RU2129375C1 - Biopreparation liquid "phytosporin" for plants protection against sicknesses - Google Patents

Abstract

FIELD: agriculture, biotechnology. SUBSTANCE: biopreparation liquid "Phytosporin" is cultural fluid containing 15-25 billion cells/ml endophytic bacterium Bacillus subtilis VNIISHM-128 (26D), antibiotics at their lytic activity 100-200 IU/ml causing lysis of phytopathogenic fungi and bacteria. Addition of stabilizing agent to a preparation and biomass stabilizing agent - humin at concentration 0.5-2% provides safety of live colony-forming cells amount in the preparation for 6-8 months and improves its properties due to enrichment with humic acids which are benefit for soil formation. EFFECT: enhanced effectiveness. 3 tbl, 5 ex

Description

 The invention relates to biotechnology, namely to biological products intended to inhibit the development of phytopathogenic fungi and bacteria.

 Currently, a dry biological preparation Phytosporin has been created based on living cells and spores of the endophytic bacterium Bacillus subtilis 26 VNIISXM 128 for protecting plants from a complex of diseases (patent N 2099947) (1).

 The preparation contains living cells and B. subtilis spores and an excipient. The drug is poured into sterile ampoules, freeze-dried and sealed. As a filler, the preparation may contain bovine serum (cattle), milk, reverse, sucrose-gelatin mixture (4% sucrose, 1% gelatin). The need for a filler is due to its protective effect for bacterial cells during freeze drying of the drug.

However, the disadvantage of the proposed form of the biological product Fitosporin - dry powder in ampoules, is:
1) the need for the acquisition and maintenance of expensive and energy-intensive devices for freeze drying, and the drying period itself is quite long (50-80 hours);
2) the inconvenience of using ampoules with the drug for the preparation of large volumes of workers diluting the drug before using them for processing plants;
3) the preparation contains only the biomass of the antagonistically active strain B. subtilis 26D and does not contain ready-made antibiotic substances that have lytic activity against pathogenic fungi that are produced by bacterial cells during cultivation and are contained in the culture fluid.

 The objective of the invention is to create a new form of the biological product Fitosporin, less expensive, convenient to use, containing ready-made antibiotic substances to protect plants from diseases caused by bacteria and fungi.

 The problem is solved by obtaining the biomass of the strain Bacillus subtilis 26 by homogeneous deep cultivation in a liquid nutrient medium containing mineral salts, an organic nitrogen source (peptone or tryptone) and carbohydrates.

Deep cultivation is carried out at 37-50 ^o C with saturation of the medium with oxygen in the range of 60-70% and stirring at a speed of rotation of the mixer 250-300 rpm The growth control of the culture is carried out by sampling every 2 hours and determining the concentration of microbial cells according to the optical turbidity standard and FEK 56M, the presence of spores is determined in smears of the culture stained by Gram, the antibiotic activity of the culture filtrate is determined by adding it to the test strains of phytopathogenic cultures fungi and is estimated by the decrease in optical density in units of lytic activity (2).

 Cultivation is carried out for 12-16 hours. The resulting culture fluid contains 15-25 billion cells / ml (70-100% of the spores) and antibiotic substances, due to which the specific activity of the liquid preparation Fitosporin is enhanced. To the obtained biomass is added a stabilizer of gumi biomass of 0.5-2%, which ensures the preservation of the number of living colony forming cells in the preparation for 6-8 months.

 The culture fluid containing the spore biomass of strain B. subtilis 26, antibiotic substances that provide lytic activity and a biomass stabilizer is used as the bacterial preparation Fitosporin in liquid form.

 The use of gumi as a stabilizer of the biomass of the drug Fitosporin improves its properties as an agricultural preparation due to the enrichment of humic acids useful for soil formation (3).

The preparation contains living cells of B. subtilis VNIISKHM 128, antibiotic substances and a stabilizer, vol.%:
B. subtilis biomass VNIISKHM 128 - (15-20) • 10 ⁹
Living microbial cells in 1 ml of culture fluid - 98.5-97
Stabilizer - 0.5-2
Antibiotic substances: lytic activity - 100-200 UNITS
The need for a stabilizer is due to its protective effect for bacterial cells during storage of the drug for 6-8 months.

 Example 1. On the basis of strain B7 subtilis VNIISHM 128 with typical morphological and cultural characteristics and biochemical properties, 6 variants of the preparation were prepared with different contents of microbial cells and different concentrations of stabilizer.

 Option 1.

A preparation containing, vol.%:
B. subtilis biomass VNIISKHM 128, - 10 • 10 ⁹
Living cells in 1 ml of culture fluid - 98.9
Biomass Stabilizer - Gumi - 0.1
Option 2

A preparation containing, vol.%:
B. subtilis biomass VNIISKHM 128, - 15 • 10 ⁹
Living cells in 1 ml of culture fluid - 98.9
Biomass Stabilizer - Gumi - 0.1
Option 3

B. subtilis biomass VNIISKHM 128, - 20 • 10 ⁹
Living cells in 1 ml of culture fluid - 98.5
Biomass Stabilizer - Gumi - 0.5
Option 4

B. subtilis biomass VNIISKHM 128, - 25 • 10 ⁹
Living cells in 1 ml of culture fluid - 98
Biomass Stabilizer - Gumi - 1
Option 5

B. subtilis biomass VNIISKHM 128, - 20 • 10 ⁹
Living cells in 1 ml of culture fluid - 97
Biomass Stabilizer - Gumi - 2
Option 6

B. subtilis biomass VNIISKHM 128, - 20 • 10 ⁹
Living cells in 1 ml of culture fluid - 96
Biomass Stabilizer - Gumi - 3
The lytic activity of the culture fluid in all variants of the drug was 100-200 UNITS.

 The manufactured versions of the drug were poured into sterile containers of 10 liters.

In the preparations made, the optimal amounts of bacterial cells of B. subtilus VNIISKhM 128 in 1 ml of culture fluid were 15 • 10 ⁹ - 25 • 10 ⁹ with a volume content of 98.9-96%. The optimal stabilizer content - gumi was 0.5-2%.

 The obtained variants of the drug were tested for harmlessness to animals, as well as specific antagonistic activity against test cultures - representatives of various groups of phytopathogenic bacteria and fungi.

To determine the safety, the contents of each variant of the drug in a volume of 1 ml was administered orally to mice. For each variant of the drug, at least 10 mice weighing 16-18 g were used. The drug was considered harmless if all the mice remained alive for 5 days of observation. The test results are presented in table. 1 (see end of description)
Tests have shown that all variants of the drug Fitosporin liquid are harmless. All animals remained alive during the observation period, none of them showed signs of disease, the initial weight of the animals increased.

 To determine the specific activity, the antagonistic activity of the drug variants was studied in relation to test cultures of phytopathogenic bacteria and fungi.

The study was carried out by the method of delayed antagonism. For this, the contents of the ampoule were dissolved in 1 ml of physiological saline. The resulting suspension was seeded with a loop in the center of a Petri dish with agarized Gauze medium No. 2 (to study the activity of bacterial test cultures) and potato agar (to study the antagonistic activity of mushroom test cultures). Crops were incubated in an incubator at a temperature of 28 ^o C for 72 hours. Then, test microorganisms were seeded to the grown culture with strokes (500 million suspensions of daily cultures of test bacteria and suspensions of fungal spores in physiological saline). Cups with test cultures of bacteria were incubated at a temperature of 37 ^o C for 18 hours, with test cultures of fungi at 28 ^o C for 5 days. The results of the experiments were taken into account by the size of the zones of inhibition of growth of test cultures. Controls were test cultures grown in parallel on Petri dishes with Gauze N 2 agar medium and potato agar without antagonist culture. The test results are presented in table. 2 and 3 (see the end of the description).

 The data obtained show that the investigated variants of the drug Fitosporin liquid are harmless to animals and are characterized by specific antagonistic activity against test cultures of phytopathogenic microorganisms.

 To study the shelf life of the drug Fitosporin liquid, a series of the drug manufactured in 1997 was stored and subjected to re-monitoring for the content of live colony-forming cells every 2 months. The number of living colony forming cells (CFU) was determined by plating on solid nutrient media: Gause 2 or potato agar and counting the grown colonies.

 In the table. 4 presents control data of 6 variants of the drug Fitosporin liquid.

 As follows from the table, when using a biomass stabilizer, living microbial cells are preserved in the preparation.

Used sources of information
1. Menlikeev M.Ya., Smirnov V.V., Baiguzina F.A. et al. Phytosporin biological product for protecting plants from diseases / Patent N 2099947, RU, C 12 N / 20, publ. 12/25/97, bull. No. 12.

 2. Kislukhina OV, Kalunyants K.A., Avizhenis V.Yu. Production technology of lysosubtilin GZ X // Abstracts of the All-Russian Research Institute of Protein Substances Biosynthesis. - 1974. - Vol. 2. - S. 77-83.

Claims (1)

 Biological product phytosporin liquid to protect plants from diseases, containing live microbial cells and bacterial spores of Bacillus subtilis VNIISKHM 128 (26D), characterized in that the number of live microbial cells in 1 ml of the drug is 15 - 25 billion and it contains antibiotic substances with lytic activity of 100 - 200 PIECES in 1 ml and biomass stabilizer - gumi 0.5 - 2%.

Images