KR100622694B1 - 200401 Streptomyces griseofuscus 200401 and a biocontrol agent of plant diseases with this strain - Google Patents

Abstract

The present invention is a Streptomyces griseofuscus 200401 strain, and relates to a plant disease control containing them, more specifically, pepper station pathogen (Phytophthora capsici) to cause severe damage when the pepper culture Streptomyces griseofuscus 200401 isolated from forest areas and non-arable soil And it is confirmed that the antibacterial effect against the anthrax ( colletotrichum acutatum ) relates to the invention to utilize the strain as a biopesticide (biopesticide, biocontrol agent) for plant disease control.

Streptomyces griseofuscus 200401 strain according to the present invention exhibits strong antimicrobial activity against pepper blight and anthrax, and thus can be used as an eco-friendly biopesticide, and as a result, it can be considered to have a high ripple effect on plant diseases such as pepper blight and anthrax. do.

Streptomyces griseofuscus, Red Pepper Bacterium (Phytophthora capsici), Anthrax (Colletotrichum acutatum), Biopesticides

Description

Streptomyces griseofuscus 200401 and a biocontrol agent of plant diseases with this strain}

The present invention is a Streptomyces griseofuscus 200401 strain, and relates to a plant disease control containing them, more specifically, pepper station pathogen (Phytophthora capsici) to cause severe damage when the pepper culture Streptomyces griseofuscus 200401 isolated from forest areas and non-arable soil And it is confirmed that the antibacterial effect against the anthrax ( colletotrichum acutatum ) relates to the invention to utilize the strain as a biopesticide (biopesticide, biocontrol agent) for plant disease control.

The type of plant disease caused by the late blight Phytophthora spp. And its degree of damage are enormous to all plant disease researchers. To date, more than 60 species of late blight are reported around the world. In Korea, 16 kinds of late blight are reported to cause economic crops. Phytophthora capsici , in particular, has a wider host range than other pests. Red peppers, watermelons, cucumbers, melons, and tomatoes are economic crops that have suffered enormous damage.

Plague germs become prevalent in low temperature and high humidity conditions, and once developed, they rapidly develop and cause enormous damage. In particular, the late blight is an aquatic bacterium, and the infection rate is very fast during rainfall. The pathogens overwinter in the pathogen residues in the soil in mycelium form. It has been classified as a fungus so far, but recently it has been newly classified as a protist. Currently, its control is metalaccil, dimethomorph, and pocetyl-A1, which are more selective for phytobacteria than general plant pathogenic fungicides. Drug efficacy is reported worldwide.

Anthrax caused by Colletotrichum actatum occurs in vegetables and fruits such as peppers, grapes, apples and peaches, causing massive damage. In particular, in the case of pepper, conventionally C. Anthrax caused by gloeosporioides has been studied, but recent changes in the major pathogens to C. actatum have been reported. Population variation of these pathogens requires the establishment of appropriate control strategies. Current anthrax control relies mainly on benzimidazole-based fungicides, including benomyl, and EBIs-based fungicides, including tebuconazole. However, the emergence of resistant strains to these fungicides are occurring all over the world, and in Korea, problems caused by the emergence of resistant strains of benzimidazole-based fungicides have emerged in various plant pathogenic fungi.

Therefore, today, a major change is needed in the strategy of plant disease control in view of the urgent solution of the above-mentioned problems and the increase of end-users' interest in the stability of foods due to the economic level and the high confidence in the stability of agricultural products. . In particular, it is urgently needed to establish a plant disease control strategy utilizing microorganisms and other biological resources without side effects.

Therefore, the present invention can greatly solve the difficulties in growing food crops (pepper) caused by late blight and anthrax, and at the same time to provide an environmentally friendly control method.

Therefore, the inventors of the present invention conducted a research project for establishing a simultaneous control strategy of phytophthora capsici and anthrax ( colletotrichum acutatum ) using microorganisms, wherein the microorganisms ( Streptomyces sp.) Isolated from forest and non-cultivated soils were pepper station. The present invention was completed by confirming that the bacterium ( Phytophthora capsici ) and anthrax ( colletotrichum acutatum ) have antibacterial activity.

The present invention as described above is characterized by using Streptomyces griseofuscus 200401 isolated from the forest area and non-cultivated soil. In addition, it is characterized by the use of Streptomyces griseofuscus 200401 as a control agent for pepper blight and anthrax.

The present inventors collected a large number of samples from forest soils and non-cultivated soils in the northwestern part of Gyeongbuk to secure microorganisms for the control of pepper blight and anthrax, which causes problems in the cultivation of food crops (pepper) and water agar medium (fungal separation) and Many microorganisms were separated using nutrient medium (bacterial separation) and selective media for actinomycetes, and then microorganisms showing high antimicrobial activity against pepper pest and anthrax were obtained. Isolated microorganisms are used indoors and greenhouses The activity was selected for assay through pot experiment.

The present inventors finally named the assay-selected microorganism as ' Streptomyces griseofuscus 200401', and deposited it with the accession number 'KFCC 11347P' at the Korea Microorganism Conservation Center.

Hereinafter, the present invention will be described in detail including specific experimental examples (examples).

1. Identification of Strains

Morphological characteristics of the final selected strains were incubated at 28 ° C. according to the International Streptomyces Project (ISP) regulations and examined under light microscopy and electron microscopy. The chain of spores showed a helical shape, and the surface of the spores was smooth and cylindrical (FIG. 1).

<Figure 1. Electron micrograph and colony photo of isolated microorganisms>

Final identification of the isolated microorganism was performed by sequencing of 16S rDNA, the method is as follows.

Isolates chromosomal DNA of this strain and then 27F (5'-AGAGTTTGATCATGGCTCAG-3 ', see SEQ ID NO: 1) and 1492R (5'-GGATACCTTGTTACGACTT-3'), which are common primers for 16S rRNA sequencing The primers were amplified by PCR under conditions of denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and polymerization at 72 ° C for 1 minute and 30 seconds. The amplified PCR product was subjected to 0.8% agarose gel electrophoresis, and then purified by using an ABI PRISM 3700 DNA Analyzer. The BLASTN program was used to identify and compare ribosomal RNA sequencing of GENEBANK and RDP (RNA database project). As a result, the isolated strain showed 99.7% homology with Streptomyces griseofuscus (see Figures 2 and 3, SEQ ID NO: 3 and 4).

<Figure 2. Strain 200401 (L) and S using BLASTN program. Comparison of nucleotide sequences of griesofuscus AY2076 (A)-see SEQ ID NOs: 3 and 4>

      <Figure 3. Strain 200401 based on the 16S rDNA nucleotide sequence of the isolated strain

Homology between Streptomyces griseofucus AY207605>

2. Fatty Acid Analysis

Gas chromatography (GC, HP 6890) was used to confirm the whole cell fatty acid composition of the strain. As a standard fatty acid, a calibration standard kit provided by Hewlett-Packard (HP) was used, and the cultured cells were incubated at 30 ° C. for 24 hours using Trypticase Soy Agar (BBL) medium. 1 mL of reagent 1 was added for saponification after cooling to water, and then cooled in running water at 100 ° C. for 30 minutes. 2 ml of reagent 2 was added for methylation, followed by reaction at 80 ° C. for 10 minutes and cooling in running water. For extraction, 1.25 ml of reagent 3 was added, and then slowly shaken at room temperature for 10 minutes, and then the lower layer solution was removed. Washing was slowly shaken for 5 minutes with 3 ml of reagent 4, and 500 µl of saturated NaCl solution was added to separate the layers. The separated supernatant was taken as an analysis sample. Analytical samples were analyzed using Sherlock, a strain identification program of MIDI company after analysis using GC (see Table 1 above). The major fatty acids were 17: 0 ANTE (57.9%) and 17: 0 ISO (29.5%).

3. Cultural (physiological) characteristics

In order to determine the physiological characteristics of this strain, starch hydrolysis, gelatin liquefaction, milk peptonization and coagulation ability, melanin pigmentation, phosphate hydrolysis, food The degree of flame resistance and growth temperature were investigated. The results are shown in Table 2 below.

Table 2 Physiological Characteristics

Survey Item  characteristic Starch hydrolysis + Milk Peptonation + Melanin pigment formation - Water Soluble Pigmentation - Nitrate reducing power -

4. Availability of carbon source and antibacterial activity according to carbon source

In order to investigate the carbon availability of the strain, using the Schilling-Gutribe agar medium, the carbon source capacity was investigated after 14 days incubation at 28 ℃. At this time, the growth of the bacteria and the antimicrobial activity against the above-mentioned plant pathogens of pepper blight and anthrax were observed. The results are shown in Table 3 and FIG.

Table 3. Carbon Availability and Antimicrobial Activity

Carbon source Usability Antibacterial activity (mm, low support)  Late blight  Anthrax D-glucose positivity 35 33 D-fructose positivity 27 26 Galactose positivity 20 18 Inositol positivity 18 24 Mannitol positivity 15 10 Raffinos voice - - Sucrose usually 5 5 Cellulose usually 3 7 Melibiose positivity 10 11 L-Arabinose positivity 12 11

<Antimicrobial activity in the medium using glucose as a carbon source>

5. Strain Culture

The strain is cultured in a medium containing nutrients that can be used by ordinary microorganisms. Glucose and nitrogen were used as the carbon source, peptone, yeast extract, and malt extract. The acidity of the medium was adjusted to 6.8. The culture was shaken under aerobic conditions, and the culture temperature was 28 ° C. Strain culture period for a typical experiment was 7 days.

The antimicrobial activity of Streptomyces griseofuscus 200401 isolated from the present invention was high in the bactericidal and anthrax. Hereinafter, the antibacterial activity assay process of the above strain will be described.

6. Assay of antimicrobial activity

(One) Streptomyces griseofuscus Culture of 200401

Species and production medium containing yeast extract 0.3%, malt extract 0.3%, peptone 0.5% glucose 1%, pH 6.8 was used. 100 ml each was dispensed into a 200 ml flask and sterilized for 20 minutes at 121 ° C. Species cultured by inoculating 0.01% of the culture medium precultured in this medium. Culture conditions were used by incubating for 7 days at 28 ℃, 150rpm.

(2) antimicrobial activity assay

The following is a result of the step-by-step assay of the strain activity. The first is an in-flight activity assay, the second is an assay of metabolites from the first active strain, the third is a bioassay in the room before the greenhouse experiment, and the fourth is a test of the possibility of packaging application testing. It is black for.

① 1st test-1st flight test

To determine the first antimicrobial activity of Streptomyces griseofuscus 200401 against pepper blight and anthrax, medium containing yeast extract 0.3%, malt extract 0.3%, peptone 0.5% glucose 1% and agar 1.5%, adjusted to pH 6.8 Was used. After stationary culture at 28 ° C. for 7 days, fumigation with chloroform was performed for about 1 hour to investigate the diffusion and antimicrobial activity of the metabolite in the medium, and the results are shown in FIG. 5.

<FIG. 5. First Antimicrobial Activity Assay of Streptomyces griseofuscus 200401>

② Secondary assay-Assay using secondary culture products

The secondary antimicrobial activity of Streptomyces griseofuscus 200401 against pepper blight and anthrax was used as a medium containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose, and adjusted to pH 6.8. Crude extracts were prepared by assaying freeze-dried drying at 28 ° C. for 7 days, and the final contents of the target pathogen culture medium were adjusted to 1% and 0.5%. As a result, the final antimicrobial activity against pepper germ disease was 76.2% and 61%, respectively, according to the crude extract content (Fig. 6). Final antimicrobial activity against red pepper anthrax was 88% and 75.3%, respectively, depending on the crude extract content.

③ 3rd test-indoor simple living experiment

Strain culture for tertiary antimicrobial activity against Streptomyces griseofuscus 200401 against red pepper blight was used as a medium containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose and adjusted to pH 6.8. After shaking culture at 28 ℃ for 7 days, the spores of the target pathogen were harvested, mixed with the culture strain 1: 1, and inoculated into the pepper fruit to test the infection by the pathogen, and the pepper fruit was used after surface sterilization using ethanol after purchase. It was. Inoculated pepper was incubated at 30 ° C. for 7 days and observed for disease. In the case of pathogen alone treatment group, the symptom of infection appeared in pepper fruit as a whole, but in the case of the mixed treatment group, no lesion was observed (FIG. 7).

<FIG. 7: Inhibition of reverse pathogens by a combination treatment of red pepper bacillus and Streptomyces griseofuscus 200401: 3 left photographs of pathogens alone and 3 right photographs of 'combination treatments'

④ 4th test-greenhouse test

Antimicrobial activity assay using adult plants in greenhouse against Streptomyces griseofuscus 200401 against red pepper blight bacillus contains 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose. Incubated. The culture conditions were shaken for 7 days at 28 ℃, harvesting the pathogens of the target pathogens were prepared by mixing the topsoil and mixed diseased soil (tops 80ml: 20ml, 10 ⁵ spore / ㎖). The concentration of Streptomyces griseofuscus 200401 was adjusted to 10 ⁷ cfu / ml and treated with the same amount as the suspension of pathogens (Treatment 1), and 50 ml (10 ⁷ cfu / ml) at weekly intervals in the pot where plants were planted. Three irrigation was performed. The disease was observed at 7 days intervals from 1 week after the last treatment. The final study of pathogen alone showed more than 80% of cases, but single treatment showed 13.6% of incidence and 84.3% of control. In case of three treatments, the incidence rate was about 5%, which showed more than 90% control value (FIG. 8).

<Fig. 8. Streptomyces griseofuscus 200401 in the greenhouse of adult pest control effect test: from the left ① control (pathogen), ② pathogens +200401 one-time treatment results, ③ pathogens +200401 three-time treatment results>

As described above in detail, it can be seen that Streptomyces griseofuscus 200401 strain according to the present invention exhibits strong antimicrobial activity against pepper blight and anthrax. Therefore, the new strain can be used as an environmentally friendly bio-pesticide, and as a result it is thought that it can give a high ripple effect on plant diseases such as pepper blight and anthrax.

Attach sequence list electronic file  

Claims (3)

Streptomyces griseofuscus 200401 strain (Accession No. 'KFCC 11347P'), characterized in that it has the control activity of pepper blight and anthrax. Plant disease control comprising Streptomyces griseofuscus 200401 strain (Accession No. 'KFCC 11347P'). The method of claim 2, Plant disease plant pepper disease control, characterized in that the pepper disease and anthrax.