KR100430961B1 - Serratia plymuthica antagonistic to white rot fungi sclerotium cepivorum - Google Patents

Abstract

Seratia plymutica antagonizing the leek black rot fungal nucleus according to the present invention is isolated from the root root microorganisms from the roots of the Seratia plymutica AL-1 strain antagonistic to Sclerotium cepivorum It was.

Isolate AL-1 was determined by the procedure of Floccaryotes and Burgy's mannual of systematic bacteriology, by the method of microbial ID Inc. Sherlock system, by partial nucleotide sequence of 16S rDNA (767bp). It was identified as Serratia plymuthica by a method such as homology search in a ribosomal database.

In S. plymuthica AL-1, the growth inhibition rate was 15 mm for Sclerotium cepivorum, and the pepper black pattern disease (Alternaria altrata) was 9 mm. Fungus (Colletotrichum gleosporioids) is 13 mm, bellflower stem (Phoma sp.) 10 mm, pepper Hiroctonia solani (8 mm), pepper Temperalium solani (8 mm), cucumber fungus Sclerotinia sclerotiorum had a antagonistic force of 7 mm and a watermelon of Fusarium oxysporium niveum of 7 mm, but had no antagonism in Didymella bryoniae.

Sera plymuthica AL-1 was induced to produce chitinase (3.2 units / ml) in fractions of 10,000 or more in TSB medium supplemented with 1% colloidal chitin, When heat-treated at 80 ° C. for 30 minutes, chitinase activity was lost but antagonism (6.4 mm) remained.

In addition, the fraction below 10,000 molecular weight showed no chitinase activity but showed antagonistic force (5.2mm), and even after heat treatment at 80 ℃, antagonistic force (5.0mm) remained, indicating that other bioactive substances other than enzyme existed. Confirmed.

Description

SERAATIA PLYMUTHICA ANTAGONISTIC TO WHITE ROT FUNGI SCLEROTIUM CEPIVORUM}

The present invention relates to Seratia plymutica, which antagonizes the leek black rot fungi, more specifically, Seratia plemutica AL that antagonizes the leek microorganisms from the roots of the leek sclerotium cepivorum. -1 Strain (Serratia plymuthica AL-1: Accession No. KCTC 10187BP) by using the Sclerotium cepivorum (Sclerotium cepivorum) and various pathogenic fungi to make microbiologically easy to prevent microbial disease It's about Serratia Plimutica.

Plant roots release various organic substances that feed allelochemics and rhizosphere microorganisms, and the types and amounts of substances secreted from the roots are plant type, age, temperature, light, plant nutrition, microorganisms and soil. It is greatly influenced by humidity and the like.

These substances are isolated from cortical cells by soluble secretions from the mucous sheath and droplets from the hair follicles, mainly from the root canal at the tip of the roots, soluble secretions from the cells, exfoliates produced by aging of the cell wall, and microorganisms. It exists in the substance, etc.

Allium sp. Plants also secrete persimmons and various organic substances, including vanillic acid and water-soluble alkyl cysteine sulphoxides, particularly vanillic acid. Is a lignin-degrading intermediate metabolite that induces sensitization in the roots and foliage of rye, oats, sugarcane, corn and Brachiaria mutica, or in soils grown in tobacco, sweet potatoes, pineapples and bananas. It was observed a lot, and it was confirmed that the growth of the serialized chrysanthemum was suppressed.

Host secretions appear between the allium sp. And the nucleus of Sclerotium cepivorum as an example of the specific germination of the endogenous endogenous germ.

Water-soluble alkyl cysteine sulphoxides from allium plants are diffused into the soil and converted into volatile alkyl sulphides by soil microorganisms, which are resistant to germ nuclei of S. cepivorum. By inducing action, the root environment of the leek plants dominates, causing white rot.

As a seedling control technique for plant soil diseases, rooting microorganisms that settle on leeks and leeks, such as the case of controlling disease by mixing leeks and gourds, and the control of tomato wilting by leek and leek mixing, do not settle in the roots of tomatoes. Due to the mixing of green onions and tomatoes, the disease can be controlled by allowing microorganisms present in the roots of green onions to inhabit the root of the tomato.

In order to establish antagonistic microorganisms in the root zone of the target plant, it is important to concentrate foreign useful microorganisms on the seedlings or planting areas of the plant, but most of the foreign useful microorganisms injected are killed by the persimmon or organic substances produced by the target crop. Or difficult to grow, you will not see the effect of the correct microorganisms.

Therefore, direct isolation of rhizome antagonistic microorganisms indigenous or predominantly inhabiting the rhizome of the target crop and re-introduction of these rhizome microorganisms will have more affinity for the microorganisms because they have affinity for the plant.

The present invention isolates 146 myomicrobial microorganisms from leek roots and uses Seratia plymutica AL-1 strains that antagonize Sclerotium cepivorum, and thus, Sclerotium cepivorum and various pathogenic strains of leek. The present invention provides a Seratia plymutica that antagonizes the leek black rot fungi, which can easily prevent microbiological diseases.

Figure 1a, 1b is a photograph substitute picture for screening microorganisms antagonistic to the leek black rot fungal nuclear bacterium according to the present invention,

Figure 2 is a picture substitute photograph magnified with a scanning microscope (15,000 times magnification) Seratia plymutica antagonizing the leek black rot fungal nucleus bacteria according to the present invention,

3 is a result table of identification of the strain of the present invention by the method of Burgess manual,

Figure 4 is a result table of identification of the strain of the present invention by the method of cellular fatty acid analysis,

5 is a nucleotide sequence of the strain of the present invention by the method of determining the partial base sequence (767 bp) of 16S rDNA,

Figure 6 is a diagram examining the antagonism of the strain of the present invention against pathogenic fungi,

7 is a chart showing the ability of the antagonist of Seratia plymutica to antagonize the leek black rot fungal nuclear bacterium according to the present invention;

8A and 8B are photographic diagrams showing antagonists inhabiting the roots of Serratia plymutica antagonizing the leek black rot fungal nucleus bacterium according to the present invention.

Hereinafter, a preferred embodiment of Ceratia plemutica antagonizing the leek black rot fungal nuclear bacterium according to the present invention will be described in more detail, the present invention is not limited by the following examples.

The present invention directly isolates the root zone antagonistic microorganisms indigenous or predominantly inhabiting the root crop of the target crop, and re-injecting these root zone microorganisms has affinity for the target plant.

Therefore, the present invention, as part of the experiment for microbiologically control the black rot mycosis caused mainly in the roots of the leek crops, the anti-fungal antagonist microorganisms antagonizing Sclerotium cepivorum of allium fistulosum Isolation was identified from the root of, and chitinase and bioactive substances were investigated.

First, the pathogenic fungus used in the present invention is a black rot fungal nuclear bacterium (Sclerotium cepivorum) that has been sold by the Rural Development Administration.

The pathogen is a low-temperature pathogenic fungus that grows only at 5 ° C to 20 ° C, so that the pathogen is cultured at a temperature of 20 ° C or lower in a PDA (potato dextrose agar) medium to form a bacterial nucleus, and stored at 4 ° C.

Other pathogenic fungi such as pepper black pattern bacterium (Alternaria altrata), pepper anthrax (Colletotrichum gleosporioids), bellflower sp. , Cucumber strains such as Sclerotinia sclerotiorum, Fusarium oxysporium niveum, and Didymella bryoniae were used in RDA and KCTC. Incubated at and stored at 4 ℃.

In order to isolate the root zone microorganisms inhabiting the leek roots, samples of leek roots are taken from Chilgok, Yeongcheon, Goryeong, and Seongju near Daegu, washed several times with sterile distilled water, broken into small pieces, and then placed in sterile 0.85% NaCl solution. After grinding and centrifuging for 2 minutes with a homogenizer, the supernatant was used as a fungal sample.

After diluting 1 ml of the fungal sample with 0.85% NaCl solution, bacteria were cultured with nutrient agar (NA) and tryptic soy agar (TSA), and actinomycetes were treated with 0.4% propionate sodium in YMA (yeast extract-malt extract agar) medium. (sodium propionate) and 25mg / L nalicidic acid were used, and mold was used as a PDA medium to spread 100 µl of the fungal sample solution on each medium, and bacteria were used at 20 ° C. for 3 days. , Actinomycetes were incubated in an incubator for 5 days at 30 ° C. for 7 days at 28 ° C., and firstly classified into colony, color, odor, etc. by optical microscope, morphological bacteria, actinomycetes, Secondary separation was used as mold.

In order to identify strains with chitin degrading ability, 1% of colloidal chitin was added to each of the bacterial selection medium, and then cultured, and the chitin cleavage ring generation ability was confirmed.

Selection of microorganisms antagonizing S. cepivorum, a low-temperature pathogenic fungus, was selected as the size of growth inhibitory rings by pairing plate cultures with isolated bacteria and pathogens.

Place the mass of pathogens grown in PDA medium in the center of PDA medium containing 1% colloidal chitin, and inoculate one platinum with pure microorganisms at 4 edges and incubate at 20 ℃ for 7 to 10 days. After that, the inhibitory zone (inhibition zone) of the pathogen mycelium was examined to select the isolated bacteria having a large length of the clear zone as antagonistic bacteria.

Identification of antagonistic microorganisms is done in three ways.

First, the morphological, culture, physiological and biochemical characteristics of microorganisms are examined according to the methods of Floccariotes and Burgy's mannual of systematic bacteriology.

Next, the microbial ID Inc. Sherlock system (microbial identification device, GC: HP Co., 6890 series) was automatically identified.

Next, the partial nucleotide sequence of 16S rDNA (767bp) was determined and identified by the 16S rDNA sequence method, which is a method of homology detection in a ribosomal database, wherein the PCR primer was R14 (5'-ACg ggC ggT gTg TAC-3 ') and R15 (5'-gCC AgC AgC CgC ggT A-3') were used, and sequencing data was identified by searching for homology in the ribosomal database.

In order to prepare colloidal chitin and measure the activity of chitinase, 100 g of crude chitin (Sigma Co., C-7170) was added to cold conc. After adding 2 L of HCl and stirring at 4 ° C. for 12 hours, 2 L of 95% cold ethanol was added to recover the colloidal chitin produced by centrifugation (Beckman Co., L8-55M) at 6,000 × g for 10 minutes. After dilution with distilled water, the pH was neutralized with 5N NaOH, washed several times with distilled water, and the colloidal chitin recovered was dried and used as a substrate of the enzyme.

Chitinase activity was measured by 0.5 ml of colloidal chitin suspended in 0.5 ml of phosphate buffer (0.05 M Na ₂ HPO ₄ -KH ₂ PO ₄ buffer, pH 7.5) in 1 ml of phosphate buffer. After the addition, 0.2ml of the enzyme solution was added and reacted at 45 ° C. for 1 hour and 30 minutes, and then the reduction equivalent was measured by DNS (dinitrosacylic acid) method.

One unit of enzyme activity was converted to the amount of enzyme that produces 1 μM of N-acetylglutosamine (acethylglucosamine) from the colloidal chitin per hour.

In order to investigate the antagonistic control mechanism of antagonists, inoculated TSB medium with or without 1% colloidal chitin and shaken for 3 days at 20 ° C, and centrifuged at 10,000 xg for 30 minutes to recover the supernatant, and then, YM10. After filtering on the basis of molecular weight 10,000 with (Amicon Co.) member (membrane), each of the antimicrobial activity was investigated, and the culture supernatant was heat treated at 80 ℃ for 30 minutes to investigate the residual antagonism.

The Seratia plymutica antagonizing the leek black rot fungal nucleus bacterium according to the present invention was deposited with the deposit number KCTC 10187BP in the Gene Bank of the Korea Institute of Science and Technology Biotechnology, an international depository institution under the Budapest Treaty.

First, the isolation of rhizosphere microbes living in the root of leek

Root microorganisms of leek were cultured in selective medium with the root of the leek as a fungal sample, and then colonized with different shapes, colors, and smells of colony, and observed and separated by optical microscope. A total of 146 rhizosphere microorganisms were isolated, including 98, 33 actinomycetes (GL), and 15 fungi (DL).

These 146 isolated microorganisms were cultured in each selective solid medium containing 1% colloidal chitin. As a result, 12 bacteria and actinomycetes showed chitin degradation ring. AL-32, AL-1, GL-14, etc. showed a clear zone.

In particular, the isolated bacteria were good microorganisms at low temperature below 20 ℃, especially isolates AL-32 and AL-1 were able to grow at 4 ℃, but did not grow at 40 ℃.

In general, leek, onion, garlic, etc. plants are grown at relatively low temperatures, so for microbiological control of these crops, low-temperature microorganisms that can grow at low temperatures will be advantageous. Growing bacteria were selected.

Second, screening of microorganisms antagonistic to leek black rot

As a result of screening microorganisms antagonistic to Sclerotium cepivorum using 146 kinds of leek myomicrobial microorganisms, the number of antagonist microorganisms showed similar pattern by region, and a total of 51 bacteria Among them, the antagonistic activity was the best among the AL-1 strains (15 mm (clear zone size), AL-8 (13 mm), AL-32 (12 mm), and AL-48 (10). ㎜) showed antagonism in order, and 9 kinds of actinomycetes had a longing ability. Among them, the GL-1 strain showed no antagonism in the initial culture, but showed high antagonism with 10㎜ after 4 days of culture. The antagonistic force was shown in the order of GL-7 (8 mm), GL-8 (7 mm) and the like.

The fungus showed antagonism in five species, while DL-4 (3㎜) had a relatively slight antagonism among them, while the other fungi showed very little antagonism.

Therefore, as shown in Figure 2, among the myobacteria such as bacteria, actinomycetes, fungi, etc. isolated from the leek, isolated bacterium No. 1 having the highest antagonistic force against S. cepivorum. AL-1 strain was used as the test strain.

Third, antagonist No. Identification and Characteristics of AL-1

First, the morphological, culture, physiological and biochemical characteristics of the microorganisms were examined according to the methods of floccariotes and Burgy's mannual of systematic bacteriology. As shown in FIG. Hepatococci are proliferative in anaerobic conditions, have main hair flagella and motility, indole reaction is negative, Bosprostauure test (VP) reaction is positive, citrate availability is positive, KCN medium Esau's development was partially possible.

In addition, the catalase test liquefied positive, gelatin, gelatin (DNase) activity, did not grow in Malonate (Malonate).

Lysine decarboxylase, arginine hydrolase, ornithine decarboxylase, etc. were not produced. No gas was produced in the glucose medium and no pigments were produced. As a result of surveying about 10 kinds of sugar availability, the antagonistic AL-1 was determined to be in Serratia.

Bacteria, such as Serra marcesens, of the genus Serratia or its related bacteria, produce red prodigiosin pigments in nutrient or MacConkey media, Al-1 had no pigmentation.

In particular, biochemical investigations showed the closest results to S. plymuthica among the microorganisms of the genus Serratia.

In another method, the microbial ID Inc. Sherlock system (microbial identification device, GC: HP Co., 6890 series) was automatically identified by the composition of fatty acids in the cell wall of the microorganism, and as shown in FIG. The composition of 14: 0 type was 8.03%, 15: 0 was 4.85%, 16: 0 was 29.03%, 17: 0 cyclo type was 16.88%, 18: 1 was 8.14%, and similarity was 47.9. It was relatively low in%, but was identified as Serratia fonticola.

In another method, the partial base sequence of 16S rDNA of antagonist microorganism AL-1 was determined. As shown in FIG. 5, the nucleotide sequence of 767 bp was determined, and the homology was searched in a ribosomal database. It was identified as Serratia plymuthica (93.2% similarity).

Also, among the 767bp sequences of PCR amplified 16S rDNA, there are two restriction enzymes EcoR1 site and one Xhol site.

Therefore, by combining the results of the above three methods, isolate AL-1 is Serratia plymuthica, or it is identified as a soft bacterium and named Serratia plymuthica AL-1.

Representative species of the genus Serratia are Serratia marcescens, in addition to S. liquefaciens, S. rubidaea, S. ficaria, S. adorifera and S. grimesii, but these are pneumonia and pneumonia. It has been found to cause many infectious diseases such as septicemia, sepsis, urinary tract infection, meningitis, peritonitis, and wound infection, and has attracted attention as a pathogen.

Serratia Plymutica has been isolated from the roots of wheat, oats, cucumbers, corn, oils and fats, and tomatoes.It is not recognized as a pathogenic microorganism that causes serious illness to humans. Or microorganisms that contaminate contact lenses.

Since S. plymuthica is a microorganism that is friendly to plant roots in agriculture, research is underway to use it for biological control of pathogenic fungi, and it produces histamine and sucrose isomerase. ) Is reported.

Fourth, antagonistic investigation of pathogenic fungi caused by Seratia plymutica AL-1

As a result of examining the antagonism of the leek pathogen caused by S. plymuthica AL-1, it was grown as a pathogenic fungus of leek as Sclerotium cepivorum as shown in FIG. The size of the low-ring ring was 15 mm, and the pepper black pattern disease (Alternaria altrata) was 9 mm, the pepper anthrax (Colletotrichum gleosporioids) was 13 mm, the bellflower stem (Phoma sp.) Was 10 mm, and the pepper jabber disease was (Rhizoctonia solani) is 8 mm, Stemprhlium solani is 8 mm, Sclerotinia sclerotiorum is 7 mm, and Fusarium oxysporium niveum is 7 mm. However, there was no antagonist activity in Didymella bryoniae.

The above results were obtained when Sera plymuthica AL-1 was incubated at 15 ° C. However, increasing the incubation temperature from 20 ° C to 30 ° C further reduced the antimicrobial activity. The growth of the fungus was also stopped, and the antibacterial activity was almost absent.

Studies on the antagonism of Serratia plimutica so far have been reported by Berg as Verticillium dahliae and Rhizoctonia solani, which causes tomato Verticillium wilting disease. It was reported that antagonism was high in the fungus, and Kloepper et al. (Collectotrichum orbiculare), McCullagh (Cucculagh), cucumber pitium root rot bacteria, Stanley (Stanley), etc. It has been reported to have antagonistic activity against fungi, and Thaning et al. Reported that antagonists of Sclerotinia sclerotiorum, which cause fungal diseases in potatoes, kidney beans, tobacco, and buckwheat.

In addition, McInroy et al. Were found to exist as endogenous microorganisms (endophytes) of cotton and sugar corn.

However, there are no reports of antagonism against leek S. cepivorum, pepper black pattern disease (Alternaria altreta), and Bellflower stem cell (Phoma sp.).

Fifth, antagonist production ability of antagonist Serratia plymutica AL-1

Antagonist mechanisms of antagonists include siderophores, β-1,3-glucanase, chitinase, antibiotic, cyanide, etc. Variously known.

1% colloidal to investigate the antagonist S. plymuthica AL-1, which has a high antagonist against S. cepivorum of leek The culture supernatant cultured in TSB medium with or without chitin (colloidal chitin) was divided into YM10 membranes based on molecular weight of 10,000 to examine the antimicrobial activity, as shown in FIG. 7. In the culture with chitin (colloidal chitin), antagonism was observed at more than 10,000 and antagonism at less than 10,000.

However, in the culture without the addition of 1% colloidal chitin, it showed activity only below 10,000 and almost no antibacterial activity above 10,000.

In addition, after heat-treating the material below 10,000 at 80 ° C., the antagonistic force was also investigated, suggesting that not only chitinase but also other bioactive substances having a molecular weight of 10,000 or less were present. It was confirmed that chitinase was induced by colloidal chitin).

Of course, the Seratia plymutica antagonizing the leek black rot fungal nucleus bacterium according to the present invention is a photograph showing the state of inhabiting antagonists in the root of the leek, as shown in Figure 8, Figure 8a is enlarged 100 times the root of the leek It is a photograph and FIG. 8B is a photograph which enlarged 3000 times the edge part of the root of the said leek.

Reports of the genus Serratia producing chitinase have been reported in Serratia marcescens, S. proteamaculans, and Serratia sp. JM. Although reported, S. plymuthica has never been reported.

As described above, Seratia plymutica antagonizing the leek black rot fungal nucleus according to the present invention isolates 146 myomicrobial microorganisms from the roots of the leek and antagonizes the AL-1 strain antagonizing the sclerotium cepivorum Because of this, it is possible to easily prevent microbiological diseases caused by Sclerotium cepivorum of leek, Sclerotium cepivorum, pepper black pattern disease (Alternaria altrata), pepper anthrax (Colletotrichum) gleosporioids, Phoma sp., Pseudomonas sp., Rhizoctonia solani, Stemprhlium solani, Sclerotinia sclerotiorum, Watermelon vines, Fusarium oxysporium, etc. There is an advantage that can be prevented microbiologically easily caused by pathogenic fungi.

Claims (1)

Serratia plymutica antagonizing the leek black rot fungal nucleus strain consisting of Serratia plymuthica AL-1 (Accession No. KCTC 10187BP) strain.