KR102754986B1 - New Bacillus amyloliquefaciens strain and uses thereof - Google Patents

Abstract

The present invention relates to a novel Bacillus amyloliquefaciens strain and its use for controlling the disease, and more particularly, to a novel Bacillus amyloliquefaciens strain having an excellent antagonistic effect against gray mold disease and its use for controlling the disease.
The present invention can provide a composition for controlling gray mold disease that is effective in preventing the disease while having low side effects by utilizing the above strain, and can provide a biological control agent for stable production of crops and eco-friendly cultivation.

Description

{New Bacillus amyloliquefaciens strain and uses thereof}

The present invention relates to a novel Bacillus amyloliquefaciens strain and its use, and more particularly, to a novel Bacillus amyloliquefaciens strain having antagonistic properties against gray mold disease and its use for controlling the disease.

Botrytis cinerea, a common gray mold pathogen, is a fungal microorganism that causes gray mold disease in numerous crops, ranging from vegetable crops such as lettuce, cucumbers, tomatoes, strawberries, and pumpkins, to floricultural crops such as roses and lilies, to fruits such as blueberries and raspberries, and to high value-added crops such as ginseng. This gray mold pathogen has a very wide host range, and continuous secondary infections are possible through the flight of asexually reproducing spores, causing enormous damage to the crop harvest caused by this disease. In addition, since these crops can cause gray mold disease during storage or distribution after harvest, postharvest disease management causes great economic damage.

Pesticides are being developed to control this gray mold disease, but chemical pesticides can cause environmental problems such as environmental pollution. In addition, pesticide spraying in greenhouses can actually promote excessive humidity in the greenhouse environment, which can cause more damage from infection in some cases.

There have been attempts to use antagonistic microorganisms as an environmentally friendly method for control, but much effort is needed for practical use.

Accordingly, the inventors of the present invention isolated and identified a strain having excellent antagonistic activity against gray mold disease, and completed the present invention for the use of the strain for control thereof.

Patent Registration No. 10-2306405

In order to solve the above problems, the present invention aims to provide a novel Bacillus strain that can be utilized for pest control.

In addition, the present invention aims to provide a plant control composition capable of controlling gray mold disease using the above strain.

In addition, the present invention aims to provide a method for controlling gray mold disease using the above strain.

To achieve the above-mentioned purpose, the present invention provides a novel strain having excellent antagonistic action against gray mold disease.

The above strain may be a species belonging to the genus Bacillus, preferably Bacillus amyloliquefaciens , and more preferably Bacillus amyloliquefaciens strain assigned accession number KACC 92389P.

The above Bacillus amyloliquefaciens has a 16S rRNA sequence of sequence number 1.

The strain according to the present invention is recognized to have an excellent control effect against Botrytis sp., a pathogen causing gray mold disease, and specifically, may be recognized to have a control effect against Botrytis cinerea, but is not limited thereto.

In addition, the present invention can provide a composition for control comprising the novel Bacillus amyloliquefaciens strain and/or a culture solution thereof as an effective ingredient.

The above culture solution may include the culture itself in which the strain is cultured, the culture filtrate, or an extract extracted from the culture using an extraction solvent through a conventional purification method. The above extract may be an extract obtained by centrifuging the culture and extracting the supernatant using chloroform, ethanol, or the like as an extraction solvent, but is not limited thereto.

The above-mentioned pesticide composition is not limited, but is preferably for pest control of plants, more preferably for pest control of ginseng.

The above-mentioned pesticide composition is recognized to have an excellent pesticide effect against Botrytis sp., the causative agent of gray mold disease, and specifically, may be recognized to have a pesticide effect against Botrytis cinerea , but is not limited thereto.

The strain or the composition for control according to the present invention can not only promote plant growth, but also activate an enzyme helpful for promoting plant growth. The enzyme may be at least one of protease, siderophore, and cellulase, and is preferably protease or cellulase, but is not limited thereto.

The pesticide composition according to the present invention may additionally contain, in addition to the effective ingredient, a solvent, a mixer, an additive, etc. that are commonly used in the art to prepare a pesticide composition.

In addition, the present invention provides a method for controlling plants using a novel Bacillus amyloliquefaciens strain and/or a culture solution thereof, and a composition for controlling plants comprising the novel Bacillus amyloliquefaciens strain and/or a culture solution thereof as an effective ingredient.

The present invention can provide a composition for controlling gray mold disease that is effective in preventing the disease while having low side effects by utilizing a biological agent rather than a chemical agent. In addition, the present invention can provide a composition for controlling biological disease for stable production and eco-friendly cultivation of crops including ginseng.

Figure 1 shows the results of the process of selecting antagonistic microorganisms against the pathogen causing gray mold disease.
Figure 2 shows the results of antagonism against the pathogen causing gray mold disease after culturing the microorganisms of Figure 1 on a medium (results of testing the antagonistic effects of BC-046, BC-095, and BC-100 by dose in a counterclockwise direction from the 12 o'clock direction).
Figures 3a to 3c show the results of antibiotic gene assays for BC-046, BC-095, and BC-100, respectively.
Figure 4 shows the results of an enzyme activity assay of a microorganism having antagonistic properties against the pathogen causing gray mold disease.
Figure 5 shows a photograph of a microorganism identified by scanning electron microscope (SEM) that has antagonistic properties against the pathogen causing gray mold disease.
Figure 6 shows the weather conditions and gray mold disease incidence rate according to the time of drug treatment.
Figure 7 shows commercially available agents used for evaluation against the pathogen causing gray mold disease.
Figure 8 shows the results of the evaluation of the pathogen causing gray mold disease.
Figure 9 shows the results of control of five-year-old ginseng plants against the pathogen causing gray mold disease.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

<Example> Selection of strains with antagonistic properties against gray mold disease

To isolate strains having antagonistic properties against the causative agent of gray mold disease of ginseng, soil samples from the rhizosphere of healthy growing plants in the Cheorwon ginseng cultivation area were collected after July, when the frequency of gray mold disease is high. To isolate microorganisms from the collected samples, 1 g of the soil sample was suspended in 9 ml of sterile water, diluted to a concentration of 10 ^-5 in a conical tube, and spread on NA [Nutrient agar; beef extract 0.3% (w/v), peptone 0.5% (w/v), agar 1.5% (w/v), Difco, USA] medium, and cultured at 25℃ for 2 days. After culture, single colonies of microorganisms with different shapes and colors were visually separated using a loop, and then purified again to isolate 100 points. After testing the antagonism of 100 isolated microorganisms against Botrytis cinerea, the causative agent of gray mold disease, three strains (BC-046, BC-095, and BC-100) were selected for the second round.

<Experimental Example 1> Evaluation of pathogen suppression ability

To confirm whether the prepared microorganism exhibits antibacterial activity against the pathogen causing gray mold disease (Botrytis cinerea), the mycelial growth inhibition ability was evaluated through counterculture, and the results are shown in Fig. 1 or 2.

The causative fungus of gray mold was prepared by inoculating on PDA [Potato Dextrose Agar; potato starch 0.4% (w/v), dextrose 2.0% (w/v), agar 1.5% (w/v), Difco, USA] medium and culturing at 25℃ for 3 days. The prepared pathogen was isolated from the mycelial tip using a cork borer (diameter 5 mm) and used as an agar disk, and the test microorganism was inoculated on LB Broth [Tryptone 1.0% (w/v), Yeast extract 0.5% (w/v), sodium chloride 1.0% (w/v), Difco, USA] and cultured at 25℃ for 1 day. The culture was then inoculated onto 8 mm paper disks at 20 ㎕, 50 ㎕, and 100 ㎕ for the test. To determine whether antibacterial substances are secreted extracellularly, the culture filtrate and cells were centrifuged after destroying the cell wall using a sonicator and used as intracellular liquid (Fig. 2). The experimental results showed that BC-095 had the best activity in both extracellular and intracellular assays.

<Experimental Example 2> Evaluation of antibiotic genes of selected strains

As a result of testing antibiotic (fen A, fen D, fen E, ituA1, ituD, srfAA, srfAD) genes for three antagonistic microorganisms according to the present invention, it was confirmed that BC-046 had no antibiotic genes, while BC-095 and BC-100 each had four antibiotic genes (Figs. 3a to 3b to 3c). For the antibiotic gene test, 3 ml of the culture solution cultured in LB Broth for 24 hours was extracted using the Higene ^™ Genomic DNA Prep Kit (BIOFACT, Korea) to extract the chromosomal DNA of each strain. The amplification of each antibiotic gene was performed using the primers prepared in Table 1. PCR was performed using a CFX96 ^™ Touch Thermal Cycler (BIO-RAD, USA) at 95°C for 30 s denaturation, 35 cycles of 65°C for 30 s, and 72°C for 30 s, followed by extension at 72°C for 7 minutes. The PCR products were electrophoresed on a 1% agarose gel using 1× TAE (Tris-acrtate-EDTA) buffer for 30 minutes, stained with ethidium bromide, and confirmed by bands under a UV transilluminator.

Bacillus genus antibiotic gene screening primers number Primer order Antibiotics Size (bp) Sequence number 1 fenA-F
fenA-R CCT CGC TCC GCA TGA TCT TTT GG
CGG GAG CAC GGT GGC AAT GTG Fengycin 1574 2 3 2 fenD-F
fenD-R TTT GGC AGC AGG AGA AGT TT
GCT GTC CGT TCT GCT TTT TC 2000 4 5 3 fenE-F
fenE-R GTT TCA TGG CGG CGA GCA CG
GAT TCG CGG GAA GCG GAT TGA GC 1580 6 7 4 ituA1-F
ituA1-R CGC CCG TGA AGG AGC AGC CG
GCC AGG AAG CGG GGC TTC AC Iturin A 1445 8 9 5 ituA4-F
ituA4-R CTG CCT GCG TAT ATG ATT CCG GC
CCG TGA TGA TGC CGT TCT TCA ATC C 920 10 11 6 ituD-F
ituD-R ATG AAC AAT CTT GCC TTT TTA
TTA TTT TAA AAT CCG CAA TT 998 12 13 7 srfAA-F
srfAA-R GCC CGT GAG CCG AAT GGA TAA G
CCG TTT CAG GGA CAC AAG CTC CG Surfactin 1485 14 15 8 srfAD-F
srfAD-R CCG TTC GCA GGA GGC TAT TCC
CGC CCA TCC TGC TGA AAA AGC G 1239 16 17

<Experimental Example 3> Evaluation of active enzyme production of strain according to the present invention

A medium was created to confirm whether the microbial enzyme secretion was possible by forming a clear zone for the three selected strains, and the measurement was performed. As a result of the protease production evaluation, all three strains formed a clear zone, and the protease activity was in the order of BC-095, BC-046, and BC-100 (Fig. 4).

In addition, cellulase is a cellulose hydrolyzing enzyme that directly exhibits antibacterial activity by decomposing the cell wall (composed of cellulose) of Botrytis species, the causative agent of gray mold disease. The results of the cellulase production assay were confirmed in the order of BC-095, BC-100, and BC-046.

<Experimental Example 4> Isolation and identification of strain according to the present invention

To identify the selected strains, molecular biological and biochemical methods based on 16S rRNA phylogenetic analysis were used. For 16S rDNA sequence analysis, chromosomal DNA was extracted from 3 ml of the culture solution cultured in LB Broth for 24 hours using the Higene ^™ Genomic DNA Prep Kit (BIOFACT, Korea). Partial amplification of 16S rRNA was performed using universal primers, 27F and 1492R. PCR was performed using a CFX96 ^™ Touch Thermal Cycler (BIO-RAD, USA) at 95 °C for 30 seconds, followed by 35 cycles of denaturation at 95 °C for 30 seconds, extension at 65 °C for 30 seconds, and then annealing at 72 °C for 7 minutes. The PCR products were electrophoresed for 30 minutes on a 1% agarose gel using 1× TAE (Tris-acrtate-EDTA) buffer, stained with ethidium bromide, and confirmed by a UV transilluminator. The amplified band in the 1,600 bp range was confirmed and the base sequence was requested to Macrogen, Inc. The base sequence analysis results were analyzed using the BLASTN program at NCBI (https://www.ncbi.nlm.nih.gov/). For biochemical analysis of the strains, the VITEK2 compact (Biomerieux, France) system was used according to the product manual. Colonies of selected microorganisms were cultured in LB broth at 25℃ for 1 day, centrifuged, collected with a loop, and immersed in a BCL card in sterilized saline solution until the turbidity was adjusted to 2.0 McF. The immersed card was loaded into the product cassette and then read.

As a result of the isolation and identification, all selected strains were confirmed to belong to the genus Bacillus , and various species were confirmed as shown in Tables 2 and 3, and morphologically, they were confirmed to be rods (Fig. 5). In addition, BC-095, which showed the best antagonistic effect against the gray mold pathogen, was finally identified, and the results are shown in Table 4, and it was deposited with the National Institute of Agricultural Sciences and assigned the accession number KACC 92389P.

Results of genetic identification of antagonistic microorganisms causing ginseng gray mold disease No. Strains Primer Identification Homology(%) Origin 1 BC-046 27F
1492R Bacillus thuringiensis JC-3 99.5 Soil 2 BC-095 27F
1492R Bacillus amyloliquefaciens Tr02A 99.2 Soil 3 BC-100 27F
1492R Bacillus subtilis Lac02W 99.0 Soil

Biochemical characterization and identification results for each selected strain No. Blochemical test 046 095 100 1 BETA-XYLOSIDASE - + + 2 L-LYSINE-ARYLAMIDASE - - - 3 L-Aspartate ARYLAMIDASE - - - 4 Leucine ARYLAMIDASE - - - 5 Phenylalanine ARYLAMIDASE + + - 6 L-Proline ARYLAMIDASE - - - 7 BETA-GALACTOSIDASE - - - 8 L-Pyrrolydonyl-ARYLAMIDASE + + + 9 ALPHA-GALACTOSIDASE - + (-) 10 Alanine ARYLAMIDASE + - - 11 Tyrosine ARYLAMIDASE + (+) - 12 BETA-N-ACETYL-GLUCOSAMINIDASE + + - 13 Ala-Phe-Pro ARYLAMIDASE + + + 14 CYCLODEXTRINE + - - 15 D-GALACTOSE - - - 16 GLYCOGENE - - - 17 myo-INOSITOL - - - 18 METHYL-A-D-Glucopyranoside acidification - (-) + 19 ELLMAN + - - 20 METHYL-D-XYLOSIDE - - - 21 ALPHA-MANNOSIDASE - - - 22 MALTOTRIOSE (-) - - 23 GLYCINE ARYLAMIDASE - (+) - 24 D-MANNITOL - + + 25 D-MANNOSE - + + 26 D-MELEZITOSE - - - 27 N-ACETYL-D-GLUCOSAMINE + - - 28 PALATINOSE - - + 29 L-RHAMNOSE - - - 30 BETA-GLUCOSIDASE - + + 31 BETA-MANNOSIDASE - - - 32 PHOSPHORYL CHOLINE - - - 33 PYRUVATE + + + 34 ALPHA-GLUCOSIDASE (+) - - 35 D-TAGATOSE - - - 36 D-TREHALOSE + + + 37 INULIN - - - 38 D-GLUCOSE + (-) + 39 D-RIBOSE + - + 40 PUTRESCINE assimilation - - - 41 Growth in 6.5% Nacl - + + 42 KANAMYCIN RESISTANCE + - - 43 OLEANDOMYCIN RESISTANCE - (-) - 44 ESCULIN hydrolyse + + + 45 TETRAZOLIUM RED - + - 46 PLOMIXIN B RESISTANCE + - - Identification result B. thuringiensis B. amyloliquefaciens B. subtilis

Strains Primer Identification Homology(%) Origin BC-095 27F
1492R Bacillus amyloliquefaciens Tr02A 99.2 Soil

<Experimental Example 5> Evaluation of the effect of pest control treatment using strains according to the present invention

The control effect against gray mold disease was evaluated using the BC-095 strain according to the present invention. The evaluation method was to treat 5-year-old ginseng fields with water 4 times each before and at the beginning of gray mold disease outbreak, for a total of 8 times. The weather conditions and gray mold disease outbreak rate according to the pesticide treatment period in this experiment are shown in Fig. 6.

The pesticide treatment was carried out by freeze-drying the selected strain culture and then treating at a level of ^1x106 cfu/㎖, and commercially available pesticides and eco-friendly materials were treated at the indicated dosages. The pesticides used in this experiment are shown in Figure 7. The control value was calculated by the following formula,

Control rate (%) = X 100.

The experimental results are shown in Figs. 8 and 9.

As a result of the experiment, the control value of the BC-095 strain treatment group according to the present invention was confirmed to be 60.5%, and the control values of the chemical agent and eco-friendly material groups were 67.5% and 7.5%, respectively. In addition, it was confirmed that the control value of BC-095 was approximately 8 times higher than that of commercially available eco-friendly materials.

Name of depository institution: National Institute of Agricultural Sciences

Accession number: KACC92389P

Date of Consignment: 20211111

<110> Gangwon-do <120> New Bacillus amyloliquefaciens strain and uses it <130> 21-12120 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 1454 <212> DNA <213> Bacillus amyloliquefaciens <400> 1 gggaaggcgc tgcctataca tgcagtcgag cggacagatg ggagcttgct ccctgatgtt 60 agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120 aaaccggggc taataccgga tggttgtctg aaccgcacgg ttcagacata aaaggtggct 180 tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240 ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300 ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360 ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420 aacaagtgcc gttcaaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta 480 actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540 gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600 gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660 ggtgaattgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720 ctgacgctga ggagcgatag cgtggggagc gaacaggatt agataccctg gttgtccacg 780 ctgtaaacga tgagtgctaa gtgttagggg gtctccgccc tttagtgctg cagctaacgc 840 attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900 gcccgcacaa gcggtggagc atgtggtcta attcgaagca acgcgaagaa ccttaccagg 960 tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcggggggca gagtgacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140 aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200 cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260 tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagcagga atcgctagta 1320 atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accacgagag tttgtaacac ccgaagtcgg tgaggtaacc tttaggagcc agccgccgaa 1440 gtggacaggg gggg 1454 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fenA-F <400> 2 cctcgctccg catgatcttt tgg 23 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> fenA-R <400> 3 cgggagcacg gtggcaatgt g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> fenD-F <400> 4 tttggcagca ggagaagttt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> fenD-R <400> 5 gctgtccgtt ctgctttttc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> fenE-F <400> 6 gtttcatggc ggcgagcacg 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fenE-R <400> 7 gattcgcggg aagcggattg agc 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ituA1-F <400> 8 cgcccgtgaa ggagcagccg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ituA1-R <400> 9 gccaggaagc ggggcttcac 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ituA4-F <400> 10 ctgcctgcgt atatgattcc ggc 23 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ituA4-R <400> 11 ccgtgatgat gccgttcttc aatcc 25 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ituD-F <400> 12 atgaacaatc ttgccttttt a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ituD-R <400> 13 ttattttaaa atccgcaatt 20 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> srfAA-F <400> 14 gcccgtgagc cgaatggata ag 22 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> srfAA-R <400> 15 ccgtttcagg gacacaagct ccg 23 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> srfAD-F <400> 16 ccgttcgcag gaggctattc c 21 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> srfAD-R <400> 17 cgcccatcct gctgaaaaag cg 22

Claims (8)

Bacillus amyloliquefaciens BC-095 strain with accession number KACC 92389P,
The above strain has a 16S rRNA base sequence of sequence number 1.
The above strain is a Bacillus amyloliquefaciens BC-095 strain characterized by having the ability to produce antibiotics consisting of fengycin, iturin A, and surfactin. In claim 1, the strain is a Bacillus amyloliquefaciens BC-095 strain characterized in that it activates at least one enzyme among protease and cellulase. delete A composition for controlling plant diseases, comprising the Bacillus amyloliquefaciens BC-095 strain of claim 1 or claim 2 or a culture solution thereof as an effective ingredient,
The above composition for pest control has an antagonistic effect against Botrytis cinere a.
A composition for controlling plant diseases, characterized in that the plant is ginseng. delete delete A composition for controlling plant diseases, characterized in that in claim 4, the composition for controlling plant diseases further comprises a solvent, a mixer, and an additive in addition to an effective ingredient. A method for controlling plant diseases using the strain of claim 1 or 2.