SE521706C2 - New biocontrol agent to control plant diseases caused by pathogenic fungi, composition, method and use thereof to control plant diseases - Google Patents

Abstract

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, Ab131, 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. Said isolates were deposited under the terms of the Budapest Treaty. The invention also provides a plant disease controlling composition and a method of controlling plant deseases caused by pathogenic funghi using one or more of the bacterial strains of the invention.

Description

N) fl are of the bacterial strains belonging to the group belonging to the seudo, »often the strains Ki72, Ab131, Ki353 and MF416, which have been deposited with" The National Collections of Industrial and Marine Bacteria Limited "(NCIMB), Aberdeen Scotland and has received NCIMB Deposit Nos 40936, 40940, 40941 and 40942 respectively. The said isolate has been deposited under the terms of the Budapest Treaty. " or fl are isolates of one or fl of the above-mentioned bacterial strains or a biologically pure culture thereof or a culture mash thereof, which contains one or more of said isolates or antipathogenically active / active methods / metabolites which Furthermore, the invention provides a bolite / metabolites thereof or antipathogenically active metabolic method for controlling plant diseases which are caused of pathogenic fungi, using one or more of the bacterial strains according to the invention. The use of the biocontroller according to the invention to control plant diseases, caused by pathogenic fungi, is also provided.

Brief Description of the Drawing Figures Figure 1 shows the effect of bacterial strains Ab 131, MF416 and Ki72 on seed and seedling diseases caused by Pythium ultimum. SI = disease index (0 = healthy and 4 = 100% infected roots).

Figure 2 shows the effect of the bacterial strains Ab131 and Ki72 and fungicides on the relative yield of sugar under field conditions in 1996.

Fungicides: Eup = Euparen (toly fl uanide) Tach = Tachigaren (hymexazole).

Insecticide: Montur = imidacloprid + tea fl utrin Figure 3 shows the effect of the bacterial strains Ki72 and Ki353 and fungicides on the relative yield of sugar under field conditions in 1997.

Fungicides: Tach. = hymexazole Eup. = toly fl uanide 10 15 20 25 30 3521 -706 Insecticide: lviuritur '- imidaclopfid + tea fl ütfill Figure 4 shows the effect of bacterial strains Ki353 and MF416 and fungicides on the number of plants under field conditions in 1998.

Fungicides: Hymexazole TMTD = tiram Insecticide: Montur = imidacloprid + teflutrin 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, which agent is one or more isolates of a or fl your bacterial strains belonging to the group comprising Pseudomonas spp. strains Ki7 2, Ab131, Ki353 and MF416, which have been deposited with "The National Collections of Industrial and Marine Bacteria Limited" (NCIMB), Aberdeen, Scotland and where NCIMB the deposit numbers 40936, 40940, 40941 and 40942, respectively, or is a biologically pure culture thereof or a mutant thereof having substantially 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, which composition is characterized in that it contains as active ingredient a biocontrol agent, as defined above, or contains a culture mash thereof, which contains the antipathogenically active the metabolite (s) thereof or contains the antipatogenically active metabolite (s) as such.

According to a preferred embodiment of the invention, the plant to be protected from pathogens is sugar beet.

The pathogenic fungus is preferably one belonging to the Pythium sp. Or Aphanomyces sp. Groups of fungi, in particular Pythium ultimum.

However, the biocontrol agent and the composition according to the invention are also intended for use in other commercial crops such as vegetables, for example tomato, in particular those crops which are infested by fungi belonging to the Pythium sp. Group. 10 20 lvl-l ._à \ ~ J @ Q \ i. . . . . A further aspect of the invention relates to a method for controlling plant diseases caused by pathogenic fungi, which method comprises introducing an effective dose of an antipathogenically active biocontroller in the environment in which the disease is to be suppressed, wherein the method is marked by the use of a biocontroller or a composition as defined above.

The biocontroller according to the invention can be applied to seeds, seedlings, vegetative propagating units of the plant, plants or plant parts or soil.

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

The following is a characterization of the new bacterial strains according to the invention and a description of preferred methods for stem propagation and for preparations and applications in the field or in greenhouses. Several embodiments are given for the purpose of illustrating, but not limiting, the scope of the invention to the method and composition of the invention.

Characterization of the new bacterial strains The strains can be characterized using biochemical tests, as shown in Table 1.

Table 1. Reaction of strains in API 20 NE rapid test where the designations in the table are as follows.

NO = nitrate reduction TRP = indole production 10 15 20 25 30 CI! GL = acid from glucose ADI-i = arginine dihydrolase URE = urease ESC = esculin hydrolysis GEL = gelatin hydrolysis PN = β-galactosidase GLU = glucose assimilation ARA = arabinose assimilation M = mannose assimilation MAN = mannitol assimilation NAG = M-assimilation = caprate assimilation ADI = adipate assimilation MLT = malate assimilation CIT = citrate assimilation PAC = phenyl acetate assimilation OX = cytochrome oxidase Preferred methods for stem proliferation and for field or greenhouse preparations and applications graft a sample of a pure culture of the strain into a surface shaking culture or into a fermentor containing a suitable fermentation medium. The strain can also grow on a sterile surface, for example an agar surface, and when it has grown out, the cells can be suspended in water or some other liquid medium known in the art. Growth media can in principle be any bacterial growth medium known in the art. The fermentation is carried out until a sufficient concentration of cells has been obtained, for example about 5x10 9 cfu (colony forming units) / ml for liquid cultures. The fermentation mash or bacterial suspension thus obtained can be used as such for use in plant protection or they can be treated or prepared before use. In a type of treatment, the bacterial cells in the fermentation broth may be killed, for example by heating, or centrifuged, and the resulting mash or supernatant containing bacterial metabolites may be used for plant protection purposes, with or without prior purification and / or concentration. Bacterial suspensions and fermenters can also be mixed homogeneously with one or more compounds or groups of compounds known in the art, provided that such compounds are compatible with the bacterial strain or its antipathogenically active metabolites or derivatives thereof.

Suitable compounds may be powdered additives or solid carriers such as talc, kaolin, bentonite or montmorillonite, wettable powders known in the art, carbonaceous nutrients (such as glucose, sucrose and fructose) or complex bacterial nutrients (such as yeast extract, bacteriological peptone and tryptone). , metal salts, salts of fatty acids, fatty acid esters, ionic or non-ionic surfactants, plant nutrients, plant growth regulators, fungicides, insecticides, bactericides and the like. Bacterial suspensions and fermenters can also be dried or freeze-dried before or after mixing with suitable compounds and the resulting product used for plant protection.

A suitable way of drying is, for example, air drying of vermiculite with added bacterial fermentation mash.

Bacterial and metabolite preparations can be applied in any manner known for the treatment of seeds, vegetative propagating units, plants and soil with bacterial strains. Spraying, spraying, powdering, spreading, pickling, dipping or casting can be selected in accordance with the intended purpose and prevailing circumstances. Advantageous dosages for seed treatment are normally from 1011 to 1012 cfu / ha and for spraying 1012 to 1014 cfu / ha or a corresponding amount of bacterial metabolites.

Example 1 Isolation of the bacterial strains according to the invention Excavated roots of various plant species were washed in sterile tap water to remove adhering soil. A 2-3 cm long piece was cut from a young root, which was handled under sterile conditions. The piece was taken from the region over the root tip area.

Small incisions were made in the root piece with a low-treated scalpel. The root piece was then rubbed against the surface of TSA 10 agar ("Tryptic Soy Agar", Oxoid Ltd., 10 g / liter). After the bacteria had grown on an agar plate, incubated at 70 ° C for nine days, the bacteria were picked and purified on TSA 10 at 5 ° C for five days.

Example 2 Preservation of the bacteria The pure culture was frozen in small ampoules at -70 ° C. As antifreeze, 10% glycerol was used in tap water, the pH was adjusted to 7.15 after autoclaving. After freezing at -70 ° C, the ampoules were stored at -20 ° C.

For long-term preservation - the isolate was shaken dry. After growing for 48 hours on TSA 10 agar ("Tryptic Soy Agar", Oxoid Ltd., 10 g / liter), the bacterial mat was scraped from the agar surface, mixed with a lyophilizer [50 g "Dextran T 70" (Pharmacia Fine Chemicals Ltd). 50 g Na-L-glutamate (Kebo AB) in 1000 ml distilled water], poured into small ampoules (20 ml) and placed in a "Hetosicc" freeze dryer (Heto Ltd., Denmark) for 24 hours. The ampoules were sealed gas-tight with rubber stoppers and stored at 4 ° C.

Example 3 Primary screening for activity against Pythium Inoculate production: Pythium ultimum was isolated from infected sugar beet roots and allowed to grow on "PDA" (Potato Dextrose Agar). Once the agar plate had been colonized by the fungus, it was subjected to maceration in a pot (13 x 12 x 7 cm), filled to two thirds with a (unsterilized) commercial peat mixture (Unit soil K normal), mixed with 20% (v / v) sand. 50 sugar beet seeds were sown in the pot at a depth of 1 cm. 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% by weight) to infect soil for primary screening and testing.

Bacterial production: The bacteria were applied to the sugar beet seeds as follows: 24 hour old cultures on "TSA 10", grown at 15 ° C, scraped from the agar surface in 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 allowed to dry overnight. 10 15 20 1.1 .r O) In primary screening experiments, 16 seeds were sown in pots, which were kept under the same conditions as described above. After 4 weeks, the number of plants was counted and the roots were graded for Pythium infection.

Effects of the bacterial strains on Pythium in greenhouse experiments The bioanalyses were performed in a climate chamber with the same plant conditions as above. Figure 1 shows the disease-controlling effect of the bacterial strains for control (untreated seeds sown in Pythium ultimum-infected 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.

Examples In 1996 and 1997 the bacterial strains Ki72 and Ab131 were tested under field conditions and in 1998 the bacterial strains MF416 and Ki353 were tested under field conditions. A high load of fungal pathogens was indicated during soil tests. The field trials were performed at six different locations and with 4 repetitions as randomized blocks at each location. The parcel size in all experiments was 6 rows x 12 meters. Figure 2 shows the relative yield of sugar for control, two treatments with bacterial strains + insecticide and one treatment with two different fungicides + insecticide.

Figure 3 shows the effect of the bacterial strains Ki72 and Ki353 and fungicides on the relative yield of sugar under field conditions in 1997.

Figure 4 shows the effect of bacterial strains Ki853 and MF416 and fungicides on the relative yield of sugar for control, two fungicides + insecticide, Ki353, Ki353 + insecticide, MF416 and MF416 + insecticide.

Claims (20)

10 15 20 25 u .f 20 October 1999 A new biocontrol agent for controlling plant diseases caused by pathogenic fungi, characterized in that it is one or more isolates of one or more of the bacterial strains belonging to the group comprising the Pseudomonas spp. Strains Ki72, Ab131, Ki353 and MF416, deposited with the National Collections of Industrial and Marine Bacteria Limited (NCIMB), Aberdeen, Scotland and with NCIMB Deposit Nos 40936, 40940, 40941 and 40942 respectively, or that it is a biologically pure culture thereof or a mutant thereof with essentially the same characteristics as the parent strain. Composition for controlling plant diseases caused by pathogenic fungi, characterized in that it contains as active ingredient a biocontrol agent defined according to claim 1 or contains a culture mash thereof, which contains the antipathogenically active metabolite (s) thereof. The composition according to claim 2, characterized in that the plant to be protected from pathogenic fungi is sugar beet. The composition according to claim 3, characterized in that the pathogenic fungus is one belonging to the Pythium sp. Or Aphanomyces sp. Groups of fungi. The composition according to claim 4, characterized in that the pathogenic fungus is Pythium ultimum. The composition according to any one of claims 2-5, characterized in that the active ingredient is in admixture with an agriculturally acceptable carrier or diluent. 7. The composition according to claim 6, characterized in that the active ingredient is in admixture with a liquid carrier. 10 20 25 _ f, .. - I, 531 / Uo hu The composition according to claim 6, characterized in that the active ingredient is impregnated with a solid porous material. The composition according to claim 6, characterized in that it further comprises additives which serve as adhesives. The composition according to claim 6, characterized in that it further comprises a nutrient source. A method for controlling plant diseases caused by pathogenic fungi and which involves introducing an effective dose of an antipathogenically active biocontroller in the environment in which the disease is to be suppressed, characterized in that a biocontroller is applied, as defined in claim 1. , or a composition according to any one of claims 2-10. The method according to claim 11, characterized in that the biocontroller or composition is applied to seeds. The method according to claim 11, characterized in that the biocontroller or composition is applied to vegetative plant propagation units. The method according to claim 11, characterized in that the biocontroller or composition is applied to plants. The method according to claim 11, characterized in that the biocontrol agent or composition is applied to a growth medium in which a plant grows or is to grow. The method according to any one of claims 11-15, characterized in that the plant to be protected is sugar beet. The method according to claim 16, characterized in that the pathogenic fungus causing the disease is one belonging to the Pythium sp. Or Aphanomyces sp. Groups of fungi. 524 706; y¿gpg§ ;; fi @ u Use of a biocontroller, as defined in claim 1, or k - en liompcsitíon enliff * any of claims 9-10 * Mr to _ ntrollera, growth disease nnb u n; _ -v caused by pathogenic fungi. The use according to claim 18, characterized in that the plant to be protected is sugar beet. The use according to claim 19, characterized in that the pathogenic fungus is one belonging to the Pythium sp. Or Aphanomyces sp. Groups of fungi.