 6-20%, 3: 21-50%, 4: 51% or more

 Antimicrobial plant extract Prortodioscin treatment showed 61.8% at 100 ppm, 63.2% at 250 ppm and 60.3% at 500 ppm.

Protodioscin of the present invention shows an excellent control value at a low concentration of 100 ppm, showing a high control ability.

6. Effect of Antibacterial Microbial Strains on Citrus Fungus Disease Control

1) The same experiment as described in 5.b above, but instead of the celestial extract, the filtrate of Bacillus velezensis (Bacillus velezensis) (Bacterial cycle Accession No. KACC 91217P), which is the strain belonging to the existing applicant of Bacillus, 1x10 ⁶ , 1x10 ⁷ , 1x10 ⁸ cfu / of the medium (optimal culture condition: molasses broth of pH 6) was sprayed on the citrus surface.

50.2%, 52.5% when treated with 1 × 10 ⁶ cfu / ml, 55.8% when treated with 1 × 10 ⁷ cfu / ml, and 60.5% when treated with 1 × ^{10 8} cfu / ml by treatment with the culture filtrate alone.

2) The same experiment as described in 5.b above, but the filtrate of Burkholderia vietnamiensis strain, Burkholderia sp. Strain possessed by the applicant instead of the celestial extract, 1 × 10 ⁷ , 1 × ^{10 8} , and 1 × ^{10 9} cfu / of media (optimal culture condition: molasses broth at pH 8) were sprayed onto the citrus surface.

53.1%, 59.4% when treated with 1 × 10 ⁷ cfu / ml, 71.9% when treated with 1 × ^{10 8} cfu / ml, and 90.6% when treated with 1 × ^{10 9} cfu / ml by culture filtrate alone.

7. Effect of Antimicrobial Microbial Strains and Celestial Extracts on Citrus Fungus Disease Control

1) The culture filtrate and celestial extract of Bacillus velezensis (Bacterial cycle No. KACC 91217P), which is a strain of the genus Bacillus, which the applicant has previously, to evaluate the utility of the mixed treatment of the antimicrobial microorganism strain and celestial extract Experiments were carried out by mixing treatments of 100 and 250 ppm, respectively, and using mixed solutions of 1x10 ⁷ cfu / of culture solution and 100, 250 ppm of celestial extracts, respectively. .

As shown in Table 3, 60 ~ 76% of the control value was shown by the mixed prescription, and showed an improved control value compared to the case of using the celestial extract alone.

2) Culture filtrate and celestial extract of Burkholderia vietnamiensis strain, a strain of Burkholderia sp. Possessed by the applicant, in order to evaluate the utility of the mixed treatment of the antimicrobial microorganism strain and celestial extract Experiments were carried out by mixing treatment with a composition of 100 and 250 ppm, respectively. Also, experiments were carried out under the same conditions as 5, using a mixture of 1x10 ⁸ cfu / of culture solution and 100, 250 ppm of celestial extracts.

As shown in Table 4, 72 ~ 94% of control value was shown by mixing prescription, which showed improved control value compared to the case of using celestial extracts alone. Compared to the equivalent or more effective, it shows the superiority of the mixed prescription effect, and considering the fact that the pass control price of eco-friendly microbial pesticides in Korea is only 50%, The combination of celestial extracts and antibacterial strains is expected to increase the possibility of environmentally friendly control in the future.

Claims (6)

Extract for cheonsan containing an antimicrobial component protodioscin; And a strain of Bacillus sp. Or Burkholderia sp. Showing an antimicrobial activity against citrus fungal diseases. According to claim 1, wherein the Bacillus sp. (Bacillus sp.) Strain is Bacillus velezensis (Bacillus velezensis) or Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) characterized in that the composition for controlling citrus fungus The method of claim 1,
The strain of Burkholderia sp. (Burkholderia sp.) Is a control composition for citrus fungus, characterized in that Burkholderia vietnamiensis The method of claim 1,
The composition is citrus fungus disease control composition, characterized in that the content of the protodiosin is used in the concentration range of 50 to 2000ppm The composition of claim 1, wherein the composition is used with a dispersant or an adhesive. The method of claim 1,
Citrus fungus disease control composition characterized in that the fungus is caused by a green fungal pathogen (Penicillium digitatum)

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