RU2421512C1 - Nutrient medium to cultivate entomopathogenic fungi - Google Patents

Abstract

FIELD: agriculture. ^ SUBSTANCE: nutrient medium contains ground larvae of synanthropic flies (live), wood chips and millet with the specified ratio of components. ^ EFFECT: invention makes it possible to increase yield of biomass of entomopathogenic fungi and biological activity of fungi towards live insect pests. ^ 2 tbl

Description

The present invention relates to agriculture and is intended for biological protection of cultivated plants from pests.

Due to the tense environmental situation, biological methods for controlling the number of plant pests are currently gaining priority. One of the areas of bioprotection is the use of entomopathogenic fungi. Entomopathogenic fungi have the ability in vivo to infect harmful insects and ticks, they can cause massive diseases and death (epizootics). In addition, fungi are able to grow on artificial nutrient media (PS), which allows you to create biological products on their basis.

A known nutrient medium, taken as an analogue. In particular, entomopathogenic fungi - producers of biological products (Beauveria bassiana (Bals.) Vuill., Metarhizium anisopliae (Metch.) Sor. And others) - are cultivated on solid PS, which includes ground millet and bran in a 2: 1 ratio ( Ogarkov B.N., Ogarkova G.R. Entomopathogenic fungi of Eastern Siberia. - Irkutsk, 2000. - P.134).

However, repeated reseeding of fungi on PS leads to a gradual decrease in their pathogenicity (Avanesov S.G. Manifestation of enzymatic activity as a virulence factor of the fungus Verticillium lecanii during cultivation. // Ecological problems of plant protection: abstract of a conference of young scientists. - L., 1990. - P.217; Thomas P. Hayden, Bidochka MJ, Khachatourians GG Entomopathogenicity of Several Fungi toward the English Grain Aphid (Homoptera: Aphididae) and Enchancement of Virulence with Host Passage of Paecilomyces farinosus // J. Econ. Entomol., 1992. - No. 1. - P.58-64).

To preserve the pathogenic properties of fungi, they resort to the passivation of industrial strains through the body of the host insect (Ogarkov B.N. Biological justification of the methods for creating and using mushroom preparations for controlling insects. Author. Diss. Dr. Pa. Biol. Sciences. - L., 1990 . - P.19), however, this technique is rather laborious and time-consuming.

A known method of increasing the pathogenicity of strains of fungi by introducing into the composition of the nutrient medium flour from insects. The nutrient medium for cultivation of entomopathogenic fungi, adopted as a prototype, includes flour from housefly larvae (Tomilova OG, Usova ON, Sternshis MV Modification of the environment for cultivation of entomopathogenic fungi. // Modern Mycology in Russia. - The First Congress of Mycologists of Russia. - Abstracts - Moscow, 2002. - P.214).

The inclusion of insects in the form of heat-treated flour in the nutrient medium has several disadvantages. The fungus does not develop on a living insect, but on a substrate that, after exposure to high temperatures (120-130 ° C), significantly changes its physicochemical properties (Tokarev VS, Nakozin V.A. Technology for the preparation and storage of feed flour from coprophage larvae. / / Biotechnology of organic waste processing and ecology. / Under the editorship of I.I. Gudilin, A.F. Kondratov, Novosibirsk. - Novosibirsk Book Publishing House, 1999. - P.220-228), which, naturally, will lead to a decrease pathogenic properties of the fungus in relation to live (native) insect pests. Thermal drying of larvae is an energy-intensive and expensive process. To store flour from larvae, preservatives are added that can to some extent inhibit the development of entomopathogenic fungi, enter plants, and through them into animals, humans, and the environment. The composition of nutrient media includes ingredients that are human food, which is irrational for economic and ethical reasons.

In addition, the composition of the nutrient medium, even the most optimal, does not take into account the fact that in the future (after processing the protected culture) entomopathogenic fungi will have to be on the plant for some time. As you know, a sharp change of substrate for all living organisms leads to stress, a delay in development, and even death.

In the nutrient medium adopted for the prototype, the larvae of synanthropic flies are subjected to heat treatment twice: first, when the larvae are dried to the state of flour, and the second time during autoclaving of the nutrient medium, which increases the cost of production and reduces the quality of the nutrient medium.

The technical task is to increase the virulence of entomopathogenic fungi by optimizing the nutrient medium for their cultivation and reducing its cost.

The objective is achieved by creating a nutrient medium, including a mixture of crushed larvae of synanthropic flies with sawdust and millet.

Live larvae are mixed with sawdust in a ratio of 1: 1 and crushed, the resulting mixture is dried. In this form, the larvae can be stored for up to 3 months without the use of preservatives. To the mixture of larvae with sawdust, ground millet is added in a 1: 1 ratio, the nutrient medium thus obtained is sterilized in an autoclave at 0.5 atm for 20-30 minutes, and then the spores of an entomopathogenic fungus are seeded.

If it is possible to deliver larvae of synanthropic flies live to the place of production of the nutrient medium, the process of drying the mixture of larvae with sawdust is excluded, and all components of the nutrient medium are crushed at the same time.

A decrease in the proportion of larvae of less than 20-25% in relation to other components does not lead to a significant increase in the virulence of an entomopathogenic fungus, and an increase in their share increases the cost of the nutrient medium without a significant increase in activity. A decrease in the proportion of millet in the nutrient medium less than 50% will lead to a decrease in the growth of the vegetative mass of the entomopathogen, and an increase in the proportion of this component does not increase the yield of biomass and spores, which is irrational.

Autoclaving the nutrient medium for 20-30 minutes at a pressure of 0.5 atm is most rational, since when the pressure in the autoclave is less than 0.5 atm, the required disinfection of the nutrient medium is not achieved, and an increase in pressure of more than 0.5 atm will increase the cost of production. Autoclaving for less than 20 minutes will also not provide the required disinfection of the culture medium. Autoclaving for more than 30 minutes will increase the cost of production.

The composition of the nutrient medium for the cultivation of entomopathogenic fungi, the ratio of components wt.%:

- larvae of synanthropic flies, live 20-25% - wood sawdust 20-25% - millet ground 60-50%

The claimed nutrient medium differs from the prototype in that the larvae of synanthropic flies are mixed with sawdust in a ratio of 1: 1, and then ground. In the process of grinding, sawdust absorbs moisture released from living larvae, contributing to better preservation of biologically active insect substances. In the direct preparation of PS, milled millet is added to the mixture of larvae with wood sawdust in the required amount, the medium is sterilized in an autoclave at 0.5 atm for 20-30 minutes. Unlike dry larvae, which were dried at high temperatures, autoclaving results in disinfection of the environment from extraneous microorganisms with a high degree of preservation of proteins, amino acids, chitin and other biologically active substances contained in living insects (Tokarev BC, Nakozin V.A. Preparation and storage technology feed flour from coprophage larvae // Biotechnology of organic waste processing and ecology. / Under the editorship of II Gudilin, AF Kondratov. Novosibirsk. - Novosibirsk book publishing house. 1999. - S.220-228). The use of wood sawdust in the nutrient medium reduces the consumption of millet, which is a food product, by 25%.

The use of distinctive features - mixing larvae of a synanthropic fly with wood sawdust, followed by grinding the resulting mixture in order to obtain a nutrient medium for culturing entomopathogenic fungi is unknown to the authors and applicant.

The inventive nutrient medium maximally preserves the physicochemical properties of the larvae, substances that are unstable to high temperatures (juvenile hormones, ecdysones, prostaglandins, enzymes) contained in housefly larvae, and save up to 500 l of liquid fuel per ton of larvae and 400 kW on the drying process / h of electricity.

The essence of the invention is illustrated below by the following description of the composition of the nutrient medium for the cultivation of entomopathogenic fungi, wt.%:

- larvae of synanthropic flies, live 20-25% - wood sawdust 20-25% - millet ground 60-50%

When growing entomopathogenic fungi on the claimed medium on the 14th day of growth, the titer of viable spores per unit area increases by 1.5-3.6 times (L. muscarium), by 3-5.1 times (B. bassiana) compared with analog medium (table 1).

The biological effectiveness of the fungus Lecanicillium muscarium (Petch) Zare et W. Gams (synonym for Verticillium lecanii (Zimm.) Viegas) strain F-8 (Novosibirsk) with a titer of a working suspension of 10 ⁷ spores / ml cultivated on the medium of the proposed composition increases depending on from day by 29.1-8.7% compared with the analogue medium and is located at the level of the fungus passaged through a living host (test object is an ordinary spider mite - Tetranychus urticae Koch.). When using a less pathogenic strain of the fungus Beauveria bassiana (Bals.) Vuill in relation to the galleria caterpillars - Galleria mellonella, a more significant increase in biological activity is observed (28-22.5% on the 7th and 10th day after infection) compared with the fungus cultivated on analog medium (table 2).

Table 1 The titer of viable spores of entomopathogenic fungi on the 14th day of growth, cultivated on PS of various compositions Kind of mushroom Title, spore / ml Analog medium Proposed environment Beauveria bassiana 0.65-3.68 × 10 ⁷ 3.3-11.0 × 10 ⁷ Lecanicillium muscarium 1.1-3.4 × 10 ⁷ 1.6-12.3 × 10 ⁷

table 2 Biological effectiveness of entomopathogenic fungi (titer of working suspension 10 ⁷ spores / ml) Kind of mushroom Biological effectiveness on the 7th and 10th day,% Proposed environment Analog medium Mushroom passaged through the host Beauveria bassiana * 33.2-45.5 5.2-23.0 50.0-66.3 Lecanicillium muscarium ** 88.1-96.0 59.0-87.3 92.9-98.5 Note: - test object * - Galleria mellonella, ** - Tetranychus urticae

Claims (1)

A nutrient medium for the cultivation of entomopathogenic fungi, including live shredded larvae of synanthropic flies, characterized in that it additionally contains sawdust and millet in the following ratio of components, wt.%:
synanthropic fly larvae, living 20-25% wood sawdust 20-25% ground millet 60-50%