RU2121793C1 - Strain of fungus trichoderma lignorum used against phytopathogenic fungi and bacteria - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: strain is obtained by step selection on the basis antibiotic, enzymatic activity and productivity indices. Strain is deposited in NII collection of cell culture of The State Scientific Center of virology and biotechnology "Vector" at N F5. Strain shows antagonistic activity against fungi Fusarium, Botrytis cinerea and Pseudomonas corrugata. The use of this strain for cucumber and tomato treatment ensures to decrease morbidity of vegetable cultures by 37.5-98% and increase their yield by 2.6 and 1.2 kg/1 sq.m., respectively. Strain can be used for plant protection against phytopathogens. EFFECT: new strain indicated above, enhanced effectiveness of the strain. 1 tbl, 3 ex

Description

 The invention relates to the microbiological industry, phytopathology and can be used to obtain a drug against phytopathogenic fungi and bacteria.

 Known strain Trichoderma longibrachiatum 7-26, with cellulose activity [1]. Solid-phase and deep cultivation is optimized for the strain. The strain is not picky about the nutrient medium and is able to use waste from various industries as a carbon source. The disadvantage of this strain is that the strain does not exhibit antagonistic activity to bacterial phytopathogens.

Known strain Trichoderma lignorum T13-82. The strain grows at 26 ^o C on traditional media using grain [2]. The strain exhibits antagonistic activity against phytopathogenic fungi of the genera Alternaria and Fusarium and, to a lesser extent, against bacterial strains. Used to produce trichodermine. The main disadvantages of this strain are: the strain is cultivated using only the solid-phase method; the strain does not have activity against Pseudomonas corrugata. The decrease in the phytopathogenic potential of the soil after treatment with the strain is not described.

 Closest to the claimed strain is a strain of Trichoderma lignorum [3, prototype]. Cultivation of this strain requires traditional media. The strain is effective in combating soil phytopathogens that cause root rot of cereals, cotton wilt, potato rhizoctonia, black cabbage, and a number of other diseases. The strain is especially promising when used in sheltered soil. The strain is stored in the Belarusian NIZR. The main disadvantages of the strain are: the need for constant maintenance of the strain by reseeding on fresh wort agar; constant selection for antagonistic properties; long, two-stage cultivation. The phytopathogenic potential of the soil during double treatment is reduced by 4 times.

 An object of the invention is to obtain a strain that provides a large degree of reduction in the phytopathogenic potential of the soil, stable in its antagonistic characteristics, able to retain its properties for a long time during long-term storage.

 The problem is solved by identifying and using a strain of Trichoderma lignorum.

 The strain Trichoderma lignorum was obtained by stepwise selection according to the signs of antibiotic, enzymatic activity and productivity when grown on solid and liquid nutrient media.

 Step selection is carried out using the method of monoconidial sieving on Petri dishes with Chapek medium. Among the cultures obtained, morphologically aligned and showing high antibiotic and enzymatic activity isolates were selected for the next monoconidial sieving. Cultures that did not preserve the required properties (having sectors and spots different in color from the original culture, or completely changing colony pigmentation, low antibiotic and enzymatic activity) are rejected.

 The strain stably maintains morphological properties, antibiotic and enzymatic activity for 10 passages.

 The strain Trichoderma lignorum is characterized by the following features.

Morphological and cultural characteristics: when the strain is grown at 26 ^o C in CA medium (pH 6.0), the colony is outstretched in dark green. The sods are large, first white, on 3-4 days green, later dark green, velvety. The reverse side of the colony has a light green color. Conidiophores in the form of lateral branches of hyphae of the aerial mycelium (76.0 x 2.3 microns), bottle-shaped phialides, are arranged in whorls 2-3 or single (6.2 x 1.5 microns). The apical conidia are rounded (3 x 2.5 μm), assembled into spherical heads.

Physiological and biochemical characteristics: the strain grows at a temperature of 4 - 30 ^o C, the pH of the medium is 5 - 7.2, the optimum temperature is 24 - 26 ^o C, pH 6 - 6.5. Relation to a carbon source. It absorbs glucose, sucrose, lactose. Salts of organic acids (oxalic sodium, sodium citrate) have an inhibitory effect on the growth of the fungus in culture. It is well absorbed by peptone, asparagine, satisfactory sodium nitrate and potassium. Hydrolyzes starch and fiber, dilutes gelatin, and peptones milk.

 The strain is able to accumulate up to 20 g / l of dry matter on the 4th day of deep cultivation.

The effectiveness of the strain is illustrated by test data in closed ground. Experimental batch (liquid formulation) was tested under production conditions in the area 480 m ² against Fusarium (Fusarium sp.), Gray mold (Botrytis cinerea) and pustostebelnosti tomato (Pseudomonas corrugata). The results are shown in the table.

 The resulting Trichoderma lignorum strain was deposited at the Vector Research Institute of Cell Culture Collections of the State Scientific Center for Virology and Biotechnology (Vector Research Institute of KKM SSC VB Vector) under registration number F5.

The defining difference of the proposed strain from the strain of the prototype is:
- a large degree of reduction in the phytopathogenic potential of the soil,
- stability in its antagonistic characteristics,
- the ability to maintain their strain properties for a long time during long-term storage.

 Since the proposed strain was obtained for the first time and was never used to obtain trichodermin, we can conclude that the proposed strain meets the criteria of the invention of "novelty" and "inventive step".

 The invention is illustrated by the following examples of specific performance.

Example 1. Obtaining the drug by deep cultivation. The uterine culture is prepared on a medium containing 1 part of beer wort and 2 parts of water (tap), pH 6.5, for 48 hours, then introduced into the fermenter with a capacity of 20 liters in an amount of 1: 10 to the volume of the nutrient medium. Cultivation in the fermenter is carried out on a medium containing, g / l: peptone 3, sucrose 5, corn extract 10, NaNO ₃ 2, with an aeration volume of 1: 3 at a temperature of 28 ^o C. The dry matter mass after 72 hours of growth of the fungus is 20 g / l

 The resulting preparation is a liquid containing in its composition a nutrient medium, spores and mycelium of the fungus, exotoxins. The titer of the drug is 10 billion cells / ml.

Example 2. Obtaining the drug by solid-phase fermentation on bulk nutrient media. As a carrier, barley is used. The mother crop for sowing grain is grown on a rocking chair with a circular rotation of 200 rpm on a wort diluted with tap water in a ratio of 1: 2 at 28 ^o C. The drug is grown for 5 days at 28 ^o C. The titer of the finished product 10 - 19 billion dispute / g. The drug is used in a dose of 20 kg / 1000 m ² , which allows to reduce diseases of vegetable crops of closed ground (cucumbers, tomatoes) by 37.5 - 98% and get a yield increase of 2.6 kg / m ² (cucumbers) and 1.2 kg / m ² (tomatoes).

Example 3. Tillage to improve the phytosanitary condition of soils (closed ground). After a single application of a liquid preparation in an amount of 30 g / m ^{2, the} phytopathogenic potential in the root zone of plants decreases from 9670 to 1730 colonies in 1 g of soil.

Sources of information
1. Gernet M.V. Biotechnology. 1985, N 3, p. 27 - 36.

 2. USSR author's certificate N 1717052. The strain of the fungus Trichoderma lignorum VNISXM N 37 for the production of trichodermin. BI 9, 1992.

 3. Baday S.V. Plant protection. 1986, N 5, p. 32.

Claims (1)

 The strain Trichoderma lignorum NII KKM SSC BB "Vector" F5, used against phytopathogenic fungi and bacteria.

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