Strain actinomycete streptomyces hygroscopicus subsp. smc in-4561 with fungicidal, bactericidal and insecticidal properties
The invention relates to the field of Microbiology and can be used as a means for protection of plants from diseases and pests. The strain Streptomyces hygroscopicus subsp. 24 deposited in the Central collection of microorganisms of Public concern “biological product” under SMC In-4561 isolated from soil samples in the Central region of Russia. This strain possesses a broad spectrum of action on phytopathogenic microorganisms and exhibits insecticidal properties, causing the death of larvae of the 1st age of the Colorado potato beetle Leptinotarsa decemlineata and larvae of Galleria mellonella. The strain shows the greatest biological activity. 5 tab., 1 Il.
The invention relates to the microbiological industry, and agricultural Microbiology, in particular, for the creation of plant protection products from the most harmful diseases and pests.
The literature describes actinobacteria producing antibiotics against pathogenic microorganisms and pests [1, 2]. Unlike pesticides, antibiotics are selectively suppressing pathogens and without creating tissue and embryos of plants. Antibiotics have Vysokoye and to avoid the accumulation of residues of harmful substances in food and the environment.
A known strain actinomycete Streptomyces griseochromogenae  effective to combat particularism rice, but it has a narrow spectrum of action.
Described a strain of Streptomyces casugaensis  is less effective in combating particularism of Fig. However, it selectively inhibits the bacteria of the genus Pseudomonas and not effective against other types of mushrooms.
Closest to the proposed strain is the actinomycetes Streptomyces higroscopicus var. limoneus, effective in the fight against scurf and particularism rice .
The disadvantage of this actinomycete in a narrow spectrum of action. Not detected its activity against 300 species tested fungi and bacteria.
The objective of the invention is the obtaining of a new strain actinomycete Streptomyces hygroscopicus subsp., has both fungicidal, bactericidal and insecticidal properties.
The problem is solved in that the proposed strain actinomycete Streptomyces hygroscopicus subsp. B-4561 with high antagonistic activity against pathogens that cause these diseases of plants: and Fusarium wilt cereal, oilseed and vegetable crops; wither or wilt technical and vegetables; rubber and brown potato rot; root rot of cereals; Alternaria cabbage, fruit rot and vinogradnye properties - he causes the death of larvae of the 1st age of the Colorado potato beetle and the death of the larvae of Galleria mellonella.
Strain actinomycete Streptomyces hygroscopicus subsp. deposited in the Central collection of microorganisms of Public concern "biological product" under number B SMC-4561, as a broad-spectrum fungicide with a bactericidal and insecticidal properties against the Colorado potato beetle Leptinotarsa decemlineata, and Galleria mellonella.
The proposed strain of Streptomyces hygroscopicus subsp differs from the strain of the prototype Streptomyces hygroscopicus var.limoneus antagonistic and antibiotic properties. He has a broad spectrum of action on phytopathogenic microorganisms and exhibits insecticidal activity. Produced by them complex antibiotics differs from prototype - antibiotic validamycin.
The strain Streptomyces hygroscopicus subsp. B-4561 isolated from soil samples in the Central region of Russia, identified by "the Determinant of actinomycetes" authors G. F. Gause, Etc., the Transfiguration and the “Quick keys bacteria Berga” [3, 4].
Cultural and morphological properties:
The strain Streptomyces hygroscopicus subsp. B-4561 has a well-developed substrate and aerial mycelium. Thin coenocytic hyphae with a diameter of 1.0 to 2.0 μm. Aerial mycelium Mature forms s (2-5 mm), isolated, lishaynikovye; first, the surface is relatively smooth, but later formed a smoky color of aerial mycelium, which becomes granular and easily converted into powder. Color of Mature specularpower aerial mycelium gray, which on mineral agar 1 becomes black due to hydration and autolysis of aerial mycelium.
Morphology chains dispute: chains of spores in the form of hooks, open loops. Spores smooth, oval shaped.
Mineral agar 1: Aerial mycelium (VM) gray, over time the surface becomes black due to the moistening of the air mycelium. Substrate mycelium (CM) light yellow; soluble pigment (RP) no. Colonies are lifted above the agar with wavy edges, mealy.
Glycerol-nitrate and oatmeal agar:
VM grey with white edges; SEE cream; RP is missing. The colonies are convex with smooth edges, matte to 2 mm in diameter.
VM faint whitish; SEE pinkish-beige color; SPM no. Colony folded diameter of 1-2 mm
Melanoidin pigments on peptone-yeast agar with iron, on the tyrosine agar and yeast medium with peptone does not form.
Culture propagated by prors the scopicus subsp. B-4561 disposes of the following carbohydrates: glucose, arabinose, sucrose, rhamnose, galactose, mannitol, sorbitol; not absorbed Inositol, cellobiose and xylose. Possesses proteolytic activity. Does not show amylolytic and lecithinase activity. Restores nitrates to nitrites.
The strain is resistant to antibiotics: rifampicin, methicillin, chloramphenicol, ampicillin, benzylpenicillin, weakly sensitive to polymyxin; doxycycline sensitive to gentamicin, streptomycin, tetracycline, monomitsina, cefaclor, amikacin, kanamycin, ristomycin.
Preparation of inoculum: the strain actinomycete grown in test tubes on the environment with oatmeal agar. After petisteho growing in a thermostat at a temperature of 28°With microscopy to determine the purity and homogeneity of culture. To assess the antibiotic activity of the strain was grown in liquid media of different composition. Was chosen optimal environment for growth and antagonistic manifestations and antibiotic activity of culture .
The strain Streptomyces hygroscopicus subsp. B-4561 is an antagonist of the following species of phytopathogenic fungi and bacteria: the genus Fusarium: Fus.frost, Fus.solani, Fus. oxysporum, Fus. Monil or wilt technical and vegetable crops; Oospora lactis - rubber rot of potatoes; Helminthosporium sativum root rot of cereals; Alternaria brassicae - Alternaria cabbage; Penicillium marfensi, Pen.purpurogenum-rot of fruits and grapes during storage; Aspergillus niger - aspergillosis cotton and fruit rot; Ervinia carotovora pv. Carotovora - bacterial diseases of cabbage, E. carotovora pv. chysanthemi - brown rot of potatoes; Actinomyces scabies - parse potatoes.
The strain also has insecticidal properties - it kills larvae 1 age Colorado potato beetle - Leptinotarsa decemlineata and death of larvae of Galleria mellonella.
Conducted Toxicological test strain of Streptomyces hygroscopicus subsp. B-4561 and made the conclusion about its pathogenicity on warm-blooded animals. In accordance with the guidelines N2620-82 and N4263-87 strain B-4561 Streptomyces hygroscopicus subsp. not virulent, toxic, malotoksicen, refers to non-pathogenic strains for warm-blooded animals - fourth hazard class.
Heterotroph. Currency in highly oxidative. In experiments on fermentation - acid not detected. Aerobe. In submerged cultivation is growing to the formation of bundles of hyphae that form small lumps. Culture can grow at temperatures from 28 to 37°C. the Optimum temperature for growth in submerged cultivation 28-30°C. Opti is Sanam agar:
Oatmeal 20,0 g
Agar microbiological 20,0 g
Drinking water To 1.0 l
Recommended storage: actinomycetes Streptomyces hygroscopicus subsp. B-4561 keep on beveled ovsanna agar in a biological test tubes or on plates, the edges of which are closed Skoch at a temperature of +4°C for 3-4 months; in dried form in a sealed ampoule with protective sharethelove environment.
Example 1. Cultivation of the strain and the study of its antagonistic properties.
Strain actinomycete Streptomyces hygroscopicus subsp. B-4561 grown on solid medium in biological membranes with oatmeal agar at a temperature of 28°C for 5 days. Prepared seed material is washed off with sterile water or saline. The resulting suspension make Catalogne bulb nutrient liquid volume of 50 ml and placed on a rocker at a temperature of 28°C, the number of revolutions of the stirrer is 240 rpm
Deep cultivation is used nutrient medium containing organic components:
Soy flour 10.0 g
BVK 5.0 g
Corn extract 3.0 ml
Chalk 2.0-3.0 g
Distilled water Up to 1000 ml
pH of 7.2
Time 120 hours of cultivation.
Verification of the purity culture and its staroobraidcheskii strain actinomycete Streptomyces hygroscopicus subsp. B-4561 was determined by the method of diffusion in agar wells. Biological testing was performed on a large set of test cultures of phytopathogenic microorganisms. Data on the antagonistic activity of the strain Streptomyces hygroscopicus subsp. B-4561 are shown in table 1.
The results of the biological testing culture fluid of strain is shown on the drawing. Zone of inhibition of growth of test cultures are determined by the diameter of the negative zone in mm
The greatest biological activity of the proposed strain marked against phytopathogenic fungi causing parse potatoes; fusariose and root rot of cereals and vegetable crops; decay of vegetable and fruit during storage; wilting, hommos industrial crops; Aspergillus, Alternaria vegetable, fruit crops. High activity is observed against bacterial pathogens causing bacterial diseases and rot of vegetables.
Example 2. Check the biological activity actinomycete Streptomyces hygroscopicus subsp. strain-4561 on the larvae of the Colorado potato beetle, Leptinotarsa decemlineata.
For testing was prepared by suspension culture fluid of strain in physiological solution at concentrations of 10, 2, 0.4 percent. In the control experiment ISA through the day after the birth of their clutches, collected in the wild. Each concentration of the culture fluid was tested in two replications. For the experiment in a Petri dish was placed a sheet of potatoes soaked in the suspension culture fluid (concentration indicated above), and 10 larvae of the beetle. The Cup was kept at a temperature of 28°C. the Account of the death of the larvae produced after 2 and 4 days, separately for each concentration. The biological activity of the strain, expressed in LC50, was calculated by the formula Cerberus percentage concentration of cultural liquid (QL) suspension:
where Cm is the maximum of the tested concentrations;
s is the logarithm of the relationship of each previous concentration subsequent to (the logarithm of the ratio of dilution);
SL is the sum of the values L (proportion of dead larvae from the number of subjects), found for all concentrations adjusted for mortality in the control method of the Abbot:
where ro is the proportion of dead larvae in the test culture; RK - the proportion of dead larvae in the control experiment.
The number of dead and surviving larvae indicated in the numerator and denominator, respectively.
On the basis of the results obtained by the formula Cerberus rasschitannomu Streptomyces hygroscopicus subsp.sp. B-4561: against the larvae of the Colorado potato beetle Leptinotarsa decemlineata is 1.5%.
Example 3. The effect of strain B-4561 on larvae of Galleria mellonella (Lepidoptera).
Has experienced the effect of cultural liquid (QL) at a concentration of 100% on tracks 4 age. Suspension QOL specified concentration was mixed with the prepared artificial feed in the ratio of 2 ml suspension of 10 g of feed and spread out evenly into Petri dishes. In each Cup was placed on 10 tracks. In the control experience instead of QOL was added to drinking water. Cups with larvae kept at a temperature of 28°C. the Number of dead larvae was counted on the 5th day. Mortality of larvae in relation to the control experiment was 30%. In addition, the experience was observed slow development of larvae compared to control.
Example 4. The test strain actinomycete Streptomyces hygroscopicus subsp. B-4561 against complex diseases of potatoes.
The scheme of experiment:
1. Pre-treatment of potato tubers;
2. Double treatment of vegetating plants with an interval of 10 days.
The tests were carried out on the variety Santa 2-fold repetition of 100 hives each, by the method of NIICH. Breeding the culture fluid of strain Streptomyces hygroscopicus subsp. B-4561 carried out at a ratio of 1:5. In was found by the method of NIICH.
Disease incidence was calculated by the formula:
where P is the prevalence of disease, %;
n is the number of diseased plants, units;
N is the total number of plants in the sample, units
The degree of disease was calculated by the formula:
R - the development of diseases, %;
E - (a+b) is the sum of the products of the number of diseased plants, tubers, (a) for the corresponding score of the lesion (b);
N is the total number of plants account;
It is the highest point scale into account.
The results of field tests are presented in tables.
In the conditions of the test, the spread on potato disease Alternaria was strong throughout the growing season, and the development of vituperation during the second half of the vegetation period.
Accounting data are presented in tables 3-4.
As can be seen from the table when the first consideration when processing plant QOL prevalence and disease development was at the level of the reference variant, in subsequent surveys the development of Alternaria decreased in comparison with control 1.5 times and slightly lower than the reference version.
As can be seen from the table when vtoro the degree of development of late blight in the experimental apparatus was 12.0%, in the control of 33.6%and, in the reference version is 5.9%.
The number of infected tubers in the variant with the processing of tubers and plants QOL strain In-4561 significantly less in comparison with control and standard. Strain actively suppresses alterians and phytophthoras on the plants during the growing season and to some extent inhibits rot of tubers during storage, so the harvest of healthy tubers 9% above control options.
Sources of information
1. The second all-Union seminar "Current trends in search of antibiotics", Alma-ATA, 1989
2. M. I. Petrukhin. The use of antibiotics in the fight against plant diseases. Newsletter UPU IOBC No. 12, Leningrad, 1985, page 26.
3. G. F. Gause, Etc., Preobrazhenskaya, M. A. Sveshnikov, A. P. Terekhov, T. C. Maximov. Identification of actinomycetes. M.: Nauka, 1983, page 43.
4. Quick keys bacteria Berga. M.: Mir, 1980
5. A. N. Chermen. The avermectins: biotechnological characteristics of the producer strain Str.avermitilis BKM Ac 1301." Applied biochemistry and Microbiology. 1991. so 27, vol.6, pp. 838-844.
Strain actinomycete Streptomyces hygroscopicus subsp., deposited in the Central collection of microorganisms Gominidnyye properties.
FIELD: biotechnology, microbiology, medicine.
SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.
EFFECT: valuable medicinal properties of strain.
5 cl, 8 dwg, 10 ex