RU2035145C1 - Trichodermine productiuon method - Google Patents

Abstract

FIELD: agricultural microbiology. SUBSTANCE: method involves preparation of sterile nutritive medium containing (mass %) glycerol - 0.3-2.0, green molasses - 0.5-3.0, hydrolyzate of protein-vitamin concentrate - 3-10, phosphates - 0.2-0.6, magnesium sulfate - 0.3-0.5, ammonium nitrate - o.3-0.6, and water - the rest. Fungal race Trichoderma lignorum is inoculated for aerated cultivation till maximum accumulation of mycellium or sporulation. In the process of cultivation it is expedient to add 0.3-1.0 % of glycerol as pH of the culture fluid drops naturally to 3.0-4.5. The resulting culture fluid is condenced until the content of dry matter is 6-10%. For production of liquid preparation the concentrate is supplemented with glycerol (4-10%) and polyvinyl pyrollidone (1-3%) or polyethylene oxide (0.1-1.0%). For production of dry substance, the culture liquid concentrate (dm 20-30%) is mixed with mixture of ceolyte of type NaX and montmorillonite or polygorskite (1: 2). The rate of supplementation is 10-30% (dm). The mixture is deied and granulated. EFFECT: higher product quality. 4 cl

Description

 The invention relates to agricultural microbiology and relates to the production of plant protection products against diseases, in particular a method for producing a mushroom preparation trichodermin.

Known methods for producing trichodermin by deep cultivation of a microscopic fungus of the species Trichoderma on nutrient media containing carbon sources, nitrogen and mineral salts, followed by concentration and drying of the concentrate [1]
The closest technical solution to the invention is a method for producing trichodermin by deep cultivation of a microscopic fungus in a nutrient medium containing corn extract, molasses, salt and water, concentrating the culture fluid, introducing clinoptilolite into the zeolite concentrate, granulating the mixture, drying and grinding granules [2]
The disadvantage of this method is the relatively low content in the preparation of the active principle due to the relatively low moisture-binding ability of the clinoptilolite used.

 The aim of the invention is to increase the content of active principle in the preparation, as well as the preparation of trichodermin in both dry and liquid form.

 The essence of the method is as follows.

 Prepare a sterile growth medium containing, by weight. Glycerin 0.3-2 Green molasses 0.5-3 Protein-vitamin concentrate hydrolyzate (BVK) 3-10 Potassium phosphate 0.2-0.6 Magnesium sulfate 0.3-0.5 Ammonium nitrate 0.3-0.6 Water The rest.

The medium of the strain of the fungus Trichoderma lignorum is inoculated and cultured with stirring and aeration. In the process of cultivation with a natural decrease in the pH of the culture fluid to 4.5-3.0 add glycerin in an amount of 0.3-1.0%
Cultivation is carried out for 3-5 days.

 To obtain a liquid preparation, cultivation is carried out to the maximum accumulation of mycelial mass or spore formation, to obtain a dry preparation to a maximum spore formation.

 Upon receipt of the liquid preparation, the culture fluid is concentrated to 6-10% CB and 4-10% glycerol and 1-3% polyvinylpyrrolidone or 0.1-1.0% polyethylene oxide are introduced into the concentrate.

 To obtain dry trichodermin, a mixture of NaX type zeolite and montmorillonite or palygorskite in a ratio of 1: 2 in the amount of 10-30% to dry substances is introduced into the culture fluid concentrate (CB 20-30%), the resulting mixture is granulated and dried.

PRI me R 1. Prepare 3 m ³ sterile nutrient medium containing, by weight. Glycerin 0.3 Green molasses 3.0 BVK hydrolyzate 5.0 Phosphates 0.2 Magnesium sulfate 0.3 Ammonium nitrate 0.6 Water Else
The medium is inoculated with seed at the rate of 5% of the medium volume of the strain of the fungus Trichoderma lignorum TB-10 and cultivated in a fermenter with a volume of 5 m ³ under aeration and stirring, the cultivation time is 4 days.

A culture fluid is obtained with a titer of Chlamydospores 1 × 10 ⁸ in 1 ml.

By a known method receive 0.710 ⁸ spores / ml

 The culture fluid is divided into 2 parts, one of which is used to obtain a liquid preparation, and the other dry.

 Getting trichodermin in liquid form.

The resulting culture fluid was concentrated by filtration (10% SW), yielding 70 kg of paste containing mycelium, residues of the nutrient medium and chlamydospores (titer 2 × ^{10 9} spores / g).

 10% (7 kg) of glycerol and 0.1% (0.07 kg) of wetted polyethylene oxide are added to the paste, mixed and cooled.

The resulting preparation, which is a persistent non-stratified suspension, has the following characteristic:
Chlamydospore titer 1.0 ˙ ^{10 9} spores / ml,
biological efficiency of 70% against root rot of cucumbers and tomatoes,
the shelf life of up to three months at a temperature not higher than 10 ^° C

 Getting the drug dry.

 Add to the paste (CB 20%) 2.5 kg (10%) of a mixture of NaX zeolite and montmorillonite in a 1: 2 ratio, keep under stirring to redistribute moisture and pass through a GR-75 granulator, the granules are dried, packed and marked.

The resulting preparation is a granule from light gray to dark brown in color, which during storage and transportation do not cake. Biological effectiveness of the drug against root rot of cucumbers and tomatoes 65% titer of chlamydospores 1.0˙10 ⁹ spores / g.

PRI me R 2. Prepare 3.0 m ³ sterile culture medium containing, by weight. Glycerin 2.0 Green molasses 0.5 BVK hydrolyzate 10 Phosphates 0.6 Magnesium sulfate 0.3 Ammonium nitrate 0.3 Water Else
The cultivation and concentration process is carried out as described in example 1, but in the process of cultivation with a decrease in the pH of the culture fluid to 4.5, glycerol is introduced in an amount of 0.3%, the cultivation duration is 3 days.

 A trichodermin liquid preparation is prepared from the obtained culture fluid, introducing 20 kg (10%) of glycerol and 2 kg (1%) of polyvinylpyrrolidone into the paste.

Get trichodermin, which is a stable suspension and having the following characteristic:
titer of viable chlamydospores 0.5 ˙ 10 ⁸ spores / ml,
biological efficiency of 70% against root rot of cucumbers and tomatoes.

PRI me R 3. Prepare 3.0 m ³ sterile culture medium containing, by weight. Glycerin 1.5 Green molasses 2.5 BVK hydrolyzate 3.0 Phosphates 0.4 Magnesium sulfate 0.4 Ammonium nitrate 0.5 Water Else
The cultivation and concentration process is carried out as described in example 2, but glycerol is introduced at a pH of 3.0 culture fluid in an amount of 1.0%, the cultivation duration is 5 days.

 A dry preparation is prepared from the obtained culture fluid as described in Example 1, but 15 kg (30%) of a mixture of NaX zeolite and palygorskite are introduced into the paste in a ratio of 1: 2.

Biological effectiveness of the drug against root rot of cucumbers and tomatoes 75% titer of chlamydospores 2 ˙ ^{10 9} spores / g.

As can be seen from the above examples, the proposed method allows to increase the content of active principle in a dry preparation from 0.8 × ^{10 9} to 2 × ^{10 9} spores / g and to obtain a liquid preparation that is not inferior in dry activity to biological.

Claims (3)

 1. METHOD FOR PRODUCING TRIHODERMINE by deep cultivation of microscopic fungi Trichoderma lignorum in a nutrient medium containing carbon sources, organic nitrogen, phosphates, magnesium sulfate, ammonium nitrate and water, followed by concentration of the culture fluid, characterized in that glycerol and green molasses, as a source of organic nitrogen, a hydrolyzate of protein-vitamin concentrate in the following ratio of components in the medium, wt. Glycerin 0.3 2.0
Molasses green 0.5 3.0
Protein-vitamin concentrate hydrolyzate 3.0 10.0
Phosphates 0.2 0.6
Magnesium sulfate 0.3 0.5
Ammonium nitrate 0.3 0.6
Water Else
2. The method according to claim 1, characterized in that in the process of cultivation with a natural decrease in the pH of the culture fluid to 3.0 4.5 additionally enter glycerin in an amount of 0.3 to 1 wt.  3. The method according to PP.1 and 2, characterized in that the cultivation of the fungus is carried out until the maximum accumulation of mycelial mass or spore formation, the concentration of the culture fluid is carried out to a solids content of 6 10%, 4 10 wt. glycerol and 0.1 to 1.0 wt. polyethylene oxide or 1 to 3 wt. polyvinylpyrrolidone.  4. The method according to PP.1 and 2, characterized in that the cultivation of the fungus is carried out to the maximum spore formation, the concentration of the culture fluid is carried out to a solids content of 20-30%; a mixture of a zeolite of the NaX type and montmorillonite or polygorskite in a ratio of 1 2 in an amount of 10 is introduced into the concentrate 30% solids, the resulting mixture is granulated and dried.