KR101183538B1 - Novel paenibacillus polymyxa and microorganism agent comprising the strains for preventing phytophtora capsici of plants - Google Patents

Abstract

The present invention relates to a novel microbial agent for preventing the late blight of the plant, including the novel Panicacillus polymyx, more specifically, antagonistic reaction against the late blight bacteria, which are soil pathogens that occur in various crops, such as pepper, tomato, cucumber, watermelon The present invention relates to a novel F. Panybacilli polymyxa, a microbial agent comprising the microorganism, and a method for preventing a late blight of a plant using the microbial agent.

The Fanibacillus polymix yarn No. 72 of the present invention can be used as an excellent microorganism capable of preventing plant pests, which are a problem when growing house crops and flowers, such as pepper, tomato, cucumber, and watermelon.

By using F. Bacillus polymyxa No. 72 according to the present invention as a microorganism or as a microbial preparation, it is expected to stably grow crops in house farms or flower farms, thereby not only increasing farm income but also significantly reducing labor force.

Fannybacillus polymyxa, Plague, Microbial preparation, Phytophthora genus

Description

NOVEL PAENIBACILLUS POLYMYXA AND MICROORGANISM AGENT COMPRISING THE STRAINS FOR PREVENTING PHYTOPHTORA CAPSICI OF PLANTS}

The present invention relates to a novel microbial agent for preventing the late blight of the plant, including the novel Panicacillus polymyx, more specifically, antagonistic reaction against the late blight bacteria, which are soil pathogens that occur in various crops, such as pepper, tomato, cucumber, watermelon The present invention relates to a novel F. Panybacilli polymyxa, a microbial agent comprising the microorganism, and a method for preventing a late blight of a plant using the microbial agent.

Today, agriculture has been devoted to increasing food production through methods such as breeding, improvement of soil fertility, pest control, and weed removal, but persistent pesticide use and abuse are causing serious pollution of soil and river. In addition, the use of pesticides is currently being strengthened in the world as the pesticides remain in crops, causing problems such as toxicity, environmental pollution and toxicity to humans and livestock.

As concerns about such pesticides and interest in quality of life have increased, in recent years, biological control using microorganisms or plant extracts for crop pest control has become a concern.

Biological control is a method of selectively controlling a target bottle without affecting the environment, other plants, and killing by using microorganisms or plant extracts instead of using chemical pesticides that are harmful to the environment in response to increasing demand for safe agricultural products by consumers. .

Plague bacteria are highly pathogenic and host specific, but Phytophthora nicotianae and Phytophthora cinnamomi are known to invade over 1,000 host species. According to the result of establishing a classification system of domestically-produced bacterial pathogens conducted for five years from 1996, there were 8 to 14 kinds of bacterial pathogens. Among them, P. cactorum, P. capsici, P. drechsleri, P. infestans, and P. nicitianae are generated nationwide each year. Can be.

The most damaging crops have been eggplants, gourds and vegetables, but many of them are also endemic. In open-cultivated crops, late-stage outbreaks occur during the summer months of June and September, but house crops and flowers occur all year round. In particular, in nutrient cultivation, which has recently grown cultivated area, almost all pavement is infected. Often it is a big damage.

It is an object of the present invention to provide a microbial preparation for preventing late blight of plants using the novel Panicacillus polymix.

Another object of the present invention is to provide a Paenibacillus polymyxa No. 72 (KACC 91502P) isolated from the soil.

It is also an object of the present invention to provide a Panibacillus polymix yarn No. 72 (KACC 91502P) having a nucleotide sequence represented by SEQ ID NO: 1.

It is another object of the present invention to provide a microbial preparation comprising Penicillus polymix yarn No. 72 (KACC 91502P).

Hereinafter, the present invention will be described in detail.

The present invention is Paenibacillus polymyxa No. 72 (KACC 91502P) isolated from the soil.

Hereinafter, the present invention will be described in more detail.

In the present invention, the microorganisms showing antifungal reactions were first searched and secured from soils collected in various environments.

As a result, in the present invention, a strain showing an antagonistic response to the late blight of the plant was selected by a bioassay from a sample obtained by harvesting the root zone soil of lettuce packaging grown in the Deokyang-gu, Goyang-si, Gyeonggi-do. Morphological, biochemical and NCBI blast searches were performed on the selected strains to identify new strains of Paenibacillus polymyxa .

The selected strains were named Penivacillus polymyx No. 72, and deposited on November 10, 2009 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center (Accession No. KACC 91502P).

The strain selected according to the present invention exhibited 89% homology with Fanibacilli polymyxa and was designated as a new strain with different biochemical reactions in β-xylosidase, β-mannosidase and kanamycin.

According to the present invention, the F. Bacillus polymix No. 72 grows well on a TSB (Triptic Soy Broth) medium as a culture medium, it is possible to grow in the range of 15 to 30 ℃, it grows best at 20 to 25 ℃.

The growth pH range of the Pannibacilli polymix yarn No. 72 of the present invention is 6-9, best growing at pH 7.0-8.0.

The above-described F. Bacillus polymix yarn No. 72 according to the present invention is useful for the prevention of late blight of plants.

There is no restriction | limiting in particular in the plant which prevents a late blight bacterium by the Fanibacillus polymyxa No.72 which concerns on this invention, As said plant, vegetables of eggplant family and fruit family, Preferably pepper, tomato, cucumber, lettuce or watermelon, etc. have.

In order to examine the antimicrobial activity of phytopathogens of the Pannibacillus polymyxa No. 72 of the present invention, Phytophthora capsici , as well as Phytophthora infestans, Phytophthora dreschleri and Rhizoctonia solani, Sclerotinia homoeocarpa, Sclerotinia sclerotinia, Botyritis cinerea, etc. As a result, it showed high antibacterial activity against not only pepper blight, but also major crop pathogens such as gray mold, wilted disease and anthrax.

The F. Bacillus polymix yarn No. 72 isolated by the present invention was proved to be effective against red pepper germ in the antimicrobial pot assay, and the bioassay in the field test showed 78% control value compared to the untreated group. . In addition, all three kinds of pepper blight bacteria showed a preventive effect.

Therefore, F. Bacillus polymixosa No. 72 isolated and identified by the method of the present invention can be used as a microorganism itself for the prevention of plant pathogens, and can be prepared as a microbial agent for preventing plant pathogens. In addition, it can be used in the method of preventing plant pathogens using the F. Bacillus polymix yarn No. 72 and the microbial preparation comprising the same.

Fanibacilli polymix yarn No. 72 according to the present invention can be used in farms as a microorganism capable of preventing plant late blight, or a microbial preparation comprising the same.

In addition, the Fanibacillus polymix yarn No. 72 of the present invention can be used as an excellent microorganism that can prevent in advance the plant pests that are a problem when cultivating house crops and flowers such as pepper, tomato, cucumber, watermelon.

By using F. Bacillus polymyxa No. 72 according to the present invention as a microorganism or as a microbial preparation, it is expected to stably grow crops in house farms or flower farms, thereby not only increasing farm income but also significantly reducing labor force.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples merely illustrate the present invention in detail, and the scope of the present invention is not limited to the following examples.

Example 1 Isolation of Strains

In March 2007, soils in the root zone were harvested from lettuce packages grown in a facility house in Deokyang-gu, Goyang-si, Gyeonggi-do.

After drying the collected soil in a well-ventilated room, 1 g of the soil was quantified and placed in 10 ml of sterile water and suspended well. Then, 1 ml of the suspension was taken and diluted 10 times in a test tube containing 9 ml of sterile water. . After diluting the sterile water in which the suspension was diluted 200 ul each was put in the center of the NA (Nutrient Agar) solid plate medium and evenly pushed with a sterile spread and incubated in a 25 ℃ thermostat for 3 days.

Colonies formed in each shape were taken, labeled with strain numbers, and used for antifungal reactions.

Example 2 Identification of Strains

In order to identify the antagonistic strain against the pepper disease, Phytophthora capsici , which is a soil disease, NCBI blast was searched for sequencing of 16s rDNA, which was 99% consistent with Paenibacillus polymyxa .

For morphological identification of the strains, they were pretreated and observed with a scanning electron microscope (HITACHI S-3000), and the biochemical reaction assay was confirmed using the BCH card of the bacterial tester VITEK PRO. It was confirmed. The biochemical reactions of β-xylosidase, β-mannosidase, and kanamycin showed different biochemical reactions and 89% homology with Paenibacillus polymyxa .

As a result of electron microscopy, as shown in Figure 2 it was confirmed that the form of the strain between the simple liver.

The isolated bacterium was named Paenibacillus polymyxa No.72. The bacterium was deposited on November 10, 2009 with the accession number KACC 91502P at the National Institute of Agricultural Science.

[Table 1]

Biochemical Properties of Penibacillus Polymix No.72

One β-xylosidase - 3 Lysine - 4 Aspartate - 5 Leucine + 7 Phenylalanine + 8 L-proline - 9 ? -galactosidase + 10 L-pyrrolidoneyl - 11 α-galaltosidase + 12 Alanine - 13 Tyrosine + 14 β-N-acetyl-glucosaminedase - 15 Alafeproarylamidase - 18 Cyclodextrin - 19 D-galactose + 21 Glycozin + 22 Myo-inositol - 24 Methyl-A-D-Glucopyranoside + 25 Elman - 26 Methyl-D-xyloxide - 27 α-mannosidase - 29 Maltotriose + 30 Glycine - 31 D-mannitol + 32 D-Mannose + 34 D-Eleditose - 36 N-acetyl-D-glucosamine - 37 Palatinose + 39 L-Ramnose - 41 β-glucosidase + 43 β-mannosidase - 44 Phosphorylcholine - 45 Pyruvate - 46 α-glucosidase - 47 D-tagatose - 48 D-trihalose + 50 Inulin - 53 D-glucose + 54 D-ribose + 56 Putrescine - 58 NaCl6.5% - 59 Kanamycin - 60 Olindomycin - 61 Esculinhydrolase + 62 Tetrazolium red (-) 63 Phlomycin +

Example 3 Growth conditions test of Panibacillus polymix yarn No. 72

1) temperature

In order to confirm the proper culture temperature of the bacteria, 2 ml of pre-culture was inoculated into 200 ml of TSB (Triptic Soy Broth) medium and incubated at 180 rpm for 24 hours at 10, 15, 20, 25, 30, and 35 ℃ for each temperature. The culture medium was then irradiated with UV spectrophotometer (BECKMAM DU730) at a wavelength of 600 nm to compare the growth of the bacteria to determine the proper growth temperature.

As a result, as shown in Figure 3, the best growth when incubated for 48 hours at 20 ℃ and 24 hours at 25 ℃.

2) pH

In order to confirm the acidity suitable for the growth of the bacteria, 200 ml of TSB medium was adjusted to 3,4,5,6,7,8 with NaOH and HCl, and cultured at 180 rpm for 24 hours. Absorbance was investigated at meta 600 nm wavelength to compare the growth of bacteria.

As a result, as shown in Figure 4, the growth of the bacteria was generally well at pH 7 or more.

Example 4 Antimicrobial Activity Assay of Panibacillus Polymix No. 72

1) Antimicrobial Activity Assay for Optimal Carbon Sources

Minimal medium in order to determine the carbon source necessary for the growth of the strain _{(K 2 HPO 4 0.7%,} KH 2 PO 4 0.2%, glucose _{0.5%, (NH 4) 2} SO 4 0.1%, MgSO 4? 7H 2 O 0.01%) To prepare a carbon source was excluded from glucose and each carbon source was added at a concentration of 1% of the total solution and incubated at 25 ℃ for 180 hours at 180 rpm and then the culture degree was measured.

In order to assay the antimicrobial activity, the culture medium incubated with each carbon source in PDA medium was inoculated by 100 ul on a paper disk, and the antimicrobial activity was measured by culturing against phytophthora capsici .

As a result of the assay, as shown in Table 2, starch was used as a carbon source to cultivate the best strain, but when sorbitol was used as a carbon source, the antibacterial activity against red pepper bacterium was the greatest.

[Table 2]

2) Antimicrobial Activity Assay for Optimal Nitrogen Sources

In order to determine the nitrogen source necessary for the growth of the strain, 0.5% of each nitrogen source was added instead of 0.1% of (NH ₄ ) ₂ SO ₄ in a minimum medium, and cultured at 25 ° C. for 180 hours at 180 rpm, and then cultured. The antimicrobial activity was measured by inoculating 100 ul of the culture medium incubated with each nitrogen source in the PDA medium on a paper disk and incubating the pepper against pathogens.

When the yeast extract was used as a nitrogen source, the culture of the strain was the best and the antimicrobial production was the best.

[Table 3]

Example 5 Optimal Inorganic Salts and Antimicrobial Activity Assay

In order to identify the inorganic salts affecting the growth of the strain, the inorganic salts K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.2%, MgSO ₄ ? Instead of 0.01% of 7H ₂ O, 5 mM of each inorganic salt was added thereto, followed by incubation at 180 ° C. for 24 hours at 25 ° C., and the culture degree was measured. The antimicrobial activity was measured by inoculating 20 ul each of the culture medium added with each inorganic salt in PDA medium on a paper disc and incubating against late blight.

The results of the assay are described in Table 4 below. As shown in the table, when the MgCl ₂ was used as the inorganic salts, the culture and the antibacterial activity of the strains were large.

[Table 4]

Example 6 Antifungal Assay for Phytopathogens of Antagonist Microorganisms

In order to examine the antibacterial activity of the antagonistic microorganisms of the isolated antagonistic microorganisms, a highly viable pathogen cultured for 1 week was separated by 8 mm with cork bore, and then wound on the edge of PDA agar medium and incubated at 25 ℃ for 24 hours. Microbial culture was inoculated in a paper disk 20 ul and incubated in a 25 ℃ thermostat for more than 7 days to investigate the inhibitor that mycelia can no longer grow.

As the pathogen, Phytophthora capsici as well as Phytophthora infestans, Phytophthora dreschleri and Rhizoctonia solani, Sclerotinia homoeocarpa, Sclerotinia sclerotinia, Botyritis cinerea and the like were replaced by paper disc, and the culture results are shown in FIG. 6.

As shown in FIG. 6, the F. Bacillus polymixosa No. 72 according to the embodiment showed an antimicrobial reaction against the late blight of pepper, tomato and lettuce, as well as pepper blight, asy mildew disease, wilting disease, anthrax, etc. It was confirmed that exhibiting high antimicrobial activity against crop pathogens.

[Table 5]

Example 7 Antimicrobial Pot Assay of Antagonist Microorganisms

10 ml of antagonists incubated at 25 ° C and 180 rpm for 24 hours at the branch of peppers planted in the pot of greenhouse, and 5 ml of 5 x 10 ³ pcs / ml of the fungi of different pathogenic bacteria after 3 days Was inoculated into 3 weeks later and confirmed the presence or absence of late blight bacteria. As a result, no. The inoculation of the late blight after treatment with the microbial culture did not occur, and all three kinds of pepper blight were collected in comparison with the untreated.

TABLE 6

Example 8 Package Assay of Antagonist Microorganisms

Antagonist microorganisms cultured in TSB medium at 180 rpm for 24 hours were adjusted to a concentration of 1 x 10 ⁸ cfu / ml, and the red pepper seedlings grown on seedlings were immersed for 30 minutes and planted in large pots with a diameter of 30 cm. After incubating the red pepper bacterium in Petri dishes for 5 days, the aerial mycelium was removed with a glass rod and irradiated with fluorescent lamp light at 50 cm height at room temperature for 3 days to induce the formation of the zygomatic capsule. 30 ml of sterile water was added to a Petri dish, and the capsular bag was collected with a brush, and 5 x 10 ³ drips / ml were inoculated into 5 ml of peppers. After 3 weeks, the incidence of late blight was investigated. As a result, the red peppers treated with microorganisms had a 78% protection effect compared to the untreated.

After 3 weeks, the incidence of late blight was investigated, and the results are shown in Table 7 below.

[Table 7]

The above embodiments, test examples and drawings are merely for the purpose of describing the contents of the invention in detail, and are not intended to limit the scope of the technical idea of the invention, and the present invention described above is usually in the technical field to which the present invention belongs. As those skilled in the art, various substitutions, modifications and changes are possible within the scope without departing from the spirit of the present invention is not limited to the embodiments and the accompanying drawings, as well as the claims described below It should be determined that the claims are included in their equality.

Figure 1 shows the nucleotide sequence of the Favaibacillus polymix yarn No. 72 isolated in the present invention.

FIG. 2 is a scanning electron micrograph of the Favacillus polymix yarn No. 72 isolated in the present invention.

Figure 3 is a graph of the optimum growth temperature assay of the Favaibacillus polymix yarn No. 72 isolated in the present invention.

FIG. 4 is a graph showing an optimal growth pH assay of Pannibacilli polymix yarn No. 72 isolated in the present invention.

Figure 5 is a photograph showing the results of growth of antagonist microorganisms treated with panibacillus polymix yarn No. 72 and untreated control group isolated in the present invention.

Figure 6 is a photograph showing the results showing the antibacterial response to the late blight of the plant, Pannibacillus polymix yarn No. 72 isolated in the present invention.

<110> GyeongGi-Do <120> NOVEL PAENIBACILLUS POLYMYXA AND MICROORGANISM AGENT COMPRISING          THE STRAINS FOR PREVENTING PHYTOPHTORA CAPSICI OF PLANTS <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1426 <212> DNA <213> Paenibacillus polymyxa <400> 1 taatacatgc aagtcgagcg gggttagtta gaagcttgct tctaactaac ctagcggcgg 60 acgggtgagt aacacgtagg caacctgccc acaagacagg gataactacc ggaaacggta 120 gctaataccc gatacatcct tttcctgcat gggagaagga ggaaaggcgg agcaatctgt 180 cacttgtgga tgggcctgcg gcgcattagc tagttggtgg ggtaaaggcc taccaaggcg 240 acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300 ctcctacggg aggcagcagt agggaatctt ccgcaatggg cgaaagcctg acggagcaac 360 gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgccaggga agaacgyytk 420 gtagagtaac tgctcttgaa gtgacggtac ctgagaagaa agccccggct aactacgtgc 480 cagcagccgc ggtaatacgt agggggcaag cgttgtccgg aattattggg cgtaaagcgc 540 gcgcaggcgg ctctttaagt ctggtgttta atcccgaggc tcaacttcgg gtcgcactgg 600 aaactgggga gcttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc 660 gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tgggctgtaa ctgacgctga 720 ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga 780 tgaatgctag gtgttagggg tttcgatacc cttggtgccg aagttaacac attaagcatt 840 ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg acccgcacaa 900 gcagtggagt atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc 960 cctttgaccg gtctagagat agacctttcc ttcgggacag aggagacagg tggtgcatgg 1020 ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttat 1080 gcttagttgc cagcaggtca agctgggcac tctaagcaga ctgccggtga caaaccggag 1140 gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtactac 1200 aatggccggt acaacgggaa gcgaagsmgc gatgtggagc caatcctaga aaagccggtc 1260 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaattgcta gtaatcgcgg 1320 atcagcatgc cgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccacga 1380 gagtttacaa cacccgaagt cggtgaggta accgcaaggr gccagc 1426  

Claims (6)

Paenibacillus polymyxa No.72 (KACC 91502P) isolated from soil. The Paenibacillus polymyxa No.72 (KACC 91502P) according to claim 1, having a nucleotide sequence represented by SEQ ID NO: 1. Microbial preparation comprising Paenibacillus polymyxa No.72 (KACC 91502P) according to claim 1. The microbial preparation of claim 3, wherein the microbial preparation prevents late blight of a plant. 5. The microbial agent according to claim 4, wherein the late blight of the plant is Phytophthora . The microorganism preparation according to claim 4, wherein the plant is pepper, tomato, cucumber, watermelon or lettuce.

Images