RU2192745C1 - Fungus strain entomophthora thaxteriana petch (zygomycetes: entomophthorales) № 19 (vizr) for biopreparations preparing - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: invention relates to preparing the novel fungus strain with enhanced level of formation of toxic substances and insecticide activity (as compared with the known level) against sucking pests. Strain of fungus Entomophthora thaxteriana No. 19 (VIZR) is obtained by step-by-step selection from monoconidial inoculation of natural isolate isolated from peach aphid. Selection is carried out by trait of toxin formation of isolates of this fungus under conditions of submerged culturing. Strain of fungus Entomophthora thaxteriana is deposited in collection of pure cultures VIZR at No. 19. The yield of mycoaphidine T in strain is 0.85-0.9 g and entox is 4.8-5.0 g from 1 l of nutrient medium comprising of 4% of glucose, 1% of soybean flour and water. Content of mycoaphidine in biomass is 6-6.2% and entox is 34.0-34.9%. Use of this strain ensures to decrease concentration of working emulsion of preparations obtained on its base to 0.5% and to provide 90-100% death of agriculture pests in opened and closed soil. Invention can be used in preparing two preparations for plants protection against aphids, cobweb mites and thrips. EFFECT: improved method of preparing, valuable properties. 2 tbl, 1 ex

Description

 The invention relates to the field of agricultural microbiology and can be used to obtain preparations for protecting plants from aphids, spider mites and thrips.

 The strain of the fungus Entomophthora thaxteriana Petch was selected as the closest prototype. 18 (Zygomycetes; Entomophthorales) (VIZR) (A.S. 1658432), which is a producer of toxic substances - the basis of biological products, however, in the process of preservation and repeated reseeding, this strain sharply reduced toxin formation, and its biological effectiveness against insects decreased - pests.

 The objective of the invention is to obtain a new strain of the fungus with a higher (compared with the known) level of formation of toxic substances and insecticidal activity against sucking pests. The strain Entomophthora thaxteriana 19 (VIZR) was obtained by stepwise selection from a monoconidial sieving of a natural isolate isolated from peach aphids. The selection was carried out on the basis of toxin formation of isolates of this fungus during cultivation in deep conditions. The strain Entomophthora thaxteriana was deposited in the collection of pure cultures VIZR under 19. The yield of mycoafidin T in the strain is 0.85–0.9 g, entox 4.8–5.0 g with 1 liter of culture medium consisting of 4% glucose, 1% soy flour and water (the rest) . The content of mycoafidin T in the biomass is 6-6.2%, entox 34.0-34.9%.

MORPHOLOGICAL SIGNS
The vegetative mycelium of the strain is nonseptic, colorless, decomposing after 24 hours into short straight or curved hyphae. The formation of conidiophores with conidia occurs on the substrate mycelium.

 Conidiophores are short, colorless, unbranched, with one conidia at the end.

 Spherical conidia with a papilla at the base contain one large or several small fatty inclusions.

 The size of conidia is 25-39 x 21-32 microns, an average of 32 x 26 microns. Conidia have a single-layer cell wall, a smooth surface.

 The formation of resting spores begins at 66 hours of growth. The resting spores are rounded with a double shell and a large fat drop in the center. The dimensions of resting spores are 20-25 microns, an average of 22 microns. The surface of the spores is smooth, the cell wall is three-layered.

 Cultural and morphological characteristics of the strain were studied for wort (50% un hopped beer wort), Saburo media (1% peptone and 4% glucose) and soybean (1% soy flour and 4% glucose). On all these media, the strain forms flat, smooth and colorless colonies, which under 24 hours of light are covered with a bloom of conidia, which gives the colonies a white color. No pigment is released on Wednesday.

The surface growth pattern is smooth. The colony growth rate under standard conditions (pH 6.5-7.0, growing temperature 22 ^o С) on wort and Saburo medium 3.0 mm day, on soybean medium - 2.8 mm day.

COLOR
On agarized culture media (Saburo, soybean and wort), the strain forms colorless colonies. No pigment is released on Wednesday. A well-sporulating colony is white due to abundant white plaque from conidia.

PHYSIOLOGICAL SIGNS
The pigment does not form and does not stand out on Wednesday. From sources of carbon, it absorbs glucose, maltose, starch, fructose well (they show the best growth and sporulation). On a medium with amino acids, it can grow without hydrocarbons due to the carbon part of amino acids, however, growth is weak and sporulation is absent. It grows poorly and sporulates on media containing sucrose, lactose, galactose, arabinose, xylose and alcohols.

 It absorbs mixtures of amino acids from nitrogen sources, peptides, proteins, and can grow on asparagine media. Nitrate nitrogen does not absorb; on media with ammonium nitrogen, growth is weak.

TEMPERATURE
The growth was noted in the temperature range of 3-36 ^o C. The temperature optimum for growth of 27 ^o With conidia 20-22 ^o With
MICROAEROPHILIC GROWTH
The strain is a strict aerobic. Verification of the viability of the strain is carried out by reseeding the culture on fresh wort-agar, followed by cultivation in an incubator at 22 ^o C.

METHOD OF RESET
The duration of cultivation on jambs of wort agar is 7-8 days, after which the strain can be stored without reseeding for 1-1.5 years.

METHOD OF CONSERVATION
The strain can be stored on jambs of must-agar at 4 ^o C for 1-1.5 years without reseeding. In the State collection of microorganisms IBFMAN Russia, the strain is stored in liquid nitrogen for 3 years without loss of viability.

AVAILABILITY OF MARKER SIGNS
The strain produces two types of endotoxins and has a selective toxic effect. The productivity of E. thaxteriana 19 in terms of mycoafidine T is 0.85-0.9 g / l, according to the entox 4.8-5.0 g / l of medium, respectively, the insecticidal activity is 98-100% in various variants with test insects. The level of toxin formation of the strain is a stable sign: after 16 generations, the toxigenicity of the strain and the effectiveness of drug samples remains at the initial level.

EXAMPLE
Strains of E. thaxteriana 18 and 19 are grown on a medium containing 1% soy flour and 4% glucose, water - the rest (pH 6.5-7.0) at 27 ^o C for 3 days. The biomass is filtered off, dried at 37 ^° C (to an air-dry state) and ground.

 The isolation of mycoafidia T from 1 g of biomass is carried out with ethanol for 60 minutes, entox - hydrolysis of the biomass with an alcohol solution of alkali. The resulting solutions are separated by filtration from biomass and the alcohol is evaporated until it is completely removed.

 The yield of mycoafidin T for E. thaxteriana 19 strain is 6.08%, entox 34.7% or 0.89 g / l and 4.9 g / l of culture medium, respectively.

 The yield of mycoafidin T for E. thaxteriana 18 strain is 3.99%, for entox 11.5%, or 0.25 and 0.98 g / l of culture medium, respectively.

 Insecticidal activity was determined by the express method described in the “Test procedure for mycoafidin T in laboratory, field and production conditions” (compiled by Voronina E.G., Mukamolova T.Yu. et al., Leningrad - Pushkin, 1988). The method consists in the following: aphids were placed in a Petri dish with a diameter of 40 mm on a filter moistened with 0.3 ml of the drug solution, death was recorded after 1 h of contact with the treated surface, the number of repetitions was 4-5 cups, the number of insects in 1 cup 20-30 individuals.

 The effectiveness of drugs on tobacco thrips larvae was determined by the express method similar to aphids. Spider mites are treated by dipping bean leaves with ticks in a 1% aqueous emulsion of the preparations. The leaves are then placed in Petri dishes with water. To prevent tick scattering, death is considered after 24 hours.

 In the table. 1, 2 shows the insecticidal activity and yield of the preparative forms of the strains of the fungus E. thaхteriana 18 and 19.

 Thus, the strain Entomophthora thaxteriana 19 has an increased level of toxin formation and biological effectiveness, which allows to reduce the concentration of the working emulsion of preparations obtained on its basis to 0.5% to achieve 90-100% mortality of crop pests in open and closed ground.

Claims (1)

 The strain of the fungus Entomophthora thaxteriana 19 (VIZR) for the production of biological products mycoafidin T and entox against sucking insects - pests and ticks.

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