KR100411185B1 - A new Streptomyces sp. AG-P(KCTC 8965P), and an agent for control of plant diseases using this strain - Google Patents

Abstract

The present invention relates to the strain Streptomyces sp. AG-P (KCTC 8965P) and plant disease control agents using the same, more specifically Streptomyces sp. AG-P (KCTC) 8965P) strains showed selective antibacterial activity against phytopathogenic fungi (Oomycete) such as Phytophthora sp., A plant bacterium, and Pythium sp. New strain strain Streptomyces sp. AG-P (KCTC 8965P), which can be used as a biocontrol agent for controlling plant diseases such as plant late blight and receivable diseases using strains, and plant disease control agents using the same will be.

Description

New strain of Streptomyces spp. AGP-P (PCC C655P) and plant disease control agents using the same {A new Streptomyces sp. AG-P (KCTC 8965P), and an agent for control of plant diseases using this strain}

The present invention relates to the strain Streptomyces sp. AG-P (KCTC 8965P) and plant disease control agents using the same, more specifically Streptomyces sp. AG-P (KCTC) 8965P) strains showed selective antimicrobial activity against phytopathogenic Oomycetes such as Phytophthora sp., A plant late blight and Pythium sp. New strain strain Streptomyces sp. AG-P (KCTC 8965P), which can be used as a biocontrol agent for controlling plant diseases such as plant late blight and receivable diseases using strains, and plant disease control agents using the same will be.

Plant diseases caused by the late blight Phytophthora and the stock bacteria Pisium are very numerous and the damage is enormous. Around 60 kinds of late blight and more than 1,000 late blight host plants have been reported all over the world. In Korea, 16 kinds of phytophthora are known to cause 46 crops. For example, Phytophthora infestans are primarily in potatoes and tomatoes, and Phytophthora capsici occurs in all plant species, including peppers, watermelons, cucumbers, melons, and tomatoes. It is known that one strain species occurs in multiple hosts. In particular, the phytophthora infestans and phytophthora capsaish are causing great damage to potatoes, tomatoes and peppers in Korea.

Plague germs overwinter in diseased plants in the soil in mycelial state, and after the growth of pathogens, they appear on the ground and become secondary infectious agents. Plague is one of the most important plant diseases that causes a lot of losses in crop production because the disease develops in a cool and humid environment. Rice seed rot and stocking disease caused by the pathogens in Pisium are well known all over the world, including our country, and the damage is serious. In Korea, Psidium spp. Is known as a major pathogen that causes root rot, including seedling rot of various crops such as tomatoes and cucumbers, and ginger root rot.

In addition, late blight, a type of oomycetes, is a typical soil pathogen, which has been included in the fungal taxonomy up to now, but has recently been controlled by the International Fungal Society as classified as belonging to the protist. The fungicides used for this purpose also differ in their properties, and metalaxyl, dimethomorph, and focetyl-A1, which are different from the fungal fungicides which are generally used, are used.

However, these are chemical synthetic pesticides, and very few microbial fungicides which directly use microbial strains have been developed. In addition, many crops are hydroponically grown by genetic engineering techniques during agricultural production. However, when hydroponic farming is carried out, damage caused by late blight and incoming pathogens, which prefer water, has been reported, and most existing pesticides are insoluble in water. It is reported that the control rate is very weak or ineffective.

Therefore, as the interest in environmentally friendly agriculture grows, the interest in controlling methods using biopesticides using microorganisms is increasing. Therefore, development of microbial fungicides using microbial strains or microbial cultures itself is an urgent task.

Therefore, the present inventors have isolated new strains of soil actinomycetes, which have excellent control effects against these pathogens, while researching and developing methods for controlling pests or incoming germs belonging to the cultivation by biological methods using actinomycetes as soil microorganisms. By using this strain, the present invention was completed by confirming the development potential as a plant disease control agent such as late blight and wearing disease.

Therefore, the present invention is a plant such as late blight and wearing disease, which is known to be difficult to control using the new strain Streptomyces sp. AG-P (KCTC 8965P), which selectively shows antibacterial effects on late blight and incoming pathogens. The purpose is to provide an environmentally friendly microbial agent effective for disease control.

1 is a photograph showing the Streptomyces sp. AG-P (KCTC 8965P) strain used in the present invention was grown in Bennett agar medium (Bennett's agar).

Figure 2 is a photograph showing the antimicrobial activity against the phytophthora infestans in vitro according to the dilution ratio of Streptomyces sp. AG-P (KCTC 8965P) strain culture [treatment concentration] Is the culture stock, 2 times, 4 times, 8 times, 16 times dilution treatment and control from the upper left to the right.

Figure 3 is a graph showing the inhibitory effect of the culture medium on the mycelial growth of Pythium sp., Which is the receiving pathogen of Streptomyces sp. AG-P strain culture medium [mycelial growth index is 1 To 5, with 5 indicating complete inhibition.

Figure 4 is a photograph showing the control effect of the Streptomyces sp. AG-P (KCTC 8965P) strain against phytophthora infestans in tomato pest control in greenhouse [greenhouse experiment results, Treatment concentrations were 500, 250, 125, 63, 31 ppm treatment and control from the left].

The present invention is characterized by the strain Streptomyces sp. AG-P (KCTC 8965P).

In addition, the plant has a specific antimicrobial activity against Oomycetes pathogens such as Phytophthora sp. And Pythium sp., Which are phytopathogens of the new strain Streptomyces genus AG-P. Its use as plant disease control agents such as late blight and incoming disease.

Referring to the present invention in more detail as follows.

The present inventors collected many soil samples from all over the country to search for new microorganisms for the control of plant diseases, and using the Bennett agar medium from the collected soil samples, phytopathogenic fungi such as late blight and incoming germs ( Strains showing specific activity against Oomycetes) were isolated. The isolated actinomycetes were cultured in liquid and subjected to in vivo pot experiments to select one strain having the best activity.

Referring to the identification and culture of the strain is as follows.

1. Identification of Strains

1) Morphological characteristics of bacteria

According to the International Streptomycets Project (ISP), 14 days of incubation at 28 ° C and morphological characteristics under light and electron microscopy revealed that the chains of spores showed a spiral shape and the number of spores per chain 5 to 10 or more than 10, the surface of the spores was smooth and the spores were cylindrical and the size was 0.3 to 0.5 × 0.8 to 1.0 ㎛.

2) Cultural characteristics according to various media

After culturing for 14 days at 28 ℃ in various ISP medium conditions were examined the culture physiological characteristics of the strain, the results are shown in Table 1. The growth rate of this strain was relatively good in yeast, malt agar, oatmeal agar, inorganic salt, starch agar, glycerol and asparagine agar, tyrosine agar, glucose, asparagine agar and bennet agar. And nutrient agar. On the other hand, the color of the mycelia was light yellow in most of the medium and did not secrete water-soluble pigments.

3) Physiological characteristics

In order to investigate the physiological characteristics of the strains, after incubating at 28 ° C. for 14 days using Schilling-Gertib agar medium, starch hydrolysis capacity, gelatin liquefaction capacity, milk peptonation and flocculation capacity, melanin pigment formation ability, phosphate hydrolysis ability, The degree of salt tolerance and growth temperature were investigated and are shown in Table 2 below.

4) Carbon Source Availability

In order to investigate the carbon availability of the strain, using a Schilling-Gurtrib agar medium was incubated at 28 ℃ for 14 days and the carbon source capacity was investigated. The results are shown in Table 3 below. This strain was well used glucose, arabinose, inositol, mannitol, fructose, galactose, rhamnose, melibiose among the 12 kinds of carbon sources disclosed.

5) Cell composition

The bacterial cell wall components of this strain were investigated according to Becker et al. (Becker et al., Applied Microbiology 12: 421-423, 1964). The bacterial hydrolysis component of this strain was diaminopimellic acid (LL). .

According to the results of the morphological, cultural, physiological characteristics or sugar availability, and cell composition of the strain as described above, the strain was identified as a strain belonging to Streptomyces, and this strain was identified as AG-P strain of Streptomyces genus. It was named and deposited with the Korea Institute of Biotechnology Gene Bank (KCTC) on September 27, 1999 and was given accession number KCTC 8965P.

2. Cultivation of Strains

The strain is cultured in a medium containing nutrients that can be used by ordinary microorganisms. Glucose and soluble starch were used as the carbon source, and coarse soybean meal, yeast extract, and bee extract were used as the nitrogen source. In addition, dipotassium hydrogen phosphate was used, and cultured using calcium carbonate (calcium carbonate) for pH control. As a culture method, shaking culture was carried out under aerobic conditions, and the culture temperature was 28 ° C. In addition, in the case of culture period or shaking culture, the production of the active substance reached the highest by culturing normally for 7 days.

In addition, the antibacterial activity in vitro and in vivo was investigated by culturing AG-P strain of the genus Streptomyces spp., And showed high antimicrobial activity against late blight and incoming bacterium. As a result, I found out that it is paromomycin.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.

Example 1 Genus strain Streptomyces genus ( Streptomyces sp.) Incubation of AG-P (KCTC 8965P)

PH in medium containing 20% glucose, 10% soluble starch, 25% coarse soy flour, 1% gravy, 4% yeast extract, 2% NaCl, 0.25% K ₂ HPO ₄ , CaCO ₃ as seed and production medium Used after adjusting to 7.2. 50 ml each of two 500 ml baffled flasks were sterilized for 20 minutes at 121 ° C., inoculated with agar piece of strain, and inoculated at 2 ° C. at 150 ° C. Shake culture was carried out daily and used as species culture of the incubator. A 2 L baffle flask containing 400 ml sterilized at 121 ° C. for 20 minutes was seeded with 2.5% of the seed culture solution and incubated at 28 ° C. at 140 rpm for 7 days.

Example 2: in vitro Antimicrobial activity

The antimicrobial activity against fungi was assayed using the culture solution obtained in Example 1. Colletotrichum gloeosporioides , Penicillium oxalicum , Penicillium expensum , Penicillium genus sp.), Aspergillus flavus , Aspergillus fumigatus , Mucor ambigous , Alternaria alternata , Phytophthora infestans Phytophthora infestans Antifungal activity was investigated in vitro for several fungi, including ATCC26081 and Pythium sp. Potato liquid medium was treated with fungi and Streptomyces sp. AG-P (KCTC 8965P) strain cultures selected in the present invention to a concentration of 10% in V8 juice medium to observe the growth of bacteria. As a result, the strains of the present invention were very active against phytophthora infestans , tomato and potato bacterium, which were inactive even when diluting the culture stock 8 times. Was inhibited [FIG. 2]. On the other hand, the results of the antimicrobial activity against Pythium sp. It has been shown that it can be used as a biocontrol agent.

Example 3: in vivo Antimicrobial activity

In vivo , the plant was directly tested for its effect against tomato late blight and many other plant diseases, and its activity was tested in the greenhouse. As a result, it was effective in controlling rice blight, rice blight, tomato blight and wheat rust. In particular, the control effect was also strong against tomato late blight and wheat red rust [Table 4].

As a screening method for investigating the control effect against tomato late blight , spore concentration of Phytophthora infestans was made at 10 ⁵ sporangia / ml, and it was treated at low temperature for 13 hours at 13 ° C for 2.5 hours. After the distillation, 3 ml of AG-P strain culture solution was sprayed onto the treated tomato seedlings (second to three leaf stages). Tomato seedlings were treated for 48 hours in a 20 ° C. wet room and then incubated for 3 days in a 20 ° C. constant temperature and humidity room (relative humidity above 95%), and used for the experiment. The control effect on other rice fever, rice blast disease, cucumber ash fungus, wheat red rust disease, and barley flour disease was tested in a similar manner. As shown in FIG. 4, the effect was very high in vivo against tomato late blight, and these results indicate that the phytophthora infestans, the tomato late blight bacterium, is selective as in the medium. gave. In addition, the results showed that the inhibitory and control effects were similar for Pythium sp., Which is a pathogen, and Pythium sp. It can be shown.

As described in detail above, the present invention isolates and identifies an excellent new strain exhibiting specific antimicrobial activity against the fungus (Oomycete), was found to exhibit a strong antimicrobial activity against late blight and wearing disease by culturing this strain. Therefore, the new strain can be used as an environmentally friendly biopesticide and has a very useful effect on plant diseases such as plant late blight and wearing disease.

Claims (3)

Streptomyces sp. AG-P (KCTC 8965P), characterized by having phytopathogenic and fungal control activities against fungal diseases. Streptomyces sp. Streptomyces sp. AG-P (KCTC 8965P) strain comprising a plant disease control agent comprising. The plant disease control agent according to claim 2, wherein the plant disease is plant late disease or wearing disease.