JP2002519309A - New biological control agents - Google Patents

Abstract

  (57) [Summary] The present invention has been deposited with the National Collections of Industrial and Marine Bacteria Limited (NCIMB) of Aberdeen, Scotland and has received the NCIMB accession numbers 40936, 40940, 40941, 40942 and 41010, respectively, of the Pseudomonas sp. Strains Ki72, Ab131, A practical and effective novel biology for controlling phytopathogenic attacks in commercial crops characterized by isolates and isolates of one or more bacterial strains belonging to the genus including Ki353, MF416 and Ma358 Provide a chemical control agent. The isolate was deposited under the terms of the Budapest Treaty. The present invention also provides plant disease control compositions and methods of controlling plant disease caused by pathogenic fungi using one or more bacterial strains of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of protecting plants and crops from pathogens by bacterial strains used as biocontrol agents.

[0002]

[Prior art]

During the early stages of germination, plant seeds and seedlings attack soil-borne fungi from various plant pathogens, especially soil-borne complexes containing a genus of fungi that cause major crop damage and economic failure. Very sensitive to attacks from Pythium species. Piteum species are the species that form the best microbial colonies in agricultural and horticultural soils. Culture soils around the world contain spores from at least one Ptium species. Although the fungi of this Pythium species are often necrotic vegetative parasites of crop roots, they can also grow saprophytic. Currently, there are a few commercial fungicides used on the market. Chemical fungicides are often very effective, but they can have undesirable effects on the environment, and most are dangerous to human health, so care must be taken when handling them, and Void if a resistant pathogenic strain develops.

[0003] The use of biological control agents or biological pesticides is more effective or more preferred than the use of other control methods, and such agents have therefore been widely tried. Some bacteria and fungal strains are known to inhibit the growth of various microbial pathogens. To be effective and usable, the drugs must be stable, give reproducible results in the field, and be usable under field conditions. To date, these requirements have been hardly fulfilled and therefore have not been used commercially. One such product is based on the use of Pseudomonas chlororphis strain MA342 (NCIMB 40616) to control species-borne plant fungal diseases (10 October 1995).
PCT application WO 95/28085, published on March 26).

[0004]

[Means for Solving the Problems]

The present invention is to provide a new biological control agent that is practical and effective to control the attack of phytopathogens on commercial crops. The present invention relates to the National Collections of Industrial and Marine Bacteria in Aberdeen, Scotland. Deposited with NCIMB and its NCI
40936, 40940, 40941, and 40, respectively, as MB acceptance numbers
Pseudomonas sp. Strains Ki72, Ab131, K that have received 942 and 41010
i353, MF416 and Ma358, characterized in that they are isolates and fungi of one or more bacterial strains belonging to the genus including: The isolate was deposited under the terms of the Budapest Treaty. The present invention also relates to an isolate or isolate of one or more of the bacterial strains specified above as an active ingredient, or a biologically pure culture thereof, or a culture medium containing one or more of the isolates above. Provided are plant disease control compositions comprising those antipathogenic active metabolites or biological agents that are such antipathogenic active metabolites. Furthermore, the present invention provides a method for controlling plant diseases caused by pathogenic fungi using one or more bacterial strains of the present invention. Also provided is the use of the biological agent of the present invention for controlling plant diseases caused by pathogenic fungi.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention, in one embodiment, is directed to Aberdeen, Scotland.
en) National Collections of Industrial and Marine and Marine Bacteria Li (National Collections of Industrial and Marine Bacteria Li)
one or more belonging to the genus including Pseudomonas sp. strains Ki72, Ab131, Ki353, MF416 and Ma358, deposited with NCIMB and received the following accession numbers 40936, 40940, 40941, 40942 and 41010, respectively. Caused by pathogenic fungi, characterized in that they are isolates and isolates of bacterial strains of the genus Bacteria and their biologically pure cultures or variants thereof having essentially the same characteristics as the parental strain The present invention relates to a novel biological control agent for controlling plant diseases. According to another aspect, the present invention comprises, as an active ingredient, a biological control agent as specified above, or a medium thereof containing their antipathogenic active metabolites, or such. The present invention relates to a composition for controlling a plant disease caused by a pathogenic fungus, which comprises a novel antipathogenic active metabolite.

In one preferred embodiment of the invention, the plant to be protected from pathogens is sugar beet (sugar radish). The pathogenic bacterium is preferably a bacterium belonging to the genus Piteum or Aphanomyces, particularly a bacterium belonging to Pythium ultimum. However, the biological control agents and compositions of the present invention are also contemplated for use in other commercial crops such as vegetables, for example tomatoes, especially such crops that are attacked by Pythium spp. Another aspect of the present invention is a method of controlling a plant disease caused by a pathogenic fungus and suppressing the disease by administering an effective dose of an antipathogenically active biological control agent to the environment, The method is characterized by using a biological control agent or composition as defined above. The biological control agents of the present invention can be used in seeds, seedlings, plant growth and propagation equipment, plants or plant parts or soil.

According to yet another aspect of the present invention, the biological control agent or composition as defined above is used for controlling plant diseases caused by pathogenic fungi, especially fungi of the genus Pytheum. . The characteristics of the novel bacterial strains of the present invention and the preferred methods for growing the strains and preferred formulations and uses in the field or greenhouse are described below. Several examples are provided to further illustrate, but not limit, the scope of the methods and configurations of the present invention.

[0008] Characteristics of the novel bacterial strain [0008] This strain can be characterized as shown in Table 1 using biochemical tests.

[Table 1] The indications in this table are as follows. NO ₃ = nitrate reduction, TRP = India - le generation, acid obtained from GL = glucose, ADH = arginine Jihidorora - Ze, 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 = malic acid assimilation, CIT = citrate assimilation, PAC = phenyl-acetate assimilation,
OX = cytochrome oxidase assimilation.

<Preferred growth method of strain and preferred formulation and use in field or greenhouse> The amount of the antipathogenically active strain is determined by subjecting a sample of a pure culture of the strain to liquid vibration culture or containing an appropriate fermentation medium. It is best obtained by a fermentation process that involves inoculating a fermenter. The strain can also be cultured on a sterile surface, such as agar, and once grown, the cells can be suspended in water or other liquid media known in the art. The culture medium may in principle be any bacterial growth medium known in the art. Fermentation is carried out at a sufficient cell concentration, for example, about 5-
The operation is carried out until 10 ⁹ cfu (colony-forming unit) / ml is obtained. The fermentation medium or bacterial suspension thus obtained can be used as such for use in plant protection, or can be treated or formulated before use.

In one treatment, the bacterial cells in the fermentation medium can be killed or centrifuged, for example by heating, and the resulting medium or supernatant containing the bacterial metabolites can be previously purified and / or concentrated or Can be used for plant protection without. Bacterial suspensions and fermentation media are homogenous with one or more compositions or genera of compositions provided that the compositions known in the art are compatible with the bacterial strain or its antipathogenic metabolites or derivatives thereof. Can be mixed. Suitable compositions are powder additives such as talcum, kaolin, bentonite or montmorillonite or solid carriers, wettable powders known in the art,
Carbon source nutrients (glucose, sucrose, and fructose) or complex bacterial nutrients (yeast extract, bacterial peptone and tryptone), metal salts, salts derived from fatty acids, fatty acid esters, ionic or nonionic surfactants, phytonutrients, plants It can be a growth regulator, fungicide, insecticide, fungicide, and the like. Also, the bacterial suspension and fermentation medium can be dried or lyophilized before or after mixing with the appropriate composition and the resulting product used for protection of the seedlings. A suitable method for the drying step is, for example, air cooling of the vermiculite supplied with the bacterial fermentation medium. The bacterial and metabolite formulations can be applied in any known manner for treating seeds, plant propagation equipment, plants, and soil with bacterial strains. The spray method, the spray method, the dusting method, the dispersion method, the pellet forming method, the immersion method, or the injection method can be selected according to the intended purpose and general circumstances. Advantageous seed treatment rates are usually 10 ¹¹ to 10 ¹² cfu / ha, for spraying 10 ¹² to 10 ¹⁴ cfu / ha or the corresponding amount of bacterial metabolites.

Example 1 <Isolation of the bacterial strain of the present invention> [0011] The roots of different plant species dug up to remove the adhering soil were placed in sterile tap water and washed. A 2-3 cm length was cut off from young roots and treated under aseptic conditions. A part of the root was collected from the upper part of the root tip. Several small cuts were made in the root with a scalpel that was lit. Next, the root was placed on TSA10 agar (Tr
yptic Soy Agar, Oxoid, 10 g / l). After the bacteria were cultured on an agar plate kept at 0 ° C for 9 days, the bacteria were picked on TSA10 and purely cultured at 5 ° C for 5 days.

Example 2 <Preservation of Bacteria> This pure culture was rapidly frozen at -70 ° C in a small ampule. As a frozen support, 10% glycerol was used in tap water having a pH adjusted to 7.15 after autoclaving. After freezing at -70 ° C, the ampoule was changed to -20 ° C. This isolated culture was lyophilized during long-term storage. "TSA10" agar (Tryp
After culturing for 48 hours on tic Soy Agar, Oxoid, 10 g / l), the bacterial ground was scraped off the agar surface and freeze-dried support (Dextran T).
70, Pharmacia Fine Chemicals, 50 g;
L-glutamate, Kebo AB, 50 g; and 1000 ml of distilled water), poured into small ampoules (20 ml) and placed in a lyophilizer "Hetosick" (Heto, Denmark) for 24 hours. After lyophilization, the ampoules were air-tight with a rubber stopper and stored at 4 ° C.

Example 3 <Primary Selection of Activity against Piteum> Preparation of Inoculum Pythium ultimum was isolated from infectious sugar beet roots and cultured on “PDA” (Potato Dextrose Agar: potato glucose agar). . After inoculation of the fungi on the agar plate, the plate is comminuted and filled to 2/3 with a commercially available (unsterile) peat mixture (Enhetsjord K Normal) mixed with 20% (v / v) sand. Into a pot (13 × 12 × 7 cm). Fifty sugar beet seeds were sown in the pot to a depth of 1 cm. The pot was placed in an environmental chamber under the following conditions. That is, after brightening at 17 ° C. for 16 hours, darkening was performed at 14 ° C. for 8 hours. After 4 weeks, the soil in the pot was used as an inoculant (0.2% w / w) to infest the soil for primary sorting and testing. Bacteria production Bacteria were attached to sugar beet seeds as follows. “TS” cultured at 15 ° C
A10 ”was scraped off from the agar surface of a 9 cm Petri dish 24 hours before culture on the A10”.
Mix with ml of tap water. This mixture was poured over the seeds. After 30 minutes, the excess mixture was discarded and the seeds were dried overnight. In the primary sort, 16 seeds were sown in pots placed under the same conditions as above. After 4 weeks, seedlings were counted and roots scored for infection with Piteum.

<Effect of Bacterial Strains on Piteum in Greenhouse Test> The bioassay was performed in an environmental chamber under the same culture conditions as above. Figure 1 shows the disease control effect of bacterial strains treated with control (untreated seeds sown on soil infected with Piumium urtimum), healthy soil (untreated seeds sown on healthy soil) and treated with bacterial strains. In the case of the given species is shown.

<Effects of Bacterial Strains on Piteum in Field Tests> Examples In 1996 and 1997, bacterial strains Ki72 and Ab131 were tested under field conditions, and in 1998 bacterial strains MF416 and Ki353 were tested in the field. Tested under conditions. Soil tests showed that the pathogen was highly attached. The field test was repeated four times in six different locations and in each location with a random compartment design. The dimensions of the plots for all tests were 6 rows x 12 meters. FIG. 2 shows the relative yield of sugar for the control, two treatments with the bacterial strain + pesticide, and one treatment with two different fungicides + pesticide.

FIG. 3 shows the effect of the bacterial strains Ki72 and Ki35 and the fungicide on the relative yield of sugar under field conditions in 1997. FIG. 4 shows relative bacterial yields of bacterial strains Ki353 and MF41.
6 and the effect of the fungicide as a control, two fungicides + insecticide, Ki353, Ki35
3+ insecticide, MF416, MF416 + insecticide. <Effect of Bacterial Strains and Fungicides on Relative Sugar Yield in Field Tests in 1996, 1997, and 1998> Relative Yield of Sugar in 1996, 1997, and 1998 Field Tests The effects of the bacterial strains and the fungicides on the activity are shown in Table 2 below.

[Table 2] Eup. Was replaced by TMTD. Fungicides: Eup = upalen (trifluanid), Tach = tatigalen (hymexazole), TMTD = thiram. Insecticide: Montur = imidacloprid + tefluthrin. -= Not tested.

Deposits of microorganisms The following deposits have been made under the terms of the Budapest Treaty. Microorganisms Depositary Office Deposit date Accession number Ki72 NCIMB March 30, 1998 40936 Ab131 NCIMB March 30, 1998 40940 Ki353 NCIMB March 30, 1998 40941 MF416 NCIMMB March 30, 1998 March 40992 March 1991 Sun 41010

[Brief description of the drawings]

FIG. 1 shows the effect of bacterial strains Ab131, MF416, and Ki72 on seed and seedling diseases caused by Piumium urtimum. DI = disease index (0
= Healthy, and 4 = 100% infected root).

FIG. 2. Bacterial strain A against relative sugar yield under field conditions, 1996.
The effects of b131, Ki72 and fungicide are shown. Fungicide: Eup = Euparen (Trifluanid) Tach = Tatigalen (Himexazol), Pesticide: Montur = Imidacloprid + Tefluthrin.

FIG. 3. Bacterial strain K versus relative yield of sugar under field conditions, 1997.
9 shows the effects of i72 and Ki353 and a fungicide. Fungicide: Tach = Himexazol Eup = Trifluanid Pesticide: Montur = imidacloprid + tefluthrin.

FIG. 4. Bacterial strain Ki35 against plant numbers under field conditions, 1998.
3 and the effects of MF416 and a fungicide. Fungicide: Himexazole TMTD = Tyram Insecticide: Montur = Imidaclopride + Tefluthrin.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] August 9, 2000 (200.8.9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Tanning, Christian Sweden, S- 756 49 Uppsala, Hugo Alven V. 34 F term (reference) 4B065 AA41X AC12 AC20 BA22 CA47 4H011 AA01 BA01 BB21 DA09 DA12 DC10 DD04

Claims (20)

[Claims] 1. The National Collections of Industrial and Marine Bacteria Limited (Aberdeen, Scotland)
CIMB) and their accession numbers for the NCIMB are 40936, 40
Isolates and fungi of one or more bacterial strains belonging to the genus including Pseudomonas sp. Strains Ki72, Ab131, Ki353, MF416 and Ma358 which have received 940, 40941, 40942 and 41010, respectively, and their biology. A biological control agent for controlling plant diseases caused by pathogenic fungi, characterized in that it is a chemically pure culture or a variant thereof having essentially the same characteristics as the parent strain. 2. Caused by a pathogenic fungus, characterized in that it comprises a biological control agent according to claim 1 as active ingredient or its medium containing its antipathogenic active metabolite. Compositions for controlling plant diseases. 3. The composition according to claim 2, wherein the plant protected from pathogenic fungi is sugar beet. 4. The composition according to claim 3, wherein the pathogenic bacterium is a bacterium belonging to the genus Pythium or Aphanomyces. 5. The method according to claim 5, wherein the pathogen is Pythium ultimatum.
5. The composition according to claim 4, wherein the composition is timatum). 6. A composition according to claim 2, wherein the active ingredient is a mixture with an agriculturally acceptable carrier or diluent composition. 7. The composition according to claim 6, wherein said active ingredient is a mixture with a liquid carrier. 8. The composition according to claim 6, wherein the active ingredient is impregnated into a solid porous material. 9. The composition according to claim 6, further comprising an additive acting as an adhering agent. 10. The composition according to claim 6, further comprising a nutrient source. 11. A method comprising controlling a plant disease caused by a pathogenic fungus and effectively administering an antipathogenically active biological control agent to an environment in which the disease needs to be controlled. A method comprising using the biological control agent according to claim 1 or the composition according to any one of claims 2 to 10. 12. The method according to claim 11, wherein the biological control agent or composition is used in a species. 13. The method according to claim 11, wherein the biological control agent or composition is used in a plant growth and propagation apparatus. 14. The method according to claim 11, wherein the biological control agent or composition is used in plants. 15. The method according to claim 11, wherein the biological control agent or composition is used in a growth medium in which plants are growing or to be grown. 16. The method according to claim 11, wherein the plant to be protected is sugar beet. 17. The pathogenic bacterium causing the disease is a fungal pytheum (Pyth).
17. The method according to claim 16, wherein the bacterium belongs to the genus ium or Aphanomyces. 18. Use of the biological control agent according to claim 1 or the composition according to any one of claims 2 to 10 for controlling plant diseases caused by pathogenic fungi. 19. The use according to claim 18, wherein the plant to be protected is sugar beet. 20. The use according to claim 19, wherein the pathogenic bacterium is a bacterium belonging to the genus Pythium or Aphanomyces.