MXPA96002967A - Microorganisms for biological control of plant diseases - Google Patents

Abstract

The present invention relates to biológical control of plant diseases and concerns new microorganisms belonging to the genus Nectria, as well as their use for controlling fungal infections in plants. The invention concerns also compositions comprising new strains of the genus Nectria and their use to said purpose. The invention provides also a method for screening effective control organisms from microbial strains isolated fromásoil.

Description

MICROORGANISMS £ AE_ »THE BIOLOGICAL CONTROL OF DISEASES PE PLANTAS DESCRIPTION OF THE INVENTION Field of the Invention The present invention refers to the biological control of diseases in plants and specifically concerns new microorganisms belonging to the genus Nectria, thus as to its use to control fungal infections in plants. The invention also concerns compositions comprising new strains of the genus Necium and its use for the aforementioned purpose.The invention also provides a method for screening effective control organisms from microbial strains isolated from the soil.

Crops are affected with various diseases caused by fungi, bacteria and viruses, as well as several insect pests; Many methods of control in the ^ and cultivation, technical, chemical and biological have been developed to control those evils. The purpose of such methods is to prevent the loss of crops both qualitatively and quantitatively, caused by plant diseases and pests. In general, the term biological control of phytopathologies means the control of plant pathogens by other organisms that can be called two biological control agents (CBA). The products developed from ACB, are often called biopesticides, the mechanism of biological control of plant diseases varies and in fact, is often based on the cooperative action of many different mechanisms. The control effect, can be based on the inhibitory metabolites produced by the control agent, sometimes can produce parasites, in the pathogen or compete with it, for space or, the available nutrients. The need to discover new biological control agents has been increased by the fact that many of the traditional chemical control agents have proved to be harmful to the environment and human beings and a disadvantage of chemical agents, it is also the fact that Many pests have become resistant to one or even several control agents. The development of resistance to biopesticides is on the contrary unlikely due to its effect based on several mechanisms of different types. Usually chemical agents affect more quickly and more effectively than biopesticides. Biopesticides, on the other hand, often have a longer action than chemical agents, since their effect is based on a viable and re-producible microorganism. The most important group of biopesticides, are bacterial products directed against insects, the biosynthetics based on the bacterium Bacillus thuringiensis, are the most commonly used. A biofungicide based on the actinomycete Streptomyces, is effective against a certain number of diseased fungi born in the soil and born in the seeds, which is produced in Finland * A product that is capable of preventing the development of the disease Fo is The root (caused by Heterobasidon annosu), in the forests of co niferas has been developed from a fungus, which rots the non-harmful rhine called Phlebia gigantea. Bacteria of the genus Pseudomonas, especially of the species Pseudomonas fluorescens, have been studied extensively and a large number of strains of P. fluorescens are currently known to have fungicidal activity. See, for example, published patent applications WO 92/18613, Fl 92/1722 and WOqO / 01327, as well as EP 228 57. When searching for suitable microorganisms for biological control, large quantities of microbial strains are explored for control activity or other special property. Some methods of exploration have been described in patent publications. In the US patent 4,6 ^ 7,533 a three-stage exploration method is described, wherein the bacteria are first isolated from the soil, which contains a quantity of spore-forming spores of the harmful fungus Pyt-hium. In the second stage the isolated bacteria is explored in a greenhouse by growing cereal seed in a soil containing a large amount of pyt-hium spores, with a suspension of each bacterial agent to be tested, and the control sample without it, and from this test those bacteria are selected in whose presence the plants developed larger leaves and also grew taller. In the third stage, the bacteria is selected again, in a field similar to the test in the winter, also in this stage those bacteria in which the plants grow best are selected. In the Finnish patent society 92, 1722, on the other hand, the following procedure was used. First of all, the mycelium of a pyt-hium strain is grown in a suitable medium, a layer of sterile soil is placed on top of it, to which the microorganism to be tested is added, and its effect is eliminated. on the growth of pyt-hium, in the second stage, a second sample of soil is inoculated with a strain of pyt-hium causing a lack of damping, a seed of a plant sensitive to fungal infection is sown in the soil, and the effect of the test organism on the growth of the p'anta is determined. In the following tests, those substances that are inhibitory to pyt-hium are selected in the two tests mentioned above. SUMMARY OF THE INVENTION The present invention relates to a microorganism of the genus Nextria, for example Nectria pi tyrodes Montagne which was found to be very active especially against Fusarium fungi. The invention further relates to a biofungicidal composition prepared from the microbial strain, which comprises as an active ingredient the above-mentioned strains belonging to the genus Nectria and optionally additives or carriers conventional in the art in the form of an appropriate formulation. Examples of such formulations are compositions suitable for treating the seed, compositions, powders or granules to be sprayed onto substrates that are being created or liquid formulations for treating the soil. The present invention also provides a new and efficient screening method for fungal strains where a three step test sequence is used, which combines a sand test on rotten plants and the field soil, at each step of The test will eliminate strains of fungi that appear to be inefficient. The strains that show to be better, in the greenhouse are taken to the Investigation tests in field condition. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 the dose responds to experiment 76 in vera no -92. The percentage of sick shoots using seed inoculated with the fungus F. culum. K_e n treated, B- liquor or Baytan treatment; J76-0 = suspension of J76 spore, 1.2 x 10 cpu / l; 6 J76-1 «spore suspension of J76, 1.2 x 10 cpu / ml; 5 J76-2 »J76 spore suspension, 1.2 x 10 cpu / ml ik J76-3- J76 spore suspension, 1.2 x 10 cpu / ml i Figure 2 dose responds to experiment with J76, in summer 92 The percent of outbreaks we get from using healthy seeds. Abbreviations as in Fig. 1; The Figures histograms that represent the effect of the 3a to 3d isolated fungi rejected in the different stages of the tests in the Greenhouse on the health of the shoots in field conditions. «Untreated, B - liquor or Baytan treatment i Detailed description of the invention. Further on, the fungal strains of the invention are described in detail, their isolation and character are indicated. and prove their efficiency in field conditions.

In addition, the formulations of the fungicidal compositions formed from those strains, the characteristics of the compositions and the effectiveness tests with those compositions are described. A method for exposing fungal strains isolated from soil samples is also described. Isolation of microorganisms. The soil samples, from which the fungal or fungal strains of the present invention have been isolated, were collected in the years -89, -90 and -91 »about 190.}. the samples were collected from different parts of Finland, mainly from the research stations of the MTT (Agricultural Research Center), from different types of soil, and different planting rotations. Samples were taken from the root layer, a depth of 0-015cm; several subsamples were taken from each field, which were then taken to samples of one to two liters. The isolations were made either by a dilution method (soil isolation) or by a bait plant method (relay ai sions) whose realization is described later in the Methods section. Characterization of microorganisms. The strains of fungi isolated from the soil samples were tested in the method of exploration of the invention, which is described later in the Methods section. Five strains were found that gave very good results, which were named J76, J1 * t31 , J1432, MOSI and R0S2, being characterized in Centraalbureau voor Sch? melcul tures, Baarn, Holland. These strains have been deposited according to the Budapest treaty in the DSM deposit, (Deutsche Samlung von i roorga lsmen und Zellkul turen GmbH), under deposit numbers DSM 7522 (March 15, 1993), DSM 8805 (say 10, 1993), DSM 8806 (Dec. 10, 1993), DSM 8807 (Dec. 10, 1993) and DSM 8808 (Dec. 10. 1993) respectively. The morphological characteristics of the microorganisms are the following: Morphology: Sporodoc ia without marginal steril hyphae. Conidiophores branched repeatedly, with several branches occurring in each node, the last branches carry whorls of phialides arranged in a layer similar to a slow one. The cylindrical phiallids of up to 15 mee. long with an apical pore. Conidia produced in chains or thin balls, ellipsoidal similar to a drop with a navel of seed with 7.8 x kme. of soft wall, without appendix. Habit of the Colony: Colony on oat flake agar growing to a diameter of W) mm at 22 C in seven days mycellum hyaline, areas with spores expel sporodochial, green, arranged, in concentric rings. Smell not prominent, the back of the colony is colorless, clear exudation limited.

The conidiofora structure of strain J76 is if i. lar to that of Myrothecium verrucaria, but differs from these species with the production of non-marginalized sporodochia, conidia in dry chains and without the typical appendage in the form of a fan. The representatives of the genus Gliocaldiu, differ from the new strains because it is typical for the gliocladium, to produce penidiated individual conidiophores. Strains J76, J1 31, J1 * »32, MOSI and R0S2, were thus identified as the species Nectria pityrodes Montagne. From these strains can be prepared non-perishable formulations, which are easy to spray on plantations that require disease control. A formulation can be produced, for example, in powder form. The cultivation of a microbe, can start from an inoculation that is a PDA ball, including spores that has been maintained at -80. The microbe can be cultivated, either as a liquid culture or on a solid support; the nutrient medium comprises sugar, for example, sucrose, and nitrogen, as well as small amounts of other nutrients. Cultures (non-specific nutrients, for example, corn liquor, can be used in the cultures (with Steep liquor-CSL, or a specific nutrient medium, for example, GYM (g locosa g / 1)., extract of yeast g / 1, extract of malt 10 g / 1 or PDB, broth -patata-dextrose) the pH, is approximately 6.0, adjusting in the beginning before sterilization. The culture is performed by shaking, lasts for a period of one week to two weeks, the cell mass can be separated, either by filtering through filter paper, or by centrifugation. If a liquid culture is used, the cell mass is mixed with a carrier, for example, sil lea, milk powder, CMC (carboxymethyl cellulose). The mass is dried at room temperature and ground to obtain a powder. Methods Isolation of the microbes: a) Dilution method (soil isolation) 10g of soil were mixed to 100ml, of 0.5% water agar, (Bacto-agar). Three subsamples of 10ml were taken from the mixture and two dilutions were made from each of them -1 2 (10 and 10), within the 0.5% water agar, the dilutions were shaken, (in a water bath, in a shaker 20 to 30 min, A mi, of the dilution was pipetted on a petri dish vacuum and topped 20ml, from Lítt an Oxgall Agar, the discs were incubated at room temperature for 3-7 days, and the individual fungi were then isolated in puroe cultures on PDA (potato-dextrose-agar). bait plant (root isolation) 15 pots in a series plate of pots (plate of pots in series (Ve i-VP 96 with pots of 50ml) were filled with soil from a sample wheat seeds, barley k or approximately 15 seeds were planted in a pot and 5 pots were used for each of the plant species, the seeds were covered with sand and the pots were irrigated and incubated in a growth chamber at 15 ° C, with 1 * hrs. day length After two weeks of cultivation, the plants were taken and washed. No running water or cleaned with a brush without water; From the roots, small pieces were cut and plated. The media used: LOA (10g peptone, 10g dextrose, 15g bacto-oxail O.Otg B-crystal violet, Q.Q3g streptomycin, 20g agar, aggregating to lOOOml water), PCNB (15g peptone, 1g KH P0, 0.5 g 2 MgSO .7H 0.2g avicol, 3 ppm streptomycin, 20g agar, up to k 2 tOOOml water) and PDA-streptomycin (39g PDA preparation, 300ppm streptomycin, up to lOOOml water.After a few days of incubation, the fungi isolated were isolated and subcultured on PDA plates, strains were stored in PDA pellets or pellets, in ampoules (Nalgene 5000-0012, PP Sterlle 1.2m1) at -80 * C. Performance of the greenhouse tests in the exploratory method. Sand test: Cereal seeds were sown on a layer of sand, first micelia and spores of the pathogen and then spores of the test fungus are pipetted in a suspension of water on the seeds and for the growing substrate. - After this, the seeds are covered with sand. Two and a half weeks the intensity of the symptoms of outbreak disease is examined. Other experiments reject those fungi that do not affect the intensity of the disease or that prove to be pathogenic by themselves. Moss test A cereal seed is moistened in a spore suspension of a pathogen, and dried. After this the seed is moistened with the suspension of spores of the test fungi, dried and sown. Vasporized moss is used as a growth substrate. After two and a half weeks of culture, observations were made on the health of the outbreaks. Strains of fungi that clearly prevented the disease were taken to the field test. Field tests Ground and wet crop land is placed in 1.5 1 pots and 36 cereal seeds are sown in each pot. The treatments to the seeds before sowing are done as in the moss test, and three equal pots are used for each treatment. The symptoms of the shoots are examined after four weeks of culture. The strains that passed these tests were taken to the field tests that gave the final safety of the biopesticidal effect of the selected microbial isolates. Pathogenicity of fungal strains The possible deleterious effects of the strain of 5 fungi J76 have been examined tentatively. The results showed that the fungus is not pathogenic for plants. 33 plant species were studied. EXPERIMENTS The following experiments illustrate the use of '"^ r invention In Article [A] the use of spore suspensions of Nectaria putyrodes J76 strains is described, J1431, J1432, MOS1 and ROS2 of the invention, to control plant diseases caused by Fusarium spp. In the article [B] the preparation and use of the formulations made of these strains, in article [C] the experiments are described by means of which the mode of action of fungal strain J76 was examined, and in article [D] the performance and evaluation of the method is described of examination of the invention. 20 [A] USE OF FUNGAL SCAPS AS SUSPENSIONS OF SPPORES Experiments with suspension of spores of strain J76 In the summer of 1992, the effect of suspension of spores of strain J76 at three test sites was examined. In these tests two different seeds were used: artificially inoculated wheat with Fusarium culmorum and barley naturally infected with F. nivale. The results of the tests with wheat are given in table 1. The performance results of artificially inoculated wheat are given in table 2. Observations on the shoots with barley are given in table 3. The disease has no effect statistically significant in the yield of barley. TABL? 1 The three experiments in three test sites in the summer of 1992 with artificially inoculated wheat. Rate of emergence and disease of outbreaks. Treatment Number of outbreak rate / meter diseases percentage of severely ill outbreaks JOKIOIMEN: healthy seeds 50 5.2 untreated 11 48 Baytan dressing 45 10 J76 47 7.2 MIETOINEN: healthy seeds 45 7.3 untreated 10 42 Baytan dressing 39 3.8 J76 33 13 PÁLAKÁNE: Healthy seeds 50 3.0 untreated 15 57 Baytan dressing 51 24 J76 45 11 TABLE 2 Performance results [kg / ha]. Experiment in three test sites in the summer of 1992 with artificially inoculated wheat.

Treatment JOKIOINEN MIETOINEN PALKANE Healthy seeds 3650 3190 5290 without treatment 1830 1690 1950 dressing Baytan 3270 3190 4940 J76 3530 2950 4900 TABLE 3 Emergency rate and outbreak disease. Experiment in three test sites in the summer of 1992 with infected barley [F. nivale].

Treatment Number of outbreak rate / meter disease percentage of severely ill outbreaks JOKIOIMEN: untreated 39 7.7 Baytan dressing 40 1.2 J76 40 0.9 MIETOINEN: untreated 42 7.4 Baytan dressing 42 1.4 J76 43 2.8 PÁLAKANE: untreated 42 26 Baytan dressing 42 9.4 J76 44 6.3, v- Dose response test of strain J76 in summer 1992. As J76 showed really good results in shoot observations in the early summer of 1992 , the effect of its application rate on the control effect was tested in an experiment, which was sown just before mid-summer. Both healthy wheat seeds and seeds artificially inoculated with Fusarium culmorum were used. '- Small plots of 2 m2 were used in the experiment. Symptoms of emergence and disease in the outbreaks were observed. The outbreak samples for the follow-up of the disease were collected in four different periods. Samples for each treatment were taken from four plots planted separately for each sampling time. The observed amounts of the stems are given in table 4 and the percentages of the diseased stems are given in table 5 separately for diseased and healthy seeds. The data in Table 5 are shown graphically in Figures 1 and 2.

; - TABLE 4. Experiment of dose response for J76 in the summer of 1992. Emergencies.

Treatment Number of shoots / meter Seeds infected with rusriww Untreated 18 Baytan dressing 57 Sodium suspension J76 [1.2xl07cfu / ml] 74 Sodium suspension J76 [1.2xl06cfu / ml] 58 Sodium suspension J76 [1.2xl05cfu / ml] 51 Sodium suspension J76 [1.2 xl04cfu / ml] 20 healthy seeds: '' Untreated 71 Baytan dressing 66 Sodium suspension J76 [1.2xl07cfu / ml] 80 TABLE 5. Experiment of dose response for J76 in summer 1992. Percentage of sick shoots with seeds inoculated with F. culmorum, and healthy seeds. Abbreviations of Figure 2 Treatment Days since emergence 10 17 30 44 Seed infected with Fusarium: K 75.4 82.8 79.5 85.1 B 15.9 60.9 65.3 78.2 J76-0 10.1 50.0 66.6 70.3 J76-1 23.1 51.8 55.5 62.9 J76-2 36.4 64.7 76.0 78.0 J76-3 68.6 72.8 77.1 76.2 Healthy Seeds •• K 54.7 71.8 67.7 85.1 B 8.4 50.4 58.3 73.6 J76-0 14.3 48.0 69.9 73.8 Field tests with suspension of J76 spores in the summer of 1993. _ _, The tests were performed in Jokioinen, Mietoinen and Pálkáne. In the tests samples of six seeds were used: - Wheat "Luja", naturally infected with the fungus F_j cumo mn - Wheat "Luja", artificially inoculated with the fungus F. culmorum - Wheat "Luja" healthy - Wheat "Laari", healthy - "Kustaa" barley natu rally infected with different Fusarium fungi and BJ mushroom, SQfQkin.ana olaris - "Yty" oats, naturally infected with fungus F_, avenageym Healthy wheat samples were sown only in Jokioinen, the other four tests with seed samples were made in each of the test sites. For the tests, land of 10 m2 was planted with six replicates per treatment. The treatments of the seeds were the same for each of the seed samples: K = untreated control B = chemical control dressing of Baytan I J76S = suspension of spores J76 of a culture plate [8.4 x 10 9 cfu / kg of seeds] In table 6 the intensity of the damage caused by the disease is given separately for each treatment and seed sample. The performance results are summarized in table 7. TABLE 6. Field test with J76 spore suspension. 1993. Percentage of severely ill outbreaks BARLEY AVENUE WHEAT WHEAT "LUJA" "LAARI" INF. NAT. INC. ART. JOKIOINEN K 6.0 10.1 7.4 34.2 20.6 5.7 B 0.9 2.0 2.1 1.0 1.4 0.7 J76S 0.9 1.4 0.7 1.0 3.3 5.7 MIETOINEN K 12.8 3.5 20.4 72.0 B 1.3 0.6 5.7 6.6 J76S 5.4 0 7.1 7.3 PÁLAKANE K 9.6 7.4 10.9 26.9 B 2.5 3.3 2.1 0.6 J76S 2.4 3.6 2.3 0 TABLE 7 Performance results of field tests with suspension of spores J76199 [kg / nal BARLEY AVENUE WHEAT WHEAT "LUZ" "LAARI" INF. NAT. INC. ART JOKIOINEN K 7460 6650 6060 2950 6100 6360 B 7500 6790 5870 5690 5940 6060 J76S 7840 6650 6050 6040 6150 6570 MIETOINEN K 5900 5780 4920 3890 B 6070 5200 4910 4990 J76S 6120 5440 4670 4810 4810 PÁLAKANE K 4670 5560 3630 2950 B 4550 5200 4190 4000 J76S 4710 5430 3710 3650 Tests of J76 against wilting of wheat and rotting of the size of barley In the summer of 1993, J76 was included in a field trial, where nine chemical fungicides were tested to cover grain seeds against wheat wilt [caused by Tilletia caries]. J76 was applied as suspensions of conidia and chemical substances according to their instructions of use. Land of 0.1 m2 and five replicas were used. [Table 8]. TABLE 8 Test of the effect of the treatments with the dressing in the case of wheat wilt CONTROL EFFECTIVENESS [%] (= decrease in the number of infected spikes) T Syssato S liquid 78 Baytan WS 100 Beret 050 100 Fungazil c 100 Panoctine 35 100 Raxil I powder 100 Raxil I liquid 100 Prelude LS 100 Vitavax 20 FF 100 J76 85 In Baytan the active ingredient is triadimenos.

Baytan I is a mixture that includes triadimenos and imazalil. Táyssato S consists of carboxy and imazalil. The active ingredient in Panoctine is guazatine. The treatment of seeds with J76 spores was also included in the field trial, in which the chemical control agents were tested against the stem rotator [Bipolaris sorokiniana] of the barley. In the test 10 m2 land and four replicas were used. No differences were found in the emergency between different treatments. J76 clearly reduced the symptoms [Table 9], although the pathogen is very different compared to the Fusarium fungi against which it was selected.

TABLE 9 The effect of the treatments against the rotting of barley stems.

EFFICACY OF CONTROL [%] (= decrease in the number of sick shoots) TREATMENT Prelude LS 85 Dividend 37.5 [400 mi] 84 Dividend 37.5 [200 mi] 81 Baytan I 81 Beret Special [400 ml] 74 Raxil I powder 70 Táyssato S liquid 66 5 Panoctine Plus 66 Fungazil C 66 Beret special [200 ml] 65 Raxil I liquid 64 Beret FS 050 49 ~ PNL 210 39 [B] PREPARED FORMULATIONS OF FUNGAL STRAINS, ¥ SU EFFECT ON FIELD TESTING Powder formulations of fungal strains J76 were prepared as follows: Formulation 1 The culture was performed in 1 Erlenmeyer flask with 0.5 1 of nutrient medium, which included 4 g / k of - • sucrose, 4 g / 1 of yeast extract and malt extract g / 1. The pH was adjusted before sterilization in an autoclave to 6.0. An agar pellet including spores that had been stored at -80 ° C was used as inoculum. [means of desiccation of potato agar]. The speed of the agitator revolutions was 150 rpm, the growth temperature was room temperature [22 ° C] and the culture time was 7 to 12 days. The cells were separated by means of a paper filter. Silica, milk powder and CMC were added to the cell mass [carboxymethylcellulose] as follows: 20% cells 5% silica 55% milk powder 15% CMC [7% aqueous solution] 10% The mixture is dried at room temperature in opened Petri dishes in sterile air for 2 days. The thickness of the layer was 1-2 cm. The dry mixture was ground to powder. The viability of the formulation was 107 cfu / g. [cfu = colony forming units]. The cfu [colony forming units] is a unit that is used in the determination of the viability of the 15 microbes. The diluted microbial suspension is spread on agar plates and the colonies are counted after a few days. When the dilution is known, the number of colonies or the number of microbial cells in the original sample can be counted. Formulation 2 The cells were cultured in a manner similar to formulation 1. after which the silica, milk powder, CMC and acoric acid were mixed with the bulk 25 cells as follows: 60% cells, silica 20 %, milk powder 14% CMC [7%] 3% Ascorbic acid 3% The mixture is dried in the same way as formulation 1 and ground to powder. Feasibility 107 cfu / g.

Formulation 3 Cells were cultured in a manner similar to formulation 1, and sucrose and starch were mixed with the cell mass as follows: cells 20% sucrose 25% starch 55% 15 The mixture was dried in the same manner as formulation 1 and it was ground until it was powdered. Feasibility 107 cfu / g. Formulation 4 ~ "Strain J76 was cultured directly on a solid medium including silica carrier, for the niutritive broth 20% malt extract was used [Maltaz MP , Lahden Polttimo]. 120 g of nutritive broth was mixed in a beaker with 50 grams of silica powder and autoclaved for 20 minutes at 120 ° C. The cooled medium was inoculated with 10 grams of J76 spore suspension, which was obtained by separating the spores from a PDA plate in sterile water. The medium was incubated for 20 days at 16 ° C, after which it was dried at room temperature for 2 days. The viability of the dry preparation was 107 cfu / g. The rest of the strains of the invention can be formulated similarly. The effect of powder formulations on field tests The following is a description of the tests carried out by the Plant Protection Institute at the Agronomic Research Center of Finland in the summer of 1993 with the powder formulation of strain J76 . The tests were carried out in MTT [Agronomic Research Center] in Jokioinen, Mietoinen and Pálkáne. In trials 15 samples of six seeds were used: wheat "Luja" naturally infected with the fungus F. culmorum • * '"* - wheat" Luja "artificially inoculated with the fungus F. culmorm 20 - wheat" Luja ", healthy wheat" Laari ", healthy barley" Kunstaa ", naturally infected by several fungus Fusarium and fungus Bipolaris sorokiniana oats" Yty "natuiarally infected with the fungus F _, _ avenaceum Healthy wheat samples were sown only in Jokioinen, the other tests with seed samples were performed in each of the three test zones. For the tests, 10m2 plots were planted with six replications per treatment. The seed treatments were the same for each of the seed samples: K = untreated control B = chemical control, Baytan I dressing J76PK = J76 powder as dry dressing [8.4 x 10ß cfu / kg - the largest amount taken by the seeds] J76PN = powder J76 as liquid dressing [8.4x10 * cfu / kg] Table 10 shows separately the intensity of the damage caused by the disease is given separately for each treatment and sample of seeds. The performance results are summarized in Table 11. TABL? 10 Field tests with J76 dust in 1993. Percentages of severely damaged shoots.

BARLEY AVENUE WHEAT WHEAT '"LUJA" "LAARI" INF. NAT. INC. ART . SANO SANO JOKIOINEN K 6.0 10.1 7.4 34.2 20.6 5.7 B 0.9 2.0 2.1 1.0 1.4 0.7 J76PK 2.2 1.5 2.7 7.0 6.2 2.8 J76PN 1.2 1.9 3.9 1.3 5.7 4.6 MIETOINEN K 12.8 3.5 20.4 72.0 B 1.3 0.6 5.7 6.6 J76PK 10.1 0 17.2 42.5 J76PN 9.1 0.3 13.7 14.3 PÁLAKÁNE K 9.6 7.4 10.9 26.9 B 2.5 3.3 2.1 0.6 J76PK 5.3 2.6 4.8 6.1 J76PN 3.9 3.8 3.9 2.7 TABLE 11 Performance results of field tests with J76 powder in 1993 BARLEY AVENUE WHEAT TRIGO "LUJA" "LAARI" INF. NAT. INC. ART. JOKIOINEN K 7460 6650 6060 2950 6100 6360 B 7500 6790 5870 5690 5940 6060 J76PK 7610 6940 6060 5140 6400 6320 J76PN 7610 6970 6040 6020 6000 6400 MIETOINEN K 5900 5780 4920 3890 B 6070 5200 4910 4990 J76PK 6000 5670 4770 4620 J76PN 6100 5760 4790 4950 PÁLAKÁNE K 4670 5560 3630 2950 B 4550 5200 4190 4000 J7PK 4730 5560 3600 3390 J7PN 4870 5470 3660 3760 In addition, the control effect of the different prepared formulations of strain J76 against several fungi was tested in 1993. The results of these experiments are given in Tables 12 to 18.

TABLE 12. The control effect of J76 in sand terrain inoculated with the Gaeumannomyces fungus in Polkka wheat. Test in pot, protected.

KF = solid phase preparation [formulation 4] disease index: 0 healthy 1 slightly sick 2 strongly ill 20 3 without budding TABLE 13 Control effect of J76 preparations against Fusarium culmorum fungus in Polkka wheat. Tests in pots, moss substrate. KF = growth in the solid phase [formulation 4], R = cultivation in liquid [formulation 1] M = microbe as in agar cultures 0 healthy 1 slightly sick 2 strongly ill 3 without budding TABLE 14 The effect of J76 against the fungus Pythium in cucumber. Results as average vcalores in the three pot tests [pH 6.2, pH 6.9 and pH 7.4] Disease index: 0 healthy 1 slightly sick 2 strongly ill 3 without sprouting TABLE 15 The control effect of the fungi strain J76 on cauliflower in sandy soil contaminated with the fungus RhjsogtQni sol na Test in greenhouse pots.

TABLE 16 The effect of control of fungal strain J76 on barley "Kustaa" in sandy soil inoculated with the fungus Fusarium niyale. Test in outdoor pots, protected.

Disease index: 0 healthy 1 slightly sick 2 strongly ill 3 without sprouting TABLE 17 The control effect of fungal strain J76 against the fungus Alternaria brassicicola in cauliflower. The results are average values of 0 healthy 1 slightly sick 2 strongly ill 3 without sprouting The results of the experiments on the control effect of five strains of Nectria putyrodes of the invention [J76, J1431, J1432, MOS1 and ROS2] on Fusarium culmorum in wheat are shown in Table 18. The results are given as average values of the two experiments, in one of which moss was used as substrate and in the other field of the field.

TABLE 18 The control effect of five strains of Nectaria pjtyrodes [J76, J1431, J1432, MOS1 and ROS2] against the fungus Fus rium CUlmc-rum in wheat. The results are given as average values of two tests in pots [moss and field] Disease index: 0 healthy 1 slightly sick 2 strongly ill 3 without sprouting [C] J76 ACTION MODE Preliminary observations of the p? r forms which J76 acts as an antagonist to other fungi, have been made by means of microscopy and some tests of 5 laboratory. As the interactions of the hyphae of J76 and the fungus F. culmorum are observed in a microscopy, the first distinctive reactions can be found to be very rapid. At the points of contact of miscelios ? The cells of the Fusarium hyphae begin to decompose markedly approximately one hour after the contract. First, the cell walls lose their shape, then the cells empty out and finally the cell walls decompose completely. From the points of contact, the decomposition of the Fusarium hyphae is slow, between. When J76 and F. culmorum have grown mixed for a few days, the hyphae of the Fusarium fungus can no longer be found. Also their spores [both conids and clamidiospores] decompose by the effect of 0 J76, but more slowly than hyphae. Usually the J76 hyphae spread around the Fusarium spores before their decomposition. There are no observations according to which the J76 really penetrates the Fusarium hyphae. 5 Based on microscopic observations, it can be concluded that J76 probably secretes biologically active substances around it. By nature they can be a type of enzyme or antibiotics. Its production can also be the main mode of action of the J76 because they have not been found directly parasitized and other fungi, and as it grows slowly it can not compete effectively for the nutrients. Indications have been obtained about the production of metabolites that affect the growth of Other fungi also in a cellophane test. When J76 and F. culmorum grow together in a very thin growth substrate, a zone of inhibition is formed in which the growth of Fusarium ceases. On a substrate with normal growth this it does not show up This is probably due to the small concentrations of the substances diffused in the substrate. Volatile substances secreted by J76 have also been found to have a weak growth control effect on F. culmorum. Cellophane test: A cellophane film was placed on the growth substrate and strain J76 was grown on this film. After cultivating for 10 days the film was removed and with it the J76. Substances secreted by J76 and transported through the film remained on the substrate. As control plates were used they had a simple cellophane pellet without J76. The results of the cellophane test are given in Table 19. TABLE 19 Effect of the metabolites produced by the fungus J76 on the growth of F. culmorum in the cellophane test, growth rate mm / day J76 2.5 Control ___2 __. , __ [D] PERFORMANCE OF THE METHOD OF CLASSIFICATION AND EVALUATION OF THE RESULTS TESTS IN ARENA Sowing substrate A sowing substrate was used consisting of are, grain size 0.2-0.7 mm [Kauniston Sora Oy, Loimaa]. The sand was moistened by mixing 4 parts of sand with 1 part of water. From a plate of pots in series [Vefi-VP 96] was cut A plate of 5 x 7 pots [of 50 ml] placed in a plastic box. The pots were filled with moistened sand, so that 1-1.5 cm of its upper edge was left empty. Three spring wheat seeds "Luja" were planted in each pot. Treatments Infection with Fusarium cul orujt: F. culmorum was grown on PDA plates at room temperature for approximately 1 month [until fully sporulated]. The mycelia with the eporas were separated from the plate and mixed with distilled water by means of an Ultra-Turrax homogenizer. The amount of the spores was adjusted to 10 * spores / ml. The solution was frozen in portions of 30 ml in Minigrip bags at -20 ° c. For the tests, the mixed solutions were thawed and remixed. The solution was used as such [basic solution] and as a 10"dilution.2 The pathogenicity of the Fusarium strains was maintained when circulating it by plants [wheat seeds were inoculated with a suspension of Fusarium and the pathogen was isolated from the outbreaks patients] Suspension of antagonists: A PDA pellet taken from the freezer that included the antagonist was divided into three parts and cultured on three PDA plates.The plates were incubated at room temperature [in the dark] for approximately three weeks. The basic suspension of antagonists was prepared by scraping two antagonist plates with 50 ml of distilled water, mixed with Ultra-Turraz homogenizer, 10"1 and 10" 3 dilutions were prepared from the basic solution 3. Seed treatments: 1 ml of suspension of F. culmorum was added with pipette to the seeds seeded in the plate of pots in series, and on it 1 ml of suspension of antagonist].

Control treatments: Each plate of pots used for testing one of the fungi had additionally 15 seeds in five pots, which were treated only with the basic solution of the fungus studied. In sand tests 15 to 30 fungal strains were tested at the same time. Each day the test was started, a separate potplate was also planted, with the help of which I verified the health of the seeds as well as the pathogenicity of the inoculum of F. culmorum that cause the disease. Three control treatments in a separate plate were: non-inoculated [only water], inoculated with basic solution of F. culmorum and inoculation with a dilution [10"2] of basic solution of F. culmorum For each of these three treatments 30 seeds were planted in 10 pots Growth conditions: After pipetting the fungal suspensions, the seeds were covered with moistened sand, the boxes with the plates of pots were wrapped in plasticotransparent and transferred to a growth chamber [10]. 15 ° C, 14-hour light period.] Observations After cultivating the shoots for 16 to 18 days, they were washed under running water, and observations were made on the symptoms of disease.The outbreaks in which the seeds had been obscured or coleotile cells, and non-emerging seeds that had remained hard were considered healthy, the outbreaks had different symptoms, and the non-emergent seeds that had softened were considered sick. The control treatment plants were examined first. If only healthy plants were developed from treated seeds, and with both parono treatments, different symptoms of disease were obtained, the test was accepted and then it was decided to make observations of the plants treated with fungal strains that were to be studied. In the sand test the rating given to each strain was determined by the number of plants treated with it and that are considered sick. If the emergence of the plants treated with the fungus to be studied was only distinctly reduced compared to the healthy control, or if other symptoms of disease were found, the fungus was not considered suitable for other tests. If no damage was found, each of six combinations of spore densities of the pathogen and the spore densities of the fungus to be tested were scored separately on the following scale: 0 = all healthy plants 1 = no more than 2 damaged plants 2 = 3 5 damaged plants 3 = 6-9 damaged plants 4 = 10-13 damaged plants 5 = no more than one healthy plant Based on the individual qualifications mentioned above, those of the tested fungal strains were chosen. They were used for the next stage of testing, this is the tests on moss. To continue, those strains that obtained at least three times 0 and 1 qualifications were accepted. If the fungus did not obtain a 0 rating, it was accepted to continue if it obtained at least four times the qualification 1. PROOFS IN MOSS Substrate of growth As growth substrate moss fetilized and with vaporized lime was used. Until the autumn of 1992 Torronsuo crude moss was used without sifting, after using raw moss and sifted from Eurajoki. [Fertilization: 800 g of dolomite lime and 100 g of moss Y-lannos / 100 1 of moss [Y-lannos = trademark of a universal Finnish fertilizer]. The moistened sample was expanded in plastic boxes [28.5x49.5x9.4 cm, Wibulls Robusta Mamut box, MUoviyhtymá oy] as a layer of approximately 5 cm. There was a plastic film at the bottom of the box.

Treatments T = healthy non-inoculated seed, spring wheat "Luja." Irrigated with distilled water. F = Inoculated seed of F. culmorum. The seeds were soaked in basic solution of F. culmorum [the solution was used in excess] including approximately 10 * spores / ml. { cultivation of Fusarium, sand test]. After the treatment, the seeds were allowed to dry overnight on paper. F0 = inoculation with F. culmorum as in treatment F. When the seeds had dried, they were moistened with the basic antagonist solution. The basic solution was prepared by scraping the mycelium and spores from the plate of one of the antagonists in 25 ml of distilled water. The treatment was carried out by shaking the seeds and the antogonist solution in a small plastic bottle. After the treatment the seeds were dried on paper.

F2 = Inoculation with Ft olmoru as in treatment F. Antagonist treatment as in F0 treatment, with 10 ~ 2 dilution of the basic antagonist solution. rows were planted in the moss, 30 seeds / row. The seeding sequence was as follows: protection row, F, F0, F2, T, F, F0, F2, T, protection row. After sowing the seeds were covered with fire and the boxes became wet. Growing conditions The seedlings were grown in a greenhouse at a temperature of about 15 ° C. In a dark station, additional light was provided by multi-metal lamps for 12 hours / day. When required, the cultures were moistened with water. The cultivation time was 18 days. qualification The outbreaks were washed and observations of the symptoms of the disease were made after the test with a diagnosis. Based on the observations made of the treatments T and F, it was decided if the result of the test was accepted. The healthy control [T] was not allowed to have more than 12 diseased plants in total [of 60 seeded seeds] and the pathogenic control [F] had to have at least 52 diseased plants [of 60 seeds]. A proven strain of fungi was accepted for the next test phase [field trials] if no more than 19 diseased plants developed from seeds treated with one of the two solution concentrations [of 60 seeds].

FIELD TESTING Sowing substrate The used soil [sandy clay] was obtained from the field test zone in Jokioinen. The ground or ground is milled by hand or through a 1x1 cm table screen [summer 1992-1993]. Plastic plates of 1.5 1 [14 cm in diameter were filled in such a way that 3-4 cm of the upper edge was empty. Filtering paper was placed in the bottom of the pot. Treatment 1. Healthy seed. Moistened with simple distilled water. 2. Fusarium control. The seeds were moistened with Fusarium inoculant [preparation, see tests in sand] that had approximately 10 * spores / ml, the solution was used in excess. 3. Inaculation as in the Fusarium control When the seeds had dried, they were treated with the antagonist suspension that was prepared by mixing the mycelium and spores of a plate with 25 ml of distilled water. The treatment was carried out in a plastic bottle, in which 130 seeds [120-150 seeds] were placed, and 1 ml [0 1.5 ml] of antagonist suspension. 4. Fungicidal control. Inoculation as in the control with Fusarium. When the seeds have dried they are treated with 2 g of Baytan I powder dressing per one kg of seeds. Before, the treatments with Ceresan and Táyssato S were also used. The treated seeds were planted in pots, 36 seeds / pot, three replicates / treatment. The plantings were covered with earth. Growth conditions were as in the sample test. The culture time was approximately four weeks. Examination and qualification The shoots were washed and their disease rate was evaluated: 0 = completely healthy 1 = slightly damaged by Fusarium 2 = moderate to severe disease damage 3 = shoots with widespread or dead dark spots. TESTS OF THE EFFECTIVENESS OF THE GREENHOUSE In the summer of 1993 in a wide field test the effectiveness of the selection method of the invention was proved. In the preba it was examined that the correct isolated fungal strains were taken for the following tests during the series of selection tests between October 1991 and February 1993. For the seed treatments, 60 of those strains that had been randomly selected were selected arbitrarily. abandoned in the sand tests and that had not shown to be pathogenic for wheat. Of the strains discarded in the tests in moss 92 were accepted for the test. The 58 mushrooms selected for the tests on the ground were taken to the test. 43 of those had been discarded in the ground test and 15 were selected for field tests. In addition to the 210 treatments previously mentioned, the test also included 6 treatments of "Control: Untreated [K], Baytan dressing [B] and four strains of fungi studied higher in previous tests, among others J76. In the test "Luja" wheat, naturally infected with the fungus F. culmórum, was used. as a unit observation there was a row of shoots of 1.4 m in length, for which 5.5 g of seeds were used. Six replications were planted for all 216 treatments. The randomization of the test is in accordance with the experimental cubic network design. In this way the erroneous variation attributable to soil factors. After about five weeks after the sprouts were planted they were removed from the soil, seconded and the symptoms of disease were studied. The results of the tests are made by four histograms in figures 3a to 3d.

Among the isolates discarded in the area tests and the non-pathogenic isolates discarded in the sand test, no differences could be found. The test on moss obviously worked very well. The strains taken to the following tests were on average better than the fungi discarded. In summary in the tests in sand and fire, relatively few fungi were abandoned, which should not have been discarded. On the other hand, based on the ground test a substantial amount of the best asylums were abandoned, but of those taken to the field tests only two out of fifteen had a defective effect in natural conditions. From the results it can be concluded that based on the sand and moss tests, the best antagonists can be reliably selected for subsequent studies, but in field trials until antagonistic candidates can be discarded.

Test of fungal strain J1431 in the selection tests The fungal strains of the invention were tested J1431 in all three greenhouse selection experiments. In the sand tests J1431 obtained the scores o, 0f 1, 1, 1 and 2, and it was taken for later tests. In the test in moss J1431 obtained the following results: T: 3 patients F: 36 patients FO: 3 patients F2: 7 patients In the test in soil decmapo, where J1431 was included, the percentages of the sick outbreaks in the different treatments were: healthy 66% control with Fusarium 87% dressing of Báytan I 31% J1431 19% Based on the selection tests the J1431 took a field test with another 61 fungal strains in le vernao of 1993. In the test spring wheat was used "Luja: artificially inoculated with the spore suspension of the fungus F. culmorum, the seeds were sown in a single row pl.4 m] and the test had 6 replicates. Outbreaks were extracted from the soil, washed and observations were made of their disease symptoms, thus obtaining the number of healthy shoots per meter in different treatments: "- Fusarium control 5.9 Baytan dressing I 56 J76 61 J1431 65 5 others proven strains 30-68 Tests of the fungal strain J 1432 in the selection tests The J1432 was tested in the three selection tests carried out in the greenhouse. ^ 0 In sand test J1432 scored 0, 0, 0, 1, 1 and 2 and was taken for subsequent tests. In the test in mosses J1431 obtained the following results: 15 T: 10 patients F: 60 patients FO: 10 patients F2: 37 patients In the test in soil decmapo, where J1431 was included, the percentages of the sick outbreaks in the different treatments were: healthy 56% control with Fusarium 98% dressing of Baytan I 18% 25 J1431 38% Based on the results of the field tests the J 1432 of these tests was omitted. Deposited microorganisms The following microorganisms were deposited in accordance with the Budapest Treaty in DMS-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German collection of microorganisms and cell cultures} , Mascheroder Weg 1 b, D-38124 Braunschweig, Germany. Microorganism Deposit number Deposit date J. Wectria pityrodes DSM 7522 15 March 1993 Montagne J76 Nectria pityrodes DSM 8805 10 dec. 1993 Montagne J1431 Nectria pityrodes DSM 8806 10 Dec. 1993 Montagne J1432 Nectria pityrpdes DSM 8807 10 Dec. 1993 Montagne MOS1 Nectria pityrodes DSM 8808 10 Dec. 1993 Montagne ROS2 20

Claims (12)

1. Having described the invention as above, the content of the following is claimed as property: CLAIMS 1.- A biologically pure culture of the strain of 5 fungi Nectria pityrodes Montagne J76 with the deposit number DSM 7522.
2. 2.- A biologically pure culture of the fungal strain Nectria pityrodes Montagne J1431 with the deposit number DSM 8805. _.J
3. 3.- A biologically pure culture of the fungal strain Nectria pityrodes Montagne J1432 with the deposit number DSM 8806.
4. 4.- A biologically pure culture of the fungal strain Nectria pityrodes Montagne M0S1 with the number of 5 deposit DSM 8807.
5. 5.- A biologically pure culture of the fungal strain Nectria pityrodes Montagne ROS2 with the deposit number DSM 8808.
6. 6.- A biofungicide, comprising ucnado minus a 0 strain of fungi selected from the group consisting of the fungi strains Nectria pityrodes J 76, J1431, J1432, MOS1 and ROS2 that have the deposit numbers DSM 75322M DSM 8805, DSM 8806, DSM 8807 and DSM 8808, respectively.
7. 7. A biofungicide, comprising ucnona minus a fungal strain selected from the group consisting of the fungal strains Nectria pityrodes J 76, J1431, J1432, M0S1 and R0S2 having the deposit numbers DSM 75322M DSM 8805, DSM 8806, DSM 8807 and DSM 8808, respectively, and carriers and / or additives in the art.
8. 8. The composition according to claim 7, characterized in that the carriers and additives are selected from the group consisting of silica, milk powder, carboxymethylcellulose, sucrose, ascorbic acid and starch.
9. 9. The composition according to claim 7 or 8 characterized in that it is produced by means of a process consisting of: [a] cultivating a mushroom strain according to any of claims 1 to 5 in an appropriate culture medium , separating the cell mass and binding carriers and / or additives, drying and pulverizing the dough, or [b] cultivating a fungal strain according to any of claims 1 to 5 in a suitable culture medium with silica and, if desired add carriers and / or additives, dry and pulverize the dough.
10. 10. A method for inhibiting a fungal infection in a plant consisting in applying to the plant or its seeds a fungicide according to claim 6 or a fungicidal composition according to any of claims 7 to 9 or adding it to a growing substrate before or after sowing the seeds.
11. 11.- Use as a pesticide of a strain of Nectria pityrodes according to one of claims 1 to 5.
12. 12.- A method for classifying a microorganism fgungicida of microbial samples, comprising the steps of a) perform a test in arenam that consists of sowing the cereal seeds in a layer of sand, pipetting miceluo and spores of a pathogen and subsequently spores of the fungus to be tested in an aqueous suspension on the seeds and on the growing substrate, then covering the seeds with sand , examine the intensity of the disease symptoms of the outbreaks after an appropriate time, and discard fungi that do not affect the intensity of the disease or that prove that they are pathogenic by themselves, and b) perform a moss test of the accepted fungi , which comprises wetting a cereal seed in a suspension of spores of a pathogen and drying the seed, moistening it with a suspension of spores of the fungus studied, and sowing it in vaporized moss, cultivate for an appropriate period and observe the health of the outbreaks, and take the subsequent tests isolates that clearly inhibit the disease.