WO2001052869A2

WO2001052869A2 - Medicament and method for treating sclerotinia infections

Info

Publication number: WO2001052869A2
Application number: PCT/HU2001/000005
Authority: WO
Inventors: Gyula Beszedics; József HEGÓCZKY; Gyula Jusztusz; Mihály KECSKÉS; Gábor VERECZKEY
Original assignee: Bio-Mikro Kft.
Priority date: 2000-01-21
Filing date: 2001-01-18
Publication date: 2001-07-26
Also published as: WO2001052869A3; HUP0000207A2; AU2697401A; HU0000207D0

Abstract

The invention relates to a medicament that is suitable for treating sclerotinia infections and contains branches of the species Azospirillum, Azotobacter and Pseudomonas as an active agent; optionally together with the conventional carriers and other auxiliaries. The invention also relates to a method for treating sclerotinia infections.

Description

Preparation and method for the treatment of Sclerotinia infections

The invention relates to a preparation and a method for the treatment of

Sclerotinia infections. Viewed in more detail, the subject matter of the invention is a preparation which contains a mixture of environmentally friendly bacteria and can preferably be used for the treatment of Sclerotinia infections.

The Sclerotinia spp. are common dangerous pathogens in agriculture and horticulture all over the world. With Sclerotinia spp. are fungi living in etoden, between whose hyphae threads black hard structures, the so-called sclerotia, develop. These show a considerable morphological variety, in some species they develop on or in the roots of the plants, in others in the plant tissue. They are very hardy and can survive in the soil for several years. In spring they germinate, form pharmacies, and from these grow the fruiting bodies, which have a characteristic trumpet shape and can grow up to 10 mm high. 8 ascospores form in the fruiting bodies (asci).

In the course of the fungal attack, the sclerotia attack the root neck and the parts of the stem located near the earth. This will damage the vascular bundles, causing a disturbance in the plant's nutrition. The fungal infection leads to stem rot, which can result in the plant laying down and thus shrinking.

The Sclerotinia spp. attack more than 400 plants. They are primarily dangerous for oil plants (rapeseed, soybeans, sunflower), for vegetables and for cereals. Almost every plant has its own Sclerotinia pathogen. For example, tomatoes and potatoes from S. minor, sunflowers from S. sclerotiorum are affected.

No suitable mechanical or chemical protective measures are known against Sclerotinia infections; at the most one can try to use the active substances used against botrytis (Horväth J .: A szäntόföldi növenyek betegsegei / = The diseases of the arable plants /, Mezögazdasägi Kiadό, Budapest 1995, pp. 112-114). In practice, crop rotation is used, ie something different is grown on the respective field every few years. However, because of the risk of cross infection, this protection method is not very effective.

There was therefore a need to develop a preparation that could be successfully used against Sclerotinia infections. 5 It has now been found that the bacteria originating from the soil and from the rhizosphere of the plants, in particular strains of species belonging to the genera Azospirillum, Azotobacter and Pseudomonas, can be used successfully in the form of a mixture of bacteria for protection against the Sclerotinia infections. The invention accordingly relates to a preparation for the treatment of

Sclerotinia infections, which contains strains of the genera Azospirillum, Azotobacter and Pseudomonas as active ingredient, optionally together with the usual carriers and other auxiliary substances.

The invention further relates to a method for the treatment of 5 Sclerotinia infections. According to the method, the preparation according to the invention is applied to the plants or their habitat in an effective amount.

As already mentioned, the active ingredient of the preparation according to the invention is a bacterial mixture consisting of strains of the genera Azospirillum, Azotobacter and 10 Pseudomonas.

The gram-negative species Azospirillum sp. can, under the microaerobic conditions in the soil (which means about 1-2% oxygen content) come into close contact with the roots of the plants, settle in the upper tissue layers of the roots, reduce the free atmospheric nitrogen to .5 ammonia and this ammonia of the plant to hand over. This type of microorganism, which also requires the presence of a plant to bind nitrogen, is referred to collectively as associative nitrogen binder. In nature, plants attract the A∑ospirillum strains through organic acids excreted from their roots, sugar. J0 Azospirillum not only binds nitrogen, but also produces substances

(plant hormones and growth substances) that can be useful for the development of the plant.

The gram-negative bacteria Azotobacter also live in the soil, freely or occasionally in symbiosis with plants, and - in contrast to the associative Nitrogen binders - they are also able to reduce the nitrogen in the air to ammonia, which they use to ensure the nitrogen supply to crops. The Azotobacter strains can be selected on the basis of the measurement of the activity of the enzyme nitrogenase (Dilworth, MJ: Biochim. Biophys. Acta 127, 285 (1966)). The effectiveness of nitrogen binding and the formation of other compounds that have a beneficial effect on crops have been proven by laboratory tests and experiments on small plots.

Certain microorganisms, including the strains of the genus Pseudomonas, are able to dissolve the insoluble phosphorus into a form that the plant can absorb. This is necessary because there is enough phosphorus in the soil, but in the form of inorganic phosphates that cannot be used by the plant (e.g. apatite). The sources of soluble phosphorus compounds, the phosphorus mines, are drying up all over the world; It should also be mentioned that the excessive use of phosphorus and nitrogen-rich artificial fertilizers leads to acidification of the soil, to nitrate accumulation in the water, which promotes the algae (eutrophy) of the water. The selection for the ability to dissolve phosphate can be carried out on a glucose-free or glucose-containing medium containing 1% hydroxyapatite and 10% tricalcium phosphate.

The strains of the genus Pseudomonas are able to synthesize siderophores and phytohoπnones (for example auxins and gibberellic acid). The so-called siderophores can concentrate iron even in the presence of only small amounts of iron. This process can inhibit the multiplication of other disease-producing microorganisms by depriving them of the iron that is essential for their vital functions. The production of siderophores can be examined by inhibiting the growth of an Escherichia coli strain; the selection is made on the same basis. The biosynthesis of gibberellic acid can be observed and measured from the plant hormones. Thin-layer chromatographic methods can be used for strain selection.

The morphology of the individual strains can be described as follows. Azotobacter sp.

The cells, which can be moved in one to two days, have Perittch flagella, whose wavelength is 2.5-3.3 μm, amplitude 0.37-0.76 μm. The colonies are not slimy, but they can be of different shapes depending on the amount of extracellular polysaccharides produced. Ammonium and nitrate ions can be used as the nitrogen source, and the hydrolysis can be carried out with α-amylase or β-amylase. The G + C content of the DNA is 65.8-67.6 mol%. Azospirillum sp. The cells are spiral. They are gram-negative and move in liquid media using a single polar flag, while numerous lateral flagella are formed on solid culture media. They are chemo-organotrophic, i.e. of mainly respiratory metabolism. Aerobic, micro-aerophilic. Oxidase-positive; generally also catalase positive. The cells are capable of nitrous oxide fixation under microaerobic conditions. They accumulate poly-ß-hydroxybutyrate. The G + C content of the DNA is 69-71 mol%. Pseudomonas sp.

The species has 5 biovars, which are heterogeneous in their nutritional properties. Pseudomonas fluorescens can be regarded as Biovar I and its type strain also belongs to this group. They are straight or curved, non-helical gram-negative rods with a diameter of 0.5-0.8 μm and a length of 1.5-2.8 μm. They do not form prostheses and are not surrounded by a shell. Cell wall and membrane are the characteristic of Gram-negative bacteria. They move with the help of polar flagella. Your metabolism is strictly bound to oxygen as a terminal electron acceptor. Xanthomonaddins are not produced. The bacteria do not need organic growth factors, they are oxidase and catalase positive and chemoorganotrophic; they can utilize a wide variety of organic compounds as sources of carbon, nitrogen and energy. The optimal growth temperature is 25-30 ° C, below pH 4.5 there is no growth. They contain different pigments. The best known soluble pigment, pioverdin, is abundantly formed on low-iron nutrient media, and the fluorescence changes from white to bluish green in UV light. The maximum UV The wavelength is around 400 nm. The G + C content of the DNA is 59.4-61.3 mol%. Type strain: ATCC 3525. Pseudomonas spp. are widespread in nature.

For the purposes of the invention, the following strains are particularly preferably used, which were deposited by the authors of the present invention in the strain collection of the Budapest University of Horticulture and Food Industry: NCAIM (P) 001279 Azotobacter sp. Sarret, NCAIM (P) B 001280 Azospirillum sp. Särret and NCAIM (P) B 001281 Pseudomonas sp. Särret. The bacterial mixture to be used in the sense of the invention is produced in a manner known per se. Fermentation is preferably carried out continuously. This can be done, for example, by inoculating the fermenters of 10 liters, 100 liters, 1 m ³ and 10 m volume using 10% inoculum from the inoculum. For the purpose of increasing the scale, the finished culture of the respective fermenter is the inoculum for the fermenter following in the order. The composition of the nutrient medium in the fermenters is the same, only the nutrient medium of the starter culture, which is grown in shake flasks, is different. The fermenter with 10 1 volume is sterilized in an autoclave (30 min, 1.2 bar, 121 ° C). The 100 liter fermenter and the larger ones are sterilized by direct introduction of steam (1.3 bar, 130 ° C). The glucose is sterilized as a 50% aqueous solution separately in an autoclave (30 min, 1.2 bar, 121 ° C). The fermentation time is 24 hours, the cultivation time is 24 hours, the shaking frequency is 200 min ^"1. The air requirement is 1 dm ³ / dm ³ min, for volumes> 100 liters 0.4 clm ³ / dm ³ min. Glanopon is used as a defoamer. The temperature and pH differ depending on the individual cultures: Azospirillum sp .: t = 30 ° C, pH ~ 7.4; Azotobacter sp. 504: t = 28 ° C, pH ~ 7.3; Pseudomonas sp.: T = 30 ° C, pH ~ 6.8.

At the end of fermentation, the number of cells in the fermentation broth has depending from the culture values of 5 x 10 ⁸ - reaches 10 ⁹ / cm ^3: Azospirillum sp at. and Azotobacter sp. 504 5x 10 ⁸ - 10 ⁹ / cm ³ , in Pseudomonas sp. 4 x 10 ³ - 8 x 10 ⁵ / cm ³ . After the fermentation is stopped, the fermentation broth must be cooled. It is then processed, packaged and stored under sterile conditions.

In the bacterial mixture used according to the invention, the amount of azospirillum is generally 20-30% by mass, preferably 25

% By mass, that of Azotobacter generally 20-30% by mass, preferably 25

% By mass, and that of Pseudomonas generally 40-60% by mass, preferably

50 mass%.

The preparation according to the invention can be successfully used in various plant crops, in particular crops of oil plants such as oilseed rape, soybeans and sunflowers, vegetables and cereals, to combat Sclerotinia infections.

For the treatment according to the invention, the bacterial mixture itself or in the form of a suitable preparation is applied to the plants to be treated or their habitat.

The shelf life of the bacterial mixture according to the invention can be greatly improved if the fermentation broth obtained in the course of the fermentation is processed into suitable preparations. Suitable preparations are, for example, liquid formulations, such as solutions, suspensions and concentrates, and also solid formulations, for example powders, scattering agents, wettable powders, pastes and granules.

The preparations are manufactured in a manner known per se. In the first step, the fermentation broth is generally thickened. This can be done gently, for example, by centrifugation, ultrafiltration and separation. When using a filter insert with a suitable pore size, the volume of the fermentation broth can be reduced to 10% of the original volume by means of ultrafiltration. The concentrate obtained can be stored at low temperatures for a long time (up to one year). Before use, it only needs to be diluted to the desired application concentration.

To produce a solid formulation, the concentrate is mixed with a carrier material and the mixture is dried under mild conditions. Organic materials, for example water-insoluble biopolymers, lignocelluloses, such as plant stems, straw, starch-containing plant seeds, for example rice, wheat and barley, and by-products from agriculture and the food industry are preferably used as the carrier material. However, it is also possible to use inorganic materials, for example pearlite, diatomaceous earth and sand, and also synthetic materials, for example polyurethane foam and other plastic foam, as the carrier material.

The formulations may contain the usual additives and auxiliaries as further components. Stabilizers, antioxidants, dispersants, fillers, binders and dyes can be considered as such. Microbiological nutrients, for example carbon source, nitrogen source and water, can also be used as additives, which are then used by the bacteria for the growth and production of metabolites. The formulations generally contain the bacterial mixture in an amount of 0.1-99% by mass, preferably 0.5-95% by mass.

Treatment is carried out in the usual way, for example by watering, spraying, sriihening, sprinkling or injecting into the soil.

In general, the treatment is carried out before sowing or after sowing, but before emergence. Treatment before sowing is preferred; After preparing the soil, the formulation is applied to the surface and then incorporated into the top layer of the soil.

The application amount is generally 5-50 l / ha, preferably 10-30 1 ha, based on the bacterial mixture. This amount is generally applied with 50-5001 / ha, preferably 80-300 1 / ha, of water.

The invention is explained in more detail below with the aid of examples, but the scope of protection is not restricted to the examples. Example 1: The cultivation of the microorganism 1.1 Maintenance of the strain 1.1.1 To maintain the Azospirillum sp. the following nutrient medium is used: 1 g meat extract, 2 g yeast extract, 5 g peptone, 5 g NaCl, 15 g agar, 1 dm ³ dest. Water (pH ~ 7.4), temperature 30 ° C.

1.1.2 To maintain Azotobacter sp. 504 the following nutrient medium is used: 10 g glucose, 0.1 g CaCl ₂ ^• 2H ₂ O, 0.1 g MgSO ₄ ^• 2H ₂ 0, 0.9 gK ₂ HPO ₄ , 0.1 g KH ₂ PO ₄ , 5 g CaCO ₃ , 10 mg FeSO ₄ ^• 7H ₂ O, 5 mg NaaMoO l dm ³ distilled water (pH ~ 7.3), temperature 28 ° C.

1.1.3 To maintain Pseudomonas sp. the following sewing medium is used: 1 g meat extract, 2 g yeast extract, 5 g peptone, 5 g NaCl, 15 g agar, 1 dm ³ dist. Water, 0.45 g K ₂ HPO ₄ , 2.39 g NaH ₂ PO ₄ ^• 12H ₂ O (pH ~ 6.8), temperature 30 ° C.

1.2 Cultivation in the shake flask

In Erlenmeyer flasks of 1000 cm ³ each (sterilization at 121 ° C for 30 minutes), 200 cm ^{3 of} nutrient solution are inoculated with the microorganisms grown on inclined agar cultures. The nutrient solution has the following composition: 14 g glucose, 26 g molasses, 15 g corn steep liquor (50 mass% dry matter content), 5 g yeast extract, 4 g acid casein, 2 g (NH ₄ ) ₂ SO ₄ , 2 g NH4NO3, 2 g CaCO ₃ , 2 g KH ₂ PO ₄ , 1 g NaCl, 3 g MgSO ₄ , 4.6 g trace element solution *) *) The trace element solution has the following composition: 210 mg

FeSO ₄ ^• 7H O, 11 mg FeCl ₃ ^• 6H ₂ O, 2 mg MnSO ₄ ^• H ₂ O, 2 mg CuSO ₄ ^• 5H O, 2 mg Na ₂ MoO ₄ , 2 mg CoCl ₂ ^• 6H ₂ O, 2 mg ZnSO ₄ ^• 7H ₂ O, 1 mg Na ₂ BO ₄ ^■

10H ₂ O, 0.15 mg Bi (N0 ₃ ) ₃ ^• 2H ₂ O, 0.01 mg SnCl ₂ , 0.01 mg selenium chloride, 1 mg KI, 120 mg citric acid, distilled water 1 dm ³ . The cultures are shaken at 30 ° C for 26-34 hours on the shaker table at a frequency of 200 min ^"1 , then the sterile samples are examined under the microscope.

1.3 Pre-fermentation

In order to be able to enlarge the scale, the 10% volume laboratory fermentor is inoculated using 10% inoculum from the inoculum. With the fermentation broth created at the end of the fermentation, the semi-technical fermentor with a volume of 100 liters is inoculated, the fermentation broth created in this becomes the inoculum for the pre-fermenter of Im ³ volume. The nutrient medium used in the fermenters has a different composition than that for the shake cultures.

The fermenter cultures are cultivated in nutrient medium of the following composition. For 1 m ³ : 30 kg molasses, 10 kg corn steep liquor, 5 kg CaCO ₃ , 4 kg glucose, 200 g KH ₂ PO, 200 g CaCl ₂ , 300 mg MgSO ₄ , 200 g K ₂ SO ₄ , 500 g NaCl, 50 g FeCl ₃ , 5 g H ₂ BO ₄ , 5 g (NH4) ₂ MoO, -0.5 g KI, 0.5 g NaBr, 0.2 g

ZnSO ₄ , 0.3 g Al ₂ (SO ₄ ) ₃ , 2 dm ³ defoamer.

The 10 liter volume fermenter is sterilized in an autoclave

(30 minutes, 1.2 bar, 121 ° C). The glucose is sterilized separately in the form of a 50% strength aqueous solution in an autoclave (30 min, 1.2 bar, 121 ° C.). The

The fermenters with a volume of 100 liters or 1 m ^{3 are} sterilized by directly blowing in steam (1.3 bar, 130 ° C). The fermentation time is

24 hours, the cultivation time 24 hours, the shaking frequency 200 min ^"1

Air consumption is 1 dm ³ / dm ³ min, with 1 m ³ volume 0.4 dm ³ / dm ³ min. Glanopon is used as a defoamer. The temperature and pH differ depending on the individual cultures: Azospirillum sp .: t = 30 ° C, pH ~ 7.4;

Azotobacter sp. 504: t = 28 ° C, pH ~ 7.3; Pseudomonas sp .: t = 30 ° C, pH ~ 6.8.

1.4 Main fermentation

The main fermentation is carried out in a fermenter of 10 m ³ . As an inoculum, the one produced in the pre-fermenter with a volume of 1 m ³

Bacterial culture used. The composition of the nutrient medium used in the main fermentor is the same as that used in the pre-fermentation. Sterilization is carried out by direct steam introduction (1.3 bar,

130 ° C). The fermentation time is 24 hours, the cultivation time is 24 hours, the stirring frequency is 200 min ^"1. The air requirement is 0.4 dm ³ / dm ³ min.

Glanopon is used as a defoamer. The temperature and pH differ depending on the individual cultures: Azospirillum sp .: t = 30 ° C, pH ~ 7.4;

Azotobacter sp. 504: t = 28 ° C, pH ~ 7.3; Pseudomonas sp .: t = 30 ° C, pH ~ 6.8. At the end of fermentation, the number of cells in the fermentation broth has depending from the culture values of 5 x 10 ⁸ - reaches 10 ⁹ / cm ^3: Azospirillum sp at. and Azotobacter sp. 504 5x 10 ⁸ - 10 ⁹ / cm ³ , in Pseudomonas sp. 4 x 10 ³ - 8 x 10 ⁵ / cm ³ .

After the fermentation is stopped, the fermentation broth must be cooled. It is then processed, packaged and stored under sterile conditions.

Example 2: Treatment of Sclerotinia Infection in Sunflower Cultures

The tests were carried out on sunflowers (Helianthus annuus) outdoors by treating the soil. Before the sunflowers were sown, a bacterial mixture with the germ count of 2.5 × 10 ⁸ / cm ^{3 was} sprayed onto the soil in the stated amount and then mechanically worked into the top layer of the soil. Then the sunflower seeds are put in, and the soil is infected with the sclerotia of S. sclerotiorum (20 pieces on 60 m ² ). The viability of sclerotia at the time of infection is 70%. Then the plants are cultivated under the usual culture conditions.

Untreated plants are used as a control, plants treated with the known agent Koni Standard (Bioved Kft, Szigetszentmiklόs, HU) as a reference. The active ingredient of Koni Standard is the parasitic fungus Conioterium ministrans.

The evaluation takes place in the cotyledon stage, in the stage with 2 subsequent leaves (1) and in the stage with 6-8 subsequent leaves (2). The percentage of infestation is determined by visually rating the stem rot. The viability of sclerotia is determined by a laboratory test. The results are summarized in the following table.

Claims

claims

1. Preparation for the treatment of Sclerotinia infections, containing as active ingredient strains of the genera Azospirillum, Azotobacter and Pseudomonas, optionally together with the usual carriers and other auxiliaries.

2. Preparation according to claim 1, containing as active ingredient a mixture of 20-30% by mass of Azospirillum, 20-30% by mass of Azotobacter and 40-60% by mass of Pseudomonas.

3. Preparation according to claim 1 or 2, containing the strains NCAIM (P) B 001279 Azotobacter sp. Särret, NCAIM (P) B 001280 Azospririllum sp. Särret and NCATM (P) B 001281 Pseudomonas sp. Sarret.

4. A method for the treatment of Sclerotinia infections, in which a preparation according to any one of claims 1-3 is applied in an effective amount to the plants or their habitat.

5. Use of the strains belonging to the genera Azospirillum,

Species belonging to Azotobacter and Pseudomonas for the treatment of ScleTotinia infections.