COMPOSITION AND METHOD FOR CONTROLLING PLANT DISEASES

Abstract: The invention provides the novel Pseudomonas chlororaphis strain MA 342 deposited at The National Collections of Industrial and Marine Bacteria Limited, Scotland, under the accession number NCIMB 40616 and the use thereof, or of its mutants having essentially the same characteristics or of antipathogenically active metabolites or derivates thereof in compositions and methods for controlling plant fungal diseases.

Description: Composition and method for controlling plant diseases Field of the Invention The present invention relates to plant protection products. More specifi cally, the invention relates to a novel strain of the bacterial species Pseudomona chlororaphis and the use of compositions containing this bacterial strain o antibiotic substances produced by this strain in plant production in order t protect plants against attacks by phytopathogenic microbial agents. Background of the Invention Several agents of microbial nature with the ability to induce plant disease cause considerable damages, and accordingly economic losses, in crop plants Many of them attack leaves and/or other aerial plant parts and then usuall reach new uninfected crops by airborne spores. Others are transmitted from on crop generation to the next by being seedborne and several economically impor tant disease-inducing agents are soilborne and reside more or less inactive in th soil until a susceptible crop is grown. Procedures exerted for controlling microbial disease-inducing agents in cro production are often costly, but in most crop growing systems economicall necessary. One widely used method is treatment with biostatic or biocida chemicals. They are in most cases applied as sprays on growing crops, as seed o root treatments or as soil disinfectants. Other standard methods are breeding fo resistance and management of the cropping system itself. These standard control methods all have some drawbacks. Managing of th cropping system is effective or convenient only for certain disease problems. Als the breeding of crop plants for resistance is possible or suitable only in certai cases, may take long time and the resistance obtained may be broken after som time by the appearance of new strains of the pathogen. Chemical compounds ofte are highly effective, but they may give unwanted effects in the environment require careful handling as most are risky for human health and they also ma become ineffective where resistant pathogen strains develop. The use of biological control agents or biopesticides may be more effectiv or more preferable than the use of other control methods and, thus, such agent have been extensively tried. Several bacterial and fungal strains are known t inhibit growth of various microbial disease-inducing agents. To be effective an usable they have to be stable, give reproducible results in the field and ther must be possibilities to apply them under field conditions. To date few hav fulfilled these requirements and, thus, have been used as commercial products. Brief Description of the Invention The present invention provides a biological control agent useful an effective for controlling plant pathogen attacks in commercial plant growings. novel strain (MA 342) of the bacteria Pseudomonas chlororaphis showing th desired characteristics is provided. The isolate was deposited at the Nationa Collections of Industrial and Marine Bacteria Limited (NCIMB), Aberdeen Scotland on 14th February 1994 under the terms of the Budapest Treaty and ha received NCIMB Accession No. 40616. The invention also provides a plant disease controlling compositio containing as active ingredient the novel strain MA 342 or mutants thereof wit essentially the same characteristics or antipathogenically active metabolites o derivates thereof. Further, the invention provides a method of controlling plan diseases using the novel strain MA 342 or mutants thereof with essentially th same characteristics or antipathogenically active metabolites or derivates thereof Brief Description of the Drawing Figure 1: This figure shows the fatty acid profile obtained with the Microbia Identification System (MIDI, Newark Ltd., USA) of the bacterial isolate MA 342 Detailed Description of the Invention Below follows a characterization of the novel bacterial strain and description of preferred methods for strain proliferation and for formulations an applications in the field or in greenhouses. Several examples are offered to furthe illustrate, but not to limit, the method and composition of the invention. Characterization of the novel bacterial strain MA 342 Morphological characteristics: Colony morphology on TSA 10 (10 g Tryptic So Broth (Difco Ltd.); 12 g Technical Agar (Oxoid Ltd.) in 1000 ml distilled water) i round, white, moderately convex colonies that form well visible hyaline crystal in the agar at high cell densities. It is a Gram negative rod that shows a brigh yellow fluorescence on Kings medium B (1.5 g K 2 HP0 4 ; 1.5 g MgS0 4 -7 H 2 0; 2 g Proteose Peptone (Difco Ltd.); 10 ml glycerol, 15 g Bacto Agar (Difco Ltd.) i 1000 ml distilled water). Fatty acid analysis: The fatty acid profile of the bacterium is shown in figure 1. This analysis was performed using the Microbial Identification System (MIDI Ltd., Newark, USA), version 3.7. According to this test program, MA 342 is most similar to Pseudomonas chlororaphis, with a matching index of 0.705. Biochemical characteristics: Characteristics tested Reaction of isolate MA 342 in API 20 NE* rapid test Nitrate reduction Indole production Acid from glucose Arginine dihydrolase + Urease Esculin hydrolysis Gelatin hydrolysis B-galactosidase Glucose assimilation Arabinose assimilation Mannose assimilation Mannitol assimilation + N-acetyl-glucosamine assimilation Maltose assimilation Gluconate assimilation Caprate assimilation Adipate assimilation Malate assimilation + Citrate assimilation + Phenyl-acetate assimilation Cytochrome oxidase *API System Ltd., France Characteristics tested in additional tests Levan production Xylose assimilation .? Sorbitol assimilation + ? Preferred methods for strain proliferation and for formulations and applications in the field or in greenhouses Quantities of the active strain is best obtained by a fermentation proces that comprises inoculating a sample of a pure culture of the strain into a liqui shake culture or in a fermentor containing a suitable fermentation medium. Th strain may also be grown on a sterile surface, e. g. an agar surface, and whe grown out, the cells may be suspended in water or other liquid media known i the art. Growing media may in principle be any bacterial growth medium know in the art. The fermentation is carried out until a sufficient concentration of cells, e. g. about 5-10 cfu (colony forming units)/ml for liquid cultures, is obtained. The so obtained fermentation broth or bacterial suspension may be employed as suc for use in plant protection, or they may be treated or formulated before bein used. In one type of treatment the bacterial cells in the fermentation broth ma be killed, e. g. by heating, or centrifuged down and the resulting broth o supernatant, containing bacterial metabolites, may be used for plant protectio purposes, with or without prior purification and/or concentration. Bacterial suspensions and fermentation broths may also be homogeneously mixed with on or more compounds or groups of compounds known in the art, provided such compounds are compatible with the bacterial strains or its antipathogenicall active metabolites or derivates of these. Suitable compounds may be powder additives or solid carriers, such as talcum, kaolin, bentonite or montmorillonite, wettable powders known in the art, carbon source nutrients (such as glucose, sucrose and fructose) or complex bacterial nutrients (such as yeast extract, bacteriological peptone and tryptone), metal salts, salts from fatty acids, fatty aci ester's, ionic or non-ionic surfactants, plant nutrients, plant growth regulators, fungicides, insecticides, bactericides and the like. Bacterial suspensions and fermentation broths may also be dried or freeze-dried prior to or after being mix with suitable compounds and the resulting product used for plant protection. suitable way of drying is for example air drying of vermiculite supplied wit bacterial fermentation broth. Bacterial and metabolite preparations may be applied in any mann known for treating seeds, vegetative propagation units, plants and soil wit bacterial strains. Spraying, atomizing, dusting, scattering, pelleting, dipping pouring may be chosen in accordance with the intended objective and t prevailing circumstances. Advantageous rates of application for seed treatme are normally from 10 to 10 cfu/ha and for spraying 10 to 10 cfu/ha or corresponding amount of bacterial metabolites. Example 1 Isolation of the Microorganism MA 342 The dug up roots of the plant Empetrum nigrum were washed in sterile t water to remove adhering soil. From a young root a piece, 2-3 cm long, was c out and handled under sterile conditions. The piece was taken from the regio above the root tip area. Small cuts were made in the root piece with a flame scalpel. The root piece was then rubbed against the surface of TSA 10 agar. Aft bacteria had grown out, MA 342 was picked and was pure cultured on to TSA 1 Example 2 Preservation of the Microorganism MA 342 The Pure culture was deep frozen in small ampoules at -70°C. As free protecting agent were used 10% glycerol in tap water, pH adjusted to 7.15 aft autoclaving. After freezing at -70°C, the ampoules were stored at -20°C. For long term preservation the isolate was freeze dried. After growing f 48 hours on TSA 10 agar, the bacterial lawn was scraped off the agar surfac mixed with a freeze drying protecting agent (50 g Dextran T 70 (Pharmacia Fi Chemicals Ltd.); 50 g Na-L-glutamate (Kebo AB) in 1000 ml of distilled wate poured into small ampoules (20 ml) and put in a Hetosicc freeze drier (Heto Ltd Denmark) for 24 hours. After freeze drying the ampoules were gas tightly seal with rubber stoppers and stored at 4°C. Example 3 Effect of MA 342 against Microdochium nivale in primary greenhouse screenings The bacterium was applied to the seeds of wheat as follows: 24 hours ol cultures on TSA 10, grown at 15°C, were scraped off from the agar surface of 9 cm Petri dish and mixed with 40 ml of nutrient broth (SNB: 18 g sucrose; 5 bacterial peptone (Oxoid Ltd.); 2 g yeast extract (Oxoid Ltd.); 0.5 g K 2 HP0 4 an 0.25 g MgS0 4 '7 H 2 0 in 1000 ml distilled water and pH adjusted to 7.2-7.4) an 40 ml of a 2% (w/v) solution of sodium carboxymethyl cellulose (CMC) in steril distilled water. This mixture was poured over the seeds. After 20 minutes th excess mixture was poured off and the seeds were dried under a fan overnight. For each treatment in this greenhouse screening two pots were sown wit 50 seeds in each pot. The pots were 18 cm in diameter and 4 cm high and fille to two thirds with an unsterilized commercial peat mixture (Enhetsjord Normal), mixed with 20% (v/v) sand. The winter wheat seeds (cv. "Kosack") were artificially infested with M nivale prior to the treatment with bacteria. The pathogen was cultivated fo seven days in potato dextrose broth (24 g Potato Dextrose Broth (Difco Ltd.) pe 1000 ml distilled water) at room temperature on a rotary shaker. The resultin slurry was homogenized with a kitchen blender and poured over the seeds. Afte 30 minutes the liquid was poured off and the seeds were left to dry under a fa over night. Seeds thus infested were then treated with MA 342 and sown in pot as described above. After sowing, the pots were covered with glass lids and placed in the dar at 6°C. After five days the lids were removed, each pot watered with 100 ml o water and placed inside a five litres plastic bag that was supported by two woode sticks. The pots were then placed in a greenhouse at 12-15°C for eight days. The following treatments of seeds were tested: 1. P. chlororaphis strain MA 342, mixed with SNB/CMC, on M- nival e infested seeds 2. M- nivale-infested seeds, as disease control 3. Untreated seeds as healthy control The disease suppressive effect was recorded as the percentages of emerged healthy ( = without mycelia) plants out of those sown. Results from a typical M nivale primary screening is shown in table 1. Table 1. Effect of MA 342 on emergence and disease development in winter wheat, raised from M. nivale-infested seeds. Treatment Percentage (%) Percentage (%) healthy emergence plants out of those sown 1. MA 342 87 81 2. Disease control 13 7 3. Untreated control 90 89 Example 4 Effect of MA 342 against Drechslera teres in secondary greenhouse screenings For these screenings the isolate MA 342 was grown for 48 hours in hal strength (15 g/1) Tryptic soy broth (Difco Ltd.) on a rotary shaker in the dark at 18-20°C. Seeds of the barley cultivar "Golf", naturally infected with D. teres. were then mixed with 300 ml of the resulting bacterial suspension per kg seed in a plastic bag and, after mixing, the bag was shaken for about 4 minutes. Seeds thus treated were dried under a fan at room temperature for one day and then sown in pots as described in Example 3. The sown pots, three per treatment, were placed first in the dark at 6°C for seven days and then in a greenhouse at about 20°C as described in Example 3. For reading treatment effects the frequency of germinated plants and the frequency of plants with primary attack on the first leaf were counted. The bacterial effect was related to an untreated control and seeds treated with the fungicide Panoctine Plus 400 (guazatine 150 g/1 + imazalil 10 g 1), Rhόne-Poulenc Ltd., in a dosage of 4 ml per kg seed. Table 2. Effect of MA 342 and of Panoctine Plus 400 against D. teres i barley in a typical greenhouse secondary screening. Treatment Per cent germinated Per cent plants with plants attack on the first lea Control 92.5 13.0 Panoctine Plus 400, 4 ml/kg 95.5 0.0 MA 342, 300 ml/kg 96.1 0.0 Example 5 Effect of MA 342 on plant pathogens in field experiments Field experiments, designed as randomized blocks and with three to eigh repetitions, had plot sizes varying between experiments from 0.15 m (one T caries experiment) to about 15 m (most experiments). The experiments wer placed at different localities in Sweden and in most cases on loamy soils wit about 3 per cent humus content. Treatments of seeds with bacteria and with Panoctine Plus 400 were don as described in Example 4 above. After the treated seeds had been dried with fan they were stored at room temperature for various times before they were sow in field plots. All seeds, except those infested with T. caries, were naturall infested or infected with the various diseases tested. Seeds of winter wheat (cv "Kosack") were artificially infested with spores of T. caries by mixing 2 g crushe T. caries-ears with 1 kg wheat seeds. Effect of MA 342 on Tilletia caries The effect was read as the frequency of infected ears at time of ripenin Results obtained in two trials in 1991/92 and two trials in 1992/93 are shown i Table 3. The difference between bacterial treatment and the fungicide treatmen is significant in 1992/93. Table 3. Effect of MA 342 bacterial suspension against seed borne Tilleti caries infection. Treatment Per cent infected ears 1991/92 1992/93 Control 23 65 Panoct. 400*, 4 ml/kg 2 24 MA 342, 300 mg/kg 0 9 * Panoctine 400 (guazatine 150 g/1), Rhόne-Poulenc Ltd. Effect of MA 342 on Drechslera teres, D. Graminea, D. avenae and Ustilago avenae In the field experiments with these pathogens the number of germinate plants per m and the number of infected plants per m were measured and, i addition, in most of the experiments also i) grain yield ii) thousand kernel weigh and iii) weight per hectolitre. Effect on Drechslera teres: Results from field experiments conducted in 1991-199 and where effects against D. teres-infection in barley were tested are shown i tables 4, 5 and 6. Table 4. Results from four field experiments in barley infected wit Drechslera teres in 1991. Means from four experiments conducted a Alnarp, Lanna, Strangnas and Ultuna. Treatment Yield No. of Infected Hectolitre 1000-kerne kg ha plants/m plants/m weight, kg weight, g Control 4970 361 47 64.7 41.5 Pan. Plus 400, 4 ml 5390 353 1 66.3 43.8 MA 342, 300 ml/kg 5480 353 1 66.0 43.7 Table 5 Results from five fields experiments in barley infected with Drechs lera teres in 1992. Means from experiments conducted at Svalof Nygard, Kδlback, Ultuna and Rόbacksdalen. Treatment Yield No. of Infected Hectolitre 1000-keπιe kg/ha plants/m 2 plants/m 2 weight, kg weight, g Control 4290 358 48 67.9 51.7 Pan. Plus 400, 4 ml 4380 378 1 67.8 50.5 MA 342, 300 ml/kg 4300 380 1 68.3 52.6 Table 6. Results from two field experiments in barley infected with Drechs lera teres in 1993. Means from the experiments conducted at Kόl back and Ultuna. Treatment Yield Infected kg/ha plants/m Control 6310 74 Pan. Plus 400, 4 ml 6730 1 MA 342, 200 ml/kg 6720 5 Effect on Drechslera graminea: The results from experiments with D. graminea infected seeds conducted in 1991-1993 are shown in tables 7, 8 and 9. Table 7. Results from one field experiment in 1991 conducted in Uppsala i barley infected with D. graminea. Treatment Yield Infected kg/ha plants/m Control 3440 31 Pan. Plus 400, 4 ml 4160 2 MA 342, 300 ml/kg 4390 1 Table 8. Results from five field experiments conducted in 1992 in barle infected with D. graminea. Means from experiments conducted a Svalov, Nygard, Kδlback, Ultuna and Robacksdalen. Treatment Yield No. of Infected Hectolitre 1000-kerne o o kg ha plants/m plants/m weight, kg weight, g Control 2590 383 101 66.2 40.2 Pan. Plus 400, 4 ml 3470 381 5 66.6 39.9 MA 342, 300 ml/kg 3460 368 7 66.2 39.8 Table 9. Results from two field experiments in 1993 in barley infected wit D. graminea. Means from experiments conducted at Kόlback an Ultuna. Treatment Yield Infected kg/ha plants/m Control 2810 46 Pan. Plus 400, 4 ml 4160 1 MA 342, 200 ml/kg 3990 8 Effect on Drechslera avenae: The results from experiments with D. avenae infected oats seeds conducted in 1991-1993 are shown in tables 10, 11 and 12. Table 10. Results from one field experiment in 1991 conducted in Uppsala i oats ("Puhti") infected with D. avenae. Treatment Yield Infected kg/ha plants/m Control 4940 74 Pan. Plus 400, 4 ml 4860 32 MA 342, 300 ml/kg 5090 17 Table 11. Results from four field experiments in 1992 in oats ("Puhti " an "Vital") infected with D. avenae. Means from experiments conducte at Svalov and Ultuna. Treatment Yield No. of Infected Hectolitre 1000-kerne kg/ha plants/m 2 plants/m 9 weight, kg weight, g Control 3990 428 22 57.5 35.4 Pan. Plus 400, 4 ml 4080 456 13 57.8 35.5 MA 342, 300 ml/kg 4000 445 3 57.4 35.0 Table 12. Results from a field experiment in 1993 conducted in Uppsala i oats ("Vital") infected with D. avenae. Treatment Yield Infected kg/ha plants/m Control 7570 79 Pan. Plus 400, 4 ml 7870 14 MA 342, 200 ml/kg 7680 27 Effect on Ustilago avenae: In the field experiments with U. avenae bund ears pe m or percentage of bunt ears were read at the time of ripening. Grain yield wa not measured. Results from three experiments conducted 1991-1993 are shown i Table 13. Table 13. Results from three field experiments conducted in 1991-1993 i Uppsala in oats infected with Ustilago avenae. Treatment 1991 1992 1993 9 9 Bunt ears/m % infected ears Bunt ears/m Control 7 7 1 100,,66 95 Panoctine Plus 400, 4 ml 3 3 8 8,,77 not tested MA 342, 300 1 ' ml/kε 1 1 1,,77 15 11 300 ml 1991 and 1992; 200 ml in 1993. Example 6 Application of MA 342 to seeds and other plant parts Applying aqueous mixtures containing MA 342 to seeds. Bacterial suspension produced as described in example 3 or as described in example 4 above wer mixed with each of the following substances or compounds: Talcum powder (Kebo Lab AB), 48 g/1 of bacterial suspension Bacteriological peptone (Oxoid, Ltd.), 5 g/1 of bacterial suspension Tween 20 (Merck Ltd.), 20 ml/litre of bacterial suspension Metocel (Cellulose ether, Sveda Kemi AB), 12 g per litre of bacteria suspension Lissapol (ICI Agrochemicals Ltd.), 1 g per litre of bacterial suspension Bond (Newman Agrochemicals Ltd.), 1 g per litre of bacterial suspension In other experiments the bacterial suspensions were centrifuged at 10,00 x g for about 10 min. and the resulting pellets were resuspended in either 0.1 MgS0 or in peptone water (5 g of bacteriological peptone (Oxoid, Ltd.) per litr of tap water). After thoroughly mixing, the resulting suspensions were applied to seed as described in example 4 for unmixed bacterial suspensions. Applying freeze-dried bacteria to plant seeds. MA 342 bacteria, grown in a shak culture as described in example 4 above, were centrifuged and the resulting pelle was resuspended in a skim milk solution (200 g skim milk powder, Semper AB Sweden, per litre of sterile distilled water) as a freeze drying protecting agen The mixture was shell frozen in glass jars and then freeze dried in these jars f about 48 hours in a Hetosicc freeze drier (Heto Ltd., Denmark). The resultin powder was stored at 4°C in plastic bags or in plastic flasks with a screw ca until used. For seed application the powder was either mixed in water or in oth aqueous solutions and then applied to seeds as described in example 4 f bacterial suspensions, or it was mixed with seeds in a dry condition by thoroughl shaking the powder and the seeds (about 10 g powder per kg seed) in a plast container. Pelleting seeds treated with MA 342. MA 342 bacteria, grown in a shake cultu as described in example 4 above, were mixed in volumes 1:1 with an adherent (2 w/v aqueous solution of sodium carboxy-methyl cellulose or 50% w/v aqueo solution of gummi arabicum). The seeds were treated with this mixture as i example 4 above. An excess amount of bentonite (Dresser Minerals Inc.) or talcu powder (Kebo Lab AB) was then added to the plastic bag, the bag was inflate and vigorously shaken for a couple of minutes. After this the seeds were sprea out on big trays under a fan and allowed to dry in room temperature. Spraying plant shoots with bacterial suspensions. MA 342 bacteria, grown in shake culture as described in example 4 above, were filled in plastic han sprayers or in a powered sprayer and then sprayed on to plant leaves and shoot In other treatments the bacteria were first centrifuged at about 10.000 x g for te minutes, the pellet was resuspended in tap water and this resulting bacteri suspension was used for the spraying of plant leaves and shoots. Example 7 Effects of purified metabolites from MA 342 ' against diseases caused by Drechslera teres in greenhouse experiments. The isolate MA 342 was grown for 48 hours in half strength (15 g/1) Tryptic so broth (Difco Ltd.) on a rotary shaker in the dark at 18-20°C. The resultin bacterial suspension was centrifuged at 48,000 g for 30 min. and metabolites i the supernatant was then further purified with Sep-pak C 18 cartridges (Water Associates) as follows: 1. 80 ml of supernatant was added to a Sep-pak activated with 10 methanol. 2. The Sep-pac was washed first with 5 ml 30% ethanol and then with 5 40% ethanol. 3. The metabolites were eluted by 5 ml 70 % ethanol. 4. The 70% ethanol-eluate was evaporated in a rotation-evaporator unt about 1.5 ml water solution was left. It was then diluted with tap water u to a volume of 6.5 ml. Seeds of the barley cultivar "Golf , naturally infected with D. teres. were su merged in this 6.5 ml water solution of the metabolites for 30 minutes and wer then sown in pots with 50 seeds per pot. The pots were covered with glass lids an placed in the dark at 6°C. After nine days the lids were removed and the pot were placed in a greenhouse at 15-22°C for about two weeks. Germinated plant and disease attacks were then read as described in Example 4 above. As control were used 1) untreated seeds and 2) seeds treated with supernatant not purifie with Sep-pac and containing MA 342 cells. Table 14. Effect of purified metabolites from MA 342 and supernatant co taining MA 342 cells against D. teres in barley in a typical gree house testing. Treatment Per cent germinated Per cent plants with plants attack on the first leaf Untreated control 97,0 21,6 Supernatant with MA 342 cells 99,0 1,0 Evaporated cell-free eluate 99,0 0,0 Example 8 Results of comparative tests of MA 342 isolate and 11 other isolates of Pseudomonas chlororaphis received from different culture collections Eleven different non-Swedish isolates of Pseudomonas chlororaphis having th designations stated in Table 1 were tested, together with MA 342 isolate, f effect against leaf spot disease in barley and for inducing reactions in biochemic tests according to the test system API 20 NE. In addition, the isolates wer compared for colony appearance and crystal formation on agar plates. The 11 no Swedish isolates were from different countries and are deposited in four differen well-reputed culture collections (Table 1). Test of disease-inhibiting ability in greenhouse tests The tests were conducted with Drechslera teres-infected barley, as described i Example 4, and all isolates were tested simultaneously i order to achieve a adequate comparison. As is evident from Table 1, the results show that MA 34 is clearly unique in the sense that no one of the other isolates tested herein ha the property like MA 342 in this kind of test, viz. the ability to inhibit infectio by Drechslera teres. Table 1. Designations, country of origin and effects of MA 342 and 11 othe P. chlororaphis isolates against D. teres-infection in barley i greenhouse tests. Isolate designation Country of Effect on leaf spot in greenhouse tests. Percentage of infected plant after treatment with the isolate in question MA 342 Sweden 9 USDA B2075 Czechoslovakia 53 USDA B1869 New Zealand 60 USDA B14874 USA, Colorado 56 USDA B14869 USA, Illinois 58 USDA B1854 USA, Loisiana 43 NCTC 10686 England 54 NCTC 7357 England 58 DSM 6508 Germany 56 ATCC 9446 USA 61 ATCC 17414 USA 50 ATCC 17811 USA 58 Untreated control 71 Induction of reactions in biochemical tests according to test system API 20 NE The tests were carried out as described above. The results are shown in Table They show that MA 342 also in this respect is unique can be differentiated fro the other 11 isolates tested. Some of the isolates tested may not, according to th test, be considered to be central within the species Pseudomonas chlororaphis. Table 2. Induced reactions by the different isolates tested in a number biochemical tests according to the test system API 20 NE. A " + represents a positive reaction and a "-" no/negative reaction. 0 Property tested Isolate tested in API 20 NE test MA B B B B B NCTC NCTC DSM ATCC ATCC AT 342 2075 1869 14874 14869 1854 10686 7357 6508 9446 17414 178 Nitrate reduction Indole production Acid from glucose Arginine di ydrolase + Urease Esculin hydrolysis Gelatin hydrolysis + B-galactosidase Glucose assimilation + + Arabinose assimil. + Mannose assimilation + + + + + + + Mannitol assimilation + + + + + + + + + Glucosamine assimil. + + + + + + + Maltose assimilation Gluconate assimilation + + + + Caprate assimilation + + + Adipate assimilation Malate assimilation + Citrate assimilation + + + + + + + + + + Phenyl-acetate assimil. - + + + + + + - - + Cytochrome oxidase + + + + + + + + + Comparison of colony appearance and crystal formation on agar plate The isolates were cultured in Petri dishes on TSA 10, as described above. W observed small differences in colony appearance between all different isolates bu all isolates could not be differentiated in this way. However, the MA 342 isolat was the only isolate forming typical hyaline crystals in the agar and coul therefore, by this property, be differentiated from all other isolates.

Claims: AMENDED CLAIMS [received by the International Bureau on 4 September 1995 (04.09.95) ; original claims 1 -19 replaced by amended claims 1 -18 (2 pages ) ] 1. A Pseudomonas chlororaphis strain deposited at The National Collections of Industrial and Marine Bacteria Limited, Aberdeen, Scotland with the NCIMB accession number 40616 or biologically pure cultures thereof having the ability 5 to produce antipathogenically active metabolites, or mutants thereof having essentially the same characteristics as the parent strain. 2. A composition for controlling plant diseases caused by pathogenic fungi, characterized by containing as active ingredient the microorganism claimed in claim 1 or culture broth thereof containing antipathogenically active metabolites 10 thereof. 3. The composition according to claim 2, characterized in that the pathogen is the fungus Drechslera teres. 4. The composition according to claim 2, characterized in that the pathogen is the fungus Drechslera graminea. 15 5. The composition according to claim 2, characterized in that the pathogen is the fungus Drechslera avenae. 6. The composition according to claim 2, characterized in that the pathogen is the fungus Microdochium nivale. 7. The composition according to claim 2, characterized in that the pathogen 20 is the fungus Tilletia caries. 8. The composition according to claim 2, characterized in that the pathogen is the fungus Ustilago avenae. 9. The composition according to claims 2-8, characterized in that the active ingredient is in admixture with a carrier composition acceptable in agricultural 25 practice. 10. The composition according to claim 9, characterized in that the active ingredient is in admixture with a liquid carrier. 11. The composition according to claim 9, characterized in that the active ingredient is impregnated in a solid porous material. 12. The composition according to claim 9, characterized by further comprising additives that serve as adherents. 13. The composition according to claim 9, characterized by further comprising a nutrient source. 14. A method of controlling plant diseases caused by pathogenic fungi and comprising the introduction of an effective dose of an active ingredient into the environment where the disease-inducing agent is to be inhibited, characterized in that the active ingredients is the microorganism claimed in claim 1 or culture broth thereof containing antipathogenically active metabolites or derivates thereof. 15. The method according to claim 14, characterized in that the active ingre¬ dient is applied to seeds. 16. The method according to claim 14, characterized in that the active ingre¬ dient is applied to plant vegetative propagation units. 17. The method according to claim 14, characterized in that the active ingre¬ dient is applied to plants. 18. The method according to claim 14, characterized in that the active ingre- dient is applied to a growing medium in which a plant is growing or is to be grown. STATEMENT UNDER ARTICLE 19 In order to define distinctly the claimed scope of invention claim 1 has been amended by combination of the subject matter of the original claim 1 with the subject matter of the original claim 2. Further, claim 2, original claim 3, has been supplemented with a statement that the composition also may contain a culture broth of the microorganism containing antipathogenically active metabolites, with reference to Example 7. It is respectfully submitted that the amended claims refer to a patentable invention in relation to the prior art cited in the Search Report, in view of the unambiguous results presented in Example 8 providing results of comparative tests.

Inventors: GERHARDSON BERNDT, GUSTAFSSON ANNIKA, HOEKEBERG MARGARETA, JERKEMAN TIIU, JINGSTROEM BRITT-MARIE, JOHNSSON LENNART, GERHARDSON, BERNDT, GUSTAFSSON, ANNIKA, HOEKEBERG, MARGARETA, JERKEMAN, TIIU, JINGSTROEM, BRITT-MARIE, JOHNSSON, LENNART
Applicants: GERHARDSON BERNDT, GUSTAFSSON ANNIKA, HOEKEBERG MARGARETA, JERKEMAN TIIU, JINGSTROEM BRITT MARIE, JOHNSSON LENNART, SVENSKA LANTMAENNEN RIKSFOERBU, GERHARDSON, BERNDT, GUSTAFSSON, ANNIKA, HOEKEBERG, MARGARETA, JERKEMAN, TIIU, JINGSTROEM, BRITT-MARIE, JOHNSSON, LENNART, SVENSKA LANTMAENNEN, RIKSFOERBUND EK.FOER.
Application year: 1995
Main classification: A01N63
IPC codes: A01N63, C12N1, C12R1