KR100361135B1 - Antagonistic microorganisms of phytopathogens, microbial preparations containing them and methods of controlling phytopathogens using the same - Google Patents

Abstract

The present invention relates to a microbial pesticide preparation comprising a microorganism having both chitin degrading ability and antibiotic producing ability, and a method for controlling phytopathogens using the same. Since the microbial agent of the present invention can inhibit the growth of major soil pathogens, such as Fusarium oxysporumm, F. solani, and Rhizoctonia solani, it can be used as a new type of environmentally friendly pesticide.

Description

Antagonistic microorganisms of phytopathogens, microbial preparations containing the same and methods of controlling phytopathogens using the same

The present invention relates to antagonistic microorganisms against phytopathogens, preparations using microorganisms and biological control methods of phytopathogens using the same.

Recently, biological control using antagonistic microorganisms for the control of major diseases including soil diseases of plants has been developed and put into practical use with environmentally friendly agricultural technology. Among the pathogens, many researches on biological control of phytopathogenic fungi, bacteria and nematodes have been conducted. Among them, various kinds of microbial pesticides for the control of diseases caused by soil infectious pathogens Rhizoctonia solani and Pythium sp. However, plant diseases caused by F. oxysporum, such as wilted disease, have no commercially available microbial pesticides despite their serious damage.

Biological control by antagonistic microorganisms is mainly due to antibiotics produced by these microorganisms, parasitic or phagocytosis by cell wall degrading enzymes, and reduced density or pathogenicity of pathogens through nutrient or niche competition. Or cross-protection (cross protection) using a weak pathogenic strain is known as the main mechanism.

Among them, the mechanisms by which antagonistic microorganisms secrete cell wall degrading enzymes of pathogens to suppress pathogens have been studied. In particular, there are many studies on the action of chitinase secreted by Trichoderma sp. Since most phytopathogenic cell walls contain chitin as a major component, it has been reported that Trichoderma sp. Can suppress pathogens extensively.

However, biological control was attempted by treating antagonists with specific mechanisms in the soil, but until now, treated antagonists have not survived in a microsite for a long time in competition with other microorganisms or in a bad environment. It was not suitable for the control of long-term pathogens such as Fusarium wilt. In order to compensate for this, it was attempted to formulate the organic additives, but even in this case, the amount of the additives was too difficult, and the antagonistic bacteria were also difficult to commercialize because they are not easy to mass culture. In order to solve this problem, the present inventors selected chitinolytic bacteria and used them for biological control in Korean Patent Application No. 97-26538. In this case, chitin or chitosan was added to formulate the bacteria and formulated. Reported. However, the chitin degradability alone was not enough to control the pathogens, and sufficient control effects could not be obtained.

On the other hand, Fusarium oxysporum is a versatile pathogen belonging to incomplete fungi, which causes wilting disease in many kinds of annual and perennial plants such as vegetables, flowers and cotton, tobacco and coffee trees in temperate and greenhouse growing areas. Withering fungi such as cucumbers, watermelons, tomatoes, etc. due to the fungi currently do not have an effective control method and use the varieties resistant to the disease or rely on the grafting method.

It is an object of the present invention to provide antagonistic microorganisms against soil infectious phytopathogens which have the ability to secrete antibiotics simultaneously with chitin degradability.

Still another object of the present invention is to provide a microbial agent having excellent control effect of soil infectious plant pathogens.

Still another object of the present invention is to provide a method for controlling soil infectious phytopathogens using antagonistic microorganisms of soil infectious phytopathogens.

Still another object of the present invention is to provide a method of controlling plant diseases by inducing resistance to soil infectious plant pathogens using antagonistic microorganisms of soil infectious plant pathogens.

Figure 1 shows the control effect of the microbial preparation of the present invention for cucumber vine cutting disease.

The present invention relates to a new microorganism having excellent chitin degrading ability and antibiotic producing ability.

Chitin resolution in the present specification means the ability to degrade chitin, chitosan, derivatives thereof and the monomers or oligomers constituting them. Antibiotic production capacity refers to the ability to produce antibiotics that can kill or attenuate plant pathogens.

The present invention also relates to an excellent microbial agent having a good control effect against phytopathogenic fungi composed of microorganisms excellent in chitin resolution and antibiotic production.

The formulation of the present invention can be used in the form of microbial pesticides or microbial fertilizers.

In the case of using the microbial pesticides with excellent chitin-degrading ability, it is not enough to effectively use the microbial pesticides because it often does not effectively inhibit phytopathogenic fungi when treated in soil. The present inventors have found that the use of microorganisms that simultaneously release antibiotics that can weaken pathogens, along with chitin resolution that can degrade the cell walls of soil phytopathogens, has significantly improved the pathogen growth inhibition and killing effects.

As an example, the microbial agent of the present invention may be used as a microbial pesticide. The microbial pesticide according to the present invention may further contain a carrier, in addition to a microorganism having excellent antibiotic ability as well as chitin resolution. As the carrier, one or more of chitin, chitosan or derivatives thereof may be mixed and used, and in particular, chitin, chitosan or a derivative thereof dissolved in water is preferably used as the carrier. As such, when one or more of chitin, chitosan, or derivatives thereof are mixed and used as a carrier, when the soil is treated with a microbial pesticide, the antagonistic microorganisms may be selectively grown in the soil, thereby increasing the microbial pesticide effect.

The microbial pesticides of the present invention can improve the shelf life of microbial pesticides, either alone or in combination with such carriers, for example peat moss, zeolites, talc and kaolinite, clays. And the like.

The microbial pesticide according to the present invention may further contain a substance capable of maintaining and improving the activity of the microbial pesticide or a substance necessary for storing the microbial pesticide.

In addition, the microbial pesticide according to the present invention may further contain an active ingredient for controlling phytopathogenic fungi or other phytopathogenic fungi.

On the other hand, the microbial agent of the present invention can be used as a microbial fertilizer. The microbial fertilizer according to the present invention may contain components useful for plant growth, in addition to microorganisms having excellent antibiotic ability as well as chitin resolution.

Another aspect of the invention relates to a method for controlling phytopathogenic fungi.

The control method according to the invention can be carried out in various embodiments. For example, the control method according to the present invention consists of sowing seeds after soaking in a suspension of microbial pesticides before sowing plants, for example potatoes, cucumbers, watermelons and the like. At this time, it is preferable to seed the plants after supplying chitin, chitosan or derivatives thereof to the soil to be planted, or to additionally supply chitin, chitosan or derivatives thereof to the soil after plant sowing.

As another example, the control method according to the present invention consists of mixing a microbial pesticide containing plant pathogen antagonistic microorganisms in the culture soil or soil before or after plant sowing.

If desired, chitin, chitosan or derivatives thereof may be fed to the soil before, after or with the treatment of the microbial pesticide. In particular, when the microbial pesticide does not include a carrier such as chitin, chitosan or derivatives thereof, it is thus preferable to separately supply chitin, chitosan or derivatives thereof to the soil.

In another example, the microbial pesticide of the present invention consists in treating the above-ground part of the plant, for example, leaves, fruits and berries, during the cultivation period of the plant.

In another example, the microbial pesticide of the present invention consists in treating the plant after harvesting.

In addition, the present invention is to cultivate the plant by primary culture in a soil or medium treated with a preparation containing a microorganism having both chitin resolution and antibiotic production capacity, and then the plant is transferred to a secondary culture soil, that is, packaging or medium and cultured. The present invention relates to a method of inducing resistance to soil infectious phytopathogens. By cultivating plants in the seedlings treated with the microbial preparations according to the present invention, resistance to phytopathogens can be induced, thereby significantly reducing the incidence of phytopathogens in the main fabric. Regardless of the type of plant, the method according to the present invention can be applied to plants which are first grown in seedlings and then planted in the main cell.

In one embodiment, the present invention provides a method for controlling phytopathogens comprising culturing rice seedlings grown on rice seedlings treated with a preparation containing a microorganism having both chitin resolution and antibiotic production.

Rice is grown in small seedlings on germinated rice seedlings to obtain seedlings, which are then transplanted to rice fields. In order to control various damages caused by pathogens during rice cultivation, it is necessary to control the entire rice cultivation field to increase the amount of pesticide spraying. Therefore, not only the cost of pesticides and labor costs is increased, but also the area of pesticide treatment increases the environmental pollution. It is a serious cause. In the method of the present invention, by treating the microbial preparations according to the present invention on rice seedlings without having to treat the entire bone poinone, it is possible to effectively control diseases such as leaf blight, which is fatal to rice cultivation, Costs can be effectively reduced and environmental pollution can be significantly reduced.

Hereinafter, the embodiments of the present invention will be described in detail with reference to examples so that those skilled in the art can easily implement the present invention, but the scope of the present invention is not limited to these examples.

Example 1 Antagonist Microbial Isolation

Soil and compost samples were taken from Gyeongsangnam-do, Jeollanam-do, Chungcheongbuk-do, and Jeju-do to isolate microorganisms with chitin-degrading and antibiotic-producing ability. From the collected soil, microorganisms with a specific flora were first isolated using 0.1 Tryptic Soy Agar (TSA, Difco) medium and PDA medium (potato 300g, glucose 20g, agar 18g, 1L with distilled water). Among these strains, strains showing antagonism against pathogens and growing fast in PDA medium were selected as secondary, and these were selected as inorganic salt medium (MgSO ₄ · 7H ₂ O 0.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO _4). Colloid chitin medium mixed with 0.05% (w / v) of chitin swelled in 0.3 g, FeSO ₄ · 7H ₂ O 0.01 g, ZnSO ₄ 0.001 g, MnCl ₂ 0.001 g, pH 7.0, Agar 15 g, distilled water, 1 L fit) After incubation and culture, chitin degradation activity was assayed with or without transparent zone.

Antagonistic assay of isolated bacteria was carried out by the method of replacement culture with pathogens. Fusarium oxysporum and Rhizoctonia solani were used as pathogens. Place each fungal mycelial disc in the center of TSA and PDA medium, inoculate 50μl of antagonist suspension adjusted to a concentration of 0.5% absorbance on both sides in a 5mm diameter paper disc, and incubate for 2-14 days in a 28 ℃ thermostat. The mycelial growth inhibition distance formed by was measured. The results are shown in Table 1.

TABLE 1

Bacterial Antagonistic Test against Phytopathogenic Bacteria

Antibiotic resistance was tested against strain # 730, which has excellent growth inhibitory effect against phytopathogens in real soil and has a fast growth rate. Table 2 shows the results of the growth of strain # 730 in each medium containing 50 ppm of the antibiotics shown in Table 2. Morphological and physiological properties of # 730 are shown in Tables 3 and 4, respectively.

TABLE 2

# 730 Strain Resistance to Antibiotics

TABLE 3

Morphological and Physiological Physiology of # 730

TABLE 4

Carbohydrate Availability of Ferment (730)

From the above results, strain # 730 of the present invention was identified as Paenibacillus by Bergey's Manual of Systematic bacteriology. # 730 to Paenibacillus sp. It was named YC730 and deposited on May 7, 1998 with the accession number KCTC 0467BP to the Korea Institute of Biotechnology Gene Bank (KCTC) located at 52 Eun-dong, Yuseong-gu, Daejeon, Korea.

Table 5 shows the fatty acid composition analysis of strain # 730. The major fatty acid of the genus Paenibacillus is known as anteiso-15: 0 32-81%.

TABLE 5

Example 2. Preparation of Antagonist Microbial Formulations

Isolated antagonist microorganism strains (# 76, # 224, # 233, # 398, # 636, # 639, # 701, # 730) were inoculated in 5 ml of 0.5 TSB (Tryptic Soy Broth, Difco), respectively, After shaking for 2 hours, the cells were inoculated in 500 ml of 0.5 TSB and shaken (150 rpm) for 2 days at 28 ° C. At this time, the antagonistic microbial density was 10 ¹⁰ ~ 10 ¹² cfu / ml, the culture was centrifuged at 7,000g for 15 minutes to harvest the cells.

An aqueous chitosan solution of 2% (w / v) concentration was made using 0.1 N acetic acid or hydrochloric acid solution. To 10 ml of chitosan solution diluted to 0.5% in distilled water, 5 ml of each cell suspension obtained by suspending each of the antagonist microorganisms cultured above in 10 ml of distilled water and 4 g of zeolite were added and mixed.

Example 3 Controlling Cucumber Slice Disease of Microbial Preparations

To make the inoculator of the pathogen Fusarium oxysporum, 500g of organic soil rich in soil, 50g of chopped potato pieces and 50ml of distilled water were mixed together in a 2L Erlenmeyer flask and sterilized twice for 1 hour at 121 ° C, and then incubated for 5 days in PDA. Ten oxysporum mycelial disks were inoculated aseptically and incubated at 28 ° C. for 14 days with occasional mixing. After the incubation, the culture was dried in the shade, lightly pulverized with a mortar and then sieved to collect 1 ~ 2 mm sized particles were used as inoculum while storing at 4 ℃.

3 g of the inoculum prepared and 6 g of each microbial preparation prepared in Example 2 were inoculated into 1 kg of cultured clay (flour) commercially available, and placed in a plastic pot having a diameter of 10 cm. Five pots are planted in each of five cucumber varieties (F1 summer long, Seoul seedlings), and the seeded pots are placed on plant growth where the day / night temperature is 28 ℃ / 24 ℃ and the light is controlled by fluorescent lamps and incandescent lamps. The incidence was examined 30 days after planting. At this time, the humidity of plant growth was maintained at about 70%.

6 g of the microbial preparation prepared in Example 2 and 3 g of the pathogen inoculum were inoculated in 1 kg of culture soil, placed on plant growth, sown 4 days later, and then placed for 30 days. The number of cucumbers showing typical withering symptoms was examined. The incidence was expressed in% of the total number of cucumbers, and the control value of the formulation was evaluated by the following equation.

The experimental results are shown in Table 6.

Example 4 Effect of Microbial Preparations on Control of Hairless Rock Disease

Rhizoctonia solani inoculum, a hairless rock pathogen, was prepared in the same manner as in Example 3. 6 g of the microbial agent and 2 g of the pathogen inoculum were mixed in 1 kg of culture soil, mixed in a plastic pot of 5 cm in one side, and seeded without seeding 4 days later. 10 seeds in one pot were sown in 4 cells and placed in a greenhouse for 10 days, followed by an onset investigation. The pathology was 1-4 (1; healthy, 2; only lesions on stems, 3; collapsed by stem lesions, 4; Rot before germination) and the value was converted into%. The results are shown in Table 6.

TABLE 6

Example 5 Control Effect of Cucumber Vine Splitting Disease on Different Carriers

According to the method of Example 2, bacterial bacteria obtained by culturing strains # 398 and # 730 and a antagonist microorganism agent were mixed with a chitosan solution, a colloidal chitin, pit moss, zeolite, talc, and kaolinite as a carrier. made. Chitosan solution was prepared using an aqueous solution of 2% (w / v) concentration of 0.1N acetic acid or hydrochloric acid solution. To 10 ml of chitosan solution diluted to 0.5% in distilled water, 5 ml of each cell suspension obtained by suspending each antagonist microorganism cultured in 10 ml of distilled water and 4 g of pitmos, zeolite, talc and kaolinite were added. Mixed. The mixed suspension was used for experiments while lyophilized and stored completely unsealed at 4 ° C.

The pathogen F. oxysporum inoculum was prepared according to the method of Example 3, and the control effect on cucumber slices was examined. On the 30th day of cultivation, the incidence was examined by the number of cucumbers with typical withering symptoms. At this time, the humidity of plant growth was maintained at about 70%. The incidence rate is expressed as% of the total number of cucumbers. The experimental results are shown in FIG. 1 and Table 7. The incidence was 31.0% and 32.6%, respectively, when only peat moss or zeolite was used as a carrier for the microbial agent, but when 2,500 ppm of chitosan was added, the incidence was lowered to 22.4% and 18.6%. In Figure 1 Fo: no microbial agent treatment (treatment of pathogens only), A + F: antagonist suspension + pathogen treatment, PA + F: antagonist suspension + pit moss + pathogen treatment, PAC + F: antagonist suspension + pit MOS + chitosan + Indicates pathogen treatment.

As a result, the density of chitin-degradable antagonist microorganisms was 1.7-1.9 x 10 ⁹ per 1 g of soil at the beginning of the treatment, but the density of the preparation without chitosan gradually decreased to 1.1 x 10 ⁸ over time. The chitosan additive formulation remained high at 5.5 × 10 ⁸ (Table 8).

In addition, when the throughput of antagonist microorganisms was increased to 1, 2, 4, 6, 8, 10, and 12 g per 1 kg of soil, the control value increased to 38.9, 47.1, 58.8, 71.4, 73.5, 75.9, and 75.7%, respectively. Increased to.

TABLE 7

Control Effects of Cucumber Slices with Carrier Addition

TABLE 8

Changes in Density of Chitin-degrading Antagonists by Microbial Agents

In order to know the effect persistence of antagonist microorganisms, the control effect was examined for each period after treatment in cultured soil. The results are shown in Table 8. The stability of the formulations was examined, and the formulations used in Table 9 were all stable up to 45 days after refrigeration or at room temperature and exhibited 60-70% control value up to 90 and 180 days.

TABLE 9

Sustainability of Cucumber Slice Disease Control Effects of Microbial Preparations

Example 5 Control of Potato Blemish, Blight and Sprout Rot of Microbial Preparations

Control Effect of Potato Planted in Jeju Island Pavement Using # 398, # 730

Was investigated. As shown in Table 10, in the treatment of microbial agents, the incidence of late blight caused by Rhizoctonia solani and late blight caused by Phytophthora infestans was 30% lower than untreated. In addition, in case of shoot rot which is severely developed throughout Jeju Island, the incidence rate was 9.3% and 8.8%, respectively, when the microbial agent was treated by seed immersion or soil mixing method. (Table 11).

TABLE 10

Effect of Microbial Preparations on Potato Blot Disease and Plague Control

TABLE 11

Control of Potato Sprout Rot Disease by Microbial Preparation

Example 6 Effect of Microbial Preparations on Control of Hairless Rock Disease

A hairless pathogen Rhizoctonia solani inoculum was prepared in the same manner as in Example 4. 2-6 g of the microbial agent and 2 g of the pathogen inoculum were mixed in 1 kg of culture soil, mixed in a square plastic pot of 5 cm on one side, and seeded without seeding 4 days later. 10 seeds in one pot were sown in 4 cells and placed in a greenhouse for 10 days, followed by an onset investigation. The pathology was 1-4 (1; healthy, 2; only lesions on stems, 3; collapsed by stem lesions, 4; The germination was not carried out before germination, and this value was converted into%. The results are shown in Table 12.

TABLE 12

Effect of Microorganisms on Control of Hairless Rock Disease

Example 7 Inhibitory Effect of Major Phytopathogens by Antibiotics

Fusarium oxysporum et al. Pythium ultimum, Rhizopus sp. Rhizoctonia solani, Sclerotinia sclerotiorum, Alternaria alternata, Botrytis cinerea, Botryosphaeria dothidea, Colletotrichum lagernarium, Magnaporthe grisea were used. Antagonist microorganisms # 398, # 701, # 730 strains soybean meal medium (dextrose 5g, yeast extract 4g, beef extract 1g, starch 20g, soybean wheat powder 25g, K ₂ HPO ₄ 0.1g, 2g NaCl, 1L of distilled water) was incubated for 2 days at 30 ℃, 180rpm. 50 ml of the culture filtrate was centrifuged to remove the cells, and then the supernatant was extracted three times with the same volume of butanol, dried and dissolved in 2 ml butanol, and the solution was added to a 5 mm diameter filter paper and 50 μl of the solution was described in Example 1. The inhibitory effect on the major phytopathogenic fungi was investigated. Most of mycelial growth was strongly inhibited by 25-30mm, but not Pythium ultimum (Table 13).

TABLE 13

Inhibitory Effect of Culture Filtrate Extract on Mycelial Growth of Major Plant Pathogens

Example 8

The formulation containing the antagonistic microorganism # 730 was prepared by the method described in Example 2. Seedling soil was prepared by mixing the microbial agent at 10 g / kg in paddy soil. Seeds of rice (Ilmi rice), which began to germinate by immersing in water for 3 days, were sown in three seedlings (30 x 60 cm) containing soil treated with the above preparations, and cultivated for 13 days in a greenhouse to transfer seedlings to rice fields. . The seedlings sown on the seedling plate containing the seedling soil without mixing the microbial agent was used as a control. Twenty rice plants were planted for each test plot. One aeration consisted of an average of 20 stalks. The incidence of leaf blight was investigated after 105 days of rice transplanting in rice fields. The incidence rate was expressed as the number of stems with leaf blight among the 40 irradiated stems in%. The results are shown in Table 14. In the control group not treated with the microbial agent of the present invention, the incidence of 61.5% was compared with that of the treatment group, which showed an average incidence of 30.3%, which was about 50.7%.

TABLE 14

Control Effect of Microflora on Leafy Pattern Blight

The microorganisms and microbial pesticides according to the present invention can be used to effectively prevent or significantly improve the disease progression caused by phytopathogenic fungi, in particular F. oxysporum, F. solani and R. solani.

Claims (2)

Antagonistic microorganism Paenibacillus sp. YC 730 strain (Accession No. KCTC 0467BP) Paenibacillus sp. According to claim 1 having both chitin-degrading ability and antibiotic-producing ability. Pesticides containing any one or more of YC 730 (KCTC 0467BP) microbial strains and chitin, chitosan or derivatives thereof