MXPA99007193A - Antifungal biocontrol agents, a process for preparing and treating the same - Google Patents

Abstract

The present invention relates to antifungal biocontrol agents which enhance plant growth and reduce plant diseases by suppressing fungal pathogens, and the manufacturing and application methods thereof. The antifungal biocontrol agents of the present invention consist of one of the newly isolated strains of Streptomyces sp. WYE 20 (KCTC 0341BP) and WYE 324 (KCTC 0342BP) and a delivery medium which carries and stabilizes the cells. In particular, WYE 20 and WYE 324 of the present invention exhibit strong antifungal activity against Rhizoctonia solani and Phytophthora capsici and can be used in various ways to reduce fungal diseases in plants such as cucumber (Cucumis sativus L. ), pepper (Capsicum annuum L.), and golf course turfgrasses.

Description

AGENTS OF BIOCONTROL ANTIHONGOS, A PROCESS TO PREPARE AND TREAT THEMSELVES FIELD OF THE INVENTION The present invention relates to antifungal biocontrol agents and to a process for preparing and treating them. In particular, the present invention relates to two strains of antifungal biocontrol from Streptomyces sp. , WYE 20 and WYE 324, able to reinforce the growth of plants and reduce diseases by suppressing colonized fungal pathogens in rhizosphere soils of host plants, and the method of preparation and application thereof.

BACKGROUND OF THE INVENTION It is well known that the fungal phytopathogens found in the soil cause great economic losses in the agricultural and horticultural industries. In particular, Rhi zoctonia solani is one of the most important fungal pathogens that exhibits strong pathogenicity, is associated with seedling diseases as well as foliar diseases, such as, seed rot, root rot, stalk rot, putrefaction of the leaf and stem of many species and plant varieties, resulting in a huge economic loss. For example, P1459 / 99MX Rhi zoctonia solani AG 1 (IB) causes plant diseases in crops such as cucumber (Cucumi s sa ti vus L.) and chili (Capsi cum annuum L.) as well as brown patches in creeping agrostis on golf courses, Rhizoctonia Solani AG 2-2 causes large patches of turfgrass in large areas of golf courses, resulting in huge economic losses. In addition, Phytophthora capsi ci is a fungal phytopathogen that is found in the soil, highly destructive and very widespread that causes root and crown rot, as well as blasting of the leaves, fruits and stems of chili peppers (Capsi cum annuum L.) . It is very difficult to suppress the blight by Phytophthora in chile plants if they are infected with Phi tophthora capsi ci. Phi tophthora capsi ci is a particularly destructive phytopathogen responsible for the blasting of chili plants during hot and humid rainy seasons, causing the extermination of chili plants and consequently great economic losses. As already mentioned, both Rhi zoctonia solani and Phytophthora capsi ci are the most important fungal pathogens that exhibit strong pathogenicity and produce spores that survive for a long time in extreme conditions. Therefore, they repeatedly cause plant diseases in large areas when conditions of disease incidence are optimal. P1459 / 99 X Therefore, growers in general apply a mixture of fungicides to plants on a regular basis, to control plant diseases caused by both Rhi zoctonia solani and Phytophthora capsi ci. However, it is increasingly difficult to control the blasting of chili plants by Phytophthora and the brown patches caused by Rhizoctonia in creeping agros, due to the emergence of strains of fungal resistant fungal phytopathogens. It is also increasingly difficult to control these diseases due to the disseminated incidence of fungal resistant Rhizoctonia solani on golf courses and the proliferation of Phi tophthora capsi ci resulting from the successive cultivation of chili plants. The intensive use of agrochemicals has also caused environmental problems and residual toxicity. In addition, agrochemicals are considered ineffective in controlling plant diseases that are transmitted through the soil, due to washout and the lack of an efficient application that allows effective penetration into rhizosphere soils. It is very difficult to wait for long-term protection of the plants with an application of chemical fungicides. Therefore, the use of some rhizosphere bacteria as a biocontrol agent can provide not only more effective and economical practices for the control of diseases of the P14S9 / 99MX plants, such as Phytophthora blight caused by Phytophthora capsi ci and golf course turf disease caused by Rhi zoctonia solani, but also greater environmental conservation. It has been shown that the use of antagonistic microorganisms is a very attractive way of controlling fungal pathogens (Suh, Dissertation Ph.D., University of Idaho, Idaho, USA, 1992; Crawford et al, Appl. Environ Microbiol 59: 3899 -3905, 1993; U.S. Patent No. 5,403,584). The inventors disclosed in the prior art that certain species of Streptomyces strains can be used to control plant pathogens by using a delivery medium containing marsh moss, sand and corn meal, in the fill / soil mixture or by coating the seeds of plants with sodium alginate containing the biocontrol agent, resulting in colonization of the roots (U.S. Patent No. 5,403,584). It has also been disclosed in the prior art that swamp moss containing a beneficial biocontrol agent can be used to control pathogens in plants (U.S. Patent No. 5,595,589). The methods of the administration medium in the prior art are suitable for the application of seedlings / filler blends, but are not adequate enough P1459 / 99MX for direct application to plant seeds or plant roots to achieve efficient protection against fungal pathogens. The object of this invention is to provide novel strains of the Streptomyces species that are capable of controlling the phytopathogenic fungi by direct application to the seeds of plants or plant roots, using specially designed formulations comprising administration means that contain a biologically culture pure, selected from the recently isolated strains of the Streptomyces species. Furthermore, it is an object of the invention to provide antifungal biocontrol agents and to describe their use in the protection of plants against infections caused by fungal phytopathogens that are found in the soil.

SUMMARY OF THE INVENTION The present invention has been achieved through the isolation of the species Strep tomyces WYE 20 and WYE 324 which are capable of protecting the plants against Rhizoctonia solani and Phytoph thora capsi ci. The strains, Streptomyces sp. WYE 20 and WYE 324, are effective to avoid the incidence of fungal diseases in plants and to reinforce the growth of plants in greenhouses and trials in agricultural fields. Therefore, an aspect of the invention P1459 / 99 X are the microbiologically pure cultures of Streptomyces sp. WYE 20 and WYE 324. The present invention also encompasses various suitable administration means for the treatment of plant seeds, seedling beds / pots or pot / soil filling mixtures with Streptomyces sp. WYE 20 and WYE 324. The means of administration is very useful for carrying Streptomyces s. WYE 20 and WYE 324 in order to inhibit plant diseases caused by fungal phytopathogens. In a particular embodiment according to the present invention, the administration means consists of wood sawdust, wheat bran, chitin, chitosan and Phar amedia (trademark, manufactured by The Budkeye Oilseed Products Company, Texas, USA) together with Streptomyces. sp. WYE 20 or WYE 324. In another particular embodiment, the administration means consists of pectin and colloidal chitin and water together with Streptomyces sp. WYE 20 or WYE 324. In the preferred embodiment, the administration means comprises at least 10 colony-forming units of Streptomyces sp. WYE 20 or WYE 324, per gram of the administration medium. The present invention also encompasses methods for reducing fungal infections in seeds, seedlings and / or growing plants, before or during the planting and growing seasons through P1459 / 99MX spray treatment or application in the furrow of plant seeds, seedling beds / pots or pot / soil fill mixtures.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, S trep tomyces sp. WYE 20 and WYE 324 that are effective in inhibiting fungal diseases and increasing the growth of plants such as chili, cucumber and lawns, although their scope is not limited to these examples. The present invention encompasses various administration means that are useful for carrying S trep tomyces sp. WYE 20 and WYE 324, for the inhibition of plant diseases caused by fungal phytopathogens. In a particular embodiment of this invention, the administration means "consists of 40 to 65% w / w of wheat bran, 1 to 5% w / w of chitosan, 30 to 55% w / w of wood sawdust, 1 at 3% w / w of chitin and 1 to 3% w / w of Pharmamedia, based on the total weight of the administration medium In the preferred embodiment, the administration means also consists of 0.2 to 3.5% w / w of sporulation medium as an additional component based on the total weight of the administration medium.The preferred sporulation medium is selected from the sporulation medium ATCC # 5 or extract from P1459 / 99MX yeast-glucose-mineral salts, but the presentation does not exclude other means of sporulation. In another particular embodiment, the administration means consists of 1.0 to (ILLEGIBLE)% w / w of pectin and 0.1 to 0.6% w / w of colloidal chitin in water. In the preferred embodiment, the administration means comprises 10 to 10 °, preferably 107 to 108 colony-forming units of Streptomyces sp. WYE 20 or WYE 324 per gram of administration medium. The present invention includes a process for the production of mycelium and spore inoculum of Streptomyces sp. WYE 20 and WYE 324. Viable Streptomyces s cells. WYE 20 and WYE 324 are produced by incubating vegetative cells in the yeast-glucose-mineral salts extract or the modified Bennett's liquid medium, under the appropriate conditions. A process for preparing an antifungal biocontrol agent comprising the following: Streptomyces sp. WYE 20 or WYE 324 which is prepared by incubating at 130 rpm up to 300 rpm, at a temperature of 25 ° C to 33 ° C for 3 to 7 days. After collecting the Streptomyces sp. WYE 20 or WYE 324, the cells are lyophilized or mixed directly in a medium of administration. In addition, according to the present invention, P1459 / 99MX a process for preparing an antifungal biocontrol agent comprises the following steps: preparing a delivery medium containing 40 to 65% w / w wheat bran, 1 to 5% w / w chitosan, 30 to 55% p / p of wood sawdust, 1 to 3% p / p of chitin and 1 to 3% p / p of Pharmamedia based on the total weight of the administration medium; autoclaving the resulting administration medium; incorporate Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) within the administration medium; incubate the incorporated Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp. WYE 324 (KCTC 0342BP) at 25 ° C to 33 ° C for 5 to 14 days; and aseptically drying the resulting product to obtain an antifungal biocontrol agent. Preferably, the process further comprises aseptically mixing the resulting dried product. In the above process, the administration medium is optionally pelleted and then coated with 0.2 to 3.5% w / w of the sporulation medium based on the total weight of the administration medium. Streptomyces sp. WYE 20 or WYE 324 is incorporated at a concentration of 10 -10, preferably 10 -10 colony forming units per P1459 / 99MX gram of the administration medium. The administration medium is subjected to autoclaving at a temperature of 121 ° C for 30 to 30 minutes. In another embodiment of the present invention there is provided a process for preparing an antifungal biocontrol agent comprising: preparing the administration medium consisting of 1.0 to 3.0% w / w of pectin and 0.1 to 0.6% w / w of colloidal chitin in Water; autoclaving the resulting administration medium; and incorporate Strep tomyces s. WYE 20 (KCTC 0341BP) or S trep tomyces sp. WYE 324 (KCTC 0342BP) in the administration medium. In the above embodiment of the invention, Strep tomyces WYE 20 or WYE 324 is added to the administration medium to a final concentration of 10-10, preferably 10 -10 colony forming units per gram of administration medium. Furthermore, the present invention relates to a method for treating the antifungal biocontrol agent which comprises coating, mixing, spraying or applying within the corrugations the agent in the seeds of the plants, the mixture of pots, in the plants or in the land of them. The present invention also relates to an antifungal biocontrol agent comprising a P1459 / 99MX of the recently isolated strains of Streptomyces sp. WYE 20 and WYE 324. Therefore, it is appreciated that an antifungal biocontrol agent containing microorganisms having an antifungal property equal to that of the strains together with the administration medium according to the present invention remains within the true spirit and scope of the invention. The present invention will be described below in the following examples. However, the invention is not limited to the following examples.

MATERIALS AND METHODS 1. Microorganism Culture Medium All bacterial culture media used distilled water and sterilized in an autoclave before use. All bacterial samples were treated with standard laboratory aseptic techniques to maintain purity. 1) Medium CYD (casamino acid / yeast extract / dextrose / agar) contains casamino acids (Difco: 0.5g / l), yeast extract (Difgo: 0.8g / l), D-glucose (0.4g / l), K2HP04 ( 2.0g / l; p 7.2-7.4) and agar (18g / l) in distilled water. 2) WYE medium (water / yeast extract / agar), modified from Reddi and Rao (1971) contains yeast extract (0.25g / l), K2HP04 P1459 / 99MX (2.0g / l; p 7.2-7.4) and agar (18g / l) in distilled water. 3) YGM medium (yeast extract / glucose / mineral salts) comprises 0.6% (w / v) of yeast extract (Difco Laboratories, Detroit, MI, US.), 1.0% (w / v) glucose and glucose solution. phosphate mineral salt (5.3g of Na2HP04, 1.98g of KH2P04, 0.2g of MgSO4 »7H20, 0.2g of NaCl, 0.5g of CaCl2« 2H20, plus 1.0 ml of trace elements (Pridham and Gottlieb, J. Bacteriology 56: 107-114, 1948) per liter of deionized H20, pH 7.1 to 7.2). The trace element solution consists of 0.64 g of CuS04 »5H20, 0.11 g of FeS04» 7H20, 0.79 g of MnCl2 »4H20, 0.15g of ZnS04» 7H20 in 100 distilled water. 4) CM medium (chitin / mineral salts / agar) comprises 0.4% to 0.6% (w / v) of colloidal chitin prepared by the previously known method (Hsu and Lockwood, Applied Microbiology, p422-426), 0.6% (w / v) (NH4) 2S04 and 2.0% (w / v) agar per liter of mineral phosphate salt solution described above; pH 7.0 to 7.2. 5) Laminarin agar medium comprises 0.25% (w / v) of laminarin, 0.6% (w / v) of (NH4) 2S04 and 2.0% (w / v) agar per liter of mineral phosphate salt solution described above; pH 7.2 to 7.4. 6) Bennett's modified liquid medium containing yeast extract (2g / l), beef extract (2g / l), peptone (2g / l), glucose (10g / l) and nystatin (5 g / ml, pH 6.5) -7.5) in distilled water. P1459 / 99MX 7) Bennett's modified agar medium containing yeast extract (2g / l), beef extract (2g / l), peptone (2g / l), glucose (10g / l), nystatin (5μg / ml); pH 6.5-7.5) and agar (20g / l) in distilled water. 8) Medium ISP # 2 (Difco). 9) Medium ISP # 3 (Difco). 10) Medium ISP # 4 (Difco). 11) Sporulation medium (ATCC medium # 5) containing yeast extract (1.0 g / l), beef extract (1.0 g / l), tryptose (2.0 g / 1), FeS04 (0.01 g / l), glucose (10g / l) and agar (15g / l) (17th Edition ATCC Catalog of Bacteria and Bacteriophages). 2 _; _ Identification of Streptomvces sp. WYE 20 _ WYE 324 Strains WYE 20 and WYE 324 were identified as Strep tomyces species based on the morphological, physiological and chemical characteristics of the genus Streptomyces, as defined in Bergey 's Manual of Systematic Bacteriology (1989); the International Streptomyces Project (ISP) (1974)); and Williams, et al. (1983). The summarized results of these strains with respect to the morphological, physiological and chemical characteristics are reported in Tables I to V.

P1459 / 99 X 3. Preparation of the seed culture and storage of StreOtomvces sp. WYE 20 y_ WYE 324 The spores of an inclined plate of CYD agar were inoculated with an inoculation loop in 500 ml flasks containing 50 ml of Bennett's modified liquid medium (pH 6.5-7.5). The medium was sterilized by autoclaving for 15 minutes at 121 ° C before inoculation. The inoculated flasks were incubated with shaking at 130-300 rpm at 25 ° C to 33 ° C for 1 to 4 days to provide a standard inoculum. For short-term use. Strep tomyces sp. WYE 20 and WYE 324 were incubated on inclined plates of CYD or WYE agar at 25 ° C until sporulation and then stored at 4 ° C until used. The cultures were transferred every 4 weeks. The spores were suspended in glycerol subjected to autoclaving (15% -30%) (121 ° C, 15 minutes) and used for long-term storage at -70 ° C. 4. Collection? _ Production of Mycelia. Spore in Liquid Culture The one liter flasks containing 200ml of modified Bennett liquid medium or 200ml of YGM medium (pH 6.5-7.5) were inoculated with 8ml of each seed culture for the production of viable mycelial cells and spores of Streptomyces sp. WYE 20 and WYE 324. The inoculated flasks were incubated with P1459 / 99MX shaking at 130-300 rpm at 25 ° C to 33 ° C for 3 to 7 days. Viable cells from the mycelia and spores produced through the above process were collected aseptically by centrifugation at 4,000 rpm for 10 minutes . Production of viable cells of mycelia and_ spores with the administration medium and preparation of the antifungal biocontrol agent (1) Preparation of the antifungal biocontrol agent in powder form comprising Streptomyces sp. WYE 20 or WYE 324 and a means of administration. The administration medium of the present invention consisting of wheat bran from 40 to 65% w / w, chitosan from 1 to 5% w / w, wood sawdust from 30 to 55% w / w, chitin from 1 to 3 % p / p, Pharmamedia (The Budkeye Oilseed Products Company, Fortworth, Texas, USA) from 1 to 3% w / w based on the total weight of the medium, was mixed vigorously. The resulting administration medium was pelleted and spray coated with the sporulation medium (ATCC # 5 or YGM medium) from 0.2 to 3.5% w / w and then autoclaved at 121 ° C for 30 to 40 minutes. There was no effect on cell growth when the sporulation medium was added below 0.2% w / w, while the initial cell growth was delayed when the sporulation medium was added to the cell.

P1459 / 99MX more than 3.5% p / p. From one hundred to two hundred ml of Streptomyces sp. WYE 20 and WYE 324 (105-107 cfu / ml) were inoculated into the resulting autoclaved administration medium and then incubated at 30 ° C for 5 to 14 days. Streptomyces sp. WYE 20 and WYE 324 in the administration medium was collected aseptically and dried on a laminar flow bench sterilized by U.V. The product was mixed aseptically to obtain an antifungal biocontrol agent comprising Streptomyces sp. WYE 20 or WYE 324 and the means of administration. (2) Preparation of the antifungal biocontrol agent comprising Streptomyces sp. WYE 20 or WYE 324 and a liquid administration medium. The preparation of the antifungal biocontrol agent was carried out by incorporating viable mycelial cells and spores of Streptomyces sp. WYE 20 or WYE 324 obtained in the above, within the administration medium submitted to autoclave from 1.0 to 3.0% w / w of pectin and 0.1 to 0.6% w / w of colloidal chitin in water.

F pathogenic pathogens The following fungal pathogens were used for an antifungal test: Pythi um ul timum, Pythi um gramini tail, Rhi zoctoni a solani, Rhi zoctonia solani AG 1 (IB), Rhi zoctonia solani AG 2 -2 (IV), Fusari um P1459 / 99MX oxysporum, Fusarium um sambucinctum, Fusarium um solani, Phytophthora capsi ci, Phytoph thora parasí ti ca, Scl erotini a scl erotiorum, Scl eroti um cepivorum and Verti cíli um dahl iae. All the sepals were cultivated on papa-dextrose agar (Difco) or on corn flour agar (Difco) at 25 ° C and stored at 4 ° C.

Determination of the Number or Cells in the Administration Medium One gram of the administration medium containing Streptomyces sp. WYE 20 or WYE 324 was added to 9m of sterile distilled water and mixed vigorously with a vortex apparatus (registered trademark). The resulting suspension was serially diluted and dispersed on CMA plates to determine the colony forming units (ufe) per gram. Plates were incubated at 30 ° C and colony formation was observed. The same method was used to determine the cfu / ml of the administration medium in the liquid form. 8. In Vivo Bioanalysis to Determine Antifunqal Activity of Strevtomyces sp. WYE 20 _ WYE 324 The activity of Streptomyces sp. WYE 20 or WYE 324 to strengthen plant growth and to reduce plant diseases caused by fungal pathogens was determined by treating cucumber, chili or turf for golf with Streptomyces P1459 / 99MX sp. WYE 20 or WYE 324 in a medium of administration, and then planting the treated and untreated seeds in a suitable growth medium or in an agricultural field. The activity of Streptomyces sp. WYE 20 or WYE 324 in a medium of administration as a biocontrol agent was measured in terms of emergence, growth of emerged plants, height of the plant and disease control capacity.

EXAMPLE I Isolation of Streytomyces sp. WYE 20 y_ WYE 324 The soil samples were obtained from four lands associated with different rhizospheres in Goesan, Chungbuk Province, Korea. Isolation of the strains was carried out by serial dilution / plate dispersion techniques on WYE agar plates. The obtained strains were tested in relation to the antagonistic activity against Rhi zoctonia solani and Phytoph thora capsi ci, enzymatic activities that exhibited the degradation of the fungal cell wall; colonization of the root, growth at low temperature (4 ° C, 8 ° C); and the possibility of a large production of cell mass to isolate two novel strains. The two novel strains were named Streptomyces sp. WYE 20 and WYE 324, respectively. The purification of Streptomyces sp. WYE 20 and WYE 324 was carried out by incubating the strains on the WYE plates at 25 ° C for 4 to 10 days to allow the P1459 / 99MX strains sporulate and then their colonies were isolated and streaked onto new WYE agar plates to obtain pure cultures. The pure cultures of Streptomyces sp. WYE 20 and WYE 324 were transferred to sloped plates of CYD agar, incubated at 25 ° C-30 ° C until sporulated and stored at 4 ° C. The cultures were transferred every 4 weeks.

EXAMPLE II Identification _ Characterization of 3tret > tomvces sp. WYE 20? _ WYE 324 The morphological, physiological and chemical characteristics of the microorganisms obtained in Example I were investigated based on: Bergey 's Manual of Systematic Bacteriology (1989). The results are shown in Tables I to V.

P1459 / 99MX TABLE I PICTURE IlA P1459 / 99MX P1459 / 99MX PICTURE IIB P1459 / 99MX MR Registered Trademark P1459 / 99MX TABLE IIC P1459 / 99MX P1459 / 99MX TABLE III s C? «Or H ** TABLE III (CONTINUED) t 1 TABLE II I (CONTINUED) Note: The first and second times: the cells were grown in Tryticase Soy Broth (TSB). 5 The third and fourth times: the cells were grown on Soybean Tripticase Agar (TSA) co -: no detection, nd: not determined.

TABLE IV t TABLE IV (CONTINUED) ? or TABLE IV (CONTINUED) ? Note: The first and second times: the cells were grown in Soy Broth Tripticasa (TSB). The third and fourth times: the cells were grown on Agar Soya Tripticasa (TSA) -: no detection, nd: not determined.

TABLE V 3 ? to TABLE V (CONTINUED) ? ? TABLE V (CONTINUED) s? The two strains obtained in Example I were identified with the genus Streptomyces, as a result of the analysis of the data from Tables I to V based on the Bergey's Manual of Systematic Bacteriology (1989); International Streptomyces Project (ISP) (1974)); and William et al (1983). The two strains were named WYE 20 and WYE 324, respectively. Strain WYE 20 could belong to the species Streptomyces colombi ensi s or to closely related species in cluster 61 based on the morphological, physiological and chemical characteristics of the genus Streptomyces, but the strain was identified as a noble strain of the genus Streptomyces. Strain WYE 324 could belong to the species Streptomyces goshiki ensi s or to a closely related species in cluster 61 based on the morphological, physiological and chemical characteristics of the genus Streptomyces, but the strain was identified as a noble strain of the genus Streptomyces. Strains WYE 20 and WYE 324 were deposited in the Korean Type Culture Collection (KCTC). The Coreno Bioscience and Biotechnology Research Institute, Taejon, Korea on June 18, 1997 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Procedure P1459 / 99MX Patents, assigned Accession No. KCTC 0341BP and KCTC 0342BP, respectively.

EXAMPLE III Optimum Temperature for Cell Growth of S treptomyces sp. WYE 20? _ WYE 324 To investigate the effect of temperature on the growth of Streptomyces sp. WYE 20 and WYE 324, the spores of strains WYE 20 and WYE 324 were inoculated by streaking onto modified Bennett agar plates. The striated plates were incubated at a predetermined temperature of 4 ° C, 8 ° C, 27 ° C, 37 ° C and 45 ° C for 3 weeks. The growth was recorded at 7, 14 and 21 days. The results are shown as good growth (+); slow growth (±); no growth (-) in Table VI.

TABLE VI As shown in Table 6, the WYE strain represented little growth after a week P1459 / 99MX incubation at 4 ° C and good growth after 3 weeks of incubation, while strain WYE 324 represented slow growth after 3 weeks of incubation at 4 ° C. Strain WYE 20 represented good growth at both 8 ° C and 27 ° C. However, strain WYE 824 represented slow growth after one week of incubation at 8 ° C and represented good growth after 2 weeks of incubation at 8 ° C. In addition, strain WYE 324 represented good growth at 27 ° C. Strains WYE 20 and WYE 324 represented slow growth after one week of incubation at 37 ° C and represented good growth after 2 weeks of incubation at 37 ° C, but did not grow at 45 ° C.

EXAMPLE IV Enzymatic Activity y_ Antagonism Assay (1) Enzyme Activity Assay To determine the enzymatic activity of strains WYE 20 and WYE 324, they used both chitinase and β-1,3-glucanase, which are known to be parasitic enzymes. inhibit fungal pathogens. In order to determine the activity of chitinase in strains WYE 20 and WYE 324, each strain was streaked onto CM agar plates containing colloidal chitin as the sole source of carbon, and then incubated at 30 ° C for 14 days .

P1459 / 99MX Colony formation and growth were observed. Strains WYE 20 and WYE 324 were tested showing that they had chitinase activity, in accordance with the above. To determine the β-1,3-glucanase activity of strains WYE 20 and WYE 324, each strain was seeded with striatum on laminarin agar plates containing laminarin as a single source of carbon, and then incubated at 27 ° C. for 7 days. Growth and colony formation was observed. Strains WYE 20 and WYE 324 were tested for β-1,3-glucanase activity according to the above. (2) Antagonism Test An in vitro plate assay was performed to test the antagonistic activity of strains WYE 20 and WYE 324 in terms of growth inhibition of fungal phytopathogens. Each strain was inoculated by striatum onto CMA plates and incubated at 30 ° C for 6 to 10 days. A block of PDA (Papa Dextrose Agar) containing active growing fungal mycelia was then placed aseptically in the center of the plate and incubated at 25 ° C for 24 to 192 hours. The previous PDA block was inoculated into the CMA plates that did not contain the WYE 20 or WYE 324 strains and was used as a control. The bioassay is P1459 / 99MX repeated on three plates. Antagonism was recorded as a degree of inhibition of mycelial growth of fungal pathogens as shown in Table VII TABLE VII 24 hours of incubation, 36 hours of incubation, 192 hours of incubation. Remnant: 96 hours of incubation. ++: Strong inhibition with zone of inhibition > 1.0 cm P1459 / 99MX +: Growth definitely retarded, with obvious zone of inhibition near the colony. -: Without antagonism or very weak antagonism, n.t. : not tested.

As shown in Table VII, strains WYE 20 and WYE 324 showed an obvious zone of inhibition against Pythium ultimum. Strain WYE 20 showed strong antagonism against Pythium graminicola, Fusarium oxysporum, Fusarium sambucinctum, Fusarium solani, Rhizoctonia solani, Rhizoctonia solani AG 1 (IB), Rhizoctonia solani AG 2-2 (IV), Phytophthora capsici, Phytophthora parasitica, Sclerotinia sclerotiorum, Sclerotium cepivorum and Verticillium dahliae. In addition, WYE 324 also showed strong antagonism against Pythium graminicola, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Rhizoctonia solani AG 1 (IB), Rhizoctonia solani AG 2-2 (IV), Phytophthora capsici, Phytophthora parasitica, Sclerotinia sclerotiorum, and Sclerotium cepivorum.

EXAMPLE V Preparation of Aqente Antifunqal Biocontrol in the Powder Form A medium of administration was prepared by mixing wheat bran, chitosan, sawdust P1459 / 99MX wood, chitin and Pharmamedia according to Table VIII. The administration medium was pelleted using an extruder. In the pelletizing process, the degree of pellet formation was determined and recorded as good in case of a good pellet formation without remnants or disintegration, while it was recorded as poor in case of poor pellet formation. The results are shown in Table VIII. It was also determined as poor, moderate and good, according to air penetration and cell growth of the administration medium. The results are shown in Table VIII. One hundred fifty-five ml of Streptomyces sp. WYE 20 or WYE 324 (105-107 cfu / ml) obtained in the "Materials and Methods" section were inoculated into the resulting autoclaved administration medium and then incubated at 30 ° C for 5 to 14 days. Cultures of Strep tomyces sp. WYE 20 or WYE 324 in each administration medium were collected aseptically and dried in a laminar flow hood sterilized by UV. The dried product was mixed aseptically to obtain an antifungal biocontrol agent consisting of Streptomyces sp. WYE 20 or WYE 324 and the administration medium in powder form. The number of WYE 20 or WYE 324 cells in the administration medium was adjusted to 105-1010 cfu / g P1459 / 99 X of administration medium. The resulting antifungal biocontrol agent which consisted of WYE 20 or WYE 324 and administration medium was kept at 4 ° C and 25 ° C for 3 months. The bioavailability and growth of WYE 20 or WYE 324 in the administration medium was determined at one-month intervals and recorded at 3 months. One gram of administration medium containing WYE 20 or WYE 324 was added to 9ml of sterilized distilled water and mixed vigorously by vortexing. The colony forming units per gram of the resulting suspension were determined by serial dissolution and by the plate dispersion technique and are shown in Tables VIII and IX.

P1459 / 99MX TABLE VIII s (unit: p / p% ¡ H TABLE VIII (CONTINUED) 3 X (unit: p / p%) nt: not tested TABLE IX (CONTINUED) 3 (unit: p / p%) (.

TABLE IX (CONTINUED) (unit: p / p%) As shown in Tables VIII and IX, the Administration Medium in trials 1 to 13 was good and moderate in pellet formation, air penetration and cell growth, while controls 1 to 6 with a different range of components showed to be poor in pellet formation, air penetration or cell growth. In addition, the administration medium in cases 1 to 13 formed pellets that were coated with YGM sporulation medium and determined the effects on cell growth and viability. The results are shown in Table X.

P1459 / 99MX TABLE X 3 X (unit: p / p%! It *. 8 TABLE X 3 X YGM *:% p / p of the total weight VO As shown in Tables IX and X, the cells of WYE 20 and WYE 324 were stabilized in the administration medium of tests 1 to 26 for a prolonged period of time (> 3 months). The cells of WYE 20 and WYE 324 in the administration medium 1 to 26 showed > 10 cfu / g that was required for the biocontrol activity after 3 months storage. In particular, when the administration medium in cases 14 to 26 coated with YGM medium was used, the stability of the WYE 20 and WYE 324 cells was improved.

EXAMPLE VI Preparation of Biocontrol Agents in the Form of Suspension A medium of administration comprising 20g of pectin and 2g of colloidal chitin and the remainder of water, based on a total volume of 1.0 liters of distilled water, was autoclaved at 121 ° C. ° C for 15 minutes. The preparation of a biocontrol agent in the suspension form was achieved by incorporating Streptomyces s. WYE 20 or WYE 324 as described in the "Materials and Methods" section, in the resulting administration medium autoclaved (1.2 x 107 cfu / ml). The resulting product was stored at 4 ° C. The biocontrol agent form was suitable for P1459 / 99 X the treatment of seeds of plants and seedling beds by application in stretch marks, or sprayed after dilution with water.

EXAMPLE VII Antifungal Activity Assay In vitro biocontrol assays were conducted to determine the efficacy of the biocontrol agents prepared in Examples V and VI. The biocontrol agents were tested to determine their ability to reduce fungal diseases and improve plant growth by treating plants, plant seeds, plant roots, seedling beds, pots, mixtures for flower pot or the earth. (1) Biocontrol Activity on Stake Rot by Rhizoctonia in Cucumber (Cucumi s sa tivus L.) A biocontrol assay was conducted to test the efficacy of WYE 20 as a biocontrol agent in the inhibition of spoilage. stakes for Rhizoctonia, in cucumber. The cucumber seeds were treated with 20 ml of WYE 20 (1.2 x 107 cfu / ml) prepared in Example I saw the seeds being bathed in the liquid for three hours and then planted in pots. The P1459 / 99MX seeds were bathed for three hours in sterilized distilled water and in a medium of administration, respectively; each resulting seed was used as a control. Hortus (England) was used as a mixture of soil for the pot. The potting soil mixture was autoclaved at 121 ° C for 60 minutes and placed for 12 hours at 25 ° C, and then autoclaved at 121 ° C for 60 minutes, once again. This autoclave processing cycle was repeated three times. Rhi zoctonia solani was grown in PDB (potato dextrose broth: Difco) at 25 ° C for 14 days, collected and mixed with the autoclaved potting soil mixture in order to obtain a disease incidence of approximately 60%. The cucumber seeds were treated with the biocontrol agent and each control seed was seeded in pots containing a potting soil mixture into which Rhizoctonia solani had been artificially inoculated. Disposable paper cups (9cm in diameter) were used as sowing pots. Six cups containing three seeds per cup were prepared for each test. The cups were placed in a random block design in a greenhouse. Humidity remained between 40% and 60% and was supplied P1459 / 99MX additional water, as required. The temperature was kept between 25 ° C and 30 ° C in daylight. The results of emergence and occurrence of stalk rot were recorded periodically. The final results are shown in Table XI.

TABLE XI * Media in a column followed by the same letter are not significantly different at the P = 0.05 level. D.M .: Management Medium.

As shown in Table XI, cucumber seeds treated with WYE 20 in the medium of administration as a biocontrol agent did not show stalk rot by Rhizoctonia in P1459 / 99MX the cucumber. In addition, the cucumber plants obtained from the control seeds showed a severe retardation in growth resulting from the action of Rhi zoctonia solani, while the plants of the treated seeds did not present it. The results showed that WYE 20 was effective in controlling Rhi zoctonia solani and in improving plant growth. (2) Activity of Biocontrol against Mildiú Polvoso in Cucumber (Cucumi s sa tivus L.) A biocontrol test was carried out to test the efficacy of WYE 324 (1.2 x 107 cfu / ml) in the biocontrol agent prepared in the Example VI to inhibit powdery mildew of potted cucumber plants. Disposable paper cups (9cm in diameter) were used as the sowing pots. A cucumber seed was sown in each cup containing the potting soil mixture consisting of agricultural field land and Hortus (England) (4: 1 ratio v / v). The cups were placed in a greenhouse. Additional water was supplied as required and the temperature was maintained between 25 ° C and 30 ° C during the day. Twenty ml of the biocontrol agent containing WYE 324 was sprayed onto a plant P1459 / 99MX cucumber (14 days) at the start of the trial and sprayed once more after one week. Plants sprayed with the same amounts of water were used as a control. Three cucumber plants (one cucumber plant per cup) of each treated group and the control group were prepared in the trial. To naturally induce powdery cucumber powdery mildew caused by Sphaerotheca fuliginea, three cups per group and three sick cucumber plants (one plant per cup) were used and placed in a randomized block arrangement in a greenhouse. Humidity was maintained between 70% and 90% and additional water was added as required. The temperature was kept between 25 ° C and 30 ° C in a greenhouse. The incidence of powdery mildew on cucumber was investigated. The experiment continued for two more weeks and the trial was repeated. The results are shown in Table XII.

TABLE XI I P1459 / 99 X As shown in Table XII powdery mildew was not detected in cucumber plants sprayed with the biocontrol agent containing WYE 324, while the disease was observed in control plants. The results showed that WYE 324 was effective in controlling powdery mildew in cucumber (Cucumi s sa ti vus L.). (3) Biocontrol activity of Streptomyces sp. WYE 20 or WYE 324 on Patches Parduzcos caused by Rhizoctonia in Agrostis Tracking in Golf Courses A biocontrol test was carried out to test the efficacy of Streptomyces sp. WYE 20 and WYE 324 to inhibit brownish patches caused by Rhizoctonia in creeping agrostis of golf courses. Streptomyces sp. WYE 20 and WYE 324 produced by the liquid culture described in the "Materials and Methods" section above, were used in this trial. The number of Streptomyces sp. WYE 20 or WYE 324 was adjusted to 2.0x105 cfu / ml. The test was carried out in each block treated with WYE 20 or WYE 324 or in the control. Four blocks (1.5m x 1.5m) of each treated group and each control group were prepared on the golf course. The test was carried out in a random block design. Strains WYE 20 or WYE 324 were sprayed every 7 to 10 days P1459 / 99MX in the experimental blocks from June 25, 1996 to August 23, 1996. Two and a half liters of WYE 20 or WYE 324 per block were used each time, while the control blocks were provided with the same water amount. No chemical fungicides were used in the period of the experiment. The incidence of brownish patches of Rhizoctonia was recorded periodically and is shown in Table XIII (Site 1) and Table XIV (Site 2). As shown in Table XIII (Site 1) and Table XIV (site 2), the incidence of brown patches due to Rhizoctonia was drastically reduced in the blocks treated with WYE 20 or WYE 324. The results showed that WYE 20 and WYE 324 were effective in inhibiting brownish patches due to Rhizoctonia in creeping golf course agrostis.

TABLE XI II (S ITIO 1) P1459 / 99MX * It means that in a column followed by the same letter there are no significant differences at the P = 0.05 level.

TABLE XIV (SITE 2) * It means that in a column followed by the same letter there are no significant differences at the P = 0.05 level. (4) Biocontrol Activity in Large Patches for Rhizoctonia in a Canalizo Grass (Zoysia japani ca) A control test was carried out to test the efficacy of biocontrol agents including Streptomyces sp. WYE 20 or strain IBT 678, prepared in Example VI, to inhibit large patches of Rhizoctonia in channel grass (Zoysia japonica). The number of Streptomyces sp. WYE 20 or strain 678 in a biocontrol agent was adjusted to 2.0 x 10 6 cfu / ml.

P1459 / 99MX The soil infested with Rhi zoctonia solani AG 2-2 was inoculated into a channel grass (Zoysia japani ca) and then planted in pots (25cm in diameter). Two hundred ml of the biocontrol agent was treated once a week for two weeks and three weeks in 1996 and 1997, respectively. The pots treated with the same amounts of water were used as a control group. Three pots per group were used and placed in a random block arrangement. To naturally induce the disease, the pots were placed near the channel of a golf course in early September until the beginning of October 1996, as well as in early March until the end of June 1997, respectively. The incidence of large patches of Rhizoctonia was investigated. The large patches were not detected in the pots treated in the biocontrol agent containing WYE 20, while the disease was observed in the control and in the pots treated with IBT 678 as shown in Table XV.

P1459 / 99MX TABLE XV As shown in Table XV, the results showed that WYE 20 was effective in controlling large patches of Rhizoctonia in a channel grass (Zoysia japonica). (5) Activity of Biocontrol Against Blight by Phytophthora in Chilean Plant (Capsi cum annuum.) A biocontrol assay was carried out to test the efficacy of WYE 20 or WYE 324 in the biocontrol agent of Example VI to inhibit blast bite. Phytophthora in chile seedlings, in pots. The chili seeds were bathed in sterilized water for 2 days and treated with WYE 20 or WYE 324 in the biocontrol agent of Example VI, submerging them for 18 hours (treatment group). The cell numbers of Streptomyces sp. WYE 20 and WYE 324 were adjusted to 1.2 x 107 cfu / ml and P1459 / 99MX 1.7 x 10 cfu / ml, respectively. The chili seeds were bathed in sterilized water for 2 days and then for 18 hours. They were used as a control group. On the other hand, the chili seeds were immersed in sterilized water for 2 days, they were replaced in the administration medium of Example VI for 18 hours. They were used as the other control group. As described in the cucumber trial described above, Phytoph thora capsi ci was grown in PDB (potato Dextrose broth: Difco) at 25 ° C for 14 to 21 days and was collected and incubated in a sterilized potting soil mixture (Hortus, England) to obtain an infested soil mixture with a disease incidence of 80% blight by Phytophthora for control seedlings of chili. The resulting potting mix infested with Phytophthora capsi ci was used to determine the efficacy of WYE 20 or WYE 324 in the administration medium in the inhibition of blight by Phytophthora in the chili seedlings in the assay. Disposable paper cups (9.0cm in diameter) were used as the sowing pots. Fourteen cups containing three seeds per cup were prepared in each test. The experiments were carried out in a greenhouse at a temperature of 25 ° to 32 ° C in daylight. In the greenhouse, P1459 / 99MX pots were placed in a random block arrangement. The humidity was maintained at 80% and the additional water was sprayed on top of the pots, as required. The emergence and incidence of Phytophthora blast of chili plants was recorded as shown in Table XVI P1459 / 99MX TABLE XVI 3 X It means that in a column followed by the same letter there is no significant difference at the level P = 0.05.

As shown in Table XVI there was a significant reduction in blight by Phytophthora in pepper plants in the seeds treated with WYE 20 or WYE 324 compared to the control group. The result showed that WYE 20 and WYE 324 of the present invention are effective for controlling Phytophthora blast in chili plants. (6) Test to Determine the Improvement in Plant Growth Using Chilean Seedlings A seeding trial was conducted to determine the efficacy of WYE 20 and WYE 324 in a biocontrol agent of the present invention to improve growth of Chili plants. The chili seeds were immersed in sterilized water for 2 days and treated with WYE 20 or WYE 324 in a control agent as prepared in Example V (D.M. 22) (1000 seeds per 4g of biocontrol agent). The number of Streptomyces sp. WYE 20 and WYE 324 before treatment of the seeds were adjusted to 1.2 x 10 cfu / ml and 1.7 x 10 cfu / ml, respectively. The chili seeds were immersed in sterile water and used as a control group (Control 1). Meanwhile, the chili seeds were treated with a means of administration (1000 seeds per 4g D.M.

P14? 9 / 99MX 22) that did not contain WYE 20 or WYE 324 and was used as the other control group (Control 2). The sowing of the chili seeds was carried out in the same way as that of the cucumber seedlings, indicated above. Disposable paper cups (9cm in diameter) were used as the pots for the seedlings. One hundred and twenty-one cups containing one seed per cup were prepared in each test. The experiments were carried out in a greenhouse at a temperature of 25 ° C to 32 ° C in daylight. In the greenhouse, the pots were placed in a random block arrangement. Humidity was maintained in a range of 40 to 60% and more water was sprayed on top of the pots, as required. Fifteen ml of the cell culture broth of WYE 20 or WYE 324 obtained in the section "materials and methods", were inoculated in the pot of the seedlings treated with the biocontrol agent after 4 weeks of culture. The same amount of water was applied to the control groups. The experiments continued for 9 weeks and the results are shown in Table XVII.

P1459 / 99MX TABLE XVII * It means that in a column followed by the same letter there is no significant difference at the level P = 0.05.

As shown in Table XVII, there was a significant improvement in plant growth for chili seeds treated with WYE 20 or WYE 324 compared to those plants germinated from control seeds. This indicates that WYE 20 or WYE 324 of the present invention is quite effective in improving the growth of chili plants. (7) Biocontrol Assay Using Chilean Seedlings in Agricultural Fields A biocontrol assay was conducted to determine the efficacy of a biocontrol agent of the present invention to control blight by Phytophthora and to improve the growth of plants in fields agricultural.

P14S9 / 99MX The chili seeds were bathed in sterilized water for 2 days and immersed in a biocontrol agent prepared in Example VI for 3 hours. The seeds were treated with a biocontrol agent (D.M.22) (1,000 seeds per 4g) prepared in Example V. The number of Streptomyces sp. WYE 20 and WYE 324 before treatment of the seeds were adjusted to 1.2 x 10 cfu / ml and 1.7 x 107 cfu / ml, respectively. The chili seeds were immersed in sterilized water for 2 days and three hours, once again. Therefore, chili seeds were treated with the same administration medium that did not contain WYE 20 or WYE 324 and was used as a control group. Each seed was planted in seedling beds. Water was sprayed as required and the temperature was maintained between 20 ° C and 35 ° C. When the seedlings grew to 1.5 to 2. Ocm in height, the chili seedlings were transplanted to a seedling tray consisting of 25 seedling holes (5cm x 5cm, 6cm deep) containing a mixture of potting soil, sandy , fine and WYE 20 or WYE 324 in the administration medium (O.lg per orifice for the seedling). The control chili seedlings were transplanted to the holes that contained the same amount of soil for sandy pots and the same amount of administration medium only. These P1459 / 99MX seedling pots were incubated in a greenhouse at a temperature of 18 ° C to 35 ° C and more water was supplied as required. In the greenhouse, the pots were placed in a random block arrangement. After 11 weeks of growth in the greenhouse, each plant was transplanted to the agricultural field. Before one week after transplantation, lOml of culture broth WYE 20 or WYE 324 (1.2-1.7 x 103 cfu / ml) were added per seedling orifice in the treatment group. The same amount of water was added to the control seedling pots. The incidence of the disease and the growth of the transplanted chilies was periodically observed and the average was recorded as shown in Table XVIII (Field 1) and XIX (Field 2).

P1459 / 99MX • or H TABLE XVIII (FIELD 1) 3 X OR? OR * It means that in a column followed by the same letter there is no significant difference at the level P = 0.05.

* It means that in a column followed by the same letter there is no significant difference at the level P = 0.05.

As shown in tables XVIII and XIX there was a significant improvement in the growth and reduction of blast with Phytophthora in plants from chili seeds treated with WYE 20 or WYE 324 and compared with plants germinated from control seeds . This indicates that WYE 20 and WYE 324 of the present invention are very effective in controlling blight by Phytophthora and improving the growth of the chili plant in agricultural fields. Having provided the examples of the embodiments of the present invention and the preferred embodiments, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the present invention in its broader aspects. Therefore, the P1459 / 99MX various variations, modifications and modalities should be considered within the true spirit and scope of the present invention.

REFERENCES ATCC Catalog of Bacteria and Bacteriophages, 17 edition, 1989. American Type Culture Collection, Rockville, MD. Crawford, D.L., J.M. Lynch, J.M. Whippsw, and M.A. Ousley, 1993. Isolation and characterizat ion of actinomycete antagonists of a fungal root pathogen. Appl. Environ, Microbiol, 59: 3899-3905. Hsu, S.C., and J.L. Lockwood, 1975. Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Applied Microbiogy, pp. 422-426. Kannwischer, M.E., and Mitchell, D.J. 1978. The influence of a fungicide on the epidemiology of black shank of tobáceo. Phytopathology 68: 1760-1765. Locci, R. 1989, Streptomycetes and Related Genera, In Bergeys' Manual of Systematic Bacteriology, William and Wilkens, Baltimore, Md. 4.-2451-2492. Pridham, T.G., and D. Gottlieb, 1948. The utilization of carbon compounds by some actinomysetales as an aid for species determination. J. Bacteriol, 56: 107-114. P1459 / 99MX Reddi, G.S., and A.S. Rao, 1971. Antagonism of soil actinomycetes to some soil borne plant pathogenic fungi. Indian Phytopathol, 24; 649-657. Stanghellini, M.E., and J.G. Hancock, 1970. A quantitative method for the isolation of Pythi um ul timum from soil. Phytopathology, 60: 551-552. Stasz, T.E., G.E. Harman and G.A. Marx. 1980. Time and site of resistant and susceptible germinating by seeds by Pythi um ul timum, Phytopatholoby, 70: 730-733. Suh, H.W. 1992. Production of antifungal compounds by Pi sol i thus tinctori us SMF and Stremptomyces sp. WYEC 108, and their role in biological control. Ph. D. Dissertation University of Idaho, Moscow, Idaho, U.S. A. William et al. 1983. A probability matrix for identification of Streptomyces. J. Gen. Microbiol. 129: 1815-1830. William et al. 1983. Numerical classification of Streptomyces and Related Genera, J. Gen. Microbiol, 129: 1743-1813.

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Kobayashi, et al., "Pironetin, a novel plant growth regulator from Strep tomyces. sp. , "Abstract S2-2, p34, BMP Japan 95, April 23-26, Oiso, Kanagawa, Japan (1995), Lahdenpera et al.," Mycostop-A novel biofungicide based on S trep tomyces bacteria, "published prior to 1991 Leong, "Siderophores: their biochemistry and possible role in the biocontrol plant pathogens," P1459 / 99MX Annu. Rev. Phytopathol, 24: 187-209 (1986). Liljeroth et al., "Assimilate translocation to the Rhizosphere of two wheat lines and subsequent utilization by rhizosphere microorganisms at two soil nitrogen concentrations," Soil Biol. Biochem. 22: 1015-1021 (1990). Marcos et al., "Effect of compost on rhizosphere microflora of the tomato and on the incidence of plant growth-promoting rhizobacteria (PGPR)". Appl. Environ. Microbiol. 61: 194-199 (1995). Merriman et al., "Effect of Bacillus and Streptomyces spp. Applied to seed," in E. Bruehl (ed.), Biology and control of soilborne plant pathogens, pp. 130-133 (1977). Meyer and Linderman, "Selective influence on population of rhizosphere or rhizoplane bacteria and actinomycetes by mycorrhizas formed by Glomus fasciula tum," Soil Biol. Biochem. 18: 191-196 (1986). Miller et al., "Variation and composition of bacterial population in the rhizospheres of maize, wheat, and grass cultivars," Can. J. Microbiol. 35: 656-660 (1989). Miller et al., "Fluctuations in the fluorescent pseudomonas and actinomycete populations of rhizosphere and rhizoplane during the growth of spring wheat," Can. J. Microbiol. 36: 254-258 1989). Miller et al., "The dynamics of actinomycetes and fluorescent Pseudomonads in wheat P1459 / 99MX rhizoplane and rhizosphere, "Symbiosis 9: 389-391 (1982). Panosyan et al., "The nature of physiologically active substances of actinomycetes and the effect of their metabolites on plant growth," Plant Microbe Relationships, pp. 241-245 (1965). Scrinivansan et al., "Physiology and nutritional aspects of actinomycetes"; an overview, "World Journal of Microbiology and Biotechnology 7: 171-184 (1991), Singh and Mehrota," Biological control of Rhi zoctoni a ba ta ti col a on gram by coating seed with Bacillus and Streptomyces spp. and their influence on plant growth, "Plant and Solil 56: 475-483 (1980) Stevenson," Antibiotic activity of actinomycetes in soil as demonstrated by direct observation techniques, "J. Gen. Microbiol, 15: 372-380 (1956 Sutherland and Papavizas, "Evaluation of oospore hyperparasites for the control of Phytophthora crown rot of pepper," J. Phytopathology 131: 33-39 (1991), Tahvonen, "Preliminary experiments on the use of Strep tomyces spp. isolated from pedestrian in the biological control of soil and seed-borne diseases in pedestrian culture, "Journal de la Cientific Agricultural Society of Finland 54: 357-369 (1982). Tahvonen, "Mycostop-ettbiologiski P1459 / 99MX bekampningsmedel mot svampsj ukdomar ", (Mycostop, biological formulation for control of fungal diseases), Vaxtskyddsnotiser 49: 86-90 .English summary only (1985). Tahvonen and Avikainen," The biological control of seedborne Atrenaria Brassicicola of cruciferous plants with a powdery preparation of Streptomyces sp. , "Journal of Agricultural Science in Finland 59: 199-207 (1987) .You," Hyperparasitism of Streptomyces albus on a destructive mycoparasite nectria inven. "J. Phytopathology 117: 71-76 (1986) .Turhan," A new race of Streptomyces ochracei scl ero ti cus in the biological control of some soilborne plant pathogens, "Journal of Plant Diseases and Protection 88: 422-434 (1981) Turhan and Turhan," Suppression of damping-off pepper produced by Pythi um ul timum Trow and Rhi zoctone solani Kuhn by some new antagonist in comparison with Tri choderma harzianum Rifai. "J. Phygopathology 126: 175-182 (1989)." Zuberer et al., "Populations of bacteria and actinomycetes associated with sclerotia of Phyma to tri chum omni vorum buried in Houston Black clay." Plant and Soil 112: 69-76 ( 1988) Warren et al., "Rheologies and morphologies of three actinomycetes in submerged culture," Biotechnology and antibiotic production of P14S9 / 99MX Streptomyces coeli color grown on solid medium, "Biotechnology Letter 16: 1015-1020 (1994) Fukuchi et al.," Rotihibins, novel plant growth regulators from Streptomyces gramino faci ens, "The Journal of Antibiotics 48: 1004- 1010 (1995), Liu et al., "Biological control of potato scab in the field with antagonistic Streptomyces scabi," Phytopathology 85: 827-831 (1995), Bowers et al., "Influence of disease-suppressive strains of Streptomyces. on the native Streptomyces community in soil as determined by de analysis of cellular fatty acids, "Can. J. Microbiol. 42 -. 42 -. 21-31 (1996). Bayer and Diekmann, "The chitinase system of Streptomyces sp. ATCC 11238 and its significance for fungal cell wall degradation," Appl. Microbiol. Biotechnol. 23: 140-146 (1985). Lorito et al., "Synergistic interaction between fungal cell wall degradation enzymes and different antifungal compounds enhances inhibition of spore germination.," Microbiology 140: 623-629 (1994). Mahadevan and Crawford, "Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydi cus WYEC 108," Enzyme Microbial Technol. (nineteen ninety six) . In press. Mohamed, "Physiological and antagonistic activities of Streptomycetes in rhizosphere of some P1459 / 99MX plants, "Egypt, J. Phytopathol, 14: 121-128 (1982), Sardi et al.," Isolation of endophytic Streptomyces strains from surface-sterilized roots, "Appl. Environ Microbiol. 58: 2961-2963. (1992) Sneh et al., "Parasitism of oospores of Phytophthora megasperma var sojae P. cactorum, Pythi um sp. , and Aphanomyces eutei ches in soil by oomycetes, chytriodiomycetes, hyphomycetes, actinomycetes, and bacteria. "Phytopatology 67: 622-628 (1977), Yuan and Crawford," Characterization of Streptomyces lydi cus WYEC 108 as a potential biocontrol agent against fungal root and seed rots, "Appl. Environ. Microbiol. 61: 3119-3128 (1995)." Fravel, "Role of antibiosis in the biocontrol of plant disease," Annu., Rev. Phytopathol, 26: 75-91 (1998). , "Actinomycetes in agriculture anf forestry," P.327-358, In M. Goodfellow, ST Wiliams, and M. Mordarski (ed.) Actinomycetes in biotechology, Academic Press, Ney York (1989), Merriman et al., " The effect of inoculation of seed antagonists of Rhi zoctonia solani on the growth of wheat, "Austr. J. agr. 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P1459 / 99MX

Claims (13)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A biologically pure culture of a microorganism of Streptomyces sp. WYE 20 and Streptomyces sp. WYE 324, which has the identification characteristics of KCTC 0341BP and KCTC 0342BP, respectively.
2. 2. Antifungal biocontrol agent that includes a biologically pure microorganism of Streptomyces sp. WYE 20 and WYE 324.
3. 3. The microorganism according to claims 1 and 2, wherein the microorganism is capable of protecting plants susceptible against fungal infection.
4. 4. The microorganism according to claim 3, wherein the protection is conferred against fungal pathogens that cause diseases in seedlings, which include rot of cuttings and root rot as well as foliar diseases including powdery cucumber powdery mildew, blight by Phytophthora in chile or brownish / large parts by Rhizoctonia in lawns.
5. 5. Antifungal biocontrol agent according to claim 2, wherein the biocontrol agent P1459 / 99 X comprises a biologically pure culture that includes a biologically pure microorganism of Strep tomyces sp. WYE 20 or WYE 324 and a means of administration.
6. 6. Antifungal biocontrol agent according to claim 5 wherein the administration means consists of 40 to 67% w / w of wheat bran, 1 to 5% of chitosan, 30 to 55% w / w of wood sawdust, 1 to 3 percent w / w of chitin and 1 to 3% w / w of Pharmamedia based on the total weight of the administration medium.
7. 7. Antifungal biocontrol agent according to claim 6 wherein the administration means further comprises 0.2 to 3.5% w / w of sporulation medium.
8. 8. The antifungal biocontrol agent according to claim 7, wherein the biologically pure culture of Strep tomyces sp. WYE 20 or WYE 324 comprises 10 -10 colony forming units per gram of administration medium.
9. 9. Antifungal biocontrol agent according to claim 5 wherein the administration medium consists of 1.0 to 3.0% w / w of pectin and 0.1 to 0.6% w / w of colloidal chitin in water.
10. 10. The antifungal biocontrol agent according to claim 9, wherein the biologically pure culture of Strep tomyces sp. WYE 20 or WYE 324 comprises 10 -10 colony forming units per gram of P1459 / 99MX administration medium.
11. 11. A method for making an antifungal biocontrol agent comprising an incubation process for the production of a biologically pure culture of Streptomyces sp. WYE 20 or WYE 324; and a downstream process.
12. 12. The incubation process according to claim 11, wherein the process comprises incubating 130 to 300 rpm at 25 ° C-33 ° C for 3 to 7 days. The downstream process according to claim 11, wherein the process comprises freeze-drying the pure culture harvested from Streptomyces WYE 20 or WYE 324.
13. 13. The downstream process according to claim 11, wherein the process comprises the incorporation of culture. pure collected to a suitable administration medium. 15. A method for the preparation of an antifungal biocontrol agent wherein the method comprises the steps of: preparing the administration medium consisting of 40 to 65% w / w of wheat bran, 1 to 5% w / w of chitosan, 30 to 55% w / w of wood sawdust, 1 to 3% w / w of chitin and 1 to 3% w / w of Pharmamedia based on the total weight of the administration medium; autoclave the medium of P1459 / 99MX resulting administration; incorporate the Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp WYE 324 (KCTC 0342 BP) in the administration medium; incubate the incorporated cells of Streptomyces sp. WYE 20 (KCTC 0341BP) or Streptomyces sp WYE 324 (KCTC 0342 BP) at 25 ° C 33 ° C for 5 to 14 days; and aseptically drying the resulting product. 16. A method for making an antifungal biocontrol agent according to claim 15, wherein the method comprises aseptically mixing the resulting dried product. 17. The preparation of the administration medium according to claim 15, wherein the preparation comprises pelletizing the administration medium; and coating the resulting pellet with 0.2 to 3.5% w / w of sporulation medium 18. The incorporation of the claim 15, where Streptomyces sp. WYE 20 or WYE 324 comprises 10 -101 colony forming units per gram of administration medium. 19. The process of autoclaving according to claim 15, wherein the autoclaving process is carried out at 121 ° C for 30 to 40 minutes. 20. A method for the development of agent P1459 / 99MX of antifungal biocontrol wherein the method comprises the steps of: preparation of the administration medium consisting of 1.0 to 3.0% w / w of pectin, 0.1 to 0.6% w / w of colloidal chitin and the rest is water, and autoclaving the resulting administration medium; and incorporate Streptomyces sp WYE 20 (KCTC 0341BP) or Streptomyces sp WYE 324 (KCTC 0342BP) into the administration medium. The incorporation according to claim 20, wherein Streptomyces sp WYE 20 or WYE 324 comprises 10 -10 colony forming units per gram of administration medium. 22. A method for using antifungal biocontrol agent according to claim 2 to claim 10, wherein the use comprises coating, mixing, spraying or applying in the furrow, to the seeds of plants, mixtures of pots, growing plants or soil. . P1459 / 99MX