KR100417632B1 - A novel Trichoderma harzianum YC459 active against plant fungal pathogens and process for preparation of microbial pesticide thereof - Google Patents

Abstract

The present invention is directed to a method for the isolation of novel Trichoderma microorganism strains antagonistic to major phytopathogens from soil and named it Trichoderma harzianum YC459 (KCTC 0772BP) and the preparation of microbial agents suitable for controlling plant diseases. When the spore suspension of the strain of the present invention is treated to the leaves or cultivated soil of the main crop, there is an excellent effect of suppressing more than 50% of gray mold disease, mozzarella disease, leaf fungal disease and polyposis.

Description

Novel Trichoderma harzianum YC459 active against plant fungal pathogens and process for preparation of microbial pesticide

The present invention relates to a microbial preparation for controlling plant diseases using a Trichoderma microorganism strain and a method for manufacturing the same, and more particularly, the present invention isolates the Trichoderma microorganism strain antagonistic to phytopathogenic bacteria from the soil and trichoderma Spore suspensions, named harzianum YC 459 and mass-cultured, are treated on the leaves or cultivated soils of major crops, resulting in gray mold rot, damping-off, and leaf mold diseases. And it relates to a biological control microbial agent for controlling various phytopathogenic fungi such as early blight and a method of manufacturing the same.

Crop diseases caused by phytopathogenic fungi are very limiting factors for stable production of crops along with pests and weeds. Among many phytopathogenic fungi Botrytis CeriaB. cinerea),Lijotton Solani (R. solaniMost of the germ-forming bacteria, such as), are soil contagious, causing a wide range of diseases in a wide variety of crops, causing a lot of damage. These pathogens overwinter in the state of mycobacterium and become the primary source of infection in the following year. The microbial cells can survive in unfavorable environmental conditions in diseased tissues or soil, and can be degraded by neighboring antagonists and suppressed by spores or mycelium. Because it is strong, effective control is more difficult. Leaf fungus Cradosporium full boomCladosporium fulvum), Double-pattern germ Alternaria Solani (Alternaria solani) Causes a lot of damage to various vegetable crops. In particular, various types of fungicides are currently used for the control of these pathogens, but the development of safe and effective control methods is urgently required as the effectiveness of the drug decreases due to drug resistance due to misuse and abuse of the fungicides.

As an environmentally safe and effective control method, biological control using a microbial agent is important. Since the early 1970s, many studies have been conducted by many researchers at home and abroad. Recently, more than 10 kinds of microbial products have been commercialized in the world, and research on the development of microbial products is rapidly increasing due to the change of social awareness toward environmentally safe sustainable agriculture. Currently, representative microbial agents developed and commercialized in foreign countries are Trichoderma harzianum (Trichoderma harzianum) And Trichoderma PolysporumT. polysporumBINAB T (1988, E.R. Butts International), glycodium viens which are made ofGliocladium virensGlioGard (1990, W. R. Grace Co.), Trichoderma harzianumT. harzianumF-stop (1991, Kodak), Trichodex (1997, developed in Israel) and antagonistic bacillus subtilis (Bacillus subtilisSeed treatment agent KODIAK (1990, Gustafson), Pseudomonas fluorescensPseudomonas fluorescens) Using cotton lizhatonia and phytium (Rhizoctonia,PythiumDAGGER (1990, Ecogen Inc.), Pseudomonas SephaciaP. cepaciaSeed treatment of several crops using BLUE CIRCLE (1990, Stine Microbial Products), and the antagonistic Streptomyces griseoviridis ()Streptomyces griseoviridisFusarium withFusariumMYCOSTOP (1985, Kemira Oy Biocontrol of Finland). Among them, Trichoderma harzianum (Trichoderma harzianum), Microbial preparations have excellent disease control effects and stability of products, and various countries are trying to develop strains that are suitable for local characteristics. In Korea, research on biological control of ginseng root rot has been undertaken since the early 1970s.Trichodermahas not been commercialized using sp.). Trichoderma Harzianum (T. harzianum) Is in contact with the mycelia of the pathogen, punctures and invades and kills the pathogen. Lijotton Solani (R. solani)Wow Botrytis CeriaB. cinereaIt has been shown to have an inhibitory effect on several types of phytopathogenic fungi, including. In addition, Trichoderma Biride (T. viride), Gliocladium RoseGliocladium roseum), Penicillin species (Penicilliumsp.) is also used in greenhouse and field experiments.B.cinerea),Lijotton Solani (R. solani) Has been reported to have a control effect. Trichoderma Harzianum (T. harzianumIn order to increase the residual potency in the environment, it is necessary to select and formulate a substrate which can be used uniquely by the bacterium, or to pre-treat the substrate itself so that the antagonistic microorganisms occupy enough and cannot be used by other microorganisms or pathogens. In mass production and formulation of antagonistic microorganisms, it is necessary to select culture materials in consideration of economical efficiency in production.

Antagonist microorganisms are fungi isolated from domestic soil.B. cinerea), Lijotton Solani (R. solani), Cradosporium full boom (C. fulvum), Alternaria Solani (A. solani) Showed excellent control effect. Conventional microbial preparations using antagonistic microorganisms are known to control phytopathogenic fungi, nematodes and virus diseases, pest control, and broadly weed control. According to these well-known documents, the principle of biological control by antagonist microorganisms is based on the density of pathogens through antibiotics produced by antibiotics, parasitic or phagocytosis by cell wall degrading enzymes, and nutrient competition or niche competition. The main mechanisms of action include reducing or reducing pathogenicity, cross-protection using non-pathogenic strains or weakly pathogenic strains, and inducing host resistance. For example, Trichoderma genus (Trichodermasp.) and the genus GliocladiumGliocladiumInhibition of fungal diseases by antibiotics or volatiles, toxins or special inhibitors secreted by sp.) is known. On the other hand, antagonism by parasitic or phagocytosis has been actively studied in recent years. Pathogens caused by chitinase, glucanase, and cellulase, which are fungal cell secreting enzymes secreted by antagonists Dissolution of is known as the main mechanism of action.

The present inventors have completed the present invention by preparing a new type of environment-friendly microbial preparation by separating and formulating antagonistic microorganisms excellent in inhibiting ability of the phytopathogens for effective biological control of various major plant diseases.

Therefore, the object of the present invention focuses on the antagonism as described above, conventional Botrytis cinerea ( B. cinerea ), Li footonia solani ( R. solani ), C. fulvum , Alternaria Control of gray mold, mozzarella and rice leaf blight, leaf fungus, and double-ply disease caused by A. solani is not effective due to the expression of drug-resistant pathogens. The present invention provides a novel Trichoderma microorganism strain. Another object of the present invention is to provide a microbial agent for controlling phytopathogens containing the Trichoderma harzianum YC459 strain.

The above object of the present invention is phytopathogenic fungi Botrytis Ceria (B. cinerea), Littoonia Solani (R. solani), Cradosporium full boomC. fulvum), Alternaria Solani (A. solaniIsolation of the superior antagonistic trichoderma microorganism strain with the ability to inhibitT. harzianumName it YC459 This It was achieved by culturing the pure microorganisms and then evaluating the phytopathogen control effect.

Hereinafter, the configuration and operation of the present invention.

The present invention Botrytis CeriaB. cinerea), Littoonia Solani (R. solani), Cradosporium full boom (C. fulvum), Alternaria Solani (A. solaniSeparating the microbial Trichoderma harzianum YC459 strain antagonistic) from the soil; After treating the spore suspension of the isolated Trichoderma harzianum YC459 strain to cucumber, inoculating fungal pathogens and measuring the incidence of the trichoderma harzianum YC459 strain using the plant directly assay Doing; Mass culture and formulation of the isolated antagonist Trichoderma harzianum YC459 strain; After inoculating seedlings with potato, soil and distilled water mixture to inoculate the soil, inoculate the seedlings, sowing seedlings and treating the present invention Trichoderma harzianum YC459 strain, and then investigated the incidence of seedlings Investigating the effect of inhibiting the onset of Mozolox disease of Trichoderma harzianum YC459 strain; Investigating the incidence of gray mold disease of Trichoderma harzianum YC459 strains of the present invention and the untreated group of Tricotherma harzianum YC459 strains while cultivating tomatoes in greenhouse packaging. Investigating; Investigating the incidence of leaf fungal disease of Trichoderma harzianum YC459 strain treated and untreated group of the present invention while growing tomatoes in greenhouse packaging to control the effect of leaf fungal disease on the Trichoderma harzianum YC459 strain Investigating; Investigation of the incidence of double rounded pattern disease of Trichoderma harzianum YC459 strain of the present invention by investigating the incidence of double rounded pattern disease of the Trichoderma harzianum YC459 strain treated and untreated group It consists of investigating.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention includes any invention that changes these Examples and Experimental Examples as well as fungi derived from the strain.

Example 1: Isolation and Antagonistic Assay of Antagonist Microorganisms

Experimental Example 1: Isolation identification and antagonism assay (incubator assay)

In this experiment, Botrytis Ceria (B. cinerea)Soil and compost samples were collected from five regions, including Jinju-si, Namhae-gun, and Miryang-gun, to isolate microorganisms with antagonistic activity. 5 g of the collected sample and 45 mL of sterile water were mixed and shaken at 28 ° C. for 30 minutes, followed by dilution with soil agar plate method.⁴Diluted up to 2 times, 0.1 mL of the dilutions were dispensed onto 100 ppm of potato sugar agar (PDA, Difco) containing streptomycin, and evenly spread over the medium, followed by incubation at 28 ° C. for 5 days. Single colonies of fungi grown after incubation were purely separated and transferred to PDA slope medium and stored at 4 ° C. Antagonistic test of isolated fungiB. cinerea, R. solani, C. fulvum, A. solaniThe fungal YC459 strain was selected by observing the degree of degradation and inhibition of the contact surface hyphae of pathogens and antagonists under a microscope. Microscopic observation and identification of the morphological characteristics of the flora, conidia and conidia of the selected strains Tricoderma Harzianum (Trichoderma harzianum) And this strainT. harzianumIt was named YC459 and was deposited with KIRC 0772BP dated April 29, 2000 to KIRB Gene Bank of Daejeon.

Experimental Example 2: Antagonistic Assay on Plants of Antagonist Microorganisms of the Present Invention (In Vivo Assay)

In this Experimental Example, antagonist microorganisms were examined to investigate the inhibitory effect of pathogens when the antagonist Trichoderma harzianum YC459 of the present invention isolated on the medium of Experimental Example 1 was treated on the surface of a plant. The leaves were treated first, and after 1 day, pathogens were inoculated to investigate the degree of inhibition. The isolated antagonist microorganisms were transferred to PDA medium, incubated for 10 days at 28 ° C. incubator, and then scraped together by adding 4-5 mL of sterilized distilled water to harvest spores formed on the incubator surface. At this time, the spore concentration of the spore suspension was adjusted to an average of 10 ^6-7 / mL. The spore suspension thus harvested was sprayed onto a two-leaf cucumber leaf grown in a plastic pot and left for 1 day at room temperature, followed by a spore suspension of B. cinerea (spore concentration of 10 ^{6-). 7} mL / mL) was inoculated to flow down. Pots containing cucumber seedlings inoculated with the bacterium were placed on a 20 ° C. wet place, and left for 3 days. At this time, the pathogen used was a strain (JM42) stored and stored in diseased tomato leaves in 1994 in this laboratory. The culture and spore harvesting were carried out for 14 days at 20 ° C. incubator, and the spores generated on the surface of the incubator were antagonized. It prepared like the suspension preparation method of microorganisms. The inhibitory effect (control value) of the antagonist microorganisms was calculated by the following equation, and all experiments were carried out by planting cucumbers per pot for 2 weeks and planting a total of 4 pots. Significance test was performed using Duncan's Multiple Range Test (DMRT). Inhibitory effects obtained in the laboratory were shown in Table 1.

Control value (%) = [1- (Incidence rate of treatment / Infection rate of untreated)] × 100

Inhibitory effect of antagonist micrococcus trichoderma harzianum YC459 on the development of cucumber gray mold Treatment concentration (spore count / mL suspension) Incidence (%) Control price (%) 10 ⁴ 32.9 C 66.1 10 ⁵ 40.0 C 58.8 10 ⁶ 19.1 B 80.3 10 ⁷ 9.9 A 89.8 0 (no processing) 97.0 D - [Note] * The same alphabetic character means no significant difference at p = 0.05 (DMRT).

For untreated it not treated with the present invention antagonistic microorganism incidence but abandoned almost all leaves are dying in 97% antagonistic microorganism spores processed with the higher the spore concentration was decreased the incidence of 10 ⁷ / mL suspension of the spores processed with 89.8% It showed a high onset suppression effect.

Example 2 Mass Culture and Formulation of the Antagonist of the Invention

In this example, the antagonist trichoderma harzianum YC459 of the present invention isolated in the above example was inoculated in PDA and incubated at 28 ° C. for 7 days to form a large amount of spores. Spore suspension was prepared in the same manner as 2 and used as seed inoculum for mass culture. In this culture, the same amount of agricultural by-products of wheat bran, sawdust, rice bran, rice husk, etc., are mixed with bran + chaff, sawdust + rice bran, rice bran + rice bran, rice bran + sawdust, etc., and the yeast extract 0.5% (volume ratio) is mixed well. Water was added to 40% and placed in a sterilizable 5L plastic bag or Erlenmeyer flask or sealable container and sterilized at 121 ° C for 1 hour. After sterilizing the medium, aseptically added 200mL of the spore suspension and well mixed and incubated for 7 days at 28 ℃. Shaking once every two days during the incubation process induced even growth of mycelia and large spore formation. The cultured antagonist microorganisms were dried for 3 days at room temperature, and then pulverized and filtered through a 1 mm sieve to obtain fine mycelia and spore powder passing through the sieve. After harvest, it was formulated by adding extenders such as pea moss, vermiculite or talc, zeolite, kaolinite, and the like. In this case, any of the excipients usually used in microbial preparations can be used for formulation.

Example 3: Effect of inhibiting the onset of Mozolox bacterium by antagonist microorganism

In this example, the inhibition of Mozolox bacterium was assayed using radish. For the inoculation pathogen, 50 g of potato slices, 450 g of field soil, and 100 mL of distilled water were mixed in a 2 L Erlenmeyer flask, sterilized at 121 ° C. for 1 hour, and cultured on a PDA for 5 days, followed by R. solani mycelia fragment 20 Dogs were inoculated and incubated at 28 ° C. for 14 days, dried in the shade, crushed, and stored at 4 ° C. to be used as inoculum. Mix 0.5 g of bacterial inoculum and antagonist spore suspension prepared in the same way as Experimental Example 2 with 1 liter of topsoil in a plastic bag, put them in four plastic pots (horizontal, 7 cm each), and sowing 10 radish seeds in a greenhouse. Cultivation was done for 1 week. The degree of onset after one week of cultivation is divided into four stages (1: healthy, 2: slightly visible browning, 3: onset and dead after germination, 4: onset and not germinated). The incidence rate was calculated in terms of percentage (%). The control value was calculated in the same manner as in Experiment 2. Experimental results, as shown in Table 2 below, the higher the spore suspension concentration of the added T. harzianum YC459, the onset was suppressed, 67%, 75.5% of 50mL, 100mL were added, respectively The control agent was shown.

Inhibitory Effect of Antagonist Trichoderma harzianum YC459 on the Development of Hairless Rock Disease Spore Suspension (mL / Soil L) Incidence (%) ^* Control price (%) No treatment 92.5 D - 5 75.6 D 18.3 10 60.8 C 34.3 50 30.5 B 67.0 100 22.7 A 75.5 [Note] * Same alphabetic character means no significant difference at p = 0.05 (DMRT)

* Control value (%) = [1- (Incidence rate of treatment / Infection rate of untreated)] × 100

Example 4 Greenhouse Packaging Effect by Antagonistic Microbial Treatment of the Present Invention

Experimental Example 1: Inhibitory effect of tomato gray mold fungi by the present invention antagonist treatment

In this experimental example, the cultivation of tomato to see how the antagonist Trichoderma harzianum YC459 treatment of the present invention can suppress the development of ash fungus (pathogen: B. cinerea) in the greenhouse A control experiment was performed. The greenhouse was used to make a plastic house (23.7 ^{m in} length × 7.0 ^{m in} width × 2.3 ^{m in} height and 166 ^{m 2} ) used in rural areas. Cultivation was carried out in the following manner.

Sept. 30, 1999 Sowing of Tomato

10. 10. First meal

10. 22. Second Fake

11. 2. Formal (two sets at 20cm intervals)

12. 22. Start of modification (tomato treatment)

2000. 2. 11. First antagonistic microorganism treatment (based on spray amount 300l / 10a)

2. 20. Second antagonistic microorganism treatment (based on spray amount 300l / 10a)

3. 2. Third antagonist microorganism treatment (based on spray amount 300l / 10a)

3. 22. Incidence Survey

At this time, the treatment area 1 was 5.4m ² , was carried out by three repetition, ingot method, and used as a control agent was sprayed at a commercial concentration of Smirex (Procymidone). Antagonist microbial treatment was sprayed by diluting the spore preparation prepared in Example 2 to 500 times, 1000 times with water, the incidence was investigated one by one whether all tomatoes in the treatment. Significance test was performed using Duncan's Multiple Range Test (DMRT).

Inhibitory effect on the development of tomato gray mold disease of the present invention antagonistic microbial treatment was as Table 3 below. The average morbidity of the untreated group was 17.4%, and when the antagonist microorganisms were diluted 500 and 1,000 times, the morbidity of each group was 5.4% and 6.6%, respectively. The average incidence of procymidne was 10.4%. In terms of control value, antagonist microbial treatments averaged 65.6%, which was significantly higher than untreated or Smirex treated.

Inhibitory effect of antagonist Trichoderma harzianum YC459 on the development of tomato gray mold Dilution factor Disease rate (%) Control price (%) One 2 3 Average ^* 1,000 4.5 8.3 7.1 6.6 A 62.1 500 4.6 5.6 6.1 5.4 A 69.0 Smirex 12.3 10.6 8.2 10.4 B 40.2 No treatment 18.5 19.2 14.4 17.4 C - [Note] * The same alphabetic character means no significant difference at p = 0.05 (DMRT).

* Control value (%) = [1- (Incidence rate of treatment / Infection rate of untreated)] × 100

Experimental Example 2: Inhibition effect of leaf fungal pathogen by the present invention antagonist microbial treatment

In the present experimental example 1 to see how the antagonist trichoderma harzianum YC459 treatment of the present invention can suppress the development of leaf fungal disease (pathogen: C. fulvum ) in the greenhouse Control experiment was performed while cultivating tomatoes in the same manner. Three repetitions were performed by the ingot method, and as a control drug, propineb was sprayed at a commercial concentration. The incidence survey was conducted 20 tomato per repetition, the incidence was calculated as follows.

Incidence (%) = [∑ (occurrence x coefficient) / 4x total irradiance] x 100 (coefficient 0: no outbreak, 1: about 1/4 of all leaves, 2: 1/2 of all leaves) Degree of onset and sometimes some leaves die, 3: about 3/4 of all leaves die, and about half of the leaves die completely, 4: more than 2/3 of all leaves die completely)

Inhibitory effect on the development of tomato leaf fungal disease of the present invention antagonist microorganisms was as shown in Table 4. The incidence of untreated group was 21.9% on average, and the incidence of each treatment group was 6.6% and 6.8%, respectively. In the case of the control drug propineb, the average incidence was 21.9%. In terms of control value, antagonist microbial treatments averaged 69-70%, which was significantly higher than untreated or prophylactic treatments.

Inhibitory Effect of Antagonist Trichoderma harzianum YC459 on the Development of Tomato Leaf Fungal Disease Dilution factor Incidence (%) Control price (%) One 2 3 Average ^* 1,000 6.5 6.9 7.1 6.8 A 69.0 500 6.6 6.2 7.0 6.6 A 69.9 PROFI 10.3 11.2 14.5 12.0 B 42.9 No treatment 18.8 22.4 24.4 21.9 C - [Note] * The same alphabetic character means no significant difference at p = 0.05 (DMRT).

* Control value (%) = [1- (Incidence rate of treatment / Infection rate of untreated)] × 100

Experimental Example 3: Inhibitory Effect of Double Rounded Pattern Bacteria by the Antagonism of the Present Invention

In this experimental example, to determine the extent to which the antagonist Trichoderma harzianum YC459 treatment of the present invention can suppress the development of double pattern disease (pathogen: A. solani ) in the package, A control experiment was carried out while growing. Three repetitions were performed by the ingot method, and the control agent was sprayed at a commercial concentration with ortiba (azoxystrobin). The onset was investigated 20 weeks potato per repetition, the incidence was calculated as in Experiment 2.

May 4, 2000 Sowing of Potatoes (Breed: Tajima)

6. 28. The first antagonist treatment (based on spray amount 300l / 10a)

7. 5. Second antagonist microorganism treatment (based on spray amount 300l / 10a)

7. 13. Incidence Survey

Inhibitory effect on the development of potato double round pattern disease of the present invention antagonist microorganisms was as Table 5 below. The incidence of the untreated group was 21.7% on average, and the incidence of each treated group was 9.2% and 9.5%, respectively, when the antagonist was diluted 500 and 1,000 times. The average incidence was 21.7% for the control agent azoxystrobin. In terms of control value, antagonist microbial treatments averaged 56.2-57.6, which was significantly higher than no treatment.

Inhibitory Effect of T. harzianum YC459 on Potato Double-pattern Disease Dilution factor Incidence (%) Control price (%) One 2 3 Average ^* 1,000 10.3 8.9 9.3 9.5 A 56.2 500 9.8 8.1 9.6 9.2 A 57.6 Otiva 9.3 10.2 8.2 9.2 A 57.6 No treatment 22.8 22.9 19.4 21.7 C - [Note] * The same alphabetic character means no significant difference at p = 0.05 (DMRT).

* Control value (%) = [1- (Incidence rate of treatment / Infection rate of untreated)] × 100

As described through the above Examples and Experimental Examples, the present invention antagonist microorganism strain Trichoderma harzianum ( Trochoderma harzianum ) YC459 (Accession No. 0772BP) is a plant pathogen Botrytis cinerea , Botrytis cinerea , Antagonists such as Rhizoctonia solani , Cladosporium fulvum , Alternaria solani , etc. have been shown to cause the development of gray mold, mozzarella, leaf fungus, or lamellae It is a very useful invention for the pesticide industry and environmental industry because it can be used as an environmentally friendly, pollution-free biopesticide, because it can solve the destruction of ecosystems and human toxicity.

Claims (3)

Fungi Trichoderma harzianum YC459 (Accession No. KCTC 0772BP), which is antagonistic to phytopathogens. delete Obtaining spores by inoculating the Trichoderma harzianum YC459 fungi according to claim 1 in a PDA medium and incubating the spores to obtain a spore suspension by adding sterile distilled water; Mixing the spore suspension in the same amount of two selected from bran, rice hull, rice bran and sawdust and mix well with 0.5% yeast extract and incubate in a sterilized medium by adding water to 40% water content; Harvesting fine strains and spore powders by pulverizing the cultured cells obtained by the above step and sifting; Preparation of microbial preparations for controlling phytopathogens, characterized in that prepared by adding the fine strain and spore powder harvested by the above step to any one of the extender selected from coal, vermiculite, talc, zeolite or kaolin. Way.