RU2225107C2 - Method for preparing preparative form for storage of entomopathogenic nematodes of families steinernematidae and heterorhabditidae - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: invention relates to methods for preparing the preparative form for storage of entomopathogenic nematodes Steinernematidae and Heterorhabditidae used as biological preparations for control of insect-pests. Method for preparing the preparative form for storage of entomopathogenic nematodes of families Steinernematidae and heterorhabditidae involves preparing nematodes precipitate, preparing protective solution consisting of sodium alginate and Tween- -80, dispersing nematodes precipitate into protective solution and distinguishing by that 2-trifluoromethyl-3-oxoperfluorohexanoic acid ethyl ester is added in the concentration 0.1-0,2% additionally into protective solution with nematodes before mixing with silica filler. Invention provides increasing viability retention time of entomopathogenic nematodes for storage period, transporting and to improve technology in application of nematode preparations. EFFECT: improved preparing method. 2 tbl

Description

 The invention relates to the microbiological industry, and in particular to methods for storing entomopathogenic nematodes used as biological preparations in the fight against pests.

A known method of obtaining a preparation of entomopathogenic nematodes in water-soluble hydrogel granules based on alginates, consisting of the following components: sodium alginate, water and calcium chloride (CaCl ₂ 2H ₂ O). Alginate (2 g) is dissolved in 100 ml of water by vigorous stirring for 5 minutes. Invasive larvae of nematodes are mixed with an alginate solution in a ratio of approximately 4000 nematodes per 1 ml of water. The resulting solution containing nematodes was added dropwise to a solution of calcium chloride (100 mM CaCl ₂ 2H ₂ O). Granules are formed from alginate drops in the solution, which, after hardening, are separated by size after 20-30 minutes and rinsed with deionized water. Capsules are stored in plastic bags at a temperature of + 4 ^o C. After 9 months, the death of nematodes in capsules reaches 70-90%. Capsules are introduced into the soil to control insects, while the complete release of nematodes from the capsules occurs within a week (1).

 The following should be noted as disadvantages of the nematode formulation based on hydrogel granules: 1) the complexity of the manufacturing process of the granulated nematode formulation; 2) a small number of nematodes per unit mass of the formulation (1 g of alginate granules may contain from 5 to 15 thousand nematodes) and, as a result, a large mass of preparations, which, for example, for processing 1 ha of arable land will be from 200 to 500 kg; short preservation of the viability of nematodes in granules and slow release of nematodes from them in the soil, which allows the use of such a formulation as a prophylactic rather than an active plant protection against insects.

The closest known, adopted as a prototype, is a method of obtaining a water-dispersible pseudo-dry formulation consisting of the following components: sodium alginate, Tween-80, Aerosil grade A-380 (hydrophilic silicon dioxide powder) and distilled water. In preparative form, the nematodes remain viable at a temperature of 18-22 ^o C for 7 days and 3 months at a temperature of 2-8 ^o C (2).

 The disadvantages of the prototype are determined by: 1) the complexity and the need for strict adherence to the technique in the manufacture of the formulation, since Aerosil is an extremely light particles in dry form, easily forming a persistent aerosol that can cause chronic pneumonia in the staff, resembling lung silicosis; 2) the high cost of aerosil (up to $ 8,000 per ton); 3) poor dispersibility of the formulation in water and the formation of sediment, which impedes the use of the drug by spraying equipment; 4) a short period of preservation of the viability of nematodes during storage.

 The problem solved by the invention is to increase the shelf life of entomopathogenic nematodes during storage, transportation and improvement of the technology for the use of nematode drugs.

 The proposed method for preparing a preparation for the storage of entomopathogenic nematodes is that the precipitate of an aqueous suspension of nematodes after centrifugation is first dispersed in a protective aqueous solution containing 1% sodium alginate and 0.5% Tween-80 (Polyoxyethylene sorbitan monooleate, n = 20, qualification "h").

 With stirring, 2-trifluoromethyl-3-oxoperfluorohexanoic acid perfluorocarbon-ethyl ester is additionally added to the protective solution with nematodes at a concentration of 0.1-2%. The addition of perfluorocarbon, which is the active substance of blood substitutes, helps to increase the concentration of dissolved oxygen in a protective aqueous solution and thus helps to improve the living conditions of nematodes.

BS-100 grade carbon black (non-volatile hydrophilic silica powder with a surface area of 100 m ² per 1 g of substance) is added to the resulting nematode concentrate with constant stirring in the ratio: the mass of the nematode concentrate to the mass of white carbon from 2.8 to 2.9.

 The criterion for the end of the mixing process is the bulk density of the formulation, which should be ≤0.5 g / ml, which is expressed in obtaining a loose, homogeneous pseudo-dry gray powder.

The finished formulation is placed in plastic bags, which are hermetically sealed and stored at a temperature of 2-22 ^o C.

Example 1. The preparation of the preparative form begins with the centrifugation of an aqueous suspension of invasive larvae of nematodes (2 thousand rpm), having previously determined the number of viable nematodes in it. For example, per 1 liter of the initial aqueous suspension, the number of nematodes amounted to 50 million individuals, while 750 million individuals were found in the selected 15 liters of nematode suspension (B). After centrifugation, the mass of the sediment with nematodes was 480 g (O _s ).

Next, calculate the required amount of protective solution according to the formula
0.74 • M - O _s ,
where M is the estimated final weight of the drug,
M = B: 250 (the estimated number of nematodes in 1 g of the drug), then M = 750 million .: 250 thousand = 3000 g.

 The total mass of the protective solution (1% alginate + 0.5% Tween-80) will be 0.74 • 3000 - 480 = 1740 g.

Preparation of a protective solution: a weighed portion (0.5%) of 10 g Tween-80 is dissolved with stirring in 2000 ml of water, then here, with heating (80-90 ^o C) and constant stirring (1000-2000 rpm) add a weighed portion of alginate (1%) - 20 g until complete dissolution of the alginate. In a cooled solution to room temperature (1740 g), the nematode precipitate (480 g) is dispersed with constant stirring until a homogeneous mass is obtained.

The resulting homogeneous mass of nematodes in a protective solution is divided into three parts, each of which is additionally added with stirring ethyl 2-trifluoromethyl-3-oxo-perfluorohexanoic acid in concentrations of 0.05, 0.1 and 0.2%, respectively. e. 0.37, 0.74, 1.48 g). Next, white carbon black (BS-100) is added to the obtained nematode concentrates with constant stirring (60 rpm at the beginning of the process and up to 180 rpm at the end of it), the amount of which is calculated by the formula
BS = M • O, 26,
then BS = 3000 • 0.26 = 780 g (i.e. 260 g per concentrate).

Prepared formulations are packaged on the scales in plastic bags (25 million nematodes in 1 package). The bags are sealed and stored 5 bags at room temperature (18-22 ^o C) and 5 bags in the refrigerator at a temperature of 2-8 ^o C. At certain intervals, samples of the drug are taken from the bags and the viability of the invasive nematode larvae is assessed. The sample is placed in a measuring glass with water and a glass rod is dispersed in water, after which, under the MBS-10 binocular, the nematode viability in the sample is counted and assessed. Samples are taken in 5-fold repetition.

 The results of the assessment of the persistence of entomopathogenic nematodes Steinernema carpocapsae strain "agriotos" in the manufactured formulations are shown in table 1.

After the storage period is over, the pathogenicity of invasive nematode larvae is determined by the LD ₅₀ index (the number of invasive nematode larvae causing 50% insect death - G. mellonella caterpillars) for nematode cultures before embedding them in the preparative form and after storage (Table 2 ) (3).

 Thus, the data in tables 1 and 2 indicate the efficiency of storage of entomopathogenic nematodes in the proposed preparative forms, which achieve an increase in the duration of the preservation of the viability and invasive activity of nematodes compared to the prototype.

The best preservation of invasive larvae is achieved in preparative form, in the manufacture of which perfluorocarbon in a concentration of 0.1% is additionally introduced into the protective solution with nematodes. The duration of preservation of the viability of invasive larvae in this formulation increased to one month at room temperature and up to 6 months in storage at low temperatures (2-8 ^o C). In this case, invasive larvae do not lose pathogenicity with respect to host insects.

 With a decrease in perfluorocarbon concentration, the duration of invasive larvae viability decreased. An increase in the concentration of perfluorocarbon up to 0.2% practically does not affect the duration of the preservation of the viability of nematodes. A further increase in concentration is impractical due to the poor solubility of this substance in a protective solution with nematodes.

 The cost of a new formulation is reduced tenfold compared with the prototype and the process of its preparation does not require compliance with special safety precautions.

Sources of information
1. Kaya HK, Nelsen CE Encapsulation of steinernematid and heterorhabditid nematodes with calcium alginate: a new approach for insect control and other applications. Environ. Entomol. - 1986. - Vol. 14. - P. 572.

 2. Mevlyudov R.Ya., Danilov L.G., Ayrapetyan V.G., Sychev A.E. A method of preparing a biological preparation of nematodes for storage and its subsequent use. Patent 2144289, - Bull. 2. - 2000

 3. Veremchuk G.V., Danilov L.G. Guidelines for determining the invasive activity of nematode cultures of the genus Neoaplectana (Steinernematidae). L., 1978, 7 pp.

Claims (1)

A method of obtaining a formulation for storing entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae, comprising obtaining a precipitate of nematodes, preparing a protective solution of sodium alginate and Tween-80, dispersing the precipitate of nematodes into a protective solution, combining the resulting nematode concentrate with white soot, characterized in that in protective a solution with nematodes before mixing with white soot additionally add ethyl ester of 2-trifluoromethyl-3-oxoperfluorohexanoic acid in a concentration of 0.1-0.2%.

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