KR100750566B1 - Stephanoascus ciferrii msbb and a eco-friendly products to plant-growth promotion with this strain or cultural suspension - Google Patents

Abstract

A microorganism Stephanoascus ciferrii MSBB and environment-friendly soil microbial preparations containing the same microorganism or its cultural suspension are provided to promote growth of a plant such as red pepper, and reduce occurrence of red pepper blight without harmful effects on environments. The microorganism Stephanoascus ciferrii MSBB(KACC 91297P) isolated from the forest soil increases initial growth and fruit load amount of red pepper and has antimicrobial activity against Phytophthora capsici and Bacillus anthracis. The environment-friendly soil microbial preparations contain Stephanoascus ciferrii MSBB(KACC 91297P).

Description

Stefanoascus ciferrii MSBB and a eco-friendly products to plant-growth promotion with this strain or cultural suspension}

The present invention is a stephanoascus isolated from forest soil ciferrii MSBB strain and the soil microbial agent containing the same, more specifically Stephanoascus ciferrii MSBB ( Phytophthora) causes severe damage in pepper cultivation capsici ) and anthrax ( Colletotrichum acutatum ) to show the antimicrobial effect and relates to the invention to use this strain in soil microbial agent environmentally friendly agricultural materials.

Red pepper is one of the major economic crops, accounting for more than 20% of the domestic vegetable area. However, the series according to the narrow cultivated area is Phytophthora Capsici and Colletotrichum spp. cause various disorders due to changes in soil physicochemical properties due to infestation and serialization of late blight and anthrax. The production loss due to late blight is estimated to be about 5% per year. Cultivation methods such as darby and wheat cultivation encourage the spread of the disease. 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. Especially Phytophthora In the case of capsici , the host range is wider than other pests. Red peppers, watermelons, cucumbers, melons, and tomatoes are economic crops that suffered enormous damage.

Plague germs are aquatic bacteria that transmit very rapidly during rainfall. This pathogen has recently 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. The use of soil treatment agents and microbial derived materials utilizing useful microorganisms has been studied and applied.

Colletotrichum Anthracnose caused by actatum occurs in vegetables and fruit trees such as peppers, grapes, apples and peaches, causing enormous damage. 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, in view of the urgent resolution of the above-mentioned problems and the increase of end-users' interest in food safety and economic stability, the development and distribution of agricultural materials to support eco-friendly agricultural production Is requested. In particular, there is an urgent need for a strategy for crop cultivation methods utilizing microorganisms and other biological resources without side effects.

Therefore, an object of the present invention is to provide an eco-friendly cultivation method and to reduce the loss caused by the early growth of pepper, late blight and anthrax.

Accordingly, the present inventors have studied microorganisms isolated from forest soil, while studying the simultaneous control of phytophthora capsici and anthrax ( colletotrichum acutatum ) using microorganisms ( Stephanoascus ciferrii MSBB) completed the present invention by confirming that it has antimicrobial activity in the early growth of red peppers and in the transformation of Phytophthora capsici and anthracnose mycelia ( Colletotrichum acutatum ).

The present invention as described above is stephanoascus isolated from forest soil ciferrii It is characterized by using MSBB.

The present inventors collected a large number of samples from the forest soil in the northwestern part of Gyeongbuk to secure microorganisms for controlling pepper pestilence and anthrax that cause problems in pepper cultivation, water agar medium (fungal separation) and nutrient medium (bacterial separation), actinomycetes After separating many microorganisms using the separation selection medium and potato agar medium, the microorganisms showing antimicrobial activity against the early growth of pepper and pepper blight and anthrax are separated and secured. The activity was selected for assay through pot experiment.

The present inventors finally named the assay-selected microorganism as ' Stephanoascus ciferrii MSBB', and deposited it with the accession number 'KACC 91297P' at the Korea Agricultural Microbial Resources Center on January 26, 2007.

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

1. Identification of Strains and Culture  characteristic

Morphological characteristics of the selected strains were investigated under electron microscope after incubation at 30 ° C. using YNBG (Yeast Nitrogen Base Glucose Broth) or YNBGA (YNBG + Agar) medium. The colonies were cream and membranous, and the chains of spores were helical, and the surface of the spores was smooth and round (Fig. 1).

       <Figure 1. Colony and electron micrographs of isolated microorganisms>

Final identification of the isolated microorganisms was performed by ITS-5.8S-ITS2 sequencing. The method was as follows.

After chromosomal DNA of this strain was isolated, primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3 ') and ITS4 (5'-TCC TCC GCT TAT) were used to amplify the target gene of ITS-5.8S-ITS2. TGA TAT GC-3 ') was 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. 0.8% agarose gel electrophoresis was performed on the amplified PCR product, and then amplified DNA was isolated and purified, and the nucleotide sequence was analyzed using ABI PRISM 3700 DNA Analyzer. The BLASTN program was used to identify ENE-NKS and RDP (ITS-5.8S-ITS2 sequencing). The isolated strain was Stephanoascus As compared with 492 nucleotide sequences of the ciferrii strain CBS 5295, the homology was 100%.

Figure 2. MSBB and Stephanoascus using BLASTN program Comparison of ITS-5.8S-ITS2 Sequences of ciferrii strain CBS 5295>

To investigate the physiological characteristics of this strain, 46 species including glucose were used including glucose, 10 species including nitrate were used as nitrogen source, vitamin, osmotic sensitivity, and cycloheximide sensitivity. The results are shown in the following Tables 1, 2 and 3. For carbon sources, 39 species including D-glucose were used, and 9 species including L-sorbose were not available. Seven species, including ribose, were found to be inconsistent in availability.

Table 1 Stephanoascus ciferrii Carbon source utilization characteristics of MSBB

+: Used,-: not used, ±: variable

Easily available among the disclosed nitrogen sources were ethylamine, L-lysin and cadaverine, and the other nitrogen sources were not available.

Table 2 Stephanoascus ciferrii Nitrogen Source Utilization Characteristics of MSBB

Among the vitamins listed, pyridoxine, niacin, and PABA were found to be useful. Growth was observed in the medium containing cycloheximide, but not in the medium containing acetic acid. As a result of examining the osmotic sensitivity, the growth was not constant in the medium containing 60% D-glucoserk, the growth of bacteria was not observed in the medium containing 16% NaCl.

Table 3 Stephanoascus ciferrii Vitamin, osmotic sensitivity of MSBB and

       cycloheximide susceptibility

2. Temperature and pH Growth according to

YMB (Yeast extract Malt extract Broth) was used to investigate the optimum culture temperature and pH for agricultural use of this isolate. The temperature was set at 5 ℃ intervals from 20 ℃ to 40 ℃, and the pH was from 4 to 8 Investigations were made at intervals. The growth rate was measured using a spectrometer and investigated at absorbance A640. The optimum temperature was 30 ℃ and the pH was 6-7.

Figure 3.Stephanoascus optimum incubation temperature and pH of ciferrii MSBB>

3. antimicrobial activity, plant growth promotion and Insolence  Phosphoric Acid Solubilization

3.1. Primary antimicrobial activity

Stephanoascus To measure the primary antimicrobial activity of pepper pest and anthrax of ciferrii MSBB, yeast extract 0.3%, malt extract 0.3%, peptone 0.5% glucose 1% and agar 1.5%, and the medium adjusted to pH 6.8 Used. After incubation for 7 days at 28 ℃, the inoculation of the target pathogen was incubated for 5 days at the appropriate growth temperature of the pathogen, and the antimicrobial activity was investigated by the medium diffusion of the metabolite. It was.

Figure 4.Stephanoascus Antimicrobial Activity of Ciferrii MSBB against Pepper Blight and Anthrax>

As a result (Fig. 4), the antimicrobial activity of the reverse bacteria was shown to be about 50%, anthrax mycelial growth inhibiting ability or nateu or, Stephanoascus ciferrii MSBB showed low hyphae density of the adjacent portion to be insignificant for. Mycelial strain (swelling phenomenon) of anthrax was observed through a microscope.

3.2. Assay Using Secondary Culture Products

Stephanoascus The secondary antimicrobial activity of ciferrii MSBB against red pepper blight and anthrax was contained 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose. After 7 days shaking culture at 28 ℃ lyophilized to prepare a crude extract was assayed.

Figure 5.Stephanoascus Antimicrobial Activity of Ciferrii MSBB Extracts against Red Pepper Streptococcus>

Final content in the target pathogen culture medium was carried out by adjusting to 1% and 0.5%. As a result, the final antimicrobial activity against red pepper bacterium was 40.0% and 86.9%, respectively, according to the crude extract content.

3.3. Indoor simple living experiment

Stephanoascus ciferrii msbb Strain culture for tertiary antimicrobial activity against pepper germ bacterium of 200401 contained yeast extract 0.3%, malt extract 0.3%, peptone 0.5% and glucose 1%, a medium 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 for infection by the pathogen. Used after surface disinfection. Inoculated pepper was incubated at 25 ° C. for 7 days and then examined for disease. In the case of pathogen alone treatment, pepper berries showed infection symptoms overall, but in the combination treatment group, lesions were formed on 15 fruits of the treated fruits, showing an inhibition rate of 74.1 (Table 4).

Table 4 Stephanoascus ciferrii Inhibitory Effect of MSBB on Pepper Disease

3.4. Validation of growth promoting effect in greenhouse

Stephanoascus ciferrii The effect of MSBB on early growth of red pepper was tested in the greenhouse. Strain culture for the assay contained 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose, and strains were cultured using a medium adjusted to pH 6.8. Culture conditions were investigated by shaking three times at 1 week intervals from 1 week after the red pepper formulation after 7 days shaking culture at 28 ℃. The treatment method consisted of three treatments of 100 ml irrigation, 2-fold and 50-fold dilutions. Seven days after the final treatment, the pepper length, number of nodes, stem diameter, and fruitings over two times were measured. Fruits were harvested and counted at the 1st survey, and the duplicate count of the fruit was excluded by the same method for the 2nd and 3rd surveys. The grass length was the longest in the irrigation treatment, and no significant differences were found in the number of stem and stem diameter. The final height elongation in the irrigation treatment was about 10% compared to the untreated treatment (Table 5).

Table 5 Stephanoascus ciferrii MSBB's Pepper Growth Promoting Effect

Table 6 Stephanoascus ciferrii Effect of MSBB on Pepper Yield

The amount of fruiting was the highest in foliar fertilization treatment of 50-fold dilution, and in the third study, the fruiting amount was about 48% higher than that of the control (Table 6).

3.5. Insolence  Phosphoric Acid Solubilization

Stephanoascus ciferrii The insoluble phosphate solubilization ability of MSBB was added to the final concentration of CaCl2 and K ₂ HPO ₄ based on YMBA so that 0.5% of the inoculated stephanoascus ciferrii MSBB was inoculated and incubated at 28 ℃ for 7 days. It was. A zona pellucida was formed around the Stephanoascus ciferrii MSBB flora, and the insoluble phosphate solubilizing activity was examined (FIG. 6).

Figure 6.Stephanoascus Insoluble Phosphate Solubilization of ciferrii MSBB>

Example (1) of microbial agent

Stephanoascus ciferrii containing MSBB strain

15% by weight of soil

45% by weight of ink

5% by weight of charcoal powder

Rice bran 15% by weight

15% by weight of water

5% by weight of raw milk

The finely divided powders are ground to each other, and the finely divided powders are mixed in a mixer at the above ratio.

The mixture was put into a microbial culture chamber at 28 to 30 ° C. and deposited and fermented for 7 days while supplying air.

The fermented product was placed in a fabric bag in 20 kg units and placed in 500 l of water and precipitated for 15 days at 25 ° C. for 15 days to obtain a supernatant to obtain a microorganism preparation for irrigation and foliar spraying.

As detailed above, Stephanoascus according to the present invention. ciferrii It can be seen that the MSBB strain exhibits antimicrobial activity against pepper blight and anthrax.

In addition, it is possible to see the effect of promoting the initial growth and increase the amount of fruit by spraying irrigation or foliar spray by making the microbial agent by multiplying the strain can be obtained significant effect by fertilizing in the soil.

<110> LEE, phil su <120> Stephanoascus ciferrii MSBB and a eco-friendly products to          plant-growth <140> 1020070010440 <141> 2007-02-01 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 505 <212> DNA <213> Stephanoascus ciferrii MSBB <300> <301> T.M, Pryce          S, Palladino          I.D, Kay          G.W, Coombs <302> Rapid identification of fungi by sequencing the ITS1 and ITS2 <307> 2003-12-02 <400> 1 tttttaaatg atactttgct ttggtcagac tttaattagt ctggccagag gtatacaaac 60 tccaaattta ttttaaacat gagtctgaat tgaaaaagaa taaattattc aaaactttca 120 acaacggatc tcttggttct cgcatcgatg aagaacgcag cgaaatgcga taagtaatgt 180 gaattgcaga atttcgtgaa tcatcgaatc tttgaacgca cattgcgccc cttggtattc 240 cagggggcat acgtgtatga gcgtcatttc actcttaaac cctcgggttt agtgttgaac 300 ctttccttcg atcttttttc gaaggatggc tcgaaatgaa atggcaaggc aatccagtct 360 aaagcttaca cagtgtctta ggttttacca attacgctgg gcaaagcttg cttggatgag 420 ccgggcggta caaacttgat ttaaaagttt gacctcatac acgtaagaat acccgctgaa 480 cttaagcata tcaataagcg gagga 505

Claims (6)

Stephanoascus ciferrii MSBB strain (Accession No. KACC 91297P) characterized by early growth of red pepper, improvement of fruiting yield, and antibacterial activity against late blight and anthrax. A soil treatment microbial agent comprising Stephanoascus ciferrii MSBB strain (Accession No. KACC 91297P). Leaf dispersant containing culture medium of Stephanoascus ciferrii MSBB strain (Accession No. KACC 91297P) Stephanoascus ciferrii Liquid spray for the inhibition and control of pepper blight and anthrax including MSBB strain (Accession No. KACC 91297P). Soil microbial preparations for promoting solubilization of insoluble phosphoric acid, including Stephanoascus ciferrii MSBB strain (Accession No. KACC 91297P) delete