RU2322060C1 - Composition for protection of vegetable cultures against fungal and bacterial diseases - Google Patents

Abstract

FIELD: biotechnology, agriculture.

SUBSTANCE: invention proposes a composition containing chitosan, chitin, succinic and glutamic acids, heteroauxine, and strain Bacillus subtilis M-22 or strain Trichoderma lignorum T-36. The composition possesses antagonistic activity in combination with stimulating effect on growth of plants and provides the effective protection of vegetable cultures against fungal and bacterial diseases.

EFFECT: valuable biological properties of composition.

6 tbl, 10 ex

Description

The invention relates to the field of agriculture, more specifically to the protection of plants from disease, and is a composition consisting of natural polymers and microorganisms antagonists of phytopathogens.

The invention can be widely used for pre-sowing treatment of seeds, planting material, vegetative plants, for applying to the soil before sowing (planting) crops as a means of biological protection of plants from diseases, environmentally friendly and not inferior to fungicides in efficiency.

Chitosan-based compositions are widely used in crop production as growth regulators (1) and to increase disease resistance (2).

An analogue of the claimed composition is a preparation of a similar composition - Phytochitis (chitazonium mannitol succinate), which is registered as a growth regulator (3).

The disadvantage of these compositions is the low biological effectiveness against phytopathogens.

As a prototype taken the composition according to the patent of the Russian Federation No. 2158510, decl. May 15, 1997) of the following composition (wt.%):

Chitosan 0.05-0.3 succinic acid 0.03-0.2 Glutamic acid 0.02-0.1 heteroauxin 0.006-0.05 Water up to 100 ml

The mass ratio of chitosan: acid is 1: 1.

The main disadvantage of the prototype based on chitosan in combination with biologically active substances is that their biological activity is based only on increasing disease resistance through an altered metabolism in the plants themselves, and the compositions practically do not have direct biocidal activity.

The objective of the invention is to enhance the protective effect of the composition against major diseases of vegetable crops, i.e. increasing its biological effectiveness.

The problem is solved by introducing into the composition of the known composition additional chitin and cultures of microbes antagonists of phytopathogenic fungi and bacteria in a certain ratio. It contains Bacillus subtilis M-22 or Trichoderma lignorum T-36 as antagonist microbes.

The enhancement of the protective effect of the claimed composition is due to the combination of the antagonistic properties of the antagonist microbe with the ability of chitosan, together with biologically active substances, to regulate the metabolism in plants, enhance the formation of antipathogenic compounds in them, and thereby increase the disease resistance of plants. In this case, there is an increase in the antifungal and antibacterial activity of the composition, based on the synergistic effect of the action of its constituent components.

Chitin is a natural biopolymer that is not only a raw material for producing chitosan, but is also used in agriculture as a protective agent, in particular against fusarium wilting of vegetable crops (4).

The strains of the antagonist bacteria Bacillus subtilis M-22 and Trichoderma lignorum T-36 are stored in the State collection of VIZR microorganisms and are used as producers of the biological products Gamair and Trichodermin (5,6). However, the biological effectiveness of the use of these biological products is often unstable and highly dependent on environmental conditions, which reduces their protective effect against diseases of vegetable crops. In the claimed composition, chitin and chitosan are a substrate for feeding antagonist microbes, contribute to their rapid reproduction and better survival, provide increased activity of enzymes (chitinases, chitosans, glucanases) of B. subtilis T. lignorum strains, which, in turn, increases their antagonistic activity, act as inducers of protective reactions of plants. The biological activity of antagonist microbes in the composition appears for a longer time and to a lesser extent depends on the environmental conditions for the use of the drug, which enhances their protective effect.

The claimed composition is obtained by mixing an aqueous suspension of a powder containing chitosan, chitin, succinic, glutamic acid and heteroauxin, with a culture of the microbe antagonist in a ratio of 1: 1.

Chitosan is taken with a molecular weight of 10000-150000 daltons and a degree of deacetylation of 65-97% in the following ratio of components, g:

Chitosan, M 100-150 kDa 0.03-3.0 Chitosan, M 10-15 kDa 0.007-0.7 Chitin 0.025-2.5 succinic acid 0.007-0.7 Glutamic acid 0.03-3.0 Heteroauxin 0.001-0.1 Water up to 100 g Bacillus subtilis cultures M-22 or Trichoderma lignorum T-36

The culture of the bacterium Bacillus subtiliss strain M-22 or the fungus Trichoderma lignorum strain T-36 is mixed with the composition in a ratio of 1: 1.

Antagonist microbial cultures are prepared as follows:

1. In relation to Bacillus subtiliss strain M-22 by deep cultivation of a strain of a microorganism in a nutrient medium containing 3% corn extract and 1.5% molasses for 3 days at 24-26 ° C (5);

2. For Trichoderma lignorum T-36: by deep cultivation of a microorganism strain in a nutrient medium containing 0.1% K ₂ HPO ₄ , 0.3% KCI, 0.05% MgSO ₄ , 2% starch or glucose, for 3 days at 24-26 ° C (6).

The titer of the cells of the microbial antagonist strains in the prepared compositions varies in the following ranges:

B. subtilis M-22: June ¹⁰ -10 ¹⁰ CFU / ml

T. lignorum T-36: 10 ⁵ -10 ⁸ CFU / ml

Examples of specific implementations of the invention.

The effectiveness of the composition of the claimed composition in protecting seedlings of a cucumber from a Fusarium infection (pathogen Fusarium oxysporum) is presented in Table 1.

Seeds of cucumber variety Phoenix previously decontaminated 0.1% solution of AgNO ₃ was soaked in 100 ml of a suspension of the claimed composition for 4 hours. Then the seeds were dried in air for 3-4 hours and infected with a suspension of Fusarium oxysporum fungus so that the infectious load was 0.8 × 10 ⁴ infectious units per 1 seed. After infection, the seeds were laid out in sterile wet chambers (Petri dishes) with a layer of cotton wool and filter paper moistened with standard sterile water. 20 seeds were placed in each Petri dish; the repetition of the experiment is 10 times. Cups with seeds were kept at a temperature of 25-27 ° C for 12 days. Accounting for plant damage by the disease was carried out according to the standard method (7) in comparison with the control (item 1) and the prototype (item 5), table 1.

Pre-soaking of cucumber seeds was carried out in a composition of the claimed composition, but with a different content of components in g (Table 1, pr. 2, 3, 4).

Example 1 (paragraphs 2, table 1) shows that this composition of the claimed composition reduces the incidence of fusarium infection of cucumber seedlings by 37.5% compared with the control and 15.6% more effective than the prototype (p. 5).

Example 2 (claims 3, table 1) demonstrates the high effectiveness of the claimed composition against Fusarium infection of cucumber plants. The composition reduces the damage to cucumber plants by 55.4% compared with the control and is 32.5% more effective than the prototype (claims 5).

Example 3 shows that the claimed composition is the same as in examples 1 and 2, but with a ratio of components in table 1 (paragraph 4), it is 10.0% more effective than the prototype.

The revealed concentration limits of the claimed composition are optimal in terms of the effectiveness of the protective effect of the claimed composition against fusarium infection.

A decrease in the concentrations of the components leads to a decrease in the effectiveness of the composition. The increase in the content of the components is impractical, since with an increase in the concentration of the components the biological effectiveness of the composition does not increase.

The highest biological activity was shown by the composition (claims 3), example 2. When the cucumber seeds are soaked in the claimed composition of this composition, the defeat of the seedlings and seeds of the cucumber by the Fusarium infection decreases, and the composition exceeds the prototype by 32.5% and 24.5% respectively.

The positive effect of all variants of the composition on seed germination, the length of the root against the background of infection of the seeds with infection was revealed. The composition also has the best regulatory activity (claims 3). It was found that the soaking of cucumber seeds in the claimed composition, composition (claims 3), increases germination by 13% compared with the control and by 9.7% compared to the prototype (claims 5).

= Example 4 confirms the high biological effectiveness of the claimed composition in the treatment of cucumber seeds against plant damage by the pathogen of Fusarium rot (Fusarium oxysporum), table 2. The processing of cucumber seeds (cultivar Phoenix) was carried out by soaking for 4 hours in a suspension of composition composition (g):

Chitosan, M 100-150 kDa 0.3 Chitosan, M 10-15 kDa 0,07 Chitin 0.25 succinic acid 0,07 Glutamic acid 0.3 Heteroauxin 0.01 Water up to 100 Bacillus subtilis m-22 10 ⁸ CFU / ml

A culture of Bacillus subtilis M-22 is mixed with the composition in a 1: 1 ratio.

As a reference, the fungicide TMTD (tetramethylthiuramdisulfide) 4 kg per 1 ton of seeds was used (PP.3, Table 2). Control seeds were soaked in water and sown in vessels with infected soil (10 ⁷ conidia of the fungus F. oxysporum per 1 kg of soil). Presowing seed treatment with the composition (claim 2) reduces the prevalence of Fusarium rot on the 30th day of the experiment more efficiently (55.7%) than the fungicide TMTD (15.7%) and prototype (14.3%).

Against the background of an increase in the number of affected cucumber plants in all variants of the experiment, the claimed composition more effectively inhibits the spread of the disease, reducing the number of diseased plants within 30 days by more than 2 times compared to the control and prototype

The biological effectiveness of the composition containing the bacterial strain Trichoderma lignorum T-36 as an antagonist in protecting the tomato from fusarium wilt depending on the ratio of the components of the claimed composition ranged from 46.1 to 57.9% (table 3, paragraphs 2, 3, four). A range of optimal concentrations of the components of the composition has been established, above and below which its biological effectiveness is reduced.

Upon receipt of the composition, the Trichoderma lignorum T-36 culture was mixed with the components of the composition in a 1: 1 ratio and the resulting aqueous suspension was introduced into vessels with the ground by spilling at the rate of 100 g of aqueous suspension per 1 kg of soil. After 3 days, an infection was introduced (10-14 daily culture of the fungus Fusarium oxysporum f. Lycopersici) at the rate of 10 ⁶ conidia per 1 kg of soil. After 72 hours, tomato seedlings (variety Talalikhin) were planted in the phase of 3 real leaves.

Example 5. The introduction of the claimed composition (PP.2) in the soil significantly reduced the lesion of tomato Fusarium wilt, especially in the initial stages of plant growth (table 3). On the 10th day in this experiment, the number of affected plants decreased by 5 times compared with the control and 2 times compared with the prototype. The biological effectiveness of the composition (claims 2) on the 30th day of the experiment exceeded the prototype (claims 5) by 17.0%.

Example 6. The composition of the composition of claim 4 for the effectiveness of the protective action is comparable to the composition of the composition of claim 2. Its biological effectiveness is 45.4%, which is 16.3% higher than the prototype (paragraph 5).

Example 7. The most optimal was the composition of the claimed composition in paragraphs 3 of table 3. In this experiment, the claimed composition 10 times reduced the spread of the disease compared with the control and 5 times compared with the prototype. The high efficiency of the claimed composition is maintained throughout the experiment and by 30 days exceeds the prototype by 28.8%. The claimed composition (claims 3, table 3) has not only a high protective effect, but also growth-stimulating activity, which is confirmed by biometric data in the experimental variants without infection (table 4).

Example 8 confirms the biological effectiveness of the claimed composition in the protection of cucumber plants from Fusarium infection. Test composition (g):

Chitosan, M 100-150 kDa 0.3 Chitosan, M 10-15 kDa 0,07 Chitin 0.25 succinic acid 0,07 Glutamic acid 0.3 Heteroauxin 0.01 Water up to 100 Trichoderma lignorum T-36 10 ⁶ CFU / ml

The Trichoderma lignorum T-36 culture was mixed with the components of the composition in a 1: 1 ratio and the resulting aqueous suspension was introduced into vessels with the ground by spilling at the rate of 50 g of aqueous suspension per vessel (500 g of soil). Three days later, a 10-14 day old culture of the Fusarium oxysporum fungus was introduced at the rate of 10 ⁶ conidia per 1 kg of soil and seedlings of cucumber plants (Phoenix village) were planted in the phase of the first true leaf, 5 plants per vessel (10-fold repetition). As the prototype used the same composition as in previous experiments.

An analysis of the data showed that at the initial stages of cucumber plant growth (up to 20 days), applying the composition 2.2 times reduces the development and prevalence of Fusarium infection compared to the control. According to the effectiveness of inhibiting the development of the disease, the composition exceeds the prototype (claims 3) by 1.9 times (Table 5). The studied composition does not adversely affect the growth and development of cucumber plants (Table 5).

Example 9 demonstrates the bactericidal activity of the claimed composition when studying its effect on the growth, development and affection of tomato plants by the causative agent of stem core necrosis (Pseudomonas corrugata), (vegetation experiment, table 6).

Presowing treatment of tomato seeds was carried out by soaking for 1 hour in an aqueous suspension of the claimed composition composition (g):

Chitosan, M 100-150 kDa 0.3 Chitosan, M 10-15 kDa 0,07 Chitin 0.25 succinic acid 0,07 Glutamic acid 0.3 Heteroauxin 0.01 Water up to 100 Bacillus subtilis m-22 10 ⁸ CFU / ml)

A culture of Bacillus subtilis M-22 is mixed with the composition in a 1: 1 ratio. Control plants were soaked in water.

Artificial infection of 20-day-old tomato plants was carried out by injection of an aqueous suspension of bacteria (P. corrugata) with a titer of 10 ⁶ cells / ml. The experiment was carried out in 6-fold repetition; in each repetition there were 5 plants.

Presowing treatment of seeds with the claimed composition increased by 28.3% the weight of the aerial parts of tomato plants and 1.7 times increased the wet weight of the roots compared to the control. It should be noted that the increase in root mass in plants in this experiment is associated with the formation of lateral roots. The claimed composition positively affects the growth, development of plants, stimulates root formation, increases disease resistance, reducing the spread of tomato necrosis of the stem of the tomato stem by three times compared with the control (paragraph 1). It has been experimentally shown that the claimed composition 15% more effectively inhibits the spread of the disease than the prototype (claims 3).

Example 10. A comparative assessment of the biological effectiveness of the claimed composition was carried out under the conditions of a shallow experiment in a production greenhouse of CJSC "Summer" (St. Petersburg) on tomato plantings with a total area of 1000 m ² (Table 7). Two variants of the claimed composition were made twice during the growing season by spilling the soil under a tomato plant: after transplanting seedlings to a constant place of growth and a month later. Plants cultivated according to the technology adopted at the farm served as control. Compositions of the following composition (g):

Chitosan, M 100-150 kDa 0.3 Chitosan, M 10-15 kDa 0,07 Chitin 0.25 succinic acid 0,07 Glutamic acid 0.3 Heteroauxin 0.01 Water up to 100 Bacillus subtilis m-22 10 ⁸ CFU / ml or Trichoderma lignorum T-36 10 ⁶ CFU / ml

The culture of the bacterium Bacillus subtiliss strain M-22 or the fungus Trichoderma lignorum strain T-36 was mixed with the components of the composition in a ratio of 1: 1 and introduced into the soil 100 g per plant.

During the growing season, the development of bacterial wilting (Clavibacter michiganense subsp. Michiganensis) of tomato plants and the spread of gray rot were observed. The claimed composition significantly reduces the number of plants affected by bacteriosis. Particularly effective is a composition comprising Trichoderma lignorum T-36 fungus as a microbe-antagonist, which reduces the number of tomato plants infected with bacteriosis by 3 times. Aerogenic infection (gray rot) is one of the most dangerous phytopathogens, causing significant harm to plantings of vegetable crops in protected ground. The composition had a positive effect on increasing the disease resistance of tomato plants, which was manifested in a significant decrease in the spread and degree of development of gray rot (pathogen Botrytis cinered).

The biological effectiveness of the compositions was 62.1-55.7% and exceeded the effectiveness of the prototype by 18.5-12.1%, respectively (phytopathological accounting 25.09).

The above examples indicate a high protective and growth-promoting effect of the claimed compositions with their environmental safety. High biological activity in combination with a stimulating effect on plant growth will provide effective protection of crops from fungal and bacterial infections.

Literature

1. US patent No. 4812159, the application. 03/13/87, published on 03/14/89.

2. RF patent No. 2158510, filed. 05/15/97.

3. The state catalog of pesticides and agrochemicals approved for use in the Russian Federation, Moscow, State Chemical Commission of the Russian Federation Ministry of Agriculture of Russia, pp. 243-246, 2002

4. Bell, A.A., Hubbard, J.C., Liu, L., Davis, R.M. and Sabbarao, K.V. Effects of Chitin and Chitosan on the Incidens and Severity of Fusarium Yellows of Celery // Plant Disease, v. 82, # 3, p. 322-238.

5. Titova Yu.A., Novikova II, Khlopunova LB, Korshunov DV Trichodermin based on secondary bioconversion of waste and its effectiveness against cucumber diseases // Mycology and Phytopathology, 2002, V.36, Issue 4, S.76-80.

6. Novikova I.I., Boykova I.V. New biological products for agriculture // Biotechnology - to the national economy, 2000, M., 2000, S. 47-48.

7. Guidelines for state testing of fungicides, antibiotics and seed dressers of agricultural crops, M., 1985.

table 2
The effect of presowing seed treatment on the defeat of cucumber plants with a Fusarium infection (vegetative experiment, artificial infectious background, Fusarium oxysporum) No pp Option The prevalence of the disease,% Biological efficiency,% 20 days 30 days 30 days 6 Control: artificial infection with the fungus F.oxysporum 21,4 35.7 - 7 Composition 10.5 15.8 55.7 8 TMTD 10.0 30.1 15.7 9 Prototype 12.5 30.6 14.3 HCP _.01 3.9 2,5 2.1

Table 4
The effect of the composition on the growth and development of tomato plants (vegetative experience) Example No. Experience option 10th day Plant height, cm The number of leaves Control - Water 7.7 5.7 5 Control - artificial infection with the fungus F. oxysporum 4,5 3.3 6 Composition + artificial infection with the fungus F. oxysporum 5.1 3.9 7 Composition without artificial infection with the fungus F. oxysporum 11.8 6.2 HCP _.01 0.8 0.5

Table 5
The effect of the composition on the spread and development of Fusarium infection of cucumber plants (vegetative experiment, infectious background, pathogen Fusarium oxysporum) No pp Option The spread of the disease,% The development of the disease,% Biological efficiency,% 10 days 20 days 30 days 20 days 30 days one Control + artificial infection with the fungus F. oxysporum 25.0 45.3 65.0 20.5 42.5 - 2 Composition 5,0 20,0 27,2 9,4 16.7 60.7 3 Prototype 10.0 40,0 45.4 19.6 29.6 31.8 NDS _.01 9.1 8.8

Table 6
The effect of the claimed composition on the growth, development and affection of tomato plants by the causative agent of stem core necrosis (P. corrugata), (vegetative experiment, 120th day of experiment) No pp Experience option The weight of the aerial part of the 1st plant The weight of the root of the 1st plant The prevalence of the disease,% g % to control one The control 19.8 one hundred 1,1 76,2 2 The claimed composition 25,4 128.3 1.8 25.0 3 Prototype 22.0 111.1 1,5 40,0

Table 7
The effect of the composition on the defeat of tomato plants by bacteriosis and gray rot (CJSC "Summer") No pp Option Affected plants,% The death of plants,% Biological efficiency for dead plants,% Bacteriosis (Clavibacter michiganensis) Gray Rot (Botrytis cinered) Accounting date 09/13 25.09 11.10 25.10 25.09 11.10 25.09 one The control 14.2 30.9 86.7 89.9 4.2 42.5 - 2 Prototype 18.8 15,5 60,4 62.7 1,0 35.8 43.6 3 Composition with B.subtilis M-22 (10 ⁸ CFU / ml) 8.9 11.7 40.1 52.3 0 17.3 62.1 four Composition with T.lignorum T-36 (10 ⁶ CFU / ml) 4.8 13.7 59.2 63.1 0.8 27,2 55.7

Claims (1)

Composition for protecting vegetables from fungal and bacterial diseases, including chitosan, succinic acid, glutamic acid, heteroauxin, water, characterized in that it additionally contains chitin and a strain of Bacillus subtilis M-22 or strain Trichoderma lignorum T-36 with the following components , g: Chitosan, M 100-150 kDa 0.03-3.0 Chitosan, M 10-15 kDa 0.007-0.7 Chitin 0.025-2.5 succinic acid 0.007-0.7 Glutamic acid 0.03-3.0 Heteroauxin 0.001-0.1 Water Up to 100, moreover, the strain Bacillus subtilis M-22 with a titer of 10 ⁶ -10 ¹⁰ CFU / ml or Trichoderma lignorum T-36 with a titer of 10 ⁵ -10 ⁸ CFU / ml is mixed with an aqueous suspension of the components in a ratio of 1: 1.