RU2149552C1 - Consortium of strain-antagonists for control of bacterial and fungal sickness in plants - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: invention relates to agents used for plant protection and presents consortium of strain-antagonists for these agents preparing. Consortium consists of four strains of soil bacterium-antagonists: Bacillus polymixa, Bacillus thuringiensis, Pantoea agglomerans and P. chlororatis. Consortium is designated for preparing the preparation against bacterial and fungal sicknesses in plants. Preparation can be used under conditions of open and closed soil, at all stages of plants growth for effective suppression of gray rot development and mildew in tomato and cucumber, bacterial cancer in clover, vascular bacteriosis in cabbage, angular leaf blotch in cucumber, fusariosis wilting in tomato, helminthosporosis in barley, root rot in cotton. EFFECT: enhanced effectiveness of consortium. 1 tbl, 11 ex

Description

 The invention relates to agricultural microbiology and is a consortium of antagonist strains that can be used to protect cultivated plants from phytopathogenic bacteria and fungi.

 The intensive use of chemical plant protection products in agriculture leads to environmental pollution, disturbance of the ecological balance in nature and, as a result, the emergence of new resistant forms of pathogens. In this regard, the problem of finding and developing new environmentally friendly methods of plant protection based on the environmental interaction of organisms in nature (antagonism, predation, parasitism, etc.) arises. One of such promising methods is the use of antagonist microorganisms and their vital products for the development and creation of biological products aimed at suppressing phytopathogenic microflora.

A number of strains are known and described:
Pseudomonas sp. B-3481, which reduces and prevents the development of mucous and vascular bacterioses in cabbage (Jalilov F.S.-U. et al., 1990);
Pseudomonas putida B-1743D, used to stimulate plant growth and protect plants from fungi of the genus Fusarium and bacteria Erwinia (Kochetkov V.V. et al., 1990).

Pseudomonas fluorescens CR-328D, showing antagonistic activity against fungi of the species Alternaria sp., Botrytis cinerea, Fusarium heterosporum, F. moniliforme, Helminthosporium sativum, Sclerotinia sclerotiorum, Verticillium dahlide (Hops TA et al., 1995);
Bacillus polymixa 9A, used to suppress the phytopathogenic fungus Verticillium dahleum (Kado CJ et al., 1984).

 A common drawback of the above strains is the high likelihood of resistance in phytopathogens with prolonged use of a monoculture of these antagonist bacteria. In addition, the spectrum of action of these cultures in each case is limited and does not affect, for example, such dangerous pathogens as bacteria Agrobacterium and Corynebacterium, fungus Erysifales.

 A consortium of strains of the destructive microorganisms Pseudomonas putida, Bacillus subtilis and Bacillus subtilis, known for wastewater treatment, is known and described (Roy A.A. et al., 1988). However, this consortium of bacteria can only be used in the textile industry.

 The aim of the invention is to obtain a new environmentally safe and effective microbial preparation for combating diseases of cultivated plants. To achieve this goal, a consortium of four strains of soil antagonist bacteria was proposed: Bacillus polymixa, Bacillus thuringiensis, Pantoea agglomerans, Pseudomonas chlororatis.

 The advantage of the proposed consortium of bacteria is that phytopathogens are simultaneously exposed to a complex of antimicrobial metabolites produced by antagonists, which enhances the effect. B. polymixa, P. chlororatis and Pantoea agglomerans produce antibiotic substances that are active against phytopathogenic fungi and bacteria. The P. aglomersans strain also secrete a chitinolytic enzyme that is active against fungi. Strain B.thuringiensis has a stimulating effect on plants. In addition, an important factor characterizing these strains is their ability to colonize the rhizosphere of plants.

 The strains included in the consortium do not have phytopathogenicity, as evidenced by the absence of necrotic spots on the leaves of the treated crops (tomatoes, cucumbers, cabbage, etc.) when applying a suspension of the tested strains to them.

 The strains of Bacillus polymixa, Bacillus thuringiensis, Pantoea agglomerans, Pseudomonas chlororatis are not pathogenic, as evidenced by the high LD50 values obtained in samples from mice. These strains are also not capable of invasion, which indicates their safety for animals.

To grow strains and obtain the preparation, medium 925 (Langley, Kado, 1972) of the following composition is used, g / l:
K ₂ HPO ₄ - 3; NaH ₂ PO ₄ - 1; NH ₄ Cl - 1; sucrose - 10; MgSO ₄ - 0.3; peptone - 2; tap water - 1 l.

 As a complete medium use the medium of the BCH (meat and peptone broth).

 A consortium of antagonist strains is obtained by mixing cultures of bacteria grown in 925 medium in a ratio of 1: 1: 1: 1.

 The strains are stored on nutrient jambs in the refrigerator. The resumption of culture is carried out by reseeding on fresh shoals after 4-5 months.

 The strains were identified by determinant ("Bergey's manual ..." v.1, 1984).

The isolated strains were deposited in the All-Russian collection of industrial microorganisms at the State Research Institute of Genetics under the numbers:
Bacillus polymixa - VKPM B-7539; Bacillus thuringiensis VKPM B-7540; Pantoea agglomerans - VKPM B-7541; Pseudomonas chlororatis - VKPM B-7542.

 Characterization of the strain of Bacillus polymixa VKPM B-7539.

 A new strain of B.polymixa isolated from the soil (Moscow region).

 Morphological signs. Gram-positive, straight, movable sticks, 2.0-4 microns x 0.8 microns in size. Forms elliptical or cylindrical, centrally located endospores. On a full-fledged environment, the colonies are small, rounded, the color of the colonies: light brown.

Physiological properties. Optimum growth at 32 ^o C. The strain does not grow at 5 ^o C, retains signs of growth at 45 ^o C. Optional anaerobic.

 It produces acetoin during the breakdown of glucose. It forms acid during fermentation of arabinose and xylose (mannitol is weak). Forms pectinase, gelatinase, levansaharazu. Hydrolyzes starch, indole does not form. Catalase is positive, does not produce citrase. Breaks down glucose to form acid and gas (weakly). It forms acid from glucose, lactose, mannitol, sorbitol, arabinose, maltose. Nitrate does not reduce. In 10% NaCl does not grow.

 Antibiotic resistance: amikacin (10 μg / ml), biseptol (200 μg / ml), polymyxin B (300 μg / ml), fusidine (10 μg / ml).

 Characterization of the strain of Bacillus thuringiensis VKPM B-7540.

 A new strain of Bacillus thuringiensis was isolated from chernozem soil (Saratov).

 Morphological signs. Gram-positive sticks, movable, peritrichi. Dimensions 4-6 microns x 1.0-1.2 microns. The colonies are grayish-white, smooth, pinnate, large at the edges. In the late stages of stationary growth, it forms spores in a subterminal position, forms protein crystals of a rhomboid shape.

Physiological properties. Optional anaerobic, optimum temperature growth 28-32 ^o C, does not grow at 50 ^o C, optimum pH 7.0-7.4. Starch hydrolyzes, fiber is not absorbed. It produces proteases - it dilutes gelatin, milk peptones. Absorbs glucose, sucrose, mannose, galactose, lactose does not ferment. Nitrate does not reduce.

 The strain has toxicity for caterpillars 2-3 age of wax moth (Galleria melonella), unpaired silkworm.

 Antibiotic resistance: ampicillin (10 μg / ml), benzylpenicillin (10 μg / ml), polymyxin (300 μg / ml).

 Characterization of the strain Pantoea agglomerans VKPM B-7541.

 A new strain of P. agglomerans was isolated from the roots of a vineyard affected by bacterial cancer; the soil is typical serozem, Republic of Uzbekistan.

Morphological signs. Cells are gram-negative sticks, straight, slightly curved, of medium size. The cell size is 2-4 microns x 0.6-0.8 microns. They retain mobility at temperatures from 22 ^o C to 37 ^o C. Colon color: on the medium MBP and 925 white with a dull shade, on the diagnostic environment of bismuth sulfate with brilliant green - the colonies are green and brown.

 Physiological properties. Optional anaerobic; oxidase negative, catalase positive. The fluorescent pigment does not form; it does not dilute gelatin, starch does not hydrolyze. It does not need specific growth factors. Does not form β-galactosidase, indole, hydrogen sulfide, lysine and ornithine, urease. Ferments glucose, arabinose, mannitol, sorbitol, sucrose, xylose; does not ferment adonite, maltose, inositol. When fermented, glucose forms acid (reaction with bromothymol blue and methyl red is positive); fermentation of glucose is accompanied by the formation of gas and acetoin (Voges-Motulsky reaction is positive); when fermented, lactose forms acid; during the fermentation of rhamnose, acid does not form; Levan from sucrose does not form. 3-ketolactose does not form; able to use citrate as a carbon source; do not dispose of sodium malonate; does not dispose of urea.

 Antibiotic resistance: ampicillin (10 μg / ml), benzylpenicillin (10 μg / ml), oleandomycin (10 μg / ml), lincomycin (15 μg / ml), kefzola (10 μg / ml), fusidine (10 μg / ml )

 Characterization of the strain Pseudomonas chlororatis VKPM B-7542.

 A new strain of Pseudomonas chlororatis isolated from loamy soil in the Saransk region.

 Morphological signs. Cells are straight, small sticks 3.0 μm x 0.8-1.0 μm, motile, gram-negative. The endospore does not contain. Colony color: dull gray. On diagnostic media, Kinga produces a green fluorescent pigment that turns pink over time.

Physiological properties: Aerob. Cells grow in the range from 4 to 38 ^o C, with a temperature optimum of 25-30 ^o C, the optimum pH is 7-8. Form 3-ketolactose. Prototroph. It uses glucose, fructose, sucrose, glycerin, dulcite, inositol, mannitol as carbon sources. Fermentation of these substances produces acid without gas. Mineral salts in the ammonium form, and in the organic form in the form of peptone, amino acids, are used as sources of nitrogen. Nitrates do not reduce. Starch is not hydrolyzed. Gelatin is diluted. They coagulate milk, but do not peptone. Form indole, hydrogen sulfide, ammonia. The following amino acids are decomposed with the formation of acid: l-histidine, dl-lysine, dl-ornithine, l-arginine, l-proline, dl-serine; with the formation of alkali amino acids: l-alanine, l-asparagine, 1-glutamine.

 Antibiotic resistance: ampicillin (10 μg / ml), benzylpenicillin (10 μg / ml), erythromycin (10 μg / ml).

 The following are examples showing the effectiveness of the use of antagonist bacteria against phytopathogenic fungi and bacteria, both individually and as part of a consortium of strains.

 Example 1 demonstrating the antagonism of strains of B. polymixa VKPM B-7539, B. thuringiensis VKPM B-7540, P. agglomerans VKPM B-7541, P. chlororatis VKPM B-7542 against the phytopathogenic bacterium Corynebacterium michiganese and the fungus Fusarium oxysporum.

The strains of antagonist bacteria were grown by injection on the surface of agar medium 925 at 30 ^o C for two days. After that, the colonies were killed in pairs of chloroform for 30-40 minutes and Petri dishes were kept open for 40-60 minutes under sterile conditions to completely remove chloroform residues. Then, the lawn of the test culture was applied to the surface of the plates. To obtain a lawn, a culture of C. michiganese bacteria was grown to a concentration of 10 ⁷ cells / ml in 925 medium and nutrient agar was introduced in a dilution of 1: 100. Petri dishes were cultured under conditions optimal for the growth of the test strain at a temperature of 30 ^o C for three days.

The fungus F. oxysporum was grown separately on a plate with 925 medium, after which a mycelial fragment was taken in a loop and resuspended in sterile water so as to obtain a concentration of 10 ⁸ kf / ml. Nutrient agar was added in 0.7% at a 1:10 dilution and evenly poured onto the surface of the Petri dishes. Then the injection was made by antagonist bacteria. Cups were set at 28 ^° C. and grown from 3 to 5 days until the fungus mycelium formed a uniform lawn.

 Zones of growth inhibition of phytopathogens were determined as the radius of the clarification zone around the colony of the antagonist strain (see Table A).

 Example 2, demonstrating the antagonism of strains of B. polymixa VKPM B-7539, B. thuringiensis VKPM B-7540, P. agglomerans VKPM B-7541, P. chlororatis VKPM B-7542 individually and as part of a consortium for the phytopathogenic bacterium Agrobacterium tumefaciens.

When kolanchoe plants are infected with pathogenic A.tumefaciens strains, well-distinguishable tumors, crown galls, appear on the leaves. To study the antagonistic activity, injections (10 injections per plant) were applied to the lower surface of the leaves of the colanchoe with a syringe containing: separately the oncogenic strain A. tumefaciens 1D1 or strain 1D1 mixed with antagonist bacteria or a consortium of antagonist strains. After 3-5 weeks, the percentage of gall formation was observed and counted. Complete suppression of gall formation during infection with A. tumefaciens strain was observed in the case of a consortium of antagonist strains. Individual cultures suppressed the formation of galls with the following efficiency,%:
B.polymixa - 20
B.thuringiensis - 100
P.agglomerans - 80
P. chlororatis - 85
consortium - 100
Examples 3-10, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against plant pathogens in field and vegetation experiments.

 Field and vegetation experiments on host phytopathogen models were carried out according to methodological guidelines (Dospekhov, 1985).

 Studies were carried out on a natural infectious background, as well as on an artificial infectious background, which was created according to the methodological guidelines (Chumakov et al., 1979).

где N_k - количество пораженных растений в контроле,
N_о - количество пораженных растений в опыте.The index of suppression of plant diseases by strains of antagonists was determined by the formula

where N _k is the number of affected plants in the control,
N _about - the number of affected plants in the experiment.

To obtain a preparation of individual antagonist bacteria or a consortium of antagonist strains, antagonist bacterial strains were grown on 925 medium to a density of 10 ⁹ cells / ml. Then the preparation of individual antagonist bacteria or their consortium was used in the form of a 0.2% suspension for the treatment of experimental plants (seeds) infected or diseased plants at any phase of the growing season, including the fruiting stage. The consumption rate of the drug was 0.5 l per square meter. The frequency of treatment - depended on the degree of damage to plants.

 Example 3, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against powdery mildew of cucumbers in a greenhouse.

 Among the most harmful phytopathogens affecting cucumbers in closed ground conditions are powdery mildew pathogens of cucumbers mushrooms from the order of Erysiphales. In order to study the ability of the bacterial strains selected by us to suppress the development of powdery mildew in the conditions of the greenhouse of the Moskovsky State Farm-Plant, plots were isolated on which about 100% of the leaves were affected by powdery mildew. Plants with signs of disease damage were treated 4 times at one day intervals with culture fluid of B. polymixa VKPM B-7539 antagonist strains, B.thuringiensis VKPM B-7540, P.agglomerans VKPM B-7541, Pseudomonas chlororatis VKPM B-7542 separately and as part of the consortium. Observations of the development of the disease on the studied plants and their condition were carried out 14 days after the start of treatment of plants with bacterial suspensions. The index of disease suppression by a consortium of antagonist strains was higher than in plants treated with individual strains. Treatment of cucumbers in the greenhouse with antagonist strains prevented the further development of powdery mildew on the leaves of cucumbers, while a slowdown in the growth of mushrooms existing on the leaves of the fruit bodies was observed, and in some cases, their vital activity was stopped. The index of disease suppression by a consortium of antagonist strains was higher than when treated with individual strains. The experimental results are shown in table 1.

 Example 4, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against powdery mildew of tomatoes in a greenhouse.

 The experiments were carried out analogously to example 3, but using tomatoes affected by powdery mildew as a plant model. And in this case, the index of disease suppression by a consortium of antagonist strains was higher than when treating plants with individual strains. The results are shown in table 1.

 Example 5, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against gray rot of tomatoes in a greenhouse.

 In a protected ground, the phytopathogenic fungus Botrytis cinerea, which causes the disease “gray rot”, causes significant damage to tomato plantations. To verify the biocontrol activity of a consortium of antagonist strains, experiments were carried out on diseased plants in the greenhouse of the Moskovsky state farm. At the same time, groups of plants (20-30 bushes each) with almost the same degree of stem damage by gray rot, which was evaluated in points, from 1 (superficial damage to the stem) to 5 (complete damage to the stem by sectional area) were selected for research. When processing stems of tomatoes with a suspension of cells of antagonist strains, two methodological approaches were used. In the first case, plants with signs of stem damage by spore gray rot were treated with a suspension of the corresponding bacterial strains individually or as part of a consortium. In the second case, the treatment of the wound was carried out after scraping the areas damaged by gray rot on the stems. As a control, options were used in which plants were treated with distilled water. Observations of the state of plants were carried out 40 days after the plants were treated with a suspension of cells of antagonist strains individually or as part of a consortium. The best results were obtained in the variants with the removal of damaged tissue, which was produced immediately before the treatment of plants with preparations of antagonist strains. The results are presented in table 1.

 Example 6, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against gray decay of cucumbers in a greenhouse.

 The experiments were carried out analogously to example 5, but with the use of cucumbers affected by gray rot as a model of plants. In this case, manual treatment of the affected areas was not performed. The results are shown in table 1.

 Example 7, demonstrating the effectiveness of the action of antagonist strains individually and as part of a consortium, against the angular spotting of cucumber in vegetation experiments.

The causative agent of angular spotting, the bacterium Pseudomonas syringae pv.lachrymans, affects the aboveground organs of the cucumber throughout the entire growing season, starting with the appearance of cotyledons. To study the biocontrol activity of cells of antagonist strains, the seeds of Parade cucumbers were treated with a pathogenic strain of P. syringae pv.lachrymans, and then with culture liquids of antagonist strains individually or as part of a consortium. In the control, the seeds were treated with distilled water. Germination of seeds was carried out at a temperature of 28 ^o C for 72 hours, and then for 12 to 15 days at room temperature on Petri dishes on the surface of filter paper moistened with distilled water. Calculation of the defeat of seedlings of cucumbers with angular spotting was carried out after two weeks. The results of this experiment are presented in table 1. As can be seen from the table, in this case, the index of disease suppression by the consortium of antagonist strains was 93%, and was higher than the antagonist strains separately.

 Example 8, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against vascular bacteriosis of cabbage in vegetation experiments.

 The causative agent of the disease is vascular bacteriosis of the cruciferous bacterium Xanthomonas campestris pv.campestris affects plants in almost all phases of its development. To determine the effectiveness of the antagonist strains, experiments were carried out analogously to example 7, but using cabbage seeds infected with the bacterium X. campestris pv as a model. campestris. The results are shown in table 1.

 Example 9, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against cotton root rot in vegetation experiments.

 The common root rot of cotton, caused by the phytopathogenic fungus Rhizoctonia solani, manifests itself in this crop in the early stages of its development (up to the stage of 6-8 leaves) and leads to a significant thinning of the seedlings or their complete death. To study the effect of antagonist strains, vegetation vessels filled with 2/3 of the volume of sandy loam soil (pH 7.2) were sown with cotton seeds (9-12 seeds per replicate), and then covered with a layer of soil infected with a suspension of R. solani fungus at the rate of 1.5 mg / kg of soil, after which the soil surface was treated with suspensions of individual antagonist strains or a consortium of antagonist strains. 7 days after sowing the seeds into the soil, the plants were re-treated with a suspension of bacteria. Accounting was carried out two weeks after the start of the experiment. The degree of damage to cotton seedlings was determined as the percentage of seedlings affected by root rot to the total number of seeds sown. And in this case, the index of disease suppression by the consortium of antagonist strains was maximum and amounted to 82.3%. The results are shown in table 1.

 Example 10, demonstrating the effectiveness of the antagonist strains individually and as part of a consortium against cancer of clover meadow in field experiments.

The defeat of grass clover meadow meadow pathogen Cancer Whetzelinis trifoliorum leads to severe damage to the culture and can cause complete loss of plants in the first and second years of cultivation. To study the efficiency of use of strains antagonist to protect red clover from diseases caused by the fungus were performed W.trifoliorum small plot field trials based on Institute of feed (pos. Meadow, Moskovskaya obl.) On plots area of 1.7 m ² hard infectious background. 2 weeks before sowing clover seeds in open ground, they were processed and inlaid with suspensions of antagonist bacteria individually or a consortium of antagonist strains. For effective inlaying of antagonist bacteria, a mixture of a suspension of these bacteria with a 2% solution of CMC cellulose was used. Observations of the growth and development of plants in the experimental plot were carried out for two years. The results were calculated for 2 years. The best indicator of the disease suppression index was in the case of using a mixture of bacteria included in the consortium. The results are presented in table 1.

 Example 11, demonstrating the effectiveness of a consortium of antagonist strains against helminthosporiosis of barley in field experiments in comparison with chemical pesticides.

The phytopathogenic fungus Helminthosporium sativum causes root rot of cereal plants. The drug was tested in a production environment on the basis of the collective farm. Schepkin, Yakovlevsky district, Belgorod region. on an area of 20 hectares in 1995. As reference disinfectants, fungicides raxil and baytan in standard concentrations were used. Sowing material, 5 tons of seed barley of the Romantic variety were treated with a consortium of antagonist strains in a PS-10 pickling machine and sown with a seeding rate of 240 kg / ha. The plot under the demonstration experience is heavily loamy soil, chernozem. During the growing season, precipitation did not fall, which was an additional factor provoking the development of helminthosporiosis. Data on the defeat of plants was recorded after 1.5 months at the stage of the onset of grain loading in barley. The results showed that the prevalence determined by the standard method in the experimental plots was,% in relation to the control:
Raxil - 26
Baytan - 11
Consortium of antagonist strains - 13
Thus, it should be noted that in all the examples cited, the effectiveness of the consortium of antagonist strains exceeded the efficiency of phytopathogen suppression by cultures of individual strains. In addition, the high and stable indicators of the antibacterial and fungicidal activity of the drug of the consortium of antagonist strains on various phytopathogen host models indicate the prospects for the effective use of this drug for plant protection in agriculture. Due to the fact that the pesticidal activity of the drug is comparable to the activity of chemical agents (for example, raxil, baytan), this biological product can reduce the use of chemical plant protection products and stabilize the deteriorating environmental situation.

LITERATURE
1. USSR patent N 1793878, 02/07/93.

 2. Armor B.A. Methods of field experience (with the basics of statistical processing of research results). - M .: Agropromizdat, 1985, p. 351.

 3. USSR patent N 1805849, 03/30/93.

 4. Copyright certificate of the USSR N 1592330, 09.15.90.

 5. USSR patent N 1817874, 05.20.95.

 6. Chumakov A.E., Vlasov Yu.I., Bushkova L.N., Sidorova S.F., Guskova L.A. Infectious backgrounds in phytopathology / Ed. By Yu. N. Fadeev. - M .: Kolos, 1979, p. 208.

 7. "Bergey's manual of Systematic Bacteriology". V.1, ed. Krieg N.R. et al., Baltimore / London, 1984.

 9. Kado C.J., Liu S.-T. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol., 1981, 145, 1365-1373.

 10. Kado C.J., Schhathorst W.C., Arad H.R. Method of using B.polymixa 9A to protect plants against Verticillium dahleum vilt. Patent No. 4663162, USA. Stated March 14, 84. N 589608. Publ. 05/05/1987.

 11. King E.O., Ward M.K., Raney D.E. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med., 1954, 44, 301-307.

 12. Lynch J. M. Biological control of plant diseases: achievements and prospects. In: Brighton Crop Protection Conference-Pest and Diseases, 1988, 587-596.

Claims (1)

 A consortium of antagonist strains of Bacillus polymixa VKPM B-7539, Bacillus thuringiensis VKPM B-7540, Pantoea agglomerans VKPM B-7541, Pseudomonas chlororatis VKPM B-7542 to obtain a preparation against bacterial and fungal plant diseases.

Images