A MICROBIAL PESTICIDE ACTIVE AGAINST PLANT FUNGAL PATHOGENS AND PROCESS FOR PREPARATION THEREOF

Abstract: The present invention relates to a method for isolating a new microbial strain, belonging to Trichoderma spp., which is antagonistic to pathogenic bacteria of plant and naming the said strain as Trichoderma harzianum YC459(KCTC 0772BP), and for manufacturing a microbial pesticide for controlling properly plant diseases by using the above strain. This invention is greatly effective on controlling gray mold rot, damping-off, leaf mold and early blight as treating leaf of main crops or soil with the spore suspension of the said strain.

Description: A MICROBIAL PESTICIDE ACTIVE AGAINST PLANT FUNGAL PATHOGENS AND PROCESS FOR PREPARATION THEREOF TECHNICAL FIELD The present invention relates, in general, to a novel Trichoderma species highly antagonistic to plant fungal pathogens, isolated from soil and, more particularly, to a biological fungicide highly effective against plant fungal pathogens, which is not harmful to natural ecosystems and free of mammalian toxicity Also, the present invention is concerned with a method for preparing the biological fungicide BACKGROUND ART Together with noxious insects and weeds, plant diseases are the main factors that threaten the stable production of crops Plants are taken ill by various pathogens Representative of them are fungi Most of the pathogens which form sclerotia, such as Botrytis cinerea, and Rhizoctonia solani, reside in the soil, causing a variety of diseases in a broad spectrum of crops The fungal pathogens pass the winter as survival structures of sclerotia In the next year, sclerotia act as primary infection sources They survive from year to year in the soil or plant tissues even under severe conditions Because sclerotia are more resistant to the activity of antagonistic microorganisms than are spores and mycelia, the survival structures are highly difficult to eradicate Cladosporium fulvum, which is a kind of leaf mold, and Alternaria solani, which causes early blight, exert their pathogenic activity in various vegetables At present, various germicides are available, but their misuse and abuse bring about an increase in the drug resistance of pathogens, resulting in a significant decrease in germ control Accordingly, there remains a need for a sterilizing method that is safe and effective Biological control methods taking advantage of microbial agents are environmentally safe and effective Since the early 1970s, active and extensive research has been directed to biological control methods all over the world Recently, ten or more microbial germicides have been made commercially available Further, since environmentally safe sustainable agriculture is now recognized as preferable, microbial agents have been intensively studied Representative examples of the microbial agents that have been commercially available include BINAB T, prepared from mixtures of Trichoderma harziaum and T polysporum, for use in the control of internal decay of wood utility poles (1988, E R Butts International), GlioGard, derived from Gliocladium virens, for use in the control of seedling disease of ornamental and bedding plants (1990, W R Grace Co ), F-stop (1991, Kodak) and Trichodex (1997, developed in Israel), both derived from T harziaum, KODIAK, a seed treatment derived from Bacillus subtilis (1990, Gustafson), DAGGER, derived from Pseudomanas fluorescens, for use in the control of damping-off caused by Rhizoctonia and Pythium (1990, Ecogen Inc ), BLUE CIRCLE, a seed treatment agent for various crops, derived from P cepacia (1990, Stine Microbial Products), and MYCOSTOP, useful for controlling Fusarium-caused wilt disease and damping-off, derived from the antagonistic fungus Streptomyces griseviridis (1985, Kemira Oy Bicontrol of Finland) Of them, microbial agents using Trichoderma harzianum were found to be excellent in control effect and product safety and, in many countries, much effort has been made to isolate locally adapted strains and develop them into microbial products T harzianum kills pathogens by perforating and infiltrating their mycelia when in physical contact with them In this regard, the biofungicidal microorganism secrete chitinase and β-l,3-glucanase Purified enzymes from T harzianum were found to be inhibitory of various plant pathogens, including R solani and B cinerea In addition, Trichoma viride, Gliocladium roseum, and Penicillium sp are reported to have fungicial activity against B cinerea and R solani as demonstrated in greenhouse and field tests In order to increase the persistent activity of T harizianum in various natural environments, a substrate that the microbe can characteristically utilize is needed, or there must be provided conditions necessary for the antagonistic microorganism to predominate in a substrate, lest other microorganisms or target pathogens predate it Upon mass production and formulation of antagonistic microorganisms, another consideration for selecting culture media is economy Conventional microbial agents taking advantage of antagonistic microbes are widely used for the control of plant diseases caused by fungi, nematodes, and viruses, and for the protection of the plants from vermin and weeds According to literature, the mechanisms of biological control that antagonistic microorganisms exert on their corresponding targets are elucidated by the antibiotic functions of the antibiotic materials that they secrete, the parasitic and phagocytic functions of cell wall decomposing enzymes, the decrease in the population or pathogenicity of the target organisms through the competition for nutrients and niches, and the cross protection and resistance induction in host cells by use of non-pathogens or attenuated pathogens For example, Trichoderma sp and Gliocladium sp are used for the control of fungal diseases by taking advantage of their secretions, such as antibiotic materials, volatile materials, toxins, and special inhibitory materials Recently, intensive and active attention has been paid to the antagonistic functions based on parasitic and phagocytic functions Antagonistic microbes are known to secrete chitinase, glucanase and cellulase, all being cell wall decomposing enzymes which can act to cause the lysis of noxious pathogens DISCLOSURE OF THE INVENTION Leading to the present invention, the intensive and thorough research on the control of crop diseases without concomitant environmental destruction, conducted by the present inventors, resulted in the finding that a novel fungal species isolated from the soil is highly antagonistic to plant pathogens and its mycelia or spores can be formulated into environmentally friendly biological fungicides for use in the control of various plant diseases It is therefore an object of the present invention to provide a novel fungal species antagonistic to pathogenic fungi fatal to crops It is another object of the present invention to provide a biological fungicide for use in the control of crop diseases, which is highly potent and shows no environmental problems or toxicity to higher species It is a further object of the present invention to provide a method for preparing such a biological fungicide In accordance with an aspect of the present invention, there is provided a fungal species Trichoderma harzianum YC459 (accession No KCTC 0772BP) and its mutants, antagonistic to plant pathogens In accordance with another aspect of the present invention, there is provided a biological fungicide for controlling plant pathogens, comprising the mycelia or spores of the fungus of claim 1, or a mixture thereof, as a fungicidally active ingredient In accordance with a further aspect of the present invention, there is provided a method of preparing a biological fungicide for controlling plant pathogens, in which mycelia or spores of the fungus species of claim 1 or a mixture thereof is formulated with an ordinary carrier BEST MODES FOR CARRYING OUT THE INVENTION In the present invention, a novel species antagonistic pathogenic fungi is isolated from the soil Soil samples are taken from different regions, and dilutions are made in water To grow fungal species, the dilutions were spread over potato-dextrose agar (PDA) plates containing streptomycin, followed by incubation at 28 °C Five days are required for the formation of single colonies on the plates The single colonies are aseptically transferred to PDA slant media and stored at 4 °C The fungal colonies are assayed for antagonistic activity against pathogenic fungal species, including B cinerea, R solani, C fulvum and A solani by a substitution culture method An observation is made of mycelium lysis at the contact interface between the pathogens and the putative antagonistic species under a microscope to select a fungal species which is the most inhibitory of the pathogens This fungal species was identified as being a novel mutant of Trichoderma harzianum, named T harzianum YC459, and deposited in the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) with the accession number KCTC 0772BP on April 29, 2000 It is necessary to quantitatively evaluate the inhibitory activity of the novel fungal species against pathogens Spores are collected from cultures of the fugal species and suspended in water Dilutions are made on the suspension to control the spore population Then, the controlled spore suspension is applied to plants for assaying the control of the diseases caused by the pathogens In the case of the gray mold rot caused by B cinerea, the spore suspension of the novel fungal species is sprayed over cucumber leaves After 1 day, a spore suspension of the pathogenic fungus is inoculated into the leaves The pots of the cucumber seedlings inoculated with the pathogen are placed in a moist chamber maintained at 20 °C for three days, followed by quantitative analysis of disease occurrence as a occurrence percentage of appearing spots The suppressive activity of the antagonistic microbe of the present invention against damping-off is assayed on radish In this regard, a medium comprising potato slices and farm soil mixed with sterile water is prepared for culturing R solani, a pathogen causative of damping-off The medium in which the pathogen has been cultured is dried in the shade, powdered, and stored at 4 °C before use as an inoculum A mixture of the pathogen inoculum and ordinary soil is placed, along with different amounts of the antagonistic microbe suspension, in plastic pots which are then sawed with four radish seeds After one week of cultivation in a greenhouse, quantitative analysis is made of the disease occurrence in the radish Greenhouse and field tests of the fungicidal activity of the antagonistic microbe against various pathogenic fungi are conduced For this, tomatoes are cultivated both in a greenhouse and a field The tomatoes cultivated in a greenhouse and a field are treated with the spore suspension of the novel antagonistic microbe of the present invention and then exposed to pathogenic fungi, B cinerea causing gray mold rot, C fulvum causing leaf mold, and A solani causing early blight, separately The experiment is performed according to a randomized block design with three repeat measures For comparison, procymidone for use in the control of gray mold rot, propineb for use in the control of leaf mold, and azoxystrobin for use in the control of early blight are employed Morbidity is determined by examining the infection of all tomatoes one by one or by a sampling method A significance test is carried out according to Duncan's multiple range test (DMRT) Also, the present invention pertains to the formulation of the antagonistic microbe of the present invention The antagonistic microbe T harzianum YC459 is inoculated to PDA and cultured at 28 °C for 7 days to germinate spores which is then used to prepare spore suspensions for use as seed inoculums in mass culture Wheat chaff, sawdust, rice bran and chaff are mixed in combinations, and yeast extract is added at an amount of 0 5 % by volume to each combination The mixtures are added with water and autoclaved at 121 °C for 1 hour Each of the sterilized media is aseptically mixed with 200 ml of the spore suspension, followed by culturing the microbe at 28 °C for 7 days Biomasses of the antagonistic microbe are dried for three days at room temperature in the shade, powdered, and passed through a sieve with a hole size of 1 mm The mycelium and spore powder is formulated with a bulking agent, such as peat moss, vermiculite, talc, zeolite, or kaolinite For formulation, any of the excipients that are generally used for microbial agents may be selected A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention EXAMPLE 1 Identification of Antagonistic Microorganism and Assay for Antagonistic Activity EXPERIMENTAL EXAMPLE 1-1 Isolation of Antagonistic Microorganism and Its Antagonistic Activity (in vitro Assay) In order to isolate microorganisms antagonistic to B cinerea, soils and barnyard manure were sampled in five different regions in Korea 5 g of each of the samples was suspended in 45 ml of sterile water After being agitated at 28 °C for 30 min, the suspension was diluted by a factor of up to 104 in a soil dilution agar plate method 0 1 mL-portions of the dilution were withdrawn and evenly spread over potato-dextrose agar (PDA) media (Difco) with 100 ppm of streptomycin, followed by incubation at 28 °C for 5 days Single colonies of fungi grown on the agar plates were isolated and transferred to PDA slant media which were then stored at 4 °C The isolated fungi were assayed for antagonistic activity against B cinerea, R solani, C fulvum and A solani by a substitution culture method in which the mycelium lysis at the contact face between the pathogens and the putative antagonistic species was observed under a microscope to select a fungal species which was the most inhibitory of the pathogens This fungal species was identified to belong to Trichoderma harzianum, named T harzianum YC459, and deposited in the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) with the accession number KCTC 0772BP on Apri 29, 2000 EXPERIMENTAL EXAMPLE 1-2 Assay of Antagonistic Microbe for Inhibitory Activity To evaluate in vivo inhibitory activity of the antagonistic species T harzianum YC459, isolated in Experimental Example 1, against pathogens, the novel microbe was applied to leaves 1 day before the inoculation of pathogens Then, the antagonistic effect was monitored In this regard, the isolated antagonistic microbe was cultured on PDA medium at 28 °C for 10 days in an incubator, after which spores thus formed were collected by scraping with addition of 4-5 ml of sterile water The spore suspensions thus obtained were controlled to have a spore density of 106 or 107/ml, on average, and sprayed over cucumber leaves in the two-leaf stage, in such a sufficient amount as to flow down After the leaves were let to stand at room temperature for 1 day, a spore suspension with a spore density of 106 or 107/ml of B cinerea, a pathogen causing gray mold rot, was inoculated into the leaves in such a sufficient amount as to flow down The pots of the cucumber seedlings inoculated with the pathogen were placed for three days in a moist chamber maintained at 20 °C, followed by quantitatively measuring disease occurrence as an occurrence percentage of appearing spots The pathogen used in this experiment was the strain JM42 that had been stored in the laboratory since its isolation from diseased tomato leaves in 1994 The cucumber seedlings planted in pots were cultured at 20 °C for 14 days in an incubator and spores formed on the surface of the incubator were collected in the same manner as above to prepare spore suspensions The inhibitory activity of the antagonistic microbe against the disease caused by the pathogen was calculated according to the following equation Attack rate of treated group Control Value (%) = [ 1 - (- ) ]xl00 Attack rate of untreated group All experiments were carried out with 4 pots in each of which 2 stalks of cucumber were planted A significance test was performed according to Duncan's multiple range test (DMRT) The experiment results are given in Table 1, below TABLE 1 Inhibitory Activity of T harzianum YC459 Against the Occurrence of Gray Mold Rot on Cucumber Note *the same letters mean no significant differences at p=0 05 (DMRT) Almost all members of the group not treated with the antagonistic microbe of the present invention died, showing a morbidity of 97 % On the other hand, when applying the antagonistic microbe, the attack rate was decreased with increasing of the spore density of the suspension The spore suspension of the antagonistic microbe exhibited the greatest inhibitory activity with a control value of 89 8 % at a spore density of 107/ml EXAMPLE 2 Mass Culture and Formulation of the Antagonistic Microbe The antagonistic microbe T harzianum YC459, isolated in Example 1, was inoculated to PDA and cultured at 28 °C for 7 days to germinate spores which were then used to prepare spore suspensions for use as seed inoculums in mass culture, in the same manner as in Experimental Example 2 Wheat chaff, sawdust, rice bran and chaff were mixed in combinations of wheat chaff plus chaff, sawdust plus chaff, rice bran plus chaff, and rice bran plus sawdust, at the same amount, and yeast extract was added at an amount of 0 5 % by volume to each combination The mixtures were individually added with water to a water content of 40 % and autoclaved at 121 °C for 1 hour in 5 L autoclavable vinyl packs, Erlenmeyer flasks, or sealable vessels Each of the sterilized media was aseptically mixed with 200 ml of the spore suspension, and incubated at 28 °C for 7 days The media were agitated once every other day during the incubation so as to induce uniform growth of mycelia and germinate spores in large quantities Biomasses of the antagonistic microbe were dried for three days at room temperature in the shade, powdered, and passed through a sieve with a hole size of 1 mm The mycelium and spore powder was formulated with a bulking agent, such as peat moss, vermiculite, talc, zeolite, or kaolinite For formulation, any of the excipients that are generally used for microbial agents may be selected EXAMPLE 3 Suppressive Activity of the Antagonistic Microbe Against Occurrence of Damping-Off Using radish, the antagonistic microbe was assayed for suppressive activity against damping-off 50 g of potato slices and 450 g of farm soil were mixed along with 100 ml of sterile water in a 2L Elernmeyer flask, followed by autoclaving the medium at 121 °C for 1 hour 20 fragments of the PDA on which R solani was cultured for 5 days were inoculated to the medium, which was then incubated at 28 °C for 14 days The medium was dried in the shade, powdered, and stored at 4 °C until use as an inoculum 0 5 g of the pathogen inoculum was well mixed with 1L of ordinary soil, along with different amounts of the antagonistic microbe suspension prepared in the same manner as in Experimental Example 2 The mixtures were placed in four plastic pots with a dimension of 7x7 cm, each of which was sown with four radish seeds, followed by cultivation for one week in a greenhouse After one week of the cultivation, severity of disease was discriminated according to four levels 1 sound, 2 light brown lesion, 3 died of the disease after germination, and 4 died of the disease before germination, and morbidity was calculated as a percentage Control values of the antagonistic microbe were calculated according to the equation given above The results are given in Table 2, below TABLE 2 Suppressive Effect of T harzianum YC459 on Damping-Off Note *the same letters mean no significant differences at p=0 05 (DMRT) Attack rate of treated group Control Value (%) = [1 • ) ]xl00 Attack rate of untreated group EXAMPLE 4 Greenhouse and Field Tests of Fungicidal Activity of the Antagonistic Microbe EXPERIMENTAL EXAMPLE 4-1 Inhibitory Activity of the Antagonistic Microbe Against Tomato Gray Mold Rot To determine the fungicidal activity of the antagonistic microbe of the present invention against B cinerea causing gray mold rot, tomato plants were cultivated in a greenhouse with a dimension of 23 7 m in length, 7 0 m in width and 2 3 m in height The cultivation was performed according to the following schedule. 1999, Sep 30 Seeding tomato Oct 10 1 st Temporary planting Oct 22 2nd Temporary planting Nov 2 Planting in two rows at intervals of 20 cm Dec 22 Pollination 2000, Feb 11 1st Treatment with the antagonistic microbe (spray amount 300 L/lOa) Feb 20 2nd Treatment with the antagonistic microbe (spray amount 300 L/lOa) Mar 2 3rd Treatment with the antagonistic microbe (spray amount 330 L/lOa) Mar 22 Measuring mobidity The tomato plants treated with the antagonistic microbe occupied an area of 5 4 m2 and the experiment was performed in triplicate in a randomized block design For comparison, Procymidone was sprayed as a control at a usual concentration The spore suspension of the antagonistic microbe prepared in Example 2 was added directly to water to bring about 500- and 1,000-fold dilutions of the population Morbidity was determined by examining the infection of all tomato plants one by one A significance test was performed according to Duncan's multiple range test (DMRT) Given in the following Table 3 are suppressive effects of the antagonistic microbe of the present invention on the occurrence of gray mold rot in tomatoes As seen in Table 3, the untreated group showed a morbidity of 17 4 % on average 500- and 1,000-fold dilutions of the antagonistic microbe suspension decreased the morbidity to 5 4 % and 6 6 %, respectively Upon treatment with Procymidone, the morbidity remained at a level of 10 4 % on average As for control value, it was measured to be 65 6 %, on average, for the antagonistic microbe-treated group, which was significantly higher than that of the untreated or Procymidone-treated group TABLE 3 Suppressive Effect of T harzianum YC459 on Gray Mold Rot in Tomato Morbidity (%) Dilution fold Avg Control value(%) Note * the same letters mean no significant differences at p=0 05 (DMRT) , Attack rate of treated group Control Value (%) .1 - ( —) ]xl00 Attack rate of untreated group EXPERIMENTAL EXAMPLE 4-2 Inhibitory Activity of the Antagonistic Microbe Against Leaf Mold To determine the fungicidal activity of the antagonistic microbe of the present invention against C fulvum causing leaf mold, tomato plants were cultivated in the same manner as in Experimental Example 4-1 in a greenhouse with a dimension of 23 7 m in length, 7 0 m in width and 2 3 m in height The experiment was performed in triplicate in a randomized block design For comparison, propineb was sprayed as a control at a usual concentration Morbidity was determined by examining the infection of 20 stalks of tomatoes per repeat measure and attack frequency was calculated according to the following equation ∑(Infected Stock No. x Factor) Attack frequency (%) -x 100 4 x Total No. of stocks examined. wherein the factor is set to be 0 when no leaves are infected, 1 when one forth of the total leaves are infected, 2 when half of the total leaves are infected and some of them are withered, 3 when three fourths of the total leaves are infected and half of them are withered, and 4 when two thirds are withered to death Given in the following Table 4 are suppressive effects of the antagonistic microbe of the present invention on the occurrence of leaf mold in tomatoes As seen in Table 4, the untreated group showed a morbidity of 21 9 % on average 500- and 1,000-fold dilutions of the antagonistic microbe suspension decreased the morbidity to 6 6 % and 6 8 %, respectively Upon treatment with propineb, the morbidity remained at a level of as high as 21 9 % on average As for control value, it was measured to range from 69 to 70 %, on average, for the antagonistic microbe-treated group, which was significantly higher than that of the untreated or propmeb-treated group TABLE 4 Suppressive Effect of T harzianum YC459 on Leaf Mold in Tomato Note *the same letters mean no significant differences at p=0 05 (DMRT) Attack rate of treated group Control Value (%) = [ 1 - (- ■ ) ]xl00 Attack rate of untreated group ' EXPERIMENTAL EXAMPLE 4-3 Inhibitory Activity of the Antagonistic Microbe Against Tomato Gray Mold Rot To determine the fungicidal activity of the antagonistic microbe of the present invention against A solani causing early blight, tomato was cultivated in a field The cultivation was performed in the following schedule, 2000, May 4 Seeding potato Jun 10 1st Treatment with the antagonistic microbe (spray amount 300 L/lOa) Jul 5 2nd Treatment with the antagonistic microbe (spray amount 300 L/lOa) Jul 13 Measuring mobidity The experiment was performed in a randomized block design with three repeat measures For comparison, azoxystrobin was sprayed as a control at a usual concentration Morbidity was determined by examining the infection of 20 stocks of potatoes per repeat measure and attack frequency was calculated as in Experimental Example 4-2 Given in the following Table 5 are suppressive effects of the antagonistic microbe of the present invention on the occurrence of early blight in potatoes As seen in Table 5, the untreated group showed a morbidity of 21 7 % on average 500- and 1,000-fold dilutions of the antagonistic microbe suspension reduced the morbidity to as low as 9 2 % and 9 5 %, respectively Upon treatment with azoxystrobin, the morbidity was also reduced to 9 2 % on average As for control value, it was measured to range 56 2 to 57 6 %, on average, for the antagonistic microbe-treated group, which was significantly higher than that of the untreated group TABLE 5 Suppressive Effect of T harzianum YC459 on Early Blight in Potato Note *the same letters mean no significant differences at p=0 05 (DMRT) , Attack rate of treated group ,, _ Λ „ Control Value ( % ) = [ 1 - ( —) ] xlOO Attack rate of untreated group INDUSTRIAL APPLICABILITY As described hereinbefore, the novel strain Trichoderma harzianum YC459 (accession No KCTC 0772BP) isolated from soil is antagonistic to plant fungal pathogens Botrytis cinerea, Thizoctonia solani, Cladosporium fulvum, and Alternaria solani, showing excellent suppressive activity against gray mold rot, damping-off, leaf mold, and early blight From the novel strain, there can be derived an environment-friendly biofungicide, which is not harmful to natural ecosystems and is free of mammalian toxicity Therefore, the present invention is very useful for the agricultural chemicals and environmental industries The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation Many modifications and variations of the present invention are possible in light of the above teachings Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described Applicant s or agent s file International application No reference number YL01010PCT PCT/KR01/00616 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bιs) For International Bureau use only CD This sheet was received by the International Bureau on Authorized officer

Claims: CLAIMS 1 A fungal species Trichoderma harzianum YC459 (accession No KCTC 0772BP) and its mutants, antagonistic to plant pathogens 2 A biological fungicide for controlling plant pathogens, comprising the mycelia or spores of the fungus of claim 1, or a mixture thereof, as a fungicidally active ingredient 3 A method of preparing a biological fungicide for controlling plant pathogens, in which mycelia or spores of the fungus species of claim 1 or a mixture thereof is formulated with an ordinary carrier

Inventors: CHUNG YOUNG-RYUN, KIM KI-WOONG, LEE SON-KUG, CHUNG, YOUNG-RYUN, KIM, KI-WOONG, LEE, SON-KUG
Applicants: CHUNG YOUNG RYUN, KIM KI WOONG, LEE SON KUG, CHUNG, YOUNG-RYUN, KIM, KI-WOONG, LEE, SON-KUG
Application year: 2001
Main classification: C12N1
IPC codes: A01N63, C12N1