KR20070122181A - A composition for biological control of pepper blight caused by capsicum annuum l. using trichoderma harzianum having antagonistic activity - Google Patents

Abstract

A composition comprising a microorganism Trichoderma harzianum is provided to control pepper blight caused by Capsicum annuum L. in an antagonistic manner without environmental damage. A method for manufacturing a composition for biological control of pepper blight comprises the steps of: inoculating Trichoderma harzianum in potato dextrose medium containing corn starch, glucose and inorganic nutrients, culturing it at 26-28 deg.C for 48-96 hours to obtain a cultured medium(A), culturing Bacillus subtilis in the same medium above to obtain a cultured medium(B), inoculating and culturing the cultured medium (A) and (B) in a medium containing zeolite, white carbon, magnesium hydroxide, diatomite, talc, corn starch, glucose, KH2PO4, EDTA iron, zinc sulfate and water; spreading the cultured products on the fabric and culturing them at 20-28 deg.C for 5-7 days; cutting the cultured product along the lines with a distance of 1-3 cm when white hyphae of T. harzianum are generated on the surface to promote spore formation, and further culturing them for 3-7 days; drying the cultured products at 35-40 deg.C for 5-7 days; and pulverizing the dried cultured products.

Description

A composition for biological control of pepper blight caused by Capsicum annuum L. using Trichoderma harzianum having antagonistic activity}

1 is a photograph showing a microbial agent DYMC of the present invention.

FIG. 2 is a graph showing the results of timely investigation of the density of Trichodelma harzianum while storing DYMC wrapped in tinfoil for 12 months at room temperature.

3 is a graph showing the activity findings of tri nose Del Mar Har Jia num (T. harzianum) DYMC in the processing method and indoor and concentration under greenhouse.

Figure 4 is a graph showing the results of investigating the appearance of pepper blight after 50 days after the treatment with the pot DYMC antagonist microorganisms.

The present invention relates to a microbial preparation using the antagonist trichodelma harzianum for the control of pepper blight, more specifically, as a developmental agent for biologically controlling pepper blight by using the antagonistic trichodelma harzianum. The present invention relates to a microbial agent having an excellent effect and a method for preparing the microbial agent.

Pepper ( Capsicum annuum L.) cultivation is carried out in most farms, and the damage caused by Phytophthora capcisi , a soil infectious hospital , is severe. The pathogens present long-term survival in the soil and present many difficulties in controlling them (Hwang, BK and Kim, C. H (1995) Phytophthora blight of pepper and its control in Korea, Plant Dis . 79, 221-). 227). For the control of late blight, chemical control and biological control using microorganisms are known (Cook, R. J (1990) Twenty-five years of progress towards biological control.Pl-4.In D. Hornby (ed.), Biological control of soil-borne plant pathogens, CAB Internaional, Wallingford, UK; Handelsman J. and Stabb, E. V (1996) Biocontrol of soilborn plant pathogens, Plant Cell. 8, 1855-1869). In general, chemical control costs less than microbial pesticides and can be economically controlled. However, pepper pestilization causes soil pollution due to overuse if chemical control effects fall below the economic level depending on climatic conditions. As environmental problems such as toxic and human toxicity have emerged, antagonistic microorganisms have been raised.

Biocontrol mechanisms by antagonistic microorganisms artificially increase competition, overlapping parasites, phagocytosis, antibiotic production and enzyme production that can occur between two different species in natural ecosystems. Since it has been reported that suppressed soils are formed by interactions, they have been actively conducted as part of comprehensive control methods, so that antagonistic microorganisms belonging to actinomycetes, bacteria and fungi are highlighted as microorganisms for biological control. (Elad, Y, Kapat, A (1999) The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea , Eur . J. Plant Pathol . 105 (2), 177-189; Howell, CR, Hanson, LE, Stipanovic, RD, and Puckhaber, LS (2000) Induction of terpenoid synthesis in cotton roots and control of Rhizoctonia solani by seed treatment with Trichoderma virens , Phytopathology 90 (3), 248-252; Yedidia, I., Benhamou, N., Chet, I (1999) Induction of defense responses in cucumber plants ( Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum , Appl . Environ. Microbiol . 65 (3), 1061-1070; Conway, KE, Mereddy, R., Kahn, BA, Wu, Y., and Hallgren, S. W (2001) Beneficial effects of solid matrix chemo-priming in okra. Plant Dis . 85, 535-537; Dandurand, LM., Mosher, RD, and Knudsen, GR (2000) Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens. Can. J. Microbiol./Rev . Can. Microbiol . 46 (11), 1051-1057; Dik, A., Elad, Y (1999) Comparison of antagonists of Botrytis cinerea in greenhouse-grown cucumber and tomato under different climatic conditions, Eur. J. Plant Pathol . 105 (2), 123-137; Dik, AJ, Koning, G, and Koehl, J (1999) Evaluation of microbial antagonists for biological control of Botrytis cinerea stem infection in cucumber and tomato, Eur . J. Plant Pathol , 105 (2), 115-122; Ahn, SJ and Hwang, B. K (1992) Isolation of antibiotic-producing actinomycetes antagonistic to Phytophthora capsici from pepper-growing soils, Korean J. Mycol . 20, 259-268).

It is a representative fungus that is studied at various angles for the development of microbial pesticides. Trichoderma The genus contains mechanisms such as polysaccharides, chitin and glucans, constituents of fungal fungal cell walls, overlapping parasites, antibiotic production and competition (Lo CT, Nelson EB and Harman G. E (1997) .Improved biocontrol efficacy of Trichoderma harzianum 1295-22 for foliar phases of turf diseases by use of spray applications. Plant Dis. 81,1132-1138), since it was found to be a very effective fungus for inhibiting phytopathogens, studies on the biological control of crop pathogens using Trichoderma genus have been actively conducted (Elad, Y, Kapat, A). (1999) The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea , Eur . J. Plant Pathol . 105 (2), 177-189; Yedidia, I., Benhamou, N., Chet, I (1999) Induction of defense responses in cucumber plants ( Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum , Appl . Environ. Microbiol . 65 (3), 1061-1070; Conway, KE, Mereddy, R., Kahn, BA, Wu, Y., and Hallgren, S. W (2001) Beneficial effects of solid matrix chemo-priming in okra. Plant Dis . 85, 535-537; Dik, A., Elad, Y (1999) Comparison of antagonists of Botrytis cinerea in greenhouse-grown cucumber and tomato under different climatic conditions, Eur . J. Plant Pathol . 105 (2), 123-137; Lee, In-Kyung, Chang-Jin Kim, Shin-Duk Kim, Yoo, Dong-Duk (1990) Streptomyces Red Pepper Diseases from Parvullus, 韓國 韓 用 微生物 學 物 . 18, 142-147), Trichodex, formulated with T. harzianum T-39, currently controls gray mold disease caused by Botrytis cinerea in the United States. It is used as a microbial pesticide. In Korea, research on biological control of pepper blight has been actively conducted using Pseudomonas genus and Basillus genus since the mid-1980s (Hong, SS, Park, KS, Kim, CH and Lee). , E. J (1990) Granule formulation of Pseudomonas cepacia antagonistic to Phytophthora capsici and its viability on red-pepper, Korean J. Plant Pathol . 6, 434-439; Hwang, Byung-kuk and Eun-Soo Kim (1992) Phytophthora 抑制of pepper疫病by capsici菌株,韓國植物病理學會誌 8, 1-7; Jee, HJ, Nam, CG and Kim, C. H (1988) Studies on biological control of Phytophthora blight of red pepper Ⅰ. Isolation of antagonists and evaluation of antagonistic activity in vitro and in greenhouse, Korean J. Plant Pathol . 4, 305-312; Kim, BS and Hwang, B. K (1992) Isolation of antibiotic-producing bacteria antagonistic to Phytophthora capsici from pepper growing soils and evaluation of their antibiotic activity, Korean J. Plant Pathol . 8, 241-248; Kim, CH, Kim, KD, and Jee, H. J (1991), Enhanced suppression of red-pepper Phyphthora blight by combined applications of antagonist and fungicide, Korean J. Plant Pathol . 7, 221-225; Lee, In-Kyung, Chang-Jin Kim, Shin- Duk Kim, Yoo- Duk Lee (1990) Korean Red Pepper Diseases of Streptomyces parvullus, 韓國 역 用 微生物學會 誌. 18, 142-147; Nam, CG, Jee, HJ and Kim, C. H (1988), Studies on biological control of Phytophthora blight of red-pepper II. Enhancement of antagonistic activity by soil amendment with organic materials, Korean J. Plant Pathol . 5, 1-12; Park, HH and Kim, H. K (1989) Biological control of Phytophthora crown and root rot of greenhouse pepper with Thichoderma harzianum and Enterbacter agglomerans by improved method of application, Korean J. Plant Pathol . 5, 1-12; Park, KS, Hagiwara, H. and Kim, C. H (1993) Isolation of an antibiotic substance from Pseudomonas cepacia antagonistic to Phytophthora capsici , Korean J. Plant Pathol . 9, 1-6; Park Chang-seok (1989). A Study on the Reduction of Salt Disorders in Crop Fields by Microorganisms, Nongshim Paper Collection 101-108; Harman, GE, Chet, I. and Baker R (1980) Trichoderma hamatum effects on seedling disease induced in radish and pea by Pythium spp. or Rhizoctonia solani , Phytopathology . 70, 1167-1172).

The present invention develops DYMC, a product for commercial commercialization, using antagonistic strain ( T. harzianum ) to establish a biological control method for pepper blight, and tricho at room temperature and soil to evaluate the density change of the bacteria for the product. The density of T. harzianum was investigated and the effect of pepper blight control and pepper growth was verified by greenhouse tests.

The present invention is to solve the problems of the prior art by using a antagonist trichodelma harzianum to develop a formulation that is effective in the control of pepper blight disease to provide a microbial agent excellent biological control effect.

Hereinafter, the configuration and operation of the present invention.

The present invention provides a microbial preparation using antagonistic microorganism Trichodelma harzianum for the control of pepper blight.

The present invention is a 20 to 28 ℃ in the potato dextrose medium in the frozen storage bacteria to commercially use the antagonist Trichodelma harzianum ( T. harzianum ) for the biological control of the late onset of pepper crops After primary culture for 48-96 hours at, the culture was inoculated into a culture such as zeolite, a secondary nutrient culture, to form maximum spores during secondary culture for 5-10 days at 20-28 ° C, and 35-40 ° C. It is characterized in that it is manufactured by grinding in a grinder to produce a powder form by drying for 7 to 10 days until the water content reaches about 25 to 30%.

In the present invention, starch and inorganic nutrients are added to potato dextrose medium by adding antagonist trichodelma harzianum to biological control of late blight in pepper, and at 48-26 ℃. The culture solution (A) obtained by primary culture for 96 hours, and Bacillus subtilis in PDB (potato dextrose broth) medium in a liquid medium containing corn starch, inorganic nutrients, etc. 250 ml of primary bacterial cultures (B) obtained by inoculating at 26-28 ° C. for 24 to 36 hours were prepared and then secondary cultures (based on 1 kg; zeolite 100 g-200 g, Hite carbon 100 g-400 g, and Goto hydroxide 100 g-). 300 g, diatomaceous earth 100 g-400 g, talc 50 g-400 g, corn starch 50 g-200 g, 50 g-200 g glucose, 50 g-50 g KH2PO4, 5 g-50 g EDTA iron, 1-10 g zinc sulfate and the remaining water) A step for preparing a culture of the stirred dough state system;

Dispense the culture of the above steps to a thickness of 1-2cm on a mesh cloth, such as a mineral cloth, Second culturing for 5-7 days in a culture room at 27-28 ° C .;

When the mycelia of T. harzianum bloom on the surface of the culture after the cultivation of the above step, white crosses are formed in the shape of lobe crosses at 1-3cm intervals from the surface of the culture to the bottom, and then green spores from the hyphae Continuing culturing with humidity maintained for 5-7 days until maximum formation;

After culturing in the above step, when the spore formation of trichodelma harzianum is maximized, the temperature is maintained at 36 to 40 ° C. while removing moisture from the culture chamber with a ventilator until the moisture of the culture reaches about 25 to 30%. Drying for 7 days; And

After drying, the hardened cultures are pulverized using a grinder to prepare a powder for microbial preparations, and the method of forming endogenous spores of Bacillus subtilis is also maximized.

Tri nose Del Mar Har Jia num (T. harzianum) fungi and Bacillus subtilis (Bacillus subtilis) bacterial strain used in the present invention is a mixed form are separated from each other by a possible strain produced by the above production method, or the culture well with It can be produced as a microbial agent. Specific strain isolation method used in the present invention is as follows. First, in order to separate the T. harzianum fungus and Bacillus subtilis from the soil, 5 g of the soil, which was dried in a laboratory at room temperature and passed through 2 mm in 50 ml of sterilized water, was shaken. After diluting the supernatant, the bacteria were selected from each selective medium to be purely cultured, followed by identification of the species name, followed by replacement culture in the causal bacterium, which has excellent antagonistic effect of Trichodelma harzianum ( T . harzianum) five kinds of fungi and Bacillus subtilis (Bacillus subtilis) 3 species of starting again to identify the species and tri-nose with the most excellent antagonistic effects among others Thelma Har Gia num (T. harzianum a) and one kind of fungus One species of Bacillus subtilis was finally selected to identify the species name.

As a result, the tri nose Delmar Hari Jia num (Trichoderma harzianum) is tri nose Delmar Hari Jia num (Trichoderma harzianum) was found to be the same strain as KCTC 6043, Bacillus subtilis (Bacillus subtilis) was identified as the strain, such as Bacillus subtilis (Bacillus subtilis) 1396 KCTC.

However, tri used in the present invention co-Del Mar Har Jia num (T. harzianum) and Bacillus subtilis (Bacillus subtilis) strain belonging to the genus is when it can be manufactured by the manufacturing method as well as the possible use whichever and tri nose Del Mar Har As T. harzianum, Bacillus subtilis such as KCTC 6426, KCTC 6385, and Bacillus subtilis can be obtained by using conventional strains such as KCTC 1325, KCTC 1327, KCTC 1333, KCTC 1334, and KCTC 1396. .

Hereinafter, an Example demonstrates this invention in detail. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1: Plant material and pot soil preparation

Pepper varieties used in the present invention is a giant (Heungnong Co., Ltd.), sowing seedlings in a 50-hole plug container using the topsoil was used in the greenhouse test seedlings from 6 to 7 leaves in the greenhouse. The soil of the pot used for the greenhouse test was the soil of the pavement farm in Daegu University, which causes late blight, and the size of the pot was 16 × 16 × 13 ㎝. During the test period, the greenhouse temperature was maintained at 25 to 30 ° C and at night to 18 to 20 ° C.

Example  2: pepper Late blight  Spore production

From the strains stored in V-8 juice agar medium (V-8 juice 200 mL, CaCO ₃ 2.0 g, Agar 20 g, DW 800 mL) to produce zoospoangia of the phytophthora capsici used as inoculum. After inoculating the cultured hyphae and incubating for 5 days in a 28 ℃ incubator, by pushing the airborne hyphae on the surface of the medium with a smear rod and incubated for 24 hours under a cool white fluorescent lamp of 60W to form a spore. The formed sachets were collected using a brush after adding 5 ml of sterile saline solution.

Example  3: antagonist Formulation

The results from 2000 to 2001 to identify selected from 24 regional North Gyeongsang Province commercialization tri Corde Do Har Gia num (T. harzianum) the antagonist fungus in Tri Corde Do Har Gia num (T. harzianum) identified as KCTC 6043 strain For the purpose of formulating the following process.

First, T. harzianum fungus and Bacillus subtilis bacterium (KCTC 1396) were respectively used in potato dextrose broth (PDB), corn starch, glucose, Inoculating progeny in a medium containing diphosphate phosphate and trace elements, fungi were cultured in different culture tanks for 72 hours and bacteria for 24 hours in a 27 ° C. microbial incubator to obtain a culture solution. Thereafter, the microbial culture solution (bacteria and fungi, respectively) was inoculated in the same amount in a culture having a composition as shown in Table 1 and Table 2, which is a secondary microbial culture composition for production as a commercial formulation, and then prepared as a microbial preparation. That is, the culture inoculated in the composition of Tables 1 and 2 was incubated for 2 days at 27-28 ° C. for 5-7 days, and the mycelia were white on the surface of the culture, and then lined with 1 cm-3 cm intervals from the surface to the bottom of the leaf. Gave. Then, after 2-3 days, a large number of green spores are formed on the mycelium branch. After incubating for 5-7 days until the green spores are formed to the maximum, the temperature is adjusted to about 35 ° C, and then the water is removed with a pan. After drying for 7 to 10 days until the water content of 25% was reached and ground in a crusher and packaged.

Composition for Preparation of Microbial Agents Raw material name Input ratio Microorganism ( T. harzianum ) 350 g Diatomaceous earth 250 g Zeolite 100 g Talc 50 g Hydroxide Goto 50 g White carbon 50 g KH ₂ PO ₄ 15 g EDTA Fe 5 g ZnSO ₄ 5 g Corn starch 50 g glucose 50 g Sugar 25 g

Composition for Preparation of Microbial Agents Raw material name Input ratio Microorganism ( T. harzianum ) 200 g Microorganism ( Bacillus subtilis ) 200 g Diatomaceous earth 200 g Zeolite 100 g Talc 50 g Hydroxide Goto 50 g White carbon 50 g KH ₂ PO ₄ 15 g EDTA Fe 5 g ZnSO ₄ 5 g Corn starch 50 g glucose 50 g Sugar 25 g

Prepared as described above, antagonistic microbial agent Tri nose Del Mar Har Jia num (T. harzianum) la formulation (composition in Table 1) a commercial foliar applications and irrigation is possible wettable powder tentative DYMC (Deagu Yongse Microbe Compounds) were named using the ( 1). The DYMC packaged with tinfoil was stored at room temperature for 12 months, and the density of Trichodelma harzianum was examined over time, and the decrease in the density of the bacteria was maintained without statistical difference (Fig. 2).

Antagonist Tri nose Del Mar Har Jia num DYMC of production using (T. harzianum), it is important that a mixture of cultures incubated in the primary clay mineral form a number of spores in the period in which the subculture. These spores not only maintain vitality of antagonists for a long time during product distribution, but are also closely related to the effect of the product. In general, microbial products can be formulated into solid, liquid and powdery phases depending on the characteristics of the antagonistic microorganisms. .

A tri nose Del Mar Har num Jia a powdered form developed in the present invention (T. harzianum) The fungal activity density of DYMC was 1.9 × 10 ⁹ CFU / g in the irradiation immediately after production, and showed a tendency to gradually decrease the fungus density over time (Fig. 2). Three months of examination of the samples stored at room temperature for one year showed that the stability of the commercial product could be assured by maintaining 0.9 × 10 ⁹ CFU / g of the high bacterial density. According to the provisions of the current fertilizer law, it is considered that there is no problem as a distribution product, considering that the shelf life of microbial products is one year.

Example 4 Trichodelmar Indoors and in a Greenhouse Har Jia num (T. harzianum) of active research DYMC

Antagonistic microorganisms Har Gia Tri nose Thelma num (T. harzianum) (KCTC 6043) the use by a distributor if a wettable powder form of commercial development DYMC sealed with aluminum foil on the market in order to investigate the viability of microbial packaging Packing Density After storage at room temperature for 1 year, 1.0 g of each package was taken to investigate the density of DYMC antagonists at 3 month intervals, and the density of bacteria (cfu / g) was examined by plate dilution method. In the greenhouse test, DYMC was treated well with the upper pot soil to investigate Trichoderma spp. Activity in the pot soil, or water-diluted suspension was irrigated on the pot soil. The soil and mixing treatment were well mixed with 500 g of upper soil of the pot and 2.5 g and 5 g of DYMC, respectively, and the top portion of the pot was filled. The irrigation treatment was performed by diluting 2.5 g and 5 g of DYMC in 100 ml water, respectively. After the creation was irrigation in the port. Water was watered every 3 days to prevent drying after inoculation. The microbial activity was investigated from 5 days after treatment. The soil was collected by cork boring (25 mm in diameter) and 10 g of pot soil 3 ~ 4 cm deep from the surface was collected. 25 µg / ml of PDA medium was examined by plate dilution and performed in 5 replicates.

After the commercial formulation DYMC was mixed with pot soil and treated with suspension, the density of Trichodelma harzianum was investigated over time, and the results of ANOVA analysis were shown in Table 3 below. . There were statistically significant differences in treatment methods, treatment concentrations and days of irradiation (R ² = 0.76, F = 10.5960, P = <.0001). There was no interaction between treatment method and irradiation days.

Results of the treatment method and the concentration were as shown in FIG. 3. The density of bacteria in the DYMC mixed with pot soil was higher than that in suspension diluted with the same amount of DYMC in water. . After the per port 5g DYMC a treatment After a review on the fifth day of June 8, the density of the soil and the sphere tri nose Del Mar Har Jia num (T. harzianum) the mixed treatment was ^{5.4 × 10 5 cfu / g soil} , the same The amount of water diluted with water and treated with suspension showed 4.2 × 10 ⁵ cfu / g soil.

The effect of antagonistic microbes on soil disease depends on how well the treated antagonists settle in the soil and how long the settled microorganisms remain active for a long time. In this test, the antagonistic microorganisms appeared to take about 25 days or more after treatment, and thereafter, the density change of the bacteria did not change significantly for a certain period of time. In terms of DYMC's usage, direct use on the soil works well. When the microbial agent is diluted with water and treated in suspension before use, it is difficult to explain why the antagonistic bacteria have a lower adherence than the mixed soil treatment group. It is assumed that the temperature of the soil surface is not affected by changes in temperature. Lo (Lo CT, Nelson EB and Harman G. E (1997) .Improved biocontrol efficacy of Trichoderma harzianum 1295-22 for foliar phases of turf diseases by use of spray applications. Plant Dis. According to 81,1132-1138), the density of bacteria in the soil than it is to use a spore suspension using a tri-nose Thelma Har Gia num (T. harzianum) 1295-22 for lawn disease control as granules (granule) type High. Kim et al . Observed the occupancy of antagonists in the soil root zone after direct treatment of the spore suspension in ports such as T. hamatum and T. viride with antagonistic forces. As a result, the occupancy rate of bacteria was significantly higher in the soil depth range of 1 to 3 cm, and significantly decreased at 4 cm and below. The results of this test also showed similar test results to Kim, etc., which proved that the effect varies depending on the method of use.

DYMC, a commercial formulation, was mixed with pot soil and treated with suspension, followed by ANOVA (dispersion analysis) analysis of the density of Trichodelma harzianum over time .  division df F value P> F Processing type 3 46.2055 <.0001 Duration 5 25.5180 <.0001 Replication 4  0.4106 0.8006 Treatment x Duration 15  1.2102 0.2742

Example  5: peppers in a greenhouse Late blight  Investigation of the effects on control and pepper growth

The effect of DYMC on pepper blight was examined for 50 days in a greenhouse pot. Pepper seedlings (giant varieties) of about 6-7 leaves were used on this test on March 25. DYMC treatment was performed just before the formality, and the treatment method was as follows. The soil and mixing treatment was well mixed with 500 g of the upper soil layer and 5 g and 2.5 g of DYMC, respectively, and the red pepper seedlings were planted. In the treatment of the suspension, red pepper seedlings were planted in the pot, and water was sufficiently supplied. After 1 hour, the suspension of the same amount of DYMC diluted in 100 ml of water was irrigated on the pot soil. DYMC treatment time was carried out in the late afternoon to help settle the antagonists. The temperature of the greenhouse was maintained at 20 ~ 28 ℃ for the settlement of bacteria. In order to increase the incidence of late blight, a late blight was inoculated. The inoculation of the pathogen was confirmed after 7 days of DYMC treatment, and peppers were grown normally, and then 100 ml per pot was used with a susceptible capsule suspension (1 X 10 ⁵ zoosporangia / ㎖). Irrigation was inoculated.

In order to investigate the effect of DYMC on the growth of red pepper, DYMC was treated in the pot as described above. All treatments were watered using a spray sprinkler system at the same time each morning from three days later to help the bacteria settle in the soil after treatment with DYMC. All tests were performed once with 5 repetitions and 10 ports per repetition.

Plague Inhibition Effect

After treatment with DYMC, an antagonist microorganism in the pot, ANOVA analysis was performed to investigate the disease occurrence pattern over time. There was a statistically significant difference in treatment rate and incidence of disease over time (R ² = 0.82, F = 16.4758, P = <.0001). There was no interaction between treatment method and incidence over time. The incidence of pepper blight began to appear 15 days after inoculation of pathogens (FIG. 4). On the 50th day of DYMC treatment, the incidence of the treatments was significantly lower than that of the treatments. By treatment, DYMC 5g mixed with the upper pot soil had the lowest incidence of 28%, and 62.5% of the disease control effect compared to the incidence of untreated (72%). Appeared. However, the incidence of bulbs diluted with water in 5 g of DYMC diluted in water and irrigated in suspension was 32% (bottle control: 55.5%). The incidence rate was 36-40% (control was 50-45.5%) in the experimental group treated with 2.5g per pot by reducing the amount of DYMC, and the effect of inhibiting disease was significantly low.

DYMC formulated with spores and mycelium of the antagonist Trichodelma harzianum has been shown to affect the effects of late blight on treatment according to the treatment method. Based on the ability of antagonists to settle, the success or failure of soil disease control will depend on the results of this experiment.

In this experiment, the antagonistic microorganisms were diluted with water and used as a suspension. The low effect of the antagonistic spores and mycelium on the surface of the soil was treated as if the suspension was irrigated with soil. That is, the spores are evenly may also phenomena does not inoculate the soil gravitate to one place, and most of the spores is to compete with low prices being environmental limiting factors on spore germination in the process of settling the internal soil and pathogens in the soil surface, Direct attack, lysing and hyperparasitize (Lo CT, Nelson EB and Harman G. E (1997). Improved biocontrol efficacy of Trichoderma harzianum 1295-22 for foliar phases of turf diseases by use of spray applications. Plant Dis. 81,1132-1138) due to factors such as decrease in density, it is thought that the control effect is lowered by not reducing the density of the pathogen, which is a pathogen. Lo According to such control, and that a higher effectiveness than using the resulting spore suspension was used for soil microorganisms antagonistic tri-nose Thelma Har Gia num (T. harzianum) for 1295-22 lawn disease control in the form of granules, dollar spot (dollar spot In order to simultaneously control several grass diseases such as Pythium root rot and brown patch, it is also recommended to combine the application of granules and spore suspension.

Inhibition effect of the use of the disease was found to be low in the rate of disease occurrence when treated with the same treatment method as the high concentration treatment method, which may vary depending on the pathogens to be controlled, but the disease occurs by using a certain amount of antagonistic microorganisms It can be seen from the preliminary test that the Naturally spore germination of Tri Cordell equine (Trichoderma spp.) In the soil pathogen-infected soil was called less than 7%, tri-co-Delmas (Trichoderma) The bacteriostatic phenomenon was also observed . Since 1973 Trichoderma Since the genus is known as an effective biological control agent for controlling crop diseases in packaging and greenhouses, these fungi are antagonists that have the ability to directly attack pathogen fungi or have the ability to lysate and duplicate parasitics. ( T. hamatum ) Tr-5 is known as Pythium spp. And Rhizoctonia solani ) as a redundant parasite (Harman, GE, Chet, I. and Baker R (1980) Trichoderma ) hamatum effects on seedling disease induced in radish and pea by Pythium spp. or Rhizoctonia solani , Phytopathology . 70, 1167-1172).

Although confined to greenhouse testing, the use of 5 g of T. harzianum DYMC in soil resulted in 62% bottle control, which motivated further testing in the field. In the field trials, if the results can be similar to those of greenhouses, it is considered that the results show the possibility of developing them as microbial pesticides, considering that the control value of untreated pesticides prescribed by the current microbial pesticides is 50%.

After treatment with DYMC, an antagonist microorganism, in the pot, ANOVA analysis was conducted to investigate the development of disease over time.  division df F value P> F Treatment type 4 66.0046 <.0001 Days of investigation 6 24.7353 <.0001 Replication 4 1.4414 0.2237 Treatment x Days of investigation 15 1.8973 0.0691

Pepper growth effect

As a result of investigating and analyzing the effect of DYMC on pepper growth, there was a statistically significant difference at P = 0.05 only in the stem size (Table 5). The biomass of shoots and roots and the number of peppers with abandonment showed significant differences at 0.5%, but no effect was found in root building.

On the 60th day after the pot establishment, the growth of above ground and below ground for red pepper showed a tendency to increase slightly in DYMC compared to untreated, which recognized the need for further investigation of the effects of antagonists. However, long-term pot tests showed that environmental constraints were high when investigating the effects of antagonists on pepper growth. First of all, this test showed that the change in temperature and humidity was too large, and it is thought that these effects may have been a serious limitation to the post-settlement activity. The reason for the early growth was the difference in height, etc. through visual observation, but the difference decreased with time. Trichodelma harzianum used as a powder to control several diseases on grass 1295-22 reported that not only turf disease control effects but also turf growth (Lo CT, Nelson EB and Harman G. E (1997). Improved biocontrol efficacy of Trichoderma harzianum 1295-22 for foliar phases of turf diseases by use of spray applications. Plant Dis. 81,1132-1138), it is thought that more detailed investigation in controlled environmental conditions is needed for the effect on future growth effect.

Investigation of the effect of DYMC on pepper growth Control DYMC  g / soil 2 kg pot DYMC  Suspension 100ml / soil 2 kg pot Control 2.5 g 5 g 2.5 g 5 g F P> F Length 15.1 ± 0.7 16.1 ± 0.9 17.7 ± 1.2 16.3 ± 1.0 16.9 ± 1.4 1.4059 0.169 Stem 0.77 ± 0.06 0.86 ± 0.03 0.88 ± 0.05 0.83 ± 0.10 0.85 ± 0.08 3.3443 0.0188 Fresh weight 27.6 ± 4.2 29.2 ± 6.3 31.5 ± 4.2 28.2 ± 4.3 30.5 ± 6.5 1.7918 0.1495 Dry weight 5.5 ± 0.9 5.7 ± 1.6 5.9 ± 1.2 5.6 ± 1.4 5.9 ± 1.4 1.1267 0.5678 No. of Fruit 10.8 ± 1.3 11.7 ± 1.6 12.2 ± 1.9 11.2 ± 0.7 11..8 ± 1.2 1.1174 0.3619

As described above, the microbial agent prepared using the antagonistic microorganism Trichodelma harzianum for controlling the late blight of the pepper crop of the present invention is used as a commercial microbial agent for controlling pepper blight, which is currently a problem. It is a very useful invention for the development of biopesticides for safe crop production because it is a microorganism capable of environmentally friendly control of pepper disease.

Claims (2)

Trichodelma harzianum ( T. harzianum ) was obtained by adding corn starch, glucose and inorganic nutrients to potato dextrose medium and inoculating it in the medium, followed by primary culture for 48-96 hours in a 26-28 ℃ microbial incubator. The culture solution (A) and the culture solution (B) obtained by culturing Bacillus subtilis in the same culture solution as above, each using 250 ml of zeolite 100g-200g, Hite carbon 100g-400g, Goat hydroxide 100g-300g, 1kg secondary consisting of 100g-400g diatomaceous earth, talc 50g-400g, corn starch 50g-200g, glucose 50g-200g, KH ₂ PO ₄ 50g-50g, EDTA iron 5g-50g, zinc sulfate 1-10g and residual water Inoculating the medium to prepare a culture of dough; Dispense the culture of the above step to a thickness of 1-2cm on a cloth to dry out and flatten it Secondary culture for 5 to 7 days in a culture room at 20 to 28 ° C .; If the mycelia of T. harzianum bloom on the surface of the culture after culturing in the above step, the mycelia of the mycelium are formed in the shape of lobe crosses at 1-3cm intervals from the surface of the culture to the bottom to promote sporulation. Continuing to incubate with humidity for 3-7 days until maximum green mold spores are formed; When the sporulation is maximized after the culturing in the above step, while maintaining the temperature at 35 ~ 40 ℃ to dry while removing the moisture in the culture chamber with a ventilator to dry for 5 to 7 days until the moisture of the culture reaches about 25 to 30% step; And The method of producing a microbial agent for controlling chilli blight disease, characterized in that it comprises the step of grinding the solid culture tube after drying using a grinder. The microbial agent for pepper pepper disease control prepared by the manufacturing method of claim 1.

Images