The strain of bacteria brevibacillus laterosporus with a broad spectrum fungicide and biological drug to protect potatoes from fungal diseases during storage of biomass-based this strain

 

(57) Abstract:

The invention relates to agricultural biotechnology, in particular to the production and use of biological means to control plant pathogens. The strain of Brevibacillus laterosporus isolated from natural sources, deposited in ACIM, GNII Genetika number VKPM B-7767 and has a broad spectrum of fungicidal action. Based on the strain of Brevibacillus laterosporus VKPM B-7767 developed fungicide product containing biomass of the strain of Brevibacillus laterosporus VKPM B-7767, water, tridecan, tween-80, polyvinyl alcohol, glycerin and chinosol. The strain of Brevibacillus laterosporus VKPM B-7767 and biological drug based on it inhibit the growth of phytopathogenic fungi causing diseases of potato tubers during storage. The drug is cheap and easy to use. 2 N. p. F.-ly, 2 PL.

The invention relates to agricultural biotechnology, in particular to the production and use of biological means to control plant pathogens.

One of the serious problems of agriculture is the preservation of the harvest of vegetable crops, potatoes, cereals and fruit. The disease caused by microscopic fungi that cause serious Economics of the main industrial crops. Phytopathogenic fungi infect potatoes throughout the growing season and during storage of tubers. When storing potatoes lose up to 30% of the harvest. A major cause of death of potatoes during storage are rot fungal origin. Diseases caused by phytopathogenic fungi, also reduce the quality of the food and seed potatoes. Infected planting material becomes a source of disease of potatoes during the growing season. For protection against phytopathogens conduct decontamination of chemical fungicides tubers in the process of laying the potato harvest in storage, which significantly reduces the infection of tubers by fungal diseases. As a chemical disinfectants tubers used a number of drugs, including Carotid, Tecto, Maxim. However, chemical fungicides have a number of disadvantages. The main negative factors include nespecificnomu actions, environmental pollution, accumulation in the environment, the emergence of resistance in pathogens. Are particularly vulnerable to chemical fungicides used for the processing of finished agricultural products, because we know the negative impact of x is used biological fungicide preparations based on the strains of microorganisms with antagonistic activity against various phytopathogenic fungi. Thus, the described bacterial fungicides peptide - surfactin, iturin, lipstatin, bucillamine (1-5). The mechanism of antifungal action of the peptides caused either by lysis of the cells, either by suppression of the synthesis of components of cell walls. Some strains of B. cereus produce antibiotic zwittermicin providing an inhibiting effect on phytopathogenic fungi (6).

Biological products based on microorganisms, producing antagonistic factors, can also be used to combat fitopatogene mushrooms, managing potato tubers during storage. Given that in Russia at the store is laid over 3 million tons of potato tubers, potato protection from fungal diseases is very important. Known biological drug “Backoff”, created on the basis of a strain of B. subtilis IPM 215 (7). The drug is recommended for treatment of potato tubers before putting it into storage for protection against Fusarium wilt, late blight and stem canker (8). The drug Backoff and constituting its active principle strain of B. subtilis IPM 215 consider the ASS="ptx2"> to obtain a strain of bacteria with antifungal activity against a broad spectrum of fungal phytopathogens, including those that cause disease of potato tubers;

to develop on its basis an effective drug to protect potatoes from fungal diseases during storage.

Problem solved by:

- get a mildew-resistant strain of bacteria Brevibacillus laterosporus VKPM B-7767;

- development of biological drug to protect potatoes from fungal diseases during storage, including biomass fungicidal strain of bacteria and the target additives, characterized in that it additionally contains water, as biomass fungicidal strain of bacteria includes the biomass of the strain of Brevibacillus laterosporus VKPM B-7767, and the target additives - tween-80, glycerin, tridecan, 10% polyvinyl alcohol and chinosol in the following ratio, wt.%:

the biomass of the bacterial strain

Brevibacillus laterosporus VKPM B-7767 48-57

tween-80 2,5-5,0

glycerin 10-15

tridecan 15-25

polyvinyl alcohol 10% 11-15

chinosol 0,01-0,03

water the rest

The inventive strain selected in a multistage selek who s number VKPM B-7767.

The inventive strain of Brevibacillus laterosporus VKPM B-7767 has the following characteristics:

1. Cultural and morphological characteristics.

Gram-negative motile Bacillus, peritricha size 0,5-0,7×2,5-5,0 μm, chains do not form. Well sporulated on liquid and solid nutrient media.

As a solid nutrient media used agarized medium NBY, LB and DPS.

The composition of the agar medium NBY, wt.%:

nutrient broth 0,8

yeast extract 0,3

agar 2,0

water the rest

The composition of the agar medium (LB), wt.%:

bacto-triptan 1,0

bacto-yeast extract 0.5 to

NaCl 1,0

agar 2,0

water the rest

The composition of the agar medium DPS, wt.%:

yeast 3,0

corn flour 1,5

agar 2,0

water the rest

Under cultivation on agar media NBY, LB, DPS after 48 hours at a temperature of 28 to 30°With the strain forms a rounded colonies with a diameter of 3-4 mm beige color with a rough surface and rough edges. After 72 hours of growth on these nutrient medium strain produces spores. In the process of sporulation is formed Canoeing.

2. Physiological and biochemical properties.

The strain produces catalase. Hydrolyzes casein and gelatine, not hydrolyzes starch. Urea does not break. Not sprayway sucrose, arabinose, xylose, lactose. Sprayway glucose, maltose, mannitol, fructose. Glucose sprayway without gas. Does not form acetone. Forms lecithins, tween-esterase. Grows in the presence of lysozyme.

3. Fungicidal properties of the strain.

The strain is characterized by a broad spectrum of fungicidal activity and suppresses the development of the following phytopathogenic fungi: Alternaria tenuis, Fusarium solani, Fusarium nivale, Phomopsis helianthi, Phoma solanicola, Phytophora infestans, Rhizoctonia solani, Sclerotinia sclerotiorum and does not inhibit the growth of pests of grain crops (Aspergillus niger, Fusarium frost). The analogue of the claimed us strain In-7767 strain of B. subtilis IPM 215 and developed on the basis of the drug Backfit have a much narrower range of fungicides actions. “Backoff” is recommended for treatment of potato tubers before putting it into storage for protection only from Fusarium wilt, late blight and stem canker. The proposed strain-7767, in addition to the pathogens of these diseases, inhibit the development of fungi-causative agents of superficial necrosis (Phoma solanicola) I.

4. The composition of the drug and its production method.

On the basis of the proposed strain was developed the drug to protect the tubers from fungal diseases during storage. Biological fungicide with a content of viable spores per 1 g of the drug 2,5-3,0×1010in addition, the biomass of the strain of Brevibacillus laterosporus VKPM B-7767, it additionally contains water and the number of target components, giving the drug consumer properties such as emulsion structure, adhesion, stability. The drug emulsion structure includes oil (tridecane), surfactants (tween-80), adhesive (polyvinyl alcohol) and a stabiliser (glycerol). As preservative used chinosol.

The claimed drug has the form of an aqueous emulsion, which has a number of advantages in comparison with dry forms: savings of energy costs, no need for complicated equipment and the retention of biological activity acting early in the process of preparation of the drug, as well as the convenience and simplicity in the process of its application.

The method of obtaining the drug is carried out by gradually adding ingredients in the following order: BIE 10 min at 900-1000 rpm after adding each ingredient, then with continuous stirring carry out the introduction to the tridecane drip method with a speed of 3 ml/min For the final product system continue to mix at 900-1000 rpm for a further 30 minutes

The invention is illustrated by the following examples.

Example 1.

Cultivation of a strain of Brevibacillus laterosporus B-7767.

For cultivation of strain B-7767 use standard glass conical flat-bottomed flask with a volume of 750 cm3that fill 50 ml of sterile medium DPS.

The composition of the medium DPS, wt.%:

yeast 3,0

corn flour 1,5

water the rest

pH of 6.8 to 7.0.

In 50 ml of sterile medium DPS add 2 ml of inoculum, which has a concentration of 6×108cells/ml and obtained by cultivation with shaking (250 rpm) on the DPS environment at a temperature of 28 to 30°C for 72 hours. Crop for biomass grown in the same conditions as the seed material. To 96 hours of cultivation the sporulation reaches 90%. The titer of the culture after 96 hours of cultivation is 4,0-6,0×108cells in ml.

Example 2.

Evaluation of fungicidal activity of the strain-7767 La the conditions as follows. The strain was grown as in example 1. For the analysis of antifungal activity used culture fluid containing 4×108cells/ml Testing antifungal activity was performed by the method of the hole. For this phytopathogenic fungi (table. 1) were sown on Petri dishes with agar and potato-sucrose medium. Potato-sucrose agar was obtained by adding to broth potato sucrose and agar.

Potato-sucrose agar, wt.%:

sucrose 2,0

agar 2,0

potato broth else

The cut in the agar wells of 8 mm diameter were made and 1 ml of the culture fluid of strain VKPM B-7767. The effectiveness of antifungal effect of the proposed strain was determined by the diameter of the zone of no growth of the test microorganisms. As a standard considered the diameter of the zone of no growth, equal to 4-6 mm. the Results are presented in table 1.

Example 3.

Determination of the effective concentration of the biomass of the strain VKPM B-7767 in the inventive preparation.

To determine the effective concentration of the biomass of the strain VKPM B-7767 as part of the proposed drug is used in the method of spraying. For this phytopathogenic fungi that cause diseases of the th potato-sucrose medium. After the start of growth of fungi on nutrient medium in Petri dishes were sprayed with aqueous suspensions containing different concentrations of the biomass of the strain VKPM B-7767 rate of 1 ml per Cup. In the control Petri dishes seeded with mushrooms sprinkled with water. The fungicidal effect was expected to reduce the growth of fungi treated with a slurry of biomass, relative to control, which was taken as 100%. As took the optimum concentration of biomass, causing growth suppression of phytopathogen 70-80%. Growth inhibition of phytopathogenic fungi Alternaria tenuis, Phoma solanicola, Fusarium solani and Rhizoctonia solani was achieved when the concentration of the biomass of the strain of Brevibacillus laterosporus VKPM B-7767 equal to 20 g/L.

Example 4.

The definition of the working dose of the proposed drug for its fungicidal activity.

The strain of Brevibacillus laterosporus VKPM B-7767 were cultured as in example 1, and then the biomass was besieged by centrifugation at 12000 rpm and used taking into account the results of example 3 for the preparation of the drug in the following ratio, wt.%:

The biomass of the strain of bacteria Brevibacillus

laterosporus VKPM B-7767 48

tween-80 2,5

glycerin 10

tridecan 15

10% polyvinyl the t as follows:

- a portion of the concentrated biomass is placed in a chemical beaker and stirred by the stirrer at 900-1000 rpm for 10 min;

then the biomass is injected glycerin and mixed in a mixer for 10 min at 900-1000 rpm;

to the obtained system add tween-80 and again stirred for 10 min at 900-1000 rpm;

- then in the system impose a 10% aqueous solution of polyvinyl alcohol and implement subsequent stirring for 10 min at 900-1000 rpm;

to the obtained system add chinosol, followed by stirring the resulting system for 10 min at 900-1000 rpm;

- with continuous stirring carry out the introduction to the tridecane drip method with a speed of 3 ml/min;

- for the final product system continue to mix at 900-1000 rpm for a further 30 minutes

The fungicidal activity of the obtained preparation was determined by spraying, as with the evaluation of fungicidal properties of biomass (example 3). Made the target components of the formulation did not change the efficacy and spectrum of fungicidal action of the biomass of the strain. The results obtained are consistent with the results in the example of the processing, that made it possible to calculate the dose required for protection of 1 ton of potatoes. By calculation, it was found that under conditions of storage for processing 1 ton of potatoes, you must use 80-100 g of the proposed drug in 3 l of fluid.

Example 5.

Evaluation of the effectiveness of the protection claimed by the preparation of potato tubers from fungal diseases during storage under field conditions (repository).

The fungicidal effectiveness of the drug action in the field was evaluated against rot of potato tubers during storage at 2 points of the Central area podzolic and sod-podzolic soils of the taiga forest region of the Russian Federation. For evaluation used freshly harvested batch of potatoes. Processing samples of tubers dissolved in water preparation was carried out by spraying, and no later than 2 days after harvest. Was used the rate of drug - 80 g / t fluid - 3 l/T. the Weight of one prototype was 10 kg of tubers, the number of replications - 8. Processed samples of tubers were placed in a grid and 4 of the sample (4 frequency) of each option was included in the mass of the mound of potatoes to a depth of 1 m to determine the effect and what I efficiency in the dynamics through 120, 180, 240 days after treatment. The efficacy of the drug was determined by the method of tuberous analysis taking into account all types of illnesses, natural loss of weight, mass, absolute rot and technical waste. Were used for comparison of chemical fungicide Tecto. Comparative characteristics of the effectiveness of the proposed drug and chemical drug Tecto (90 ml per tonne) is given in table 2.

Conducted field tests (storage) showed that the effectiveness of fungicidal action proposed biofungicide has almost the same chemical drug Tecto that can be seen from the results given in table 2. Thus, the resulting mildew-resistant strain of bacteria Brevibacillus laterosporus VKPM B-7767 suppressing the development of a broader spectrum of phytopathogenic fungi causing diseases of potato tubers during storage, compared with the known strains. On the basis of the developed biological drug that is on the spectrum of fungicidal activity does not yield the claimed strain, and thanks to its composition, stable and high-tech. Selected for its use of the form of an aqueous emulsion makes the product attractive to the consumer due to the ease of its application and the

1. Influence of culture conditions on lipopeptide production by Bacillus subtilis. Appl. Biochem. Bioctechnol. 91-93: 551-561.

2. Klich, M. A., Lax, A. R., Bland, J. M. 1991. Inhibition of some mycotoxigenic fungi by iturin A, A peptidolipid produced by Bacillus subtilis. Mycopathology. 116:77-80.

3. F. Ahimou, Jacques P., Dejeu M. 2000. Surfactin and iturin A effect on Bacillus subtilis surface hydrophobility. Enzyme Microb. Technol. 27:749-754.

4. Volpon L., F. Besson, Lancelin, J. 2000. NMR structure plipastatins A and In from Bacillus subtilis inhibitors of phospholipase A2. FEMS Lett. 485: 76-80.

5. Volpon L., F. Besson, Lancelin, J. 1999. NMR structure of active and forms of the steroldependet antifungal antibiotic bacillomycin L. Eur. J. Biocem. 263:1-12.

6. Milner J. L., S. J. Raffel, Lethbridge B. J., Handelsman J. 1995. Culture condition that influence the accumulation of zwittermicin A by Bacillus cereus UW85. Appl. Environ. Biotechnol. 43: 685-691.

7. RF patent 2019966.

8. “List of pesticides and agrochemicals permitted for use on the territory of the Russian Federation. 2001.”. Supplement to the journal “Protection and quarantine of plants” No. 3, pages 106-108.

1. The strain of bacteria Brevibacillus laterosporus VKPM B-7767, with a broad spectrum of fungicidal action.

2. Biological preparation to protect potatoes from fungal diseases during storage, including biomass fungicidal strain of bacteria and the target additives, characterized in that it additionally contains water, as biomass fungicidal strain of bacteria it includes BIOS alcohol and chinosol in the following ratio of components, wt.%:

The biomass of the bacterial strain

Brevibacillus laterosporus

VKPM B-7767 48-57

Tween-80 2,5-5,0

Glycerin 10-15

Tridecan 15-25

Polyvinyl alcohol 10% 11-15

Chinosol 0,01-0,03

Water The Rest



 

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