RU2226547C1 - Fungus strain entomophthora thaxteriana (petch - Google Patents

Abstract

) (ZYGOMYCETES; ENTOMOPHTHORALES) 9 (VIZR) AS PRODUCER OF BIOPREPARATION ENTOX. FIELD: biotechnology, agricultural microbiology. SUBSTANCE: invention can be used in preparing preparations used for protection of plants against aphids, spider mites and thrips. The strain Entomophthora thaxteriana (Petch. ) 9 (VIZR) is obtained by step-by-step selection from monoconidial plating the natural isolate isolated from peach aphid. Selection is carried out by the trait of toxin formation entox by isolates of this fungus in culturing under submerged conditions. The strain Entomophthora thaxteriana (Petch. ) is deposited in the collection of pure cultures VIZR at 9. The yield of entox by this strain is 8.39-8.45 g per 1 l of nutrient medium consisting of 4% of glucose, 1% of soybean meal and water. Content of entox in biomass is 70-71%. The prepared fungus strain shows higher level for formation of toxic substances (base of the biopreparation entox) as compared with the known strain and insecticide activity against suctorial pests. EFFECT: valuable properties of fungus strain. 2 tbl, 1 ex

Description

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

Known strains of the fungus Entomophthora thaxteriana Fetch. No. 18, 35-MP and 19-11 (Zygomycetes; Entomophthorales) (VIZR) (AS No. 1345385 and No. 2150836), which are producers of toxic substances - the basis of biological products. However, in the process of preservation and repeated reseeding, strain No. 35-MP lost its toxin-forming properties and reduced its biological effectiveness in relation to insect pests. Strain 19-11 was selected as the producer of mycoafidin T and therefore has a very low entox productivity.

Strain No. 18 (AS No. 1658432), which is also a producer of toxins, was taken as a prototype; over time, its toxin-forming properties and the activity of toxins significantly decreased.

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 (the basis of the biological product entox) and insecticidal activity against sucking pests. Strain Entomophthora thaxteriana (Petch.) No. 9 (VIZR) was obtained by stepwise selection from a monoconidial sieve of a natural isolate isolated from peach aphids. The selection was carried out on the basis of the formation of toxins - entox by isolates of this fungus during cultivation in deep conditions. The strain Entomophthora thaxteriana (Petch.) Was deposited in the collection of pure cultures VIZR under No. 9. The yield of entox is in the strain 8.39-8.45 g with 1 liter of culture medium consisting of 4% glucose, 1% soy flour and water (the rest). Entox content in biomass is 70-71%.

Morphological features

The vegetative mycelium of the strain is non-centered, colorless, decaying 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.

Conidia homers 25-39 × 21-32 microns, an average of 32 × 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% non-hopped beer wort), Saburo medium (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-hour illumination, are covered with a bloom of conidia, which gives the colonies a white color. Pigment on Wednesday is not allocated.

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

Color

On agarized nutrient media (Saburo, soybean and must), the strain forms colorless colonies. Pigment on Wednesday is not allocated. 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 there is no snorulation. It grows poorly and sporulates on media containing sucrose, lactose, galactose, arabinose, xylose and alcohols.

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

Temperature

Growth was observed in the temperature range + 3-36 ° С. The optimum temperature for growth is 27 ° C for conid formation 20-22 ° C.

Microaerophilic growth.

The strain is a strict aerobic. Checking the viability of the strain is carried out by reseeding the culture on freshly prepared wort agar, followed by cultivation in an incubator at 22 ° C.

Reseeding Method

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

Conservation Method

The strain can be stored on jambs of wort-agapa at + 4 ° C for 1-1.5 years without reseeding. In the State collection of microorganisms VIZR Russia, the strain is stored for 8 years without loss of viability.

The presence of marker signs

The strain produces two types of endotoxins (mycoafidin T in a smaller amount and entox in a larger one) and has a selective toxic effect. Productivity of Entomophthora thaxteriana (Petch.) No. 9 but for entox is 8.39-8.48 g / l of medium, 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 No. 35-MP, 18, 19-11 and No. 9 are grown on a medium containing 1% soy flour and 4% glucose, water - the rest (pH 6.5-7.0) at 27 ° C for 3 days. The biomass is filtered off, dried at a temperature of 37 ° C (to an air-dry state) and ground.

Isolation of entox from 1 g of biomass is carried out by hydrolyzing it with an alcohol solution of alkali. The resulting solutions were separated by filtration from biomass and the alcohol was evaporated until it was completely removed.

The yield of entox for strain E. thaxteriana No. 9 is 71% or 8.482 g / l of culture medium, respectively.

The yield of entox for strain E. thaxteriana No. 35-MP is 45% or 3.5 g / l of culture medium, respectively.

The yield of entox for strain E. thaxteriana No. 18 is 11.5% or 0.35 g / l of culture medium, respectively.

The yield of entox for strain E. thaxteriana No. 19-11 is 14.5% or 1.885 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. L. - Pushkin, 1988). The method consists in the following: aphids were placed in a Petri dish 40 mm in diameter 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. The treatment of spider mites is carried out by dipping bean leaves with mites in a 1% aqueous emulsion of the preparations. The leaves are then placed in Petri dishes with water to prevent ticks from moving from the treated surface; death is counted after 24 hours.

In the table. 1, 2 shows the insecticidal activity and yield of the preparative forms of strains of the fungus E. thaxteriana No. 18 and No. 9.

Thus, the strain Entomophthora thaxteriana (Рetch.) No. 9 has an increased level of toxin formation and biological effectiveness, which allows to reduce the concentration of the working emulsion of the preparations obtained on its basis to 0.5% to achieve 90-100% death of crop pests in open and closed ground.

Claims (1)

The strain of the fungus Entomophthora thaxteriana (Petch.) (Zygomycetes; Entomophthorales) No. 9 (VIZR) to obtain the biological product of Entox against sucking insect pests and ticks.