KR20080089324A - Growing methods of kiwi using streptomyces cultivation mixture - Google Patents

Abstract

Culture methods of Kiwi by using microorganism is provided to inhibit growth of Botryosphaeria dothidea resulting in fruit blight and Pseudomonas syringae resulting in blossom blight by using Streptomyces-cultured medium without agricultural chemicals, so that environment-friendly cultivation of kiwi is accomplished. A microorganism Streptomyces CH0110(KCTC 11359BP) antagonizes fruit blight and blossom blight-causing bacteria including Botryosphaeria dothidea and Pseudomonas syringae, and is isolated from the soil. A biological composition for preventing fruit blight and blossom blight comprises the cultured medium of Streptomyces CH0110(KCTC 11359BP). The fruit blight and the blossom blight of Kiwi are prevented by spraying the biological composition to flowers and fruits of Kiwi.

Description

Growing methods of Kiwi using Streptomyces cultivation mixture

The present invention relates to an environmentally friendly cultivation method of sesame seed using Streptomyces genus soil actinomycetes. More specifically, streptococcus soil radiobacterium culture medium having high antagonism against fruit rot pathogens and flower rot pathogens of sea stalks is dispensed to the petals and ripening of the growing sea stalks to prevent overgrowth and flower rot. The present invention relates to a method for growing green tuna in an environmentally friendly manner without using pesticides.

Recently, the government recognized the importance of environmental agriculture and established the basic foundation of environmental agriculture by 2000 and the dissemination stage by 2005, aiming to reduce 50% of pesticides and 40% of chemical fertilizers to reduce the environmental pollution of agriculture. It plans to fully settle down by 2010 and is promoting eco-friendly agricultural policy. Jeollanam-do, which occupies approximately 52% of the country's national production, is striving to build eco-friendly agriculture and spread awareness with a long-term plan to cultivate 30% of all farms as eco-friendly agricultural producers.

In order to create an eco-friendly production base for sesame, it is possible to replace existing chemical fertilizers and pesticides, and it is necessary to establish and disseminate eco-friendly agricultural materials that can be easily purchased and used safely by farmers. In particular, the development of biological products that can control pests in an environmentally friendly manner is urgently needed. Overseas, about 20 kinds of bacterial strains for biocontrol have been developed, including Pseudomonas sp., Bacillus sp., Streptomyces sp. And Agrobacterium sp. It is marketed.

P. aeruginosa, P. putida , P. fluorescens , P. fluorescens , Mesorhizobium loti , Bacillus subtilis , and P. aeruginosa Streptomyces coelicolor ( Streptomyces coelicolor ), etc. in the genetic unit to increase the activity of the strain is being studied. Typical antimicrobial materials they produce include biscosinamide, lipopeptide, pyoluteorin, 2,4-diacetylphloroglucinol, and 2-hydroxyphenazene compounds. This is being reported.

Bacterial blossom blight is a bacterial Pseudomonas syringe pv. Since it is reported to be caused by Syringe ( Pseudomonas syringae pv. Syringae ), it occurs in 30-40% of the flowers depending on the year of occurrence, which is a disease that greatly affects the production, such as falling or fruit malformed. In this aspect, the present invention by selecting and identifying actinomycetes as antagonists against overgrown rot pathogens and flower rot pathogens in sesame fruit in order to secure basic data for eco-friendly cultivation in agricultural sites of the sea stalks Development of an eco-friendly cultivation method of the green tea without the use of pesticides to complete the present invention.

The problem to be solved by the present invention is the use of pesticides by controlling actinomycetes and flower rot disease by selecting and identifying actinomycetes as antagonists against overgrown rot pathogens and flower rot pathogens that occur in the fruit of the sea cucumber for eco-friendly cultivation in the agricultural field of the tuna The purpose of this study was to develop an eco-friendly cultivation method of green tuna.

It is an object of the present invention to provide a microorganism Streptomyces CHO110 (KCTC 11359BP) that antagonizes the overripe rot and flowering rot pathogens of blue tuna.

In addition, to provide a biological composition for controlling the ripening rot and flower rot of the sesame seed containing the Streptomyces CHO110 (KCTC 11359BP) culture as an active ingredient.

In addition, another object of the present invention is to provide a method for cultivating environmentally friendly sesame seeds by dispensing to the petals and ripening of the sesame as the biological composition for the control to prevent over-rot and flower rot.

The effect of the present invention is to select and identify actinomycetes as an antagonist against overgrowth rot pathogens and flower rot pathogens that occur in the fruit of the larvae for eco-friendly cultivation in the agricultural field of the sea stalk. It is to provide a method of cultivating sesame seeds.

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

1. Isolation of Over-Meat Rot Pathogens and Flower Rot Pathogens

In order to separate the overgrown rot pathogens and flower rot pathogens generated from the fruits of the sea cucumbers produced in the southwestern region of Jeonnam, including Goheung, Suncheon, Jangheung, and Haenam, the fields were collected by disease and sign by exploring the fields of the field of angyo Botryosphaeria dothidea , a causative agent of overgrowth rot, was isolated from Pseudomonas syringae , a causative agent of flowering rot.

The re-inoculation of isolated overgrowth rot and flower rot pathogens showed the same symptoms as the overgrowth rot and flower rot. That is, in the case of fruit rot, the first part of the fruit surface was recessed concave in the fruit, and gradually progressed thereafter, the center turned to milky white to sulfur yellow, and the surrounding was dark green. When cutting the fruit infected with the pathogen, the inner part showed soft green soft symptom and rotted to the pulp core. In case of flower rot disease, the disease occurred severely from April to June, mainly in the flower buds and firearms before and after flowering. The disease showed browning of the surgery, and in severe cases, the petals browned and the flowers did not bloom. Because of the invasion and infection of bacteria by invasion of the base of the operation, ovary and flower bed, it was not pollinated and degraded.

2. Isolation of Single Strains from Nature

Separation of single strains from nature was carried out as follows. In order to select antagonistic root zone bacteria with high growth inhibitory ability against overgrown rot and germ rot pathogens, 250 single strains were isolated from the soil of the tuna grove in southwestern Jeonnam province. Over 250 single strains isolated as single strains were inoculated in nutrient agar medium to form a flora, and then refrigerated and used for the antimicrobial replacement culture experiments with the sedible overripe rot pathogen and flower rot pathogen.

3. Replacement of Pathogens and Antagonists and Selection of Antagonists

As a result of screening the antagonistic strain against the fruiting rot pathogen ( Botryosphaeria dothidea ) generated from the fruit of the tuna, as shown in Figure 3, the antagonistic strain was classified into two groups, bacteria and actinomycetes. Antagonists # 110 and # 120, considered to be actinomycetes, showed high antagonism of more than 90%. In particular, antagonists # 110 had the best antagonism of 96%. The cross streak test was used for the antibacterial test of isolated strains # 110 and # 120, which are considered to be actinomycetes among rot fungus.

Assay bacteria include line A: Bacillus subtilis [gram (+) bacteria], line B: Escherichia coli [Gram (-) bacteria], line C: Pseudomonas syringe pv. Line D: Pseudomonas genus [plant pathogen] was used. Actinomycetes were inoculated on a plate medium capable of growing both actinomycetes and flower rot pathogens and assay bacteria, and incubated at 27 to 30 ° C for 2 days. Incubation was carried out at 24 ° C. for 24 hours to determine the length of the growing jersey. Isolate antagonist # 110 showed potent inhibition against all assays, including rot pathogens. Therefore, # 110 was finally selected as an antagonist against rot fungus flower rot disease (FIG. 3, FIG. 4 and Table 1).

4. Culture and Morphological Characteristics of Isolated Strains

The culture characteristics of # 110, a strain with excellent antagonistic activity against fruit rot and flower rot, were found to be good in most media except tyrosine agar (ISP medium 7). Yeast extract-malt Mycelial growth and spore formation were good in oatmeal agar (ISP medium 3) and inorganic salt-starch agar (ISP medium 4), including extract agar (ISP medium 2) (Table 2).

The color of the hyphae was mostly gray in ISP medium 4, the back color was grayish yellow in glycerol-asparagine agar (ISP medium 5) medium, melanin formation was observed only in ISP medium 6, Observed only in ISP medium 5 and negative. After culturing 14 days of # 110 highly antagonistic against the aging and pathogenic rot pathogens of the fruit of the sea salt in the inorganic salt starch agar medium, the size was about 0.58 × 0.91 ㎛ as observed by scanning microscope and phase contrast microscope. The spores were spherical and the surfaces were smooth, and the spore chains were arranged rectiflexible (Table 3).

5. Physiological Biochemical Properties

The availability of carbon source of strain # 110, a highly antagonistic strain against fruit ripening rot and flowering rot pathogens, was found to be excellent in the availability of xylose, cellobiose, rhamnose and arabinose, and fructose, raffinose, and meso-. Sugar availability of inositol, sucrose and mannitol was found to be insufficient (Table 4). In addition, antagonist # 110 was found to be able to grow in a wide range of pH in the culture medium of pH 4-11. Casein hydrolysis, esculin urea and starch resolution were positive, melanin production was negative, and no soluble pigment was produced. As a result of examining the culture, morphology, physiology and biochemical characteristics of the strain, it was determined that # 110 was a strain of Streptomyces genus. In addition, the strain # 110 spore form, the growth state in various culture medium and the strain colors and because it appears similar to the Streptomyces genus strains listed in the International Streptomyces Project in terms of physiological characteristics finally gilhanggyun # 110 Streptomyces sp. Named # 110. The strain was deposited in the microbial deposit center of the Korea Research Institute of Bioscience and Biotechnology, 111, Gwahak-ro, Yuseong-gu, Daejeon, in accordance with the Budapest Treaty with accession no. KCTC 11359BP dated June 24, 2008.

6. Growth Inhibition Effect of Rotten Pathogens of Root Flower in Media Containing Antagonist Culture

Isolated antagonistic strain Streptomyces sp. To determine whether the antibacterial substance secreted by CHO110 (KCTC 11359BP) is a protein or non-protein, sterilization of the culture medium of the strain for 15 minutes at 121 ℃ to examine the antimicrobial activity of the rot fungus and flower rot pathogenic pathogens The inhibitory power was confirmed to be close to 100%, through which the antimicrobial material in the culture material of the strain was confirmed to be a non-protein component without degeneration due to heating. Streptomyces sp. CHO110 (KCTC 11359BP) was cultured in SD + B + P medium for 3 days, centrifuged at 10,000 rpm for 8 minutes to remove cells, mixed with PDA for culture, and sterilized for 15 minutes at 121 ° C. After removal, the medium prepared by the heat treatment method was mixed with PDA medium and sterilized to examine the antibacterial effect on the pathogen of the sterilized culture solution subjected to high temperature heat treatment.

7. Effect of Controlling Anti-Mature Overgrowth and Flower Rot Disease on Antagonism Treatment

Table 6 shows the results of the treatment of the culture solution of bacterial strains with high antimicrobial activity in vitro on the control of M. rot and flower rot. The control group treated with the cultivation of wild rot and germ rot pathogen was very severe and the occurrence of disease was very rare in the test tract treated with antagonists such as # 110 and # 120. It was investigated that there is no possibility of developing biopesticides using these strains.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1 Isolation of Over-Meat Rot Pathogens and Flower Rot Pathogens

Field visits and flower rot pathogens (Pseudomonas syringe pv. Syringe) infected with fungus pathogens (Botrios paeria dotidea), while visiting the tuna cultivation complex and storage area in southwestern Jeonnam, Haenam, Jangheung and Goheung. The diseased pathogens were collected, and pathogens were separated and reported by symptom and sign. Isolation of the over-rotted rot pathogen and flower rot pathogen was carried out as shown in FIG. In other words, the diseased area of the fruit was wet-treated for 3 days, and then surface sterilization of the diseased tissue with 70% ethanol and 5% NaOCl, followed by washing with 0.85% physiological saline to place the lesion on the selective medium for separating pathogens. Pathogens which formed a flora were isolated by incubating for 2-3 days in an incubator with a relative humidity of 90% or more.

Isolation of isolated overgrowth rot pathogen and flower rot pathogen on the larvae fruit, the same symptom as the scabbard fruit rot disease and the flower rot disease, and the cultivation of the rapeseed fruit rot pathogen (Botriospaeria dotidea) and flowers received from the plant pathology laboratory of Sunchon National University After confirming the same symptoms as rot pathogens (Pseudomonas syringe pv. Syringe) was used as a public pathogen.

Example 2 Effective Antagonist Microbial Isolation

Separation of effective antagonist microorganisms was carried out in 100 mL of Tris-HCl buffer (pH 7.5) from soil samples of 5-20 cm from the sesame orchard surface in southwestern Chonnam, and cultured for 10 minutes. the soil suspension was diluted × 10 ³ ~10 ^8-fold with physiological saline (0.85%, NaCl), collected in a 200 mL double inoculation were plated at different concentrations on nutrient agar media (nutrient agar plate). The plated inoculated nutrient agar medium was incubated for 30 to 36 hours in a 30 ℃ incubator and then separated into a single strain based on the color and shape of the bacterial flora and stored in a slope medium (Fig. 2).

Example 3 Selection of Antagonist Strains

The selection of antagonist strains against overgrown rot pathogens was selected as antagonists by forming overgrown rot pathogens (Botrios paeria dotidea) of the fruit of the larvae and the formation of a large inhibitory zone through the replacement of effective microorganisms. Growth inhibition rate for pathogens was expressed as a percentage compared to the non-treated group after measuring the low zone of the antagonists formed against the pathogen flora. On the other hand, the isolation of strains with high antimicrobial activity against rot fungus pathogen was selected by cross-stroke test. That is, inoculated with a single strain isolated on a plate medium capable of growing both the isolate and the rot flower rot pathogens and assay bacteria, incubated for 2 days at 27-30 ℃ and at right angles from the isolate strain After inoculating the assay bacteria in a straight line and incubating at 30 ℃ for 24 hours to measure the length of the growing jersey was selected as a high-low strain antagonistic bacteria.

Example 4 Culture Morphological and Physiological Characteristics of Isolated Antagonists

The cultivation and physiological and biochemical characteristics of the isolated microorganisms were examined to identify the antagonistic bacteria with excellent antagonistic activity against the ripening rot and flower rot pathogens in the oyster fruit of southern Chonnam. Soluble cultivation of antagonists by medium type such as glucose asparagine agar, Czapek sucrose agar, Bennett agar and International Streptomyces project (ISP) medium for 14 to 21 days Whether the pigment was produced or not was investigated.

In addition, the branched form of mycelium, the shape, size, and donor status of the mycelium on the inorganic salt starch agar medium (ISP medium 4) were incubated for 21 days at 30 ° C. by an inclined cover-slip method. The microscope was scanned with a phase contrast microscope and a scanning microscope. The physiological characteristics of the isolated antagonists were investigated for the carbon source availability of the finally selected antagonistic bacteria against over ripening rot pathogens (Botrios paeria dotidea) and flowering rot pathogens (Pseudomonas syringe pv. Syringe). Starch decomposition power and nitrate reducing power were observed.

Example 5 Chemical Identification and Characterization of Isolated Antagonists

Finally, the diaminopimelic acid (DAP) isomer and cell wall amino acid in the cell wall of the isolated antagonists were analyzed. In other words, the dried cells were hydrolyzed with 6N HCl at 100 ° C. for 18 hours, and then filtered through a filter paper to perform cellulose thin layer chromatography.

Example 6 Pathogen Growth Inhibition of Antagonistic Culture Solution

The antagonists were incubated for 3 days in SD + B + P medium (5% sugar, 3% soy sauce, 0.2% beef extract, 0.2% peptone) and centrifuged at 10,000 rpm for 8 minutes to remove cells and Mixed with PDA and sterilized for 15 minutes at 121 ℃. In addition, the medium prepared by filtration of the antagonistic culture medium was prepared by sterilizing PDA (fourfold concentration) and 1/4 culture supernatant for the entire culture and then mixing 3/4 culture supernatant filtered with 0.45 μm membrane filter. . The prepared culture medium was poured onto a plate and hardened, and then inoculated with botanical overgrowth rot pathogen (Botrios paeria dotidea) and flower rot pathogen (Pseudomonas syringe pv. Syringe) and incubated in a 25 ° C. incubator for 7 days. Was compared with the control inoculated with PDA.

Example 7 Examination of the Effect of Antagonistic Treatment on the Control of Overgrowth Rot Disease and Flower Rot Disease

The effect of the culture of antagonists on the biological control of the overripe rot pathogen (Botrios paeria dotidea) and the rot pathogen (Pseudomonas syringe pv. Syringe) was examined in vitro. That is, after spraying the angular rot fungi and flower rot pathogens respectively, the antagonist culture was diluted to a concentration of 5.0 × 10 ⁷ cfu / ㎖ irradiated at 30 days after treatment while keeping the plants in the growth. The control effect of rot fungus flower rot according to antagonistic treatment was divided into very severe (+++), severe (++), slightly occurring (+) and non-infectious and no disease occurrence (-).

1 is a pathogenic bacterial strain Boturios paella dotidea and Pseudomonas syringe pv, which causes ripening rot and flowering rot of lychee fruit. It shows separation of syringes.

Figure 2 shows the isolation of a single bacterial strain derived from sesame orchard soil samples in southwestern Chonnam region.

Figure 3 shows the antimicrobial activity of the antimicrobial bacterial strains (# 110 and # 120) against rotted rotting disease, Botryos paeria dotidea (BD), infected in the lychee fruit on potato dextrose agar (PDA) plate at 28 ° C. for 5 days. will be.

FIG. 4 is a flower rot disease Pseudomonas syringe pv infected in the rapeseed fruit on a yeast malt extract agar (YM) plate at 30 ° C. for 1 day. It shows the inhibitory effect of the antibacterial strain on the syringe.

[# 110 and # 120: antagonistic bacteria,

Line A: Bacillus subtilis <gram bacteria>,

Line B: Escherichia coli <gram bacteria>,

Line C: Pseudomonas syringe pv. Syringe <flower rot>,

Line D: Pseudomonas <Plant Pathogen>]

Claims (3)

Microorganism Streptomyces CHO110 (KCTC 11359BP) Antagonists of Overgrowth and Flower Rot Pathogens Biological composition for control of over ripening rot and flower rot of sesame seed comprising Streptomyces CHO110 (KCTC 11359BP) culture of claim 1 as an active ingredient A method for cultivating environmentally friendly tuna according to claim 2, by dispensing on the petals and ripening of the tuna, and preventing over-rot and flower rot.

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