RU2032345C1 - Strain of bacterium bacillus licheniformis for preparing of preparation against sunflower white rot - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: strain Bacillus licheniformis B-5 is isolated from sclerotia of sunflower white rot. Strain is deposited in Collection VIZR at number B-5. Strain of bacterium is grown on sterile sunflower husk, dried and milled. Obtained preparation has a titer 6·10⁹. It is used with sticker - polyvinylpyrrolidone. EFFECT: detection of strain indicated above. 4 dwg, 5 tbl

Description

 The invention relates to plant protection, it relates to strains of microorganisms used for the production of bacterial preparations used to control pathogens of plants, namely the white rot of sunflower.

The known strains of sunflower white rot antagonist fungi Coniothyrium minitans, Gliocladium catenulatum, Trichoderma viridae, Penicillium vermiculatum (Talaromyces flavus), Chaetomium olivaceum (Huang HS Biological of sсlerotinia wilt in p. 24-14, Canada. 12, Canada) Maclaren DZ Huang HS Rimmer SP Hyperparasitism of Sclerotinia sclerotiorum by Talaromyces flavus, Canada Journal of plant Pathology, 1986, N 8, p. 43-48;
Tikhonov O.I. Maslienko L.V. Antagonists of sclerotia white rot of sunflower, Scientific and Technical Bulletin of the All-Union Scientific-Research Institute of Oilseeds, 1985 IV (91), p. 25-29).

 Some information is known about the bacterial strains Bacillus subtilis and Bacillus cereus antagonists of white rot of sunflower (Afanasyeva L.V. Skvortsova I.N. Prospect for the use of bacteria against the causative agent of white rot of sunflower. Microbiological method of protecting plants from pests, diseases and weeds, Chisinau, "Stiinets ", 1989, pp. 86-91).

 Attempts are also made to use the bacteria Bacullus subtilis and Bacillus cereus antagonists of the sunflower white rot pathogen (Zazzerini A. Tosi L. Prove di antagonisto de aveum isolati battrini nei confronti della Scl. Sclerotirum Lid de Bary, Inf. Fitopatol. 1985, Vol. 35 N 1 , p. 27-30).

 However, studies to test these strains were carried out at the level of laboratory and vegetation experiments and their effectiveness has not been confirmed by field trials.

 The purpose of the invention is the selection of an active strain of the bacterium antagonist of the pathogen of white rot of sunflower, which has an effective protective effect and an expanded spectrum of antifungal effects (ashy rot, phomopsis).

 The strain Bacillus licheniformis B-5 was isolated from sclerotia of white rot of sunflower collected from the affected sunflower plants in the experimental farm OPKh "Kruglik" NGO "Oilseeds".

White rot sclerotia was washed in running water, then dried on filter paper, kept for 1 min in alcohol, cut with sterile glass using a scalpel and laid out in Petri dishes with potato-sucrose agar. Petri dishes were placed in a thermostat, t 25 ^о С. When bacteria appeared around sclerotia, they were sifted out into clean Petri dishes with potato-sucrose agar, followed by double culture testing for their antagonistic activity (Huang, 1979). The most active antagonistic effect is the strain of the bacterium B-5.

 The strain of you, licheniformis B-5, was determined at the Department of Soil Microorganisms at Moscow State University and deposited in the microorganism culture collection of the All-Union Institute for Plant Protection.

 Morphological signs.

 The cells are rod-shaped (1.5 x 3) x (0.6 x 0.8) μm, the spores are oval, gram-positive, motile, sporangia do not swell.

 The growth ability of Bac bacteria was determined. licheniformis B-5 on common nutrient media: potato-sucrose agar, meat and peptone broth and agarized wort. We studied the growth of culture on plant substrates to identify affinity for them: slices of potatoes, carrots, cabbage agar.

 On the potato-sucrose agar of the colony, Bac.licheniformis B-5 bacteria are round, smooth, off-white in color, spreading, with an uneven edge. On meat-peptone broth gives abundant growth, forms a thin leathery white film, growing on the walls of the tube. On agarized wort, the colonies are off-white in color, round, with an uneven edge. There was good growth on cabbage agar. On potatoes and carrots it grows without fermentation.

 Physiological signs.

Facultative anaerobe with optimum temperature of 30-35 ^{° C,} with an optimum pH of 6.8-7.0. Minimum growth temperature 30 ^C, maximum 55 ° ^C.

 Attitude towards carbohydrates: glucose, arabinose and mannitol breaks down with the formation of acid without the formation of gas.

 Starch hydrolyzes, dilutes gelatin, and milk peptones. There is an increase of 2.5 and 7% NaCl in dextrose agar. Culture growth in the presence of casein as the sole source of carbon and nitrogen is characterized by increased spore formation. The morphological and cultural properties of the strain Bacillus licheniformis B-5 are given in table 1.

 Antagonistic properties.

 The strain Bscillus licheniformis B-5 has antagonistic activity to Sclerotinia sclerotiorum, Macrophomina faseolorum, Diaporthe helianthi, Diaporthe faseolorum. To determine the antagonistic activity of the strain Bacillus licheniformis B-5, the double culture method was used (Huahg, 1979). The pathogen cultures and the B-5 bacterial strain were sown simultaneously at diametrically opposite points of the Petri dish on potato-sucrose agar. The control was pure cultures of bacteria and pathogens, seeded separately. Accounting was carried out every three days for 10 days of cultivation. Marked growth of pathogens and antagonist, the nature of their interaction.

 The strain of bacteria Bac. licheniformis B-5 develops faster than pathogens and forms zones of 1-1.5 cm with sclerotinia and phomopsis (Fig.1, 2) and 0.5-1 cm with ashy rot (Fig.3), which last for a month . Mycelium of white rot and phomopsis at the border of the zone with the bacterial strain blackens, lyses, sclerotia is almost not formed.

PRI me R 1. The experiment was carried out in laboratory conditions in Petri dishes. Artificially grown sclerotia of white rot was soaked in a suspension of Bacillus licheniformis B-5 with a density of 10 ⁶ colony forming units per 1 ml of water for 24 and 48 hours. Then they were washed with sterile water, dried on filter paper, cut and laid out in Petri dishes on potato-sucrose agar . The control was sclerotia without treatment with a bacterial suspension. Accounting was carried out 20 days after the bookmark experience. Soaking sclerotia in a bacterial suspension for 24 and 48 hours was effective. The death of sclerotia in these variants was 90-100% in the control of 6-14% (Table 2).

PRI me R 2. The experiment was carried out in laboratory conditions in Petri dishes. Sunflower seeds were treated with a suspension of a strain of the bacterium Bacillus licheniformis B-5 with a density of 10 ⁶ colony forming units per 1 ml. The treatment time was 4 hours. Then the seeds were washed with water, laid out in Petri dishes on filter paper and a piece of white rot mycelium grown on agarized potato-sucrose medium was applied to them. Control1 seeds were soaked for 4 hours in sterile water and infected with a white rot pathogen. Control 2 seeds without infection by the pathogen. The filter paper in Petri dishes was moistened daily with sterile water. Accounting for germination of seeds and their defeat by the causative agent of the disease was carried out after 10 days (figure 4).

 The experimental data are given in table.3.

PRI me R 3. The experiment was carried out in the field in the foothill zone of the Krasnodar Territory, on the collective farm named after XX Party Congress of the Otradnensky district against a background of high natural damage to sunflower crops with white rot and in the mountainous zone of the Adygea region at the Dakhovsky state farm at artificial infection with a pathogen (introduction of sclerotia into the wells during sowing). The plot area is 30 m ² , the repetition is three times. Sunflower variety VNIIMK 8883. Before sowing, sunflower seeds were treated with powder made on the basis of the Bac bacterial strain. licheniform grown on a sterilized husk of sunflower, followed by drying and grinding in a mill. The titer of the drug 6 ^. 10 ⁹ , the consumption rate of 10 g per 1 kg of seeds, with moistened seeds with 30 ml of water and an adhesive 20% PVP (polyvinyl pyrrolidol). Control of sunflower seeds without treatment with a biological product and without making an infection (against the background of natural rot damage) and making an infection (against the background of artificial infection with a pathogen). Standard sunflower seed treated with fungicide. The experimental data are presented in tables 4 and 5.

 The results shown in the tables show the effective action of the isolated strain of the bacterium Bacillus licheniformis-5 against white rot of sunflower. In addition, this strain inhibits ash rot and phomopsis, i.e. has an extended spectrum of action.

Claims (1)

 The bacterial strain Bacillus licheniformis VIZR B-5 to obtain a drug against white rot of sunflower.

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