KR102093197B1 - Method for controlling pathogenicity of pathogenic bacteria by treating 2S,3S-Butanediol - Google Patents

Abstract

The present invention relates to a method for controlling pathogenicity of pathogenic bacteria by treatment with 2S, 3S-butanediol, and through the present invention, 2S, 3S-butanediol is very useful in related industries by confirming that it can be used as an effective antimicrobial microbial agent. .

Description

Method for controlling pathogenicity of pathogenic bacteria by treating 2S,3S-Butanediol}

The present invention relates to a method for controlling pathogenicity of pathogenic bacteria by treatment with 2S,3S-butanediol.

Chemical pesticides are widely used in agriculture to control plant pathogens. However, in recent years, the use of chemical pesticides has raised many issues, including the possibility of causing environmental toxicity and antibiotic-resistant microorganisms. Accordingly, biological pesticides made from secondary metabolites from microorganisms or plants are more preferred, and 2,3-butanediol may be one of them.

2,3-butanediol is a chemical raw material that is widely used as a printer ink, perfume, moisturizer, softener, plasticizer, food and medicine material, etc., especially because it can be used as a raw material for synthetic rubber by chemical conversion to butadiene. As a representative biochemical raw material that can be replaced, it has recently attracted attention. In addition, the utilization of 2,3-butanediol is expected to increase in the agricultural field due to its physiologically active functions such as promoting crop growth.

2,3-butanediol, previously known as a plant immunity enhancing substance, has been reported to reduce the occurrence of plant diseases by enhancing plant immunity, but 2,3-butanediol directly inhibits pathogenicity of pathogenic bacteria. For the first time in the present invention, it was confirmed, and will be reported on this.

On the other hand, Korean Patent Registration No. 148242323 discloses a composition for inhibiting pathogens of the genus Fectobacterium including actinomycetes extract, and Korean Patent Registration No. 1589139 discloses a composition for controlling plant diseases and a method for manufacturing the same. , The method for controlling the pathogenicity of pathogenic bacteria by treatment with 2S,3S-butanediol of the present invention is not disclosed.

The present invention is derived from the above requirements, and the effect of reducing the occurrence of plant diseases caused by fungi, bacteria and viral pathogens by enhancing the immunity of plants by 2,3-butanediol, which is known as a plant immunity enhancing substance, has been Although reported, it was first confirmed in the present invention that 2,3-butanediol directly inhibits the pathogenicity of pathogenic bacteria.

In the present invention, as a result of treatment with 2R,3R-butanediol in the plant pathogen Pectobacterium carotovorum , rsmC , a modulator for reducing pathogenicity, and rsmA expression of the gene was increased, the control factor that increases the virulence and gacA rsmB, motility regulators and flhC flhD , and plant cell wall degrading enzymes pelA , pehA , pnl, prtW And It was confirmed that the expression of celS decreased, resulting in a decrease in the pathogenicity of Fectobacterium carotoborum.As a result of treatment with 2S,3S-butanediol in another plant pathogen, Ralstonia solanacearum, bacterial It was confirmed that the pathogenicity of green blight bacteria was reduced.

Also, Pseudomonas aeruginosa ), bacterial pneumoniae ( Klebsiella pneumoniae ), Staphylococcus aureus , and Acinetobacter baumannii by treatment with 2R,3R-butanediol or 2S,3S-butanediol It was confirmed that the pathogenicity was reduced.

Accordingly, the present invention was completed by confirming that 2R,3R-butanediol or 2S,3S-butanediol can be used as an antibacterial microbial agent without changing the density of bacteria with a differential effect from existing antibiotics through the present invention.

In order to solve the above problems, the present invention provides a composition for reducing pathogenicity of plant pathogens containing 2S,3S-butanediol as an active ingredient.

The present invention relates to Pectobacterium carotoborum (Pectobacterium), which is the causative agent of plant soft rot exposed to 2,3-butanediol either directly or in a gaseous state. carotovorum subsp. carotovorum , As a result of inoculating Pcc21) strain into potatoes or Arabidopsis, in particular, in the case of Pcc21 treatment exposed to 2R,3R-butanediol (R-type), it was confirmed that the signs of soft rot were reduced. Also, Pseudomonas aeruginosa ), bacterial pneumonia ( Klebsiella pneumoniae ), Staphylococcus aureus , and Acinetobacter Baumani ( Acinetobacter) baumannii ) as a result of treatment with 2R,3R-butanediol or 2S,3S-butanediol in animal pathogens, the pathogenicity of animal pathogens is reduced, and as a result of treatment with 2S,3S-butanediol in bacterial green blight ( Ralstonia solanacearum ), bacterial It was confirmed that the pathogenicity of green blight bacteria was reduced. Therefore, 2R,3R-butanediol or 2S,3S-butanediol can be used as an effective antimicrobial microbial agent, which is very useful in related industries.

In addition, expression of budA and budC genes involved in the synthesis of 2S,3S-butanediol (S-type) in pathogenic bacteria Fectobacterium carotoborum (Pcc21) exposed to 2R,3R-butanediol (R-type) decreased. As a result, the S-type butanediol (S-type) synthesis is inhibited, and the production of pectinase, a plant cell wall degrading enzyme, which is a decisive factor of pathogenicity, is reduced. It is possible to provide information on the mechanism of regulation of 2,3-butanediol secretion.

1 shows a schematic diagram of an experiment for a method of inoculating and exposing 2,3-butanediol to vegetable rot pathogens.
Fig. 2 shows the results of symptom investigation in potato and Arabidopsis thaliana by 2R,3R-butanediol. (A) Potato slice pathogenicity test, (B) Arabidopsis pathogenicity test
3 is a result of confirming the inhibition of pathogenicity of vegetable soft pathogens (PCC21) by 2R,3R-butanediol. (A) Pathogenic control mechanism, (b) motility factor, pathogenic factor and pathogenic regulator gene expression survey, (c) motility measurement, (d) pectin-degrading enzyme activity measurement.
4 is a result of the investigation of the number and gene expression of the vegetable soft rot pathogen (PCC21) exposed to 2R,3R-butanediol in Arabidopsis thaliana. (A) The number of vegetable soft rot pathogens (PCC21) per leaf slice of Arabidopsis thaliana, (B) The amount of ribosome expression of vegetative soft rot pathogens compared to the amount of actin gene expression of Arabidopsis thaliana. (C) a vegetable soft rot Arabidopsis leaves impressions on the 2,3-butanediol Won Kyun (PCC21) and acetonitrile is raised dicarboxylate (budA) expression level of the control, (D) expression of pectin hydrolase (pelA).
FIG. 5 is a result of investigation of symptoms of vegetable soft pathogens (PCC21) by 2R,3R-butanediol, 2S,3S-butanediol, and meso-butanediol, which are isomeric forms of 2,3-butanediol. RR; 2R,3R-butanediol, SS; 2S,3S-butanediol, meso; A mixture of 2S,3R butanediol and 2R,3S-butanediol, BVCs, Bacillus volatile substances synthesizing 2R,3R-butanediol, Control; Untreated control
6 is a result of investigation of growth of vegetable soft pathogen (PCC21) and expression of pathogenic factor genes by treatment with 2R,3R-butanediol and 2S,3S-butanediol.
Figure 7 shows the genes budA and 2,3 encoding alpha-acetolactate dicarboxylase, an enzyme of the 2S,3S-butanediol synthesis metabolic pathway (A) and 2S,3S-butanediol synthesis metabolic pathway of vegetable soft pathogen (PCC21). -This is the result of gene expression investigation (B) of the gene budC encoding butanediol dehydrogenase.
8 shows the degree of pathogenicity inhibition according to the concentrations of 2R,3R-butanediol and 2S,3S-butanediol in vegetable soft rot pathogens. S; 2S,3S-butanediol, R; 2R,3R-butanediol, 1= 1 μM, 0.1=0.1 μM, 0.01=0.01 μM 2,3-butanediol concentration, BTH is a substance that suppresses disease by enhancing plant immunity, Con; Control without 2,3-butanediol treatment
9 shows the degree of inhibition of pathogenicity according to the concentration of 2R,3R-butanediol. The upper drawing shows the survival rate (%) of pathogens when 2R,3R-butanediol is treated by concentration, and the lower drawing shows the survival rate of pathogens over time when 2R,3R-butanediol is treated by concentration (%).
FIG. 10 is an insect model by 2R,3R-butanediol using a honeybee fantail moth. This is the result of confirming the inhibition of pathogenic bacteria of veterinary pathogens.
11 shows the inhibition of pathogenicity of bacterial green blight bacteria by 2S,3S-butanediol.
Figure 12 is by 2S,3S-butanediol using the honey bee sprout moth Veterinary pathogen pathogenic inhibition is shown.
13 shows the degree of inhibition of pathogenicity of Pseudomonas aeruginosa according to the concentration of 2S,3S-butanediol.

In order to achieve the object of the present invention, the present invention is a plant pathogen by treating 2R,3R-butanediol (2R,3R-butanediol) or 2S,3S-butanediol (2S,3S-butanediol) to a plant pathogen pathogenic regulator, It provides a method for reducing the pathogenicity of plant pathogens comprising the step of regulating the expression of a gene encoding a motility regulator or a plant cell wall degrading enzyme protein.

In general, pathogenic regulation of plant pathogens reduces or enhances pathogenicity by regulating the expression of genes encoding them by various regulators such as pathogenic regulators, motility regulators, or plant cell wall degrading enzymes. The regulation of the pathogenic factor of the vegetable soft pathogen (PCC21) known in the previous paper is reduced by inhibiting the plant cell wall degrading enzyme when the regulator RsmA increases, and the amount of RsmA is suppressed by the other regulator, rsmB. Therefore, as the amount of rsmB increases, the amount of RsmA decreases and the activity of plant cell wall degrading enzymes cannot be suppressed, resulting in strong pathogenicity. In addition, pathogenic control is related to the motility of vegetable soft pathogens. When FlhCD, a motility regulator, increases, motility becomes active and affects pathogenicity. In addition, FlhCD, a motility modulator, influences pathogenicity by activating the pathogenic modulator rsmB through another modulator, GacA. This motility modulator FlhCD complex is inhibited by the modulator RsmC. RsmC can also reduce pathogenicity by directly increasing the expression of RsmA.

In the method according to an embodiment of the present invention, the pathogenic regulator coding gene is gacA , rsmA , rsmB , rsmC , pelA , pehA , pnl , prtW or celS , and the motility regulator coding gene is flhC or flhD , and the plant cell wall The degrading enzyme coding gene is pelA , pehA, pnl, prtW, or celS , but is not limited thereto.

In the method according to an embodiment of the present invention, the rsmC And The expression of the rsmA gene is increased, gacA , rsmB , flhC , flhD , pelA , pehA , pnl , prtW And celS The expression of the gene is reduced to reduce the pathogenicity of plant pathogens, but is not limited thereto.

In the method according to an embodiment of the present invention, the plant pathogen may be Pectobacterium carotovorum , Dikeya dadantii , or Ralstonia solanacearum . , Is not limited thereto. In particular, in Dikeya dadantii , the genes budA and 2,3-parts encoding alpha-acetolactate dicarboxylase, an enzyme in the synthetic metabolic pathway of 2S,3S-butanediol by treatment with 2R,3R-butanediol. It was confirmed that the expression of budC, the gene encoding tanediol dehydrogenase, was suppressed, thereby reducing pathogenicity (FIG. 7).

In addition, the present invention provides a composition for reducing pathogenicity of plant pathogens containing 2R,3R-butanediol (2R,3R-butanediol) or 2S,3S-butanediol (2S,3S-butanediol) as an active ingredient.

The composition includes 2R,3R-butanediol (2R,3R-butanediol) or 2S,3S-butanediol (2S,3S-butanediol) as an active ingredient, and the 2R,3R-butanediol (2R,3R-butanediol) or 2S ,3S-butanediol (2S,3S-butanediol) can reduce the pathogenicity of plant pathogens by treating plant pathogens.

In the method according to an embodiment of the present invention, the plant pathogen is Pectobacterium carotovorum , Dikeya Dadanti (Dikeya dadantii ) or Ralstonia solanacea room (Ralstonia solanacearum ), but is not limited thereto.

In the method according to an embodiment of the present invention, the Pectobacterium carotovorum and Dikeya dadantii are pathogenic by 2R,3R-butanediol (2R,3R-butanediol) treatment. It is reduced, Ralstonia solanacearum (Ralstonia solanacearum) is reduced pathogenicity by 2S,3S-butanediol (2S,3S-butanediol) treatment, but is not limited thereto.

In the method according to an embodiment of the present invention, the 2S,3S-butanediol (2S,3S-butanediol) may reduce pathogenicity by regulating the expression of a gene encoding a pathogenic regulator protein of a plant pathogen, but is limited thereto. It doesn't work.

In the method according to an embodiment of the present invention, the pathogenic regulator gene may be rsmB ( regulator of secondary metabolites B ) or pehA ( polygalacturonase A ), but is not limited thereto.

In addition, the present invention provides a method of reducing the pathogenicity of animal pathogens by treating animal pathogens with 2R,3R-butanediol (2R,3R-butanediol) or 2S,3S-butanediol (2S,3S-butanediol).

In the method according to an embodiment of the present invention, the animal pathogen is Pseudomonas aeruginosa , bacterial pneumonia ( Klebsiella pneumoniae ), Staphylococcus aureus , or Acinetobacter baumannii , but is not limited thereto.

In addition, the present invention provides a composition for reducing pathogenicity of animal pathogens containing 2R,3R-butanediol (2R,3R-butanediol) or 2S,3S-butanediol (2S,3S-butanediol) as an active ingredient. The composition includes 2R,3R-butanediol (2R,3R-butanediol) or 2S,3S-butanediol (2S,3S-butanediol) as an active ingredient, and the 2R,3R-butanediol (2R,3R-butanediol) or 2S ,3S-butanediol (2S,3S-butanediol) can reduce the pathogenicity of animal pathogens by treating animal pathogens.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

A. 2R,3R-butanediol treatment of pathogenic bacteria expression control method

purpose

Control of pathogenic bacteria by controlling the pathogenicity of animal/plant pathogenic bacteria by treatment with butanediol isomers 2R,3R-butanediol and 2S,3S-butanediol isomers

Materials and methods

1. Control of pathogenicity of plant pathogens by treatment with 2R,3R-butanediol

(1) Vegetable soft pathogen, a plant pathogen ( Pectobacterium carotovorum strain PCC21, hereinafter PCC21) Method of exposing 2,3-butanediol to bacteria and inoculation method

(A) Plant pathogen culture method

The vegetable soft pathogen PCC21 was a single colony grown in Luria-Bertani (LB; 1% tryptone, 0.5% yeast extract, 1% NaCl) solid medium was inoculated into the LB liquid medium, followed by inoculation at 30° C. at 250 rpm for 16 hours.

(B) 2,3-butanediol exposure method

Two methods of exposing the 2R,3R-butanediol of vegetable soft pathogens to bacteria were directly put into the bacterial culture medium and treated in a gaseous state using an I-plate. The direct treatment method was to put 5ml of the bacterial culture solution prepared in (A) into a 50ml tube, add 2R,3R-butanediol at a concentration of 1uM, incubate at 30°C for 5 hours, then centrifuge to discard the supernatant, and collect and use the precipitated bacterial culture. . In addition, the method of treating in a gaseous state using an I-plate was plated with 2 ml of a bacterial culture solution cultured by the method of (A) on a small Petri dish (60 mm dish) in which LB agar medium was poured. As shown in Fig. 1, a small Petri dish inoculated with a vegetable softening pathogen was put on only one side of the I-plate, and 100 μl of 2,3-butanediol at a concentration of 1 uM was dropped on the other side. 2R,3R-butanediol was exposed to vegetable soft pathogens at 30° C. for 15 hours. 2R,3R-butanediol (Cat no. 237639-1G, Sigma) was used in the experiment.

(C) Inoculation method of vegetable soft pathogen (PCC21) in plants

Potato slice pathogen treatment method: Potatoes were cut into 0.5mm thick, immersed in 30% NaOCl for 10 minutes, and washed 5 times with sterilized water. It was prepared by covering it with 3M paper moistened so as not to dry out. The prepared potatoes were placed on a 125mm square plate with 3M paper, and the vegetable soft pathogen (PCC21) exposed to 2,3-butanediol ^{was diluted to 10 5} in the middle of the potato, and 10 μL was dropped and inoculated. To maintain humidity, the lid was covered and only the edges were covered with plastic wrap. Symptoms were observed at 30° C. for 24 hours.

Method for treating pathogens in Arabidopsis thaliana: Arabidopsis thaliana was prepared by culturing in soil for 3 weeks, and PCC21 exposed to 2R,3R-butanediol was prepared at 10 ⁸ cfu/ml and inoculated by spray method. After ensuring that 100% humidity was well maintained, symptoms were observed for 24-48 hours.

(2) gene expression investigation

Gene expression was investigated by qRT-PCR method by extracting RNA of vegetable soft pathogen (PCC21) exposed to 2R,3R-butanediol. RNA was extracted by the method of RNeasy Plus mini Kit (Cat. No 74134, Qiagen), and qRT-PCR was performed using Superscript III First strand cDNA synthesis kit (Cat. No 18080051, Invitrogen) and iQ SYBR GREEn Supermix (Cat. No 170- 8880, BioRad) was used.

Plant cell wall degrading enzyme (PCWDE), a pathogenic gene of vegetable soft pathogens (PCC21), pectin degrading enzymes ( pelA , pehA , and pnl ), protease ( prtW ), and fibrinolytic enzyme ( celS ) and regulators that regulate the expression of pathogenic factors gacA , rsmA , rsmC And rsmB In addition, the expression of flhCD , a motility-related gene related to pathogenicity, was also investigated (Fig. 2). In addition, the expressions of the 2S,3S-butanediol synthetic metabolic pathway genes budA (alpha-acetolactate dicarboxylase) and budC (2,3-butanediol dehydrogenase) genes were also investigated. The primers used for gene expression investigation are shown in Table 1.

(3) Investigation of plant cell wall pectin hydrolase activity

In order to measure the pectin hydrolase activity, a Pel (pectate lyase) reaction solution containing 0.1M Tris-HCl (pH8.5), 2.2mM CaCl ₂ and 5.75mg/ml of PGA (polyglacturonic acid) was prepared. 990 μl of Pel reaction solution and 10 μl of vegetable soft pathogen culture solution were mixed well and reacted at room temperature, and the change in absorbance was measured for 10 minutes at a wavelength of 235 nm using a spectrophotometer (Activity = A ₂₃₅ h ^-1 *OD600nm ^-1) . Converted). Vegetable soft pathogens used in the reaction include vegetable soft pathogens (PCC21) cultured for 16 hours in LB liquid medium (PCC21), added to 5 ml of new LB liquid medium, and exposed to 2R,3R-butanediol for 9 hours, and 2R,3R -A control that was not exposed to butanediol was prepared and the activity was compared.

(4) Investigation of motility of vegetable soft pathogen

In order to confirm the motility of vegetable soft pathogens, a motility irradiation medium was prepared by putting 0.3% agar in LB medium on one side of the I-plate. In addition, 2R,3R-butanediol was dropped on the other side to confirm whether 2R,3R-butanediol had an effect on motility. As a control, water was added instead of 2R,3R-butanediol. In the center of the motility irradiation medium, 1 ul of the vegetative soft pathogen was added to the culture medium and incubated at 30° C. for 24 hours, and the degree of movement was measured.

2. Control of pathogenicity of animal pathogenic bacteria by treatment with 2R,3R-butanediol

(1) Animal pathogenic bacteria culture method and 2R,3R-butanediol exposure method

(A) Animal pathogenic bacteria culture method

Pseudomonas (Pseudomonas) as an animal pathogen aeruginosa ), bacterial pneumonia ( Klebsiella pneumonia ), Staphylococcus arueus , Acinetobacter Baumani (Acinetobacter) baumannii ) culture was used by inoculating a single colony grown in Luria-Bertani (LB) solid medium into LB liquid medium and culturing it at 37°C at 250 rpm for 16 hours.

(B) 2R,3R-butanediol exposure method

1.5ml of animal pathogenic bacteria pre-cultured in LB liquid medium was added to 150ml of LB liquid medium, and after incubation at OD600=0.3 at 250 rpm at 37°C, 2R,3R-butanediol was added and reacted for 6 hours to be used.

(2) Killing assay using honeybee fantail moth

The pathogenicity of animal pathogens was investigated using 3-4 year old bee fantail moth. Five animals were used for each treatment group, and three repetitions were performed for each treatment group. Animal pathogens exposed to 2R,3R-butanediol were ^{prepared to be 10 6} cfu/2 ul, and infected with a syringe into the inside of the epidermis of the honey bee moth. Incubation at 30° C. was observed to observe the survival rate every 6 hours.

Primer sequence used for gene expression Gene Primer (5 -> 3') (SEQ ID NO) pehA Forward CCA GCA TAG TTT GCC AGT TTA TC (1) Reverse GGT CAA TGG CGT TCG GTA TAG (2) pelA Forward CTG GAA GAG CAC CGG TAA AT (3) Reverse CCA GCA TAG TTT GCC AGT TTA TC (4) pnl Forward TAC CTG GGA GCT GCG TAA TA (5) Reverse ACG TAG GGC TTG GAA TCT TTA TC (6) prtW Forward CAT CAC GGC GAT CCA ATA TCT (7) Reverse GGC TAT TGC TGG TAG TGG TAT AG (8) celS Forward GTG CCG GTA GAT TTG ATG GA (9) Reverse CAC TGG ACG GCA GGT TAT AC (10) gacA Forward CAC TGG ACG GCA GGT TAT AC (11) Reverse ATG TCC ATC AGG ACA ACA TCT AC (12) rsmA Forward CAT GAT CGG CGA TGA GGT AA (13) Reverse TCT TCA CGG TGG ACA GAA AC (14) rsmB Forward CCG AGA TAG AGA CAT CGA AGA ATT AG (15) Reverse GCA GAA TAG AGC AGG TAG CAT AG (16) rsmC Forward CAT GAT CGG CGA TGA GGT AA (17) Reverse TCT TCA CGG TGG ACA GAA AC (18) flhC Forward ACT CAC GCT CAT CAA CCT AAA (19) Reverse TTC ATC CAG CAG TTG AGG TAT T (20) flhD Forward TAC TGG CGC AAC GCT TAA T (21) Reverse CAA TTT CAC CAT CTG CGG TAA AG (22) budA Forward CCA GCT TAC ACT CAG GGA ATT A (23) Reverse GCA ATC ACA CCA AAC GTC AG (24) budC Forward GCG AAC AGG CAT TGA TGA TTT (25) Reverse ACG TTG TCG ATC GCG TTT A (26)

B. 2S,3S - Butanediol Method of inhibiting pathogenic bacteria by treatment

purpose

Control of pathogenic bacteria by controlling the pathogenicity of animal/plant pathogenic bacteria by treatment with 2S,3S-butanediol isomers

Materials and methods

1. Control of pathogenicity of plant pathogens by treatment with 2S,3S-butanediol

(1) Plant pathogen Bacterial green blight ( Ralstonia solanacearum ) exposure to 2S,3S-butanediol and inoculation method

(A) Plant pathogen culture method

Bacterial green blight bacteria were cultured at 30° C. for 16 hours in CPG (0.1% casamino acid, 1% Peptone, 0.5% Glucose, 2% agar) solid medium.

(B) 2S,3S-butanediol exposure method

Bacterial green blight was prepared by dissolving bacteria cultured in CPG solid medium in CPG liquid medium well. Put 2S,3S-butanediol in CPG medium and spread on the solidified medium with 2S,3S-butanediol mixed with bacterial green and dry pathogen culture turbid solution. After incubation at 30° C. for 12 hours, the cultured cells are recovered in a solid medium. _{The collected cultured cells were diluted in a 1mM MgCl 2} solution containing 2S,3S-butanediol, reacted for 3 hours, and then used for plant inoculation. In the experiment, 2S,3S-butanediol (Cat. No. B1343, TCI) was used.

(C) Inoculation method of bacterial green blight bacteria in plants

2S,3S-butanediol-treated bacterial blight bacteria were drenched in the roots of tobacco plants cultured for 7 weeks at a concentration ^{of 10 8 cfu/ml.} Symptoms were observed for 3 weeks after inoculation with the bottle.

2. Control of pathogenicity of animal pathogenic bacteria by treatment with 2S,3S-butanediol

(1) Animal pathogenic bacteria culture method and 2S,3S-butanediol exposure method

(A) Animal pathogenic bacteria culture method

Pseudomonas (Pseudomonas) as an animal pathogen aeruginosa ), bacterial pneumonia ( Klebsiella pneumonia ), Staphylococcus arueus , Acinetobacter Baumani (Acinetobacter) baumannii ) culture was used by inoculating a single colony grown in Luria-Bertani (LB) solid medium into LB liquid medium and culturing it at 37°C at 250 rpm for 16 hours.

(B) 2S,3S-butanediol exposure method

1.5ml of animal pathogenic bacteria pre-cultured in LB liquid medium was added to 150ml of LB liquid medium, and after incubation at OD600=0.3 at 250 rpm at 37°C, 2S,3S-butanediol was added and reacted for 6 hours to be used.

(2) Killing assay using honeybee fantail moth

The honey bee moth is widely used in the study of pathogenicity and pathogenic factors for insect pathogenic nematodes, pathogenic fungi and bacteria. Compared to using vertebrate animals such as mice as model animals, it is easier to obtain the number of individuals required for the experiment, and the cost is low and the breeding cycle is short, so results can be obtained quickly. Used for pathogenicity test.

The pathogenicity of animal pathogens was investigated using 3-4 year old bee fantail moth. Five animals were used for each treatment group, and three repetitions were performed for each treatment group. Animal pathogens exposed to 2R,3R-butanediol were ^{prepared to be 10 6} cfu/2 ul, and infected with a syringe into the inside of the epidermis of the honey bee moth. Incubation at 30° C. was observed to observe the survival rate every 6 hours.

Example One. 2R,3R - Butanediol Exposed Vegetable softness pathogen ( Pectobacterium carotovorum PCC21, hereinafter PCC21) Bacteria are reduced in symptoms in potatoes and Arabidopsis.

Vegetable soft pathogen (Pectobacterium carotovorum PCC21) exposed to 2R,3R-butanediol and not exposed to potato and Arabidopsis thaliana were inoculated to observe symptoms. Potatoes were cut into 0.5mm thick, sterilized with NaOCl, and washed 5 times with sterilized water. 10 ul of ^{the bacterial culture solution diluted to 10 5} cfu/ml of PCC21 was dropped onto the potato and inoculated. The inoculated potatoes were placed in an airtight container to maintain 100% humidity and observed for 24 hours at 30°C. The area of the area where the symptom appears was measured, and the state of the symptom was observed, and the degree of the symptom was indicated.

2R,3R-butanediol-treated vegetable soft pathogen (PCC21) was inoculated with a small symptom area, but 2R,3R-butanediol-treated control (Control) was found to have a large disease area (Fig. 2). . PCC21 exposed to 2R,3R-butanediol ^{was prepared in Arabidopsis thaliana at a concentration of 10 8} cfu/ml and inoculated by spraying evenly. 100% humidity was maintained and symptoms were observed for 48 hours. Arabidopsis thaliana treated with 2R,3R-butanediol showed almost no symptoms, but in the control group, the granules melted and fell off the stem. It was found that the symptoms were reduced in PCC21 treated with 2R,3R-butanediol in both potato and Arabidopsis thaliana.

Example 2. 2R,3R - Butanediol is Vegetable soft rot (PCC21) It inhibits the expression of pathogenic genes and the activity of pectin hydrolase, a pathogenic factor of bacteria.

The pathogenicity of PCC21 is known to be due to the secretion of plant cell wall degrading enzymes. As for the hydrolase of plant cell wall, fibrinase (celS), pectinase ( pelA , pehA , pnl ), and proteolytic enzyme ( prtW ) are known. The expression of these genes is influenced by various regulators. The regulation of pathogenic factors of vegetable soft pathogens known in the previous paper is that when the regulator RsmA increases, the pathogenicity decreases by inhibiting the plant cell wall degrading enzyme. The amount of RsmA is suppressed by the other regulator, rsmB. Therefore, as the amount of rsmB increases, the amount of RsmA decreases and the activity of plant cell wall degrading enzymes cannot be suppressed, resulting in strong pathogenicity.

In addition, pathogenic control is related to the motility of vegetable soft pathogens. When FlhCD, a motility regulator, increases, motility becomes active and affects pathogenicity. In addition, FlhCD, a motility modulator, affects pathogenicity by activating the pathogenic modulator rsmB through another modulator, GacA. This motility modulator FlhCD complex is inhibited by the modulator RsmC. RsmC can also reduce pathogenicity by directly increasing the expression of RsmA. This control mechanism is shown in Figure 3 (a).

Therefore, in order to check whether the reduction in pathogenicity in the vegetable soft pathogen (PCC21) exposed to 2R,3R-butanediol affects the pathogenic control mechanism of Fig.3 (a), pathogenic modulators ( gacA, rsmA , rsmB , rsmC ), motility The expression of factors ( flhC, flhD ) and pathogenic factors ( pelA , pehA , pnl, prtW , celS ) was investigated, and the motility and plant cell wall degrading enzyme activity of vegetable soft pathogens were also measured. As a result, expression of rsmC and rsmA genes, which are regulators that reduce pathogenicity, increased, and gacA , which is a regulator of pathogenicity , and flhC , flhD, and plant cell wall degrading enzymes pelA , pehA , pnl , prtW , and celS . Was confirmed to have decreased (Fig. 3(b)), and rsmB, a modulator for increasing pathogenicity, was found to be as high as 1.1 when exposed to 2R,3R-butanediol compared to that of not exposed to 2R,3R-butanediol. It was confirmed that it appeared similarly, and through FIG. 6, it was analyzed that the expression level would decrease compared to the control after this time.

In order to confirm whether the decrease in the expression of the motility regulators flhC and flhD decreased the motility, an LB medium containing 0.3% agar, a motility investigation medium, was prepared on one side of the I-plate and was inoculated with a vegetable soft pathogen (PCC21). On the other side, 2R,3R-butanediol was dropped and then motility was investigated. As a control, water was dropped instead of 2R,3R-butanediol to investigate. As a result, it was confirmed that the motility of the vegetable soft pathogen exposed to 2R,3R-butanediol was reduced compared to the control as shown in (c) of FIG. 3. It was confirmed that the activity of the plant cell wall degrading enzyme PelA (pectate lyase) was also decreased in the vegetable soft pathogens exposed to 2R,3R-butanediol compared to the control (Fig. 3D).

In addition, on the 0th, 1st, and 2nd days after inoculation of the vegetable soft pathogen into Arabidopsis, the number of vegetable soft pathogens and the expression levels of the pathogenic factors aceto, dicarboxylase (budA) and pectin hydrolase (pelA) were investigated. Results The number of vegetable soft pathogens (PCC21) exposed to 2R,3R-butanediol was treated on the second day compared to that treated with vegetable soft pathogens (PCC21) not exposed to 2R,3R-butanediol (PCC21). It was confirmed that the amount of expression of and pathogenic factors was reduced (FIG. 4).

In addition, 2R,3R-butanediol, 2S,3S-butanediol, meso-butanediol, and 2R,3R-butanediol synthesized Bacillus volatile substances were treated to inhibit the pathogenicity of vegetable soft pathogens (PCC21). When treated with 3R-butanediol, it was confirmed that the inhibitory effect on pathogenicity of vegetable soft pathogens (PCC21) was remarkably high (FIG. 5).

In addition, 2R,3R-butanediol and 2S,3S-butanediol did not affect the growth of vegetable soft pathogen (PCC21) (top view), and gene expression was induced by 2S,3S-butanediol and pathogenic genes rsmB and pehA. Although the gene expression of the 2S,3S-butanediol synthesis gene budA was higher than that of the control, the gene expression did not increase at the beginning of treatment by 2R,3R-butanediol treatment, and the pathogenic regulator rsmB was treated at 3 hours after treatment with 2R,3R-butanediol. The expression of was decreased compared to the control, which was analyzed to induce a decrease in the expression of pathogenic factors after 3 hours (Fig. 6).

In addition, it has been reported that the 2S, 3S-butanediol synthesis metabolic mutant in Dikeya dadantii, another soft pathogen, has decreased pathogenicity. Therefore, as a result of investigating whether 2R,3R-butanediol has a pathogenic inhibitory effect by inhibiting the synthesis of 2S,3S-butanediol, 2R,3R-butanediol is an enzyme in the synthesis metabolic pathway of 2S,3S-butanediol, alpha-acetolactate dicarboxyl. and verified that the gene encoding the inhibitory raised budA and 2,3-diol di-section Taneja expression of the gene encoding the recombinase to budC to hydro (Fig. 7).

In addition, it was confirmed that 2S,3S-butanediol has a pathogenic inhibitory function at a low concentration (0.01 μM), and 2R,3R-butanediol has a pathogenic inhibitory function at various concentrations (1 μM and 0.01 μM) (FIG. 8 ).

Pseudomonas aeruginosa ) in 2R,3R-BDO was cultured at a concentration of 10 μM-100pM, and 10 ² CFU/2 μl was injected into the larvae of honey bees. After 24 hours, the survival rate of the honey bee fantail moth larvae increased by 7-14 times compared to the untreated group. After 24 hours, the survival rate of bee larvae in the 2R,3R-BDO 100nM treatment group was the highest at 86%. It was confirmed that pathogenicity was reduced when 2R, 3R-BDO was treated with Pseudomonas aerugosa (FIG. 9).

Example 3. Inhibition of pathogenicity of animal pathogens by 2R,3R-butanediol

It was confirmed that the pathogenicity of animal pathogens was inhibited by 2R,3R-butanediol. The animal pathogen used in the experiment was Pseudomonas aeruginosa ), bacterial pneumonia ( Klebsiella pneumoniae ), Staphylococcus aureus , Acinetobacter Baumani ( Acinetobacter) baumannii ) was used. Each animal pathogenic bacteria was exposed to 2R,3R-butanediol, and then infected with the honeybee fantail moth larvae, and the pathogenicity was investigated through the survival rate.

The honey bee moth is widely used in the study of pathogenicity and pathogenic factors to insect pathogenic nematodes, pathogenic fungi and bacteria. Compared to using vertebrate animals such as mice as model animals, it is easier to obtain the number of individuals required for the experiment, and the cost is low, and the breeding cycle is short, so results can be obtained quickly. This is a model system that is attracting attention as an alternative to model animals. Used for pathogenicity test. As a result, animal pathogens Pseudomonas aeruginosa , bacterial pneumonia ( Klebsiella pneumoniae ), Staphylococcus aureus and Acinetobacter baumannii were confirmed that pathogenicity was inhibited by 2R,3R-butanediol (FIG. 10).

Example 4. 2S,3S - Butanediol Caused by bacterial Green blight ( Ralstonia solanacearum ) Pathogenicity inhibition

Bacterial green blight bacteria exposed to 2S,3S-butanediol were inoculated with a 7-week-old tobacco plant root drench. After 3 weeks, it was confirmed that the withering symptoms were reduced compared to the control of bacterial green blight bacteria exposed to 2S,3S-butanediol (FIG. 11).

Example 5. Inhibition of pathogenicity of animal pathogens by 2S,3S-butanediol

It was investigated whether 2S,3S-butanediol also affects animal pathogens. The animal pathogen used was Pseudomonas aeruginosa ), bacterial pneumonia ( Klebsiella pneumoniae ), Staphylococcus aureus , and Acinetobacter baumannii were used. Each animal pathogenic bacteria was exposed to 2S,3S-butanediol, and then infected with the honeybee fantail moth larvae, and the pathogenicity was investigated through the survival rate. When exposed to 2S, 3S-butanediol in Pseudomonas aeruginosa, bacterial pneumonia, Staphylococcus aureus, and Acinetobacter Baumani, it was confirmed that the survival rate of the honeybee fantail moth was increased compared to the control (FIG. 12 ).

In addition, Pseudomonas aeruginosa according to the concentration of 2S,3S-butanediol aeruginosa ), as a result of examining the degree of inhibition of pathogenicity, Pseudomonas aeruginosa ) in 2S,3S-BDO was incubated at a concentration of 10 μM-100pM, and 10 ² CFU/2 μl was injected into the larvae of bee moth. After 24 hours, the survival rate of the larvae of honey bee larva increased by 7-12 times compared to the untreated group. After 24 hours, the survival rate of larvae of honey bee moth in the 2S,3S-BDO 100pM treatment was the highest at 73%. When Pseudomonas aeruginosa was treated with 2S, 3S-BDO, pathogenicity was reduced (FIG. 13).

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Claims (4)

As a composition for reducing pathogenicity of Ralstonia solanacearum containing 2S, 3S-butanediol (2S, 3S-butanediol) as an active ingredient,
The composition for reducing pathogenicity of Ralstonia solanacearum having the ability to reduce the withering symptoms of plants by reducing the pathogenicity of Ralstonia solanacearum by exposing Ralstonia solanacearum to the composition for reducing pathogenicity .
The method of claim 1, wherein the 2S, 3S-butanediol (2S, 3S-butanediol) is to reduce the pathogenicity by regulating the expression of the gene encoding the pathogenic regulator protein of Ralstonia solanacearum . Composition characterized by. The composition of claim 2, wherein the pathogenic regulator gene is rsmB ( regulator of secondary metabolites B ) or pehA ( polygalacturonase A ). A method for reducing the pathogenicity of Ralstonia solanacearum comprising the step of adding 2S, 3S-butanediol to Ralstonia solanacearum,
At this time, by exposing Ralstonia solanasearum to 2S, 3S-butanediol, the withering symptoms of plants are reduced by reducing the pathogenicity of Ralstonia solanasearum,
How to reduce the pathogenicity of Lalstonia Solanasearum.

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