KR20040091954A - Bacillus subtilis HN12 inhibiting the growth of Cylindrocarpon destructans and the method and composition to protect the rust of plant roots using this strain - Google Patents

Abstract

PURPOSE: A microorganism, Bacillus subtilis HN12, inhibiting the growth of Cylindrocarpon destructans and a method and composition for protecting the rust of plant roots using the same are provided, thereby inhibiting the rust of plant roots without toxicity. Therefore, repeated cultivation of ginseng can be carried out. CONSTITUTION: The microorganism, Bacillus subtilis HN12(KCCM 10481), inhibiting the growth of Cylindrocarpon destructans is provided, wherein Bacillus subtilis HN12(KCCM 10481) is isolated from the soil. The method for protecting the rust of plant roots comprises spraying the cultured medium of Bacillus subtilis HN12(KCCM 10481) into the plant roots or the surrounding soil thereof, wherein the cultured medium of Bacillus subtilis HN12(KCCM 10481) contains 400000 to 400000000 cfu/ml. The composition for protecting the rust of plant roots comprises Bacillus subtilis HN12(KCCM 10481).

Description

Bacillus subtilis HN12 inhibits growth of Cylindrocarpon destructans and methods for controlling and suppressing root rot (Bacillus subtilis HN12 inhibiting the growth of Cylindrocarpon destructans and the method and composition to protect the rust of plant roots using this strain}

The present invention relates to a new strain of Bacillus subtilis that inhibits the growth of Cylindrocarpon destructans, the causative agent of root rot of ginseng.

In the past, plant root rot research mainly focused on a broad-scale root rot disease caused by complex root rot and Fusarium solani. Root rot disease by Cylindrocarpon destructans has been intensively studied since 1992, and it is known that this pathogen is mainly caused by trees and bulbs such as daffodils, cyclamen, alfalfa and ginseng.

Although many studies have been conducted on the control of plant root rot caused by cylindrocarpon destructans, the mechanism of the occurrence of root rot caused by cylindrocarpon destructans and the physiology and ecological basic research of pathogens are still insufficient. Prevention and control methods are unknown, but only prevent the spread of root rot caused to the surrounding area.

〈Ginseng Rot Rot Disease〉

In ginseng cultivation, harvesting ginseng and cultivating other crops for 7-10 years and then cultivating ginseng again, and cultivating ginseng after harvesting ginseng, do not cultivate other crops, and managing ginseng again for 1-2 years To cultivate. Ginseng is a representative crop that causes obstacles due to crops, and it is necessary to secure cropland. However, in major ginseng producing areas, cropland is not enough due to the increase of cultivated area. Therefore, so-called cultivation is carried out by moving to other regions to cultivate cropland, but such long-distance business trips increase the production cost and cause management problems along the remote areas. Lack of adaptation may cause a decrease in the productivity and quality of ginseng.

The symptom of softening of ginseng is caused by root rot which causes roots to rot to dark brown and leaves become prematurely reddened. As a pathogen related to root rot of ginseng, Phytophthora cactorum, Cylindrocarpon destructans, and Fusarium solani have been reported. Among these pathogens, it is known as a causative agent of serial disturbance, and the damage caused by cylindrocarpon destructans, which form a thick film spore and causes disease due to soil infection, has been observed. The isolation and pathogenicity of interest has increased interest in these pathogens.

It is known that about 50% of the crops are harvested in six crops of grassland, and the occurrence of root rot in these crops is generally considered to be part of the 50% rate of decay during cultivation. . However, when cultivating or re-creating ginseng within 4-5 years after harvesting ginseng, root rot occurs frequently in ginseng packaging.In the field where the rot occurred, the initial rate of root rot caused by root rot within 30 years was 30%, Severe cases reach up to 80%, and then progress rapidly to 100% absent from 3-4 years old ginseng.

After Cylindrocapon destructans was identified as the causative agent of root rot of ginseng, the physiological characteristics of the pathogen and the utilization and secretion of amino acids in culture were actively studied. Initially, the bacterium was named C. radicicola, and was renamed Cylindrocarpon Distractans by Scholten. Differences in the mycelial growth and toxin production in the growth medium of cyndrocarpon destructans and the effects of temperature on the growth of this bacterium have been reported. Differences in growth due to various factors, such as characteristics, culture temperature, light, pH of the medium, the type of medium has been reported. Subsequently, reports related to growth characteristics were confirmed by re-separation of the syringdrocarpon destructans as root rot fungi related to the intestinal rot of ginseng. In order to develop the selective medium of this pathogen, the growth characteristics of the culture conditions such as temperature and pH and the addition of antimicrobial substances have been reported.

As a method of reducing the density of soil pathogens present in the soil, conventional methods of desalination in soil cultivated with ginseng and a method of using a soil fumigant are known. When we examined the density of soil microorganisms after treating the ginseng harvested soil in the fall, the bacteria that had a high growth rate recovered to the level after 20 days of treatment. It was reported to decrease and then return to normal at the next year of ginseng transplantation. Root rot occurred when the root rot pathogen was inoculated 30 cm from the soil surface and ginseng was transplanted, but it did not occur when the root rot pathogen was inoculated at 50 cm or less. Fumigation to the depth is expected to have a control effect, but this fumigation method is not applicable to root rot due to root rot during ginseng cultivation.

In addition, chemical pesticides have been used to control root rot of ginseng, but the control is still low, and in some arable lands, chemical pesticides are used indiscriminately to seriously contaminate cultivated ginseng as well as surrounding water and soil. In some cases. The use of such chemical pesticides needs to be regulated in terms of environmental pollution, and in the case of foreign countries, red ginseng products are also subject to restrictions on pesticides that are prohibited from use. If you need to use caution, but this is actually ignored in the cultivated farm. Apart from these import restrictions, there are serious damages caused by organic synthetic pesticides and environmental pollution, and the development of low-toxic, eco-friendly biopesticides is urgently required at this time.

<Bacillus subtilis>

On the other hand, Bacillus subtilis (Bacillus subtilis) is a bacterium that is aerobic and forms apoptosis as G (+) and is mostly present in soil. Morphologically, it is a rod-shaped fungi with flagella around the cells. The follicle is ovate, centrally located, and the expansion of the cylindrical spores and spore sac is observed. In general, the cell width is 0.9 μm or less and there is no lipid globule.

In physiologically unfavorable environments (overdrying, poor nutrition, high temperature, influx of harmful components, etc.), spores form and enter a dormant life, and endogenous spores are released to the outside in poor environment, and growth conditions are good. Germinate the ground. It has excellent protein and fiber degrading ability, produces 2,3-butanediol, a crop growth promoting substance, and antagonizes pathogenic microorganisms by secreting antibiotic subtilin.

The fields of use known to date are as follows.

1) Plasmid

It is very important to use as a plasmid of recombinant DNA technology in genetic engineering.

2) microbial agents

B. subtilis strains are mainly used, and breeding is good even under malnutrition conditions, and it is easy to formulate as heat resistant spores, but low temperature resistance is weak. It secretes antibiotics (bacitracin, bacilycin, bulbiformin, fengymycin) to suppress pathogens such as warehousing and black pulmonary nucleus disease, and it produces enzymes to promote organic decomposition and to produce organic acids to generate cations of poorly soluble phosphorus compounds in soil. By chelate binding, phosphate ions are made as possible. In addition, it secretes physiologically active substances such as vitamins, nucleic acids and amino acids to promote growth and disease resistance of crops. In addition, the growth rate is fast, depleting the essential nutrients of the pathogen also acts to inhibit the growth of bacteria. If soil or environment with low organic content is not suitable, growth promotion and pest control may not be shown.

3) Combined enzyme activity improves feed efficiency

Strains that exhibit complex enzyme activities such as proteases, phytase, cellulase, xylanase, α-amylase, and lipase are used to improve feed efficiency. Its fast growth rate is particularly effective during short digestion times of chickens, and it is excellent in acid resistance, bile resistance and heat resistance because it forms endospores.

4) Environment

It is used for food leachate treatment system by pure culture using complex components, mineral, basic, amino acid, alcohol and digestive enzyme.

5) Meju fermentation

It is distributed evenly on the surface and inside of Meju and has strong protein and carbohydrate degrading enzyme.

6) Used for MS farming

MS farming is a new concept of eco-friendly farming that is differentiated from modern chemical farming and organic farming. It is a farming method that can normalize diseased land that has been fertilized with chemical fertilizers and pesticides. MS means microorganisms such as Bacillus subtilis, fungi, lactic acid bacteria, photosynthetic bacteria, yeast bacteria and actinomycetes, which are effective among the soil microorganisms in the wild and domestic fields. Special methods for screening these excellent microorganisms: MS 115 through the selection of patents 115425, 0207061, 0202285) and then various kinds of wild grasses (myeongju, mosquito rod, injection mugwort, etc.), herbal medicines (Peak, Sinseoncho, Hwanggi, etc.) A functional bioactive substance that can be widely used in crops, horticulture, fisheries, animal husbandry, etc. as a fermentation agent extracted after metabolizing and using functional raw materials and domestic abundant resources as a basic medium. -2000-0044427).

Aerobic microorganisms and anaerobic microorganisms coexist in closed areas such as the ground and the water, respectively, through the assimilation or catabolism to inhibit the generation of free radicals in living and moving animals, and to prevent damage and to prevent the oxidation of metals such as iron. And the ability to remove rust.

The present inventors have searched for antagonistic microorganisms that can control the root rot of ginseng from soil microbial resources with the aim of developing environmentally friendly biopesticides to control the root rot of ginseng, which is a problem when growing or reworking ginseng. The present invention has been led.

The present invention provides a novel Bacillus subtilis HN12 strain (KCCM-10481), which is a antagonistic microorganism against cyrindrocarpon distractans, a causative agent of root rot of ginseng. Bacillus subtilis HN12 strain, because it was found in the soil of Korea, is suitable for direct use in the soil and does not have toxicity to the environment and human body can be used as an environmentally friendly bio pesticide.

In addition, the present invention provides a root rot inhibitor and a method for controlling root rot caused by Sirandrocarpon distractans using Bacillus subtilis HN12 strain.

Figure 1 shows the results of gas chromatography analysis of intracellular fatty acid of Bacillus subtilis HN12.

2 is an electron microscope (× 8,800) photograph of Bacillus subtilis HN12.

Figure 3 is a photograph showing the growth inhibitory activity against the Syringrocarpon destructhans strain of Bacillus subtilis HN12 in YPD medium.

4 is a photograph showing the root rot control effect of ginseng treated with Bacillus subtilis HN12.

In the present invention, in the process of searching for antagonistic microorganisms that inhibit the growth of root rot pathogens of ginseng from domestic soil microbial resources, it exhibits growth inhibitory activity against root rot pathogens, ie, cyndrocapon destructans, from soil samples of Chungcheongbuk-do. Antagonistic microbes were explored.

The microorganisms searched were identified by the 16S rRNA sequencing method of the microorganism, which is the most used method for identifying the position of the new strain. The base sequence of the gene encoding the 16S rRNA of the microorganism explored in the present invention is shown in SEQ ID NO: 1. As a result of comparing the base sequence data of the 16S rRNA of the known strain, the searched microorganism of the present invention was classified into Bacillus subtilis, and the constituents and ratios of fatty acids, cell appearance, etc. On the grounds it was named Bacillus subtilis HN12 (deposit institution: Korea Microorganism Conservation Center, deposit date: April 4, 2003, accession number: KCCM-10481).

Bacillus subtilis HN12 strain of the present invention inhibited the growth of Sirandrocarpon distractans in experiments with YPD medium (FIG. 3), and also showed the results of improving root rot of ginseng even in experiments applied to ginseng farmland. (Figure 4).

Based on these results, the present invention provides a root rot control agent by the Syringro carpon destructhans containing Bacillus subtilis HN12 strain as an active ingredient.

As used herein, the term "Bacillus subtilis HN12 strain" refers not only to the Bacillus subtilis HN12 strain itself, but also to the product produced by the strain and the material separated and extracted from the strain.

In addition, the present invention provides a method for controlling root rot caused by the Syringro carpon destructhans comprising spraying the Bacillus subtilis HN12 strain to the root of the plant or the soil adjacent thereto. In this case, the Bacillus subtilis HN12 strain is preferably sprayed intensively on the root of the plant or adjacent soil, and the number of microorganisms per ml of the culture of the Bacillus subtilis HN12 strain to be sprayed is about 4 × 10 ⁶ to 4 × 10 ⁸ . It is preferable to adjust so that.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by the following examples.

Example 1

<Selection of antagonistic microorganisms>

In order to search for antagonistic microorganisms from the soil, soil samples were gradually diluted in distilled water, plated in a separation medium, and each strain was purely separated.

Sterilize the microbial separation medium (glucose 2%, yeast extract 1%, polypeptone 1%, potassium hydrogen phosphate monophosphate 0.05%, potassium diphosphate 0.05%, magnesium sulfate seven times crystallized water 0.02%, agar 1.8%, hydrogen ion concentration 7.2) After the temperature was lowered to about 50 ^o C, a colistin solution (5 mg / l) dissolved in distilled water and a nalidixic acid solution (10 mg / l) dissolved in methanol were used to suppress the growth of ordinary bacteria. ) 1 ml per 1 liter of sterilized separation medium was mixed well and hardened and used as a separation medium. Soil samples were gradually diluted and plated in the prepared separation medium and separated while incubating at 28 ^° C. In order to search for microorganisms that antagonize the bacteria, the causative agent of root rot, Cylindrocarpon dextantans, is cultured on YPD agar medium, and the strain to be searched next to it inhibits the growth of Syringrocaphone strain. By observing, finally, microorganisms with growth inhibition ability were selected for the cyndrocarpon strain.

Example 2

<Identification of Exploded Antagonists>

In order to identify the strains selected in Example 1, 16S rRNA sequences of microorganisms, which are the most used method for identifying the position of the new strains, were analyzed and compared with data of known strains.

The base sequence of the gene encoding the new strain of the present invention, that is, 16S rRNA of Bacillus subtilis HN12 strain is shown in SEQ ID NO: 1. Comparing this with the 16S rRNA sequences of the known strains Bacillus subtilis AB07589 and AB017590 showed 99% similarity with AB017589 and 99% consensus with AB017590. According to the above results, the new strain of the present invention was classified as Bacillus subtilis.

In addition, the characteristics of the intracellular fatty acid of the Bacillus subtilis HN12 strain of the present invention was analyzed by gas chromatography. As a result, the Bacillus subtilis HN12 strain of the present invention was Bacillus lentimorbus. ) And 58.4% match with Bacillus subtilis.

In addition, as a result of confirming the form of the new strain of the present invention by an electron microscope, the size of the strain is 0.6-0.7 × 1.5-1.6 ㎛ on average less than the size of the general Bacillus subtilis 1.2-2.5 ㎛, flagella Dogs were, and their length was about five times the size of the cells (Fig. 2).

According to the above results, the new strain of the present invention was classified as Bacillus subtilis, and named as Bacillus subtilis HN12 based on the characteristics of the components and ratio of the fatty acids and the cell appearance. It was deposited on April 4, 2003 with the Korea Microorganism Conservation Center (Accession No .: KCCM-10481).

Example 3

<Proliferation of Bacillus subtilis HN12>

The Bacillus subtilis HN12 strain of the present invention isolated and identified above was cultured by shaking culture at 28 ° C. for 24 hours in YPD medium (casein 1%, peptone 1%, glucose 2%, yeast extract 1%, salt 0.5%). Was mixed with 20% glycerin in a ratio of 1: 1, and put into 2 ml frozen vials and stored frozen at -70 ° C and used for culturing microorganisms.

In order to produce Bacillus subtilis HN12 microorganisms, an active microorganism production medium (glucose 2%, yeast extract 1%, ammonium sulfate 0.1%, potassium phosphate 0.1%, magnesium sulfate 7 times crystallized water 0.05%) in a 1-liter Erlenmeyer flask , Calcium chloride 0.5%, sodium chloride 1%, initial pH 7.0) to 100 ml of the medium, and the frozen vial was inoculated and incubated at 28 ℃ 48 hours, 200rpm shaking culture. This cultured HN12 strain was used in the experiments of Examples 4 and 5 below.

Example 4

〈Growth Inhibitory Effect on Cylindrocapon Destructurals〉

In order to confirm that the Bacillus subtilis HN12 strain of the present invention inhibits the growth of the Cylindrocarpon distractans strain, the growth inhibitory activity of the Syringrocarpon distractans strain was analyzed in YPD medium.

Syringocarpon distractans incubated at 37 ° C. for 72 hours in YPD medium were taken 3 × 3 mm with sterile spatula and placed on YPD medium to be observed for 72 hours, followed by sterilization of Bacillus subtilis HN12 strain. It was taken with platinum teeth and side by side dated and incubated for 5 days at 28 ° C. As a result of the culture, the HN12 strain was inoculated as shown in Figure 3, it was confirmed that the growth of the Syringro carpon destructhans strain.

Example 5

〈Ginseng root rot inhibitory effect of Bacillus subtilis HN12 strain〉

Ginseng root rot inhibitory activity of the Bacillus subtilis HN12 strain was tested in the farmland of Jeungpyeong, Chungcheongnam-do, in order to identify the ginseng root rot.

At the time of testing in May 2002, the presence of root rot fungi was confirmed in the fallow ginseng arable land, and two year old ginseng was transplanted to one of the 5,000 arable land (Note: The size of one cell is 1.8 × 0.9 m and was used for the test. The ginseng number is about 500 roots), 3/4 of the ginseng roots are covered with soil containing root rot fungi, and the roots above are adsorbed on the same amount of vermiculite to the Bacillus subtilis HN12 strain cultured by the method of Example 3. After spraying the adsorbate evenly, the ginseng was grown by covering the soil with root rot fungi. In August of the same year, ginseng was harvested to investigate the improvement of root rot of ginseng against HN12 strain. As a result of visual inspection, as shown in the photograph of FIG. 4, the root portion of the root not treated with the adsorbate adsorbed on the vermiculite was rotted as it is, but the root portion of the root treated with the adsorbate improved root growth. Was confirmed to be good.

As confirmed by the above examples, the Bacillus subtilis HN12 strain of the present invention inhibits the growth of cyrindrocarpon distractans, a causative agent of root rot of ginseng, effectively controlling root rot of ginseng. It is possible to improve the root rot of cultivated ginseng by applying to the cultivated site as well as the cultivated ginseng.

In addition, the Bacillus subtilis HN12 strain of the present invention, which has been searched from the soil of Korea, is suitable for direct use in the soil, and has the advantage of being an environmentally friendly pesticide without toxicity to the environment and human body.

Root rot control and control method of ginseng using the new strain of the present invention is expected to contribute significantly to the cultivation of ginseng by enabling the production or re-production of ginseng, US ginseng, Chinese ginseng, etc. It can also be widely used to cultivate other bulbs where root rot caused by tans is a problem.

<110> Eurekagene company <120> Bacillus subtilis HN12 inhibiting the growth of Cylindrocarpon          destructans and the method and composition to protect the rust of          plant roots using this strain <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1443 <212> DNA <213> Bacillus subtilis HN12 <400> 1 gcctaataca tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60 cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120 ctaataccgg atgcttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc 180 acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga 240 cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300 tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360 ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc 420 cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480 cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540 tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600 gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660 cgtagagatg tggaggaaca ccagtggcag aggcgactct ctggtctgta actgacgctg 720 aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780 atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840 tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900 agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960 cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg 1020 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080 atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140 aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200 atggacacaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1260 agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320 cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380 gtttgtaaca cccgaagtcg gtgaggtaac ctttatggag ccagccgccg aaggtgggac 1440 aga 1443

Claims (4)

Bacillus subtilis HN12 strain (KCCM-10481) which inhibits the growth of Sirandrocarpon distractans. Root rot preventive agent by Sirandrocarpon distractans containing Bacillus subtilis HN12 strain (KCCM-10481) as an active ingredient. A method for controlling root rot caused by Sirindrocapon destructhans, which comprises spraying Bacillus subtilis HN12 strain (KCCM-10481) to the root of a plant or adjacent soil thereof. The method according to claim 3, wherein the number of microorganisms per ml of culture of the Bacillus subtilis HN12 strain to be sprayed is 4 × 10 ⁶ to 4 × 10 ⁸ .