Background of the Invention Plant seeds and seedlings are in the early stage after seed germination very sensitive to attacks from different soil-borne fungal plant pathogens, especially from the soil-borne complex of Pythium species that includes a group of fungi which causes severe crop losses and economical drawbacks. Pythium species are among the most succesful microbial colonists in agricultural and horticultural soils. Probably all cultivated soils in the world contains spores from at least one Pythium species. The Pythium species group of fungi are often necrotrophic parasites of roots of crops but they can also grow saprophytically.

Presently, there are a few commercial fungicides used on the market.

Chemical fungicides often 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 may become ineffective where resistent pathogen strains develop.

The use of biological control agents or biopesticides may be more effective or more preferable than the use of other control methods and, thus, such agents have been extensively tried. Several bacterial and fungal strains are known to inhibit growth of various microbial disease-inducing agents. To be effective and usable they have to be stable, give reproducable results in the fields and there must be possibilities to apply them under field conditions. To date few have fulfilled these requirement and, thus, have been used as commercial products.

One such product is based upon the use of the Pseudomonas chlororaphis strain MA342 (NCIMB 40616) for controlling seed-borne plant fungal diseases (PCT-application W095/28085 published on October 26,1995).

Brief Description of the Invention The present invention provides a novel biocontrol agent useful and effective for controlling plant pathogenic attacks in commercial crops,

characterized by being an isolate or isolates of one or more of the bacterial strains belonging to the group containing the Pseudomonas spp. strains Ki72, Ab 131, Ki 353,1IF416 and Ma 358 deposited at The National Collections of Industrial and Marine Bacteria Limited (NCIMB), Aberdeen, Scotland and having received the NCIMB accession numbers 40936,40940,40941,40942 and 41010, respectively. Said isolates were deposited under the terms of the Budapest Treaty.

The invention also provides a plant disease controlling composition comprising, as active ingredient, a biocontrol agent being an isolate or isolates of one or more of the bacterial strains specificly mentioned above or a biologically pure culture thereof, or a culture broth thereof containing one or more of said isolates or antipathogenically active metabolite (s) thereof, or the antipathogeni- cally active metabolite (s) as such. Further, the invention provides a method of controlling plant diseases caused by pathogenic fungi using one or more of the bacterial strains of the invention. The use of the biocontrol agent of the invention for controlling plant diseases caused by pathogenic fungi is also provided.

Brief Description of the Drawings Figure 1 shows the effect of the bacterial strains Ab 131, MF 416 och Ki 72 against seed and seedling diseases caused by Pythium ultimum. DI = disease index (0 = healthy and 4 = 100 % infected roots).

Figure 2 shows the effect of bacterial strains Ab 131 and Ki-72 and fungicides on the relative yield of sugar under field conditions in 1996.

Fungicides: Eup = Euparen (Tolyfluanid) Tach = Tachigaren (Hymexazol), Insecticide: Montur = Imidacloprid + Tefluthrin Figure 3 shows the effect of bacterial strains Ki 72 and Ki 353 and fungicides on the relative yield of sugar under field conditions in 1997.

Fungicides: Tach. = Hymexazol Eup. = Tolyfluanid Insecticide:

Montur = Imidacloprid + Tefluthrin Figure 4 shows the effect of bacterial strains Ki 353 and MF 416 and fungicides on the number of plants under field conditions in 1998.

Fungicides: Hymexazol TMTD = Tiram Insecticide: Montur = Imidacloprid + Tefluthrin Detailed Description of the Invention The present invention relates, in one aspect, to a novel biocontrol agent for controlling plant diseases caused by pathogenic fungi, characterized by being an isolate or isolates of one or more of the bacterial strains belonging to the group containing thePseudomonas spp. strains Ki72, Abl31, Ki 353, MF416 and Ma 358 deposited at The National Collections of Industrial and Marine Bacteria Limited (NCIMB), Aberdeen, Scotland and having received the NCIMB accession numbers 40936,40940,40941,40942 and 41010, respectively, or being a biologically pure culture thereof or a mutant thereof having essentially the same characteristics as the parent strain.

According to a further aspect, the invention relates to a composition for controlling plant diseases caused by pathogenic fungi, characterized by con- taining, as active ingredient, a biocontrol agent as defined above or containing a culture broth thereof containing the antipathogenically active metabolite (s) thereof or containing the antipathogenically active metabolite (s) as such.

In one preferred embodiment of the invention, the plant to protect from pathogens is sugar beet.

The pathogenic fungus is preferably one belonging to the Pythium sp. or Aphanomyces sp. groups of fungi, particularly Pythium ultimum.

However, the biocontrol agent and composition of the invention is also contemplated for use in other commercial crops such as vegetables, e. g. tomato, particularly such crops attacked by the Pythium sp. group of fungi.

A further aspect of the invention relates to a method for controlling plant diseases caused by pathogenic fungi and comprising the introduction of an effective dose of an antipathogenically active biocontrol agent into the environ- ment where the disease is to be suppressed, the method being characterized by

applying a biocontrol agent or a composition as defined above.

The biocontrol agent of the invention can be applied to seeds, seedlings, plant vegetative propagation units, plant or plant parts or soil.

According to yet another aspect of the invention, the use of the biocontrol agent or composition, as defined above, is provided for controlling plant diseases caused by pathogenic fungi, particularly the Pythium sp. group of fungi.

Below follow characterizations of the novel bacterial strains of the inven- tion and a description of preferred methods for strains proliferation and for formulations and applications in the field or in greenhouses. Several Examples are offered to further illustrate, but not to limit in scope, the method and composition of the invention.

Characterization of the novel bacterial strains The strains may be characterized using biochemical tests as shown in Table 1. Isolat NO TR GL AD UR ESC GEL PN GL A M MAN NA MA GNT CAP AD ML CIT PAC OX p IH E U R G L I T Ab - - - + - - + - + + + + + - + + - + + - + 131 Ki 72 - - - + - - + - + + + + + - + + - + + - - MF + + + + + ++ + + + + + + + 353- Isolat NO, TR GL ADH UR ESC GEL PN -p I E'U L I T Ma 358 - - - + - - + - + + + + + - + + - + + - + Tab. 1. Reaction of strains in API 20 NE rapid test where the denotations in the table are as below.

N03 = Nitrate reduction TRP = Indole production GL = Acid from glucose ADH = Arginine dihydrolase URE = Urease ESC = Esculin hydrolysis

GEL = Gelatin hydrolysis PN = B-galactosidase GLU = Glucose assimilation ARA = Arabinose assimilation M = Mannose assimilation MAN = Mannitol assimilation NAG = N-acetyl-glucosamine assimilation MAL = Maltose assimilation GNT = Gluconate assimilation CAP = Caprate assimilation ADI = Adipate assimilation MLT = Malate assimilation CIT = Citrate assimilation PAC = Phenyl-acetate assimilation OX = Cytochrome oxidase Preferred methods for strain proliferation and for formulations and applications in the field or in greenhouses Quantities of the antipathogenically active strain is best obtained by a fermentation process that comprises inoculating a sample of a pure culture of the strain into a liquid shake culture or in a fermentor containing a suitable fermen- tation medium. The strain may also be grown on a sterile surface, e. g. an agar surface, and when grown out, the cells may be suspended in water or other liquid media known in the art. Growing media may in principle be any bacterial growth medium known in the art. The fermentation is carried out until a sufficient concentration of cells, e. g. about 5-109 cfu (colony forming units)/ml for liquid cultures, is obtained. The so obtained fermentation broth or bacterial suspension may be employed as such for use in plant protection, or they may be treated or formulated before being used.

In one type of treatment the bacterial cells in the fermentation broth may be killed, e. g. by heating, or centrifuged down and the resulting broth or supernatant, containing bacterial metabolites, may be used for plant protection purposes, with or without prior purification and/or concentration. Bacterial suspensions and fermentation broths may also be homogeneously mixed with one or more compounds or groups of compounds known in the art, provided such

compounds are compatible with the bacterial strains or its antipathogenically active metabolites or derivates of these. Suitable compounds may be powdery 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 acid esters, 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 mixed with suitable compounds and the resulting product used for plant protection. A suitable way of drying is for example air drying of vermiculite supplied with bacterial fermentation broth.

Bacterial and metabolite preparations may be applied in any manner known for treating seeds, vegetative propagation units, plants and soil with bacterial strains. Spraying, atomizing, dusting, scattering, pelleting, dipping or pouring may be chosen in accordance with the intended objective and the prevailing circumstances. Advantageous rates of application for seed treatment are normally from 10ll to 1012 cfu/ha and for spraying 10 12 to 1014 cfu/ha or a corresponding amount of bacterial metabolites.

Example 1 Isolation of the Bacterial Strains of the Invention The dug up roots of different plant species were washed in sterile tap water to remove adhering soil. From a young root a piece, 2-3 cm long, was cut out and handled under sterile conditions. The piece was taken from the region above the root tip area. Small cuts were made in the root piece with a flamed scalpel. The root piece was then rubbed against the surface of TSA 10 agar (Tryptic Soy Agar. Oxoid Ltd., 10 g/litre). After bacteria had grown out on an agar plate incubated at 0 °C for nine days, bacteria were picked and were pure cultured on to TSA 10 at 5 OC for five days.

Example 2 Preservation of the Bacteria The pure culture was deep freezed in small ampoules at-70°C. As freeze support 10% glycerol were used in tap water, pH adjusted to 7.15 after auto-

claving. After freezing at-70°C, the ampoules were transferred to-20°C.

For long term preservation the isolate was freeze dried. After growing for 48 hours on"TSA 10"agar (Tryptic Soy Agar, Oxoid Ltd., 10 g/litre), the bacterial lawn was scraped off the agar surface, mixed with a freeze drying support (Dextran T70, Pharmacia Fine Chemicals Ltd., 50 g; Na-L-glutamate, Kebo AB, 50 g; and 1000 ml of distilled water), 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 sealed with rubber stoppers and stored at 4°C.

Example 3 Primary Screenings for Activity against Pythium Inoculumpreparation: Pythium ul, imum was isolated from infected sugar beet roots and grown on"PDA" (Potato Dextrose Agar). When the agar plate was colonized by the fungus it was macerated into a pot (13 x 12 x 7 cm) filled to two thirds with a (unsterilized) commercial peat mixture (Enhetsjord K Normal), mixed with 20% (v/v) of sand. 50 sugar beet seeds were sown in the pot at 1 cm depth. The pot was placed in a climate chamber under the following conditions: 16 hours of light at 17°C followed by 8 hours of darkness at 14°C. After 4 weeks the soil in the pot was used as an inoculum (0.2% w/w) to infest soil for primary screening and trials.

Bacteria production: The bacteria were applied to the seeds of sugar beets as follows: 24 hours old cultures on"TSA 10", grown at 15°C, were scraped off from the agar surface of a 9 cm Petri dish and mixed with 15 ml of tap water. This mixture was poured over the seeds. After 30 minutes the excess mixture was poured off and the seeds were dried over niaht.

In primary screenings, 16 seeds were sown in pots placed under the same conditions as described above. After 4 weeks, the number of plants was counted and roots were scored for infection by Pythium.

Effects of the bacterial strains on Pythium in greenhouse tests.

The bioassays were performed in a climate chamber with the same growing conditions as above. In Figure 1 the disease control effect of the bacterial strains is shown for control (untreated seeds sown in Pythium ultimum infested

soil), healthy soil (untreated seeds sown in healthy soil) and for seeds treated with bacterial strains.

Effects of the bacterial strains on Pythium in field trials.

Example In 1996 and 1997 the bacterial strain Ki 72 and Ab 131 were tested under field conditions and in 1998 the bacterial strain MF 416 and Ki 353 were tested under field conditions. A high loading of fungal pathogens was indicated by soil tests. The field trials were conducted at six different locations and with 4 repetitions in randomized block design at each location. Plot size in all trials were 6 rows x 12 meter. In Figure 2 the relative yield of sugar is shown for control, two treatments with bacterial strains + insecticide and one treatment with two different fungicides + insecticide.

In figure 3 the effect of bacterial strains Ki 72 and Ki 353 and fungicides on the relative yield of sugar under field conditions in 1997 is shown.

In figure 4 the effect of bacterial strains Ki 353 and MF 416 and fungicides on the relative yield of sugar is shown for control, two fungicides + insecticide, Ki 353, Ki 353 + insecticide, MF 416, MF 416 + insecticide.

Effect of bacterial strains and fungicides on the relative yield of sugar in field trials in the years 1996,1997 and 1998 The effect of bacterial strains and fungicides on the relative yield of sugar in field trials in the years 1996,1997 and 1998 is shown in Table 2 below.

Table 2

Treatment 1996 1997 1998 Sugar Yield rel Sugar Yield rel Sugar Yield rel Untreated control 100 100 100 Eup. + Tach. + 106 111 119* Montur Ab 131 + Montur 103 L 18--107 L 18 + Montur--109 Ki 72 + Montur 105 106- MF 416--114 MF 416 + Montur--114 Ki 353--112 Ki 353 + Montur 107 111 121 Ma 358--120 Ma 358 + Montur-109 115 * Eup. replaced with TMTD Fungicides: Eup = Euparen (Tolyfluanid), Tach = Tachigaren (Hymexazol), TMTD = Tiram Incecticide: Montur = Imidacloprid + Tefluthrin -= Not tested Depositions of Microorganisms The following depositions have been made under the terms of the Budapest Treaty: Microorganism Depository Authority Deposition Date Accession No.

Ki 72 NCIMB 30 March 1998 40936 Ab 131 NCIMB 30 March 1998 40940 Ki 353 NCIMB 30 March 1998 40941 MF 416 NCIMB 30 March 1998 40942 Ma 358 NCIMB 10 March 1999 41010