RU2538157C1 - Bacillus subtilis B 93 VIZR BACTERIAL STRAIN FOR PROTECTION OF POTATOES FROM ILLNESSES DURING STORAGE - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: Bacillus subtilis strain - I5-12/23 has the antagonist activity with reference to phytopathogenic funguses and bacteria. The strain is reposited in GNU VIZR State Collection of Microorganisms under registration number Bacillus subtilis 93 VIZR also can be used for protection of potatoes against illnesses during storage.

EFFECT: improved preservation of potato tubers.

5 tbl

Description

The invention relates to agricultural microbiology and is a strain producing a complex of biologically active substances with fungicidal and bactericidal action, can be used to obtain a biological product against fungal and bacterial diseases of potatoes during storage.

Potato is one of the most valuable crops. However, its preservation in fresh form from harvest to harvest is associated with certain difficulties associated with the defeat of tubers by phytopathogenic microorganisms, causing significant damage to potatoes even when refrigerated.

Currently, the chemical method occupies a leading position in the protection of fruits and vegetables from phytopathogens during cultivation and storage. However, the global use of chemical protective agents leads to a number of negative consequences - disturbances in the structure of biocenoses, a decrease in their ability to self-regulate, and the accumulation of pesticides in soil, water, and food. In addition, resistance to them is increasing in populations of pests and pathogens that reduce the effectiveness of their use.

The analysis of domestic and foreign scientific and technical literature showed that biological control can successfully compete with chemical protective equipment, without at the same time possessing their shortcomings. The microbiomethod also has significant advantages in terms of saving materials, energy and using renewable resources. A relatively new direction in the development of biocontrol is the use of biologics based on active antagonist strains during storage of crop production.

Known strains of Bacillus subtilis - producers of a number of biological fungicides - Alirina-B, Gamair, Bactofita, Albit, etc. (“The state catalog of pesticides and agrochemicals approved for use on the territory of the Russian Federation”, 2013). However, they have low biological effectiveness in conditions of vegetable stores and do not provide effective protection of crop products from diseases during storage. In the "State catalog of pesticides and agrochemicals approved for use on the territory of the Russian Federation" there are no biological products recommended for the protection of potatoes from diseases in a storage facility.

The use of Bacillus subtilis strain 413 to scientifically substantiate the reduction of carrot losses from infectious diseases and the improvement of product quality indicators during long-term cold storage has been scientifically substantiated (Samusenko, 2001).

The closest to the proposed one is B. subtilis strain B-10 VIZR, the producer of the drug Alirin-B for the protection of crops from fungal and bacterial diseases ("State catalog of pesticides and agrochemicals approved for use in the Russian Federation", 2013). But this strain has a narrower spectrum of action and insufficient biological effectiveness against phytopathogenic fungi and bacteria at low temperatures during long-term storage of crop products. Alirin-B is recommended for use on potatoes only during the growing season against the "black leg", rhizoctonia, late blight and alternariosis.

The objective of the invention is the creation of a strain with a wide spectrum of activity and high antagonistic activity against pathogens of fungal and bacterial diseases of potatoes and promising for the protection of tubers from diseases during storage.

The task was accomplished by selecting a strain of Bacillus subtilis I5-12 / 23, producing a complex of biologically active substances with high antagonistic activity against pathogens of fungal and bacterial diseases of potatoes during storage. The strain is deposited in the State collection of microorganisms pathogenic for plants and their pests GNU VIZR under the number B-93 VIZR. The strain was isolated from Indian soils (Delhi) by signs of antagonistic activity against phytopathogenic fungi and bacteria. Identification of the strain was carried out according to the determinant of the bacteria of Bergey (Holet D., Krieg N. et al. The determinant of bacteria of Bergey. In 2 volumes. 9th edition. Publishing house "Mir". 1997).

CHARACTERISTICS

1. Morphology: Straight sticks with rounded ends, often form chains.

2. Size: 0.8-0.9 × 2.5-3.5 microns.

3. Cell wall and Gram stain: gram-positive, cell wall thick gram-positive type (analysis method - staining with gentian dye and Gram iodide solution). It does not form capsules.

4. Spore formation: endospores are oval, with a central position, heat-resistant, highly resistant to many adverse environmental conditions. No more than one spore is formed in a cell. Endospores are ellipsoidal, located centrally and do not go beyond the size of sporangium. Near-peroral inclusions were not found. Spore formation is not suppressed in air.

5. Type of division: transverse binary.

6. Simple: none.

7. Mobility: movable.

8. Flagella: peritrichous flagella.

9. Colony pigmentation: colonies are creamy, matte, flat, with an uneven edge, rapidly spreading over the surface of the nutrient medium, later the colonies become denser, glossy and acquire a brownish-beige hue.

10. The pigment is not allocated on Wednesday.

11. There are no selective media.

12. Oxidase: positive.

13. Catalase: positive.

14. Attitude to oxygen: aerob. In anaerobic agar does not grow.

15. Chemorganotroph, respiratory metabolism. Sources of carbon used for aerobic growth: glucose, sucrose, mannitol, maltose and lactose. When using glucose, arabinose, xylose and mannitol forms acid and acetoin, it does not form gas. Forms hydrolases. Hydrolyzes starch and casein. Restores nitrate to nitrite.

16. Under aerobic conditions, sorbitol and inositol do not grow.

17. Inorganic substrates do not oxidize.

18-20. Under anaerobic conditions does not grow.

21. Relation to pH: the strain grows in the pH range from 4.5 to 8.5. The optimum pH is 6.5-7.5.

23. Attitude to NaCl: the culture does not grow at 10% NaCl.

24. Relation to temperature: the maximum growth temperature is 45-50 ° C, the minimum is 2-4 ° C, the optimal is 22-26 ° C.

25. Attitude to antibiotics: forms polypeptide and polyene antibiotics.

26. Cytochromes: not determined.

27. Menaquinones: not determined.

28.% HC: not determined.

29. Need for growth factors or special additives: not required.

30. Additional information: strain producing a biological product to protect potato tubers from phytopathogenic fungi and bacteria - pathogens during prolonged storage. Method for determining product activity: biological for suppressing the growth of test cultures of phytopathogenic fungi and bacteria.

31. The method of storage of strain:

a) jambs with SPA or MPA,

b) in a lyophilized state.

32. Shelf life: reseeding after 6 months.

33. Relation to phages: no phage were isolated.

34. Genetic features: the strain was genotyped using the DRIM method (double cleavage, selective labeling).

Antimicrobial spectrum: inhibits the growth of phytopathogenic fungi and bacteria.

Zones of growth retardation of test cultures of phytopathogenic fungi and bacteria (radius in mm):

Alternaria brassicicola - 20.0

Colletotrichum atrawenta - 15.0

Verticillium dahliae - 24.5

Sclerotinia sclerotiorum - 22.0

Phoma exigua -20.5

Fusarium oxysporum - 20.5

Bipolaris sorokiniana - 19.0

Helmintosporium solani - 25.0

Pythium debarianum - 15.0

Ascochyta melonis - 14.0

Septoria nodorum - 20.5

Rhizoctonia solani - 25.0

Phytophthora infestans - 20.5

Clavibacter michiganensis var. sepedonicum - 22.0

Compared with the prototype, the proposed strain has not only a wider spectrum of action against pathogens of fungal diseases, but also a high antagonistic activity against phytopathogenic bacteria (table 1).

Methods of using the strain are described in the examples, and the results are presented in tables.

Example 1. The cultivation of strain B. subtilis B 93 VIZR was carried out in depth conditions in Erlenmeyer flasks for 72 hours on a circular shaker at 28 ° C until the completion of the sporulation phase.

The composition of the liquid nutrient medium (g / l) (workshop on microbiology, ed. N. Egorova, 1976):

Sprat pancreatic hydrolyzate - 10.05

Sodium Chloride - 4.95

Glucose - 10.0

Water - up to 1 liter

pH 7.1 ± 0.2

0.5 / 30 min

The antagonistic activity of the 3-day old culture fluid obtained by deep growing the strain was determined using the standard microbiological well method (Microbiology Workshop, edited by N.S. Egorov, 1976; Guide to Practical Training in Microbiology, edited by N.S. Egorova, 1995). The diameter of the growth inhibition zone was measured after 3-5 days, depending on the growth rate of the test culture. The effectiveness against phytopathogenic fungi and the spectrum of action compared to the prototype are presented in table 1.

Table 1 Comparative characteristics of the spectrum of antagonistic activity of B. subtilis B-10 VIZR and B. subtilis B 93 VIZR strains (culture fluid) No. Types of phytopathogenic microorganisms The diameter of the lysis zone of the test culture, mm B. subtilis B 93 VIZR B. subtilis B-10 (prototype) one Clavibacter michiganense subsp. michiganense 24 0 2 Clavibacter.michiganensis var. sepedonicum 22 0 3 Xanthomonas campestris pv. vesicatoria eighteen 0 four Pseudomonas corrugata 24 0 5 P. syringae pv. tomato thirty 0 6 Erwinia carotovora subsp. carotovora 24 0 7 Alternaria solani twenty eighteen 8 Alternaria brassicicola 22 10-15 9 Fusarium graminearum 34 25-30 10 Fusarium oxysporum twenty 20-30 eleven Ascochyta fabae eighteen eighteen 12 Colletotrichum lagenarium 24 twenty 13 Rhizoctonia solani 24 eighteen fourteen Bipolaris sorokiniana 24 5-7 fifteen Sclerotinia libertiana 24 5-7 16 Septoria nodorum 15-20 18-20 17 Phytophthora infestans 10-20 20-21 eighteen Phoma exigua 14-21 20-22 19 Helmintosporium solani 15-20 25-28

As can be seen from table 1, the strain B. subtilis B 39 VIZR, in contrast to the prototype, has not only antifungal, but also high antibacterial activity. Significantly higher antagonistic activity of the proposed strain against phytopathogenic fungi Colletotrichum lagenarium, Rhizoctonia solani, Bipolaris sorokiniana and Sclerotinia libertiana.

Example 2. The culture fluid of strain B. subtilis B 93 VIZR was obtained in the same way as in example 1. The study of biological effectiveness in model experiments and in the potato storage was carried out in accordance with standard methods outlined in the following manuals: "Methods of research on potato culture", M., 1967; "Methods of field experience", M., 1985; "Guidelines for state testing of fungicides, antibiotics and seed dressers of agricultural crops", M., 1965; "Methodology of research on the protection of potatoes from diseases, pests, weeds and immunity", M., 1995; “Guidelines for registration testing of fungicides in agriculture”, St. Petersburg, 2009.

Laboratory experiments were performed taking into account the dynamics of the development of diseases in the tuber embankment in the potato storage during the main storage period. For the experiments, 10 tubers were selected for repetition with signs of disease. The tubers were processed according to the experimental design and placed in wet chambers.

In laboratory experiments, to determine the effect of the strain on the development of sporulation of silver scab, tubers were treated with dilutions of the culture fluid with a titer of viable cells of 10 ⁸ and 10 ⁹ CFU / ml. In the standard, the tubers were treated with Maxim fungicide, KS (0.2 l / t), in the control, with water. After processing, the tubers were placed in wet chambers. After 2-7 days, we took into account the intensity of sporulation of the fungus that causes silver scab of potatoes (Helmintosporium solani Dur.ex.Mont).

The experimental data are statistically processed by analysis of variance.

The results obtained indicate the promise of using the selected strain to reduce the disease damage of potatoes during storage (table 2).

In the fight against silver scab (table 2), the strain showed higher biological activity in comparison with the prototype and standard. During the first survey, no sporulation experience of Helmintosporium solani Dur.ex.Mont was noted in any of the variants. The results of the second survey showed that the development of the disease when using strain B. subtilis B 93 VIZR at both concentrations of the working solution was 2 times lower compared to strain B. subtilis B-10 (prototype) and 4.5 times compared to control. The biological efficiency in these experimental variants reached 77.8%, the indicator of biological efficiency exceeded the efficiency of the chemical standard.

table 2 The biological effectiveness of B. subtilis strain B 93 VIZR in relation to the sporulation of silver scab on potato tubers Variants of the experiment, the titer of cells in dilutions of the culture fluid of the strain, CFU / ml The second account (12.28.2012) Biological efficiency,% the number of tubers with sporulation, P,% the development of the disease, R,% R, % R% Control, water 90.0 36.0 0 0 B. subtilis B-10, 10 ⁹ (prototype) 70.0 16.0 22.2 55.6 B. subtilis B 93 VIZR 10 ⁹ 40.0 8.0 55.6 77.8 B. subtilis B 93 VIZR, 10 ⁸ 40.0 8.0 55.6 77.8 Maxim, KS, standard, 0.2 l / t 60.0 12.0 33.3 67.7 NDS _0.5 8.0 2.2

Example 3. The culture fluid of strain B. subtilis B 93 VIZR was obtained in the same way as in example 1 and 2. The laying of samples of potato tubers for long-term storage in order to assess the biological effectiveness of the strain in relation to the complex of diseases was carried out in the potato storage facility of GNU VNIIKH (Moscow region, Lyubertsy district, p. Kraskovo, Korenevo microdistrict).

In the experiment, tubers of the Sante variety were used (medium early, universal use). Productivity is high. The variety is resistant to potato cancer (the causative agent of Synchytrium endobioticum), the golden potato cyst nematode (Globodera rostochiensis), and viral diseases, and is susceptible to late blight to the late blight. It is medium tolerant to common scab, susceptible to rhizoctonia and fomosis. A set of material for the experiments was made 3 days after harvesting. Before processing and laying for storage, a tuber analysis was performed to determine the phytopathological state of potatoes. Before laying for storage, the tubers were treated with dilutions of the culture fluid of the strain with a titer of viable cells of 10 ⁸ and 10 ⁹ CFU / ml. In the standard, the tubers were treated with Maxim fungicide, KS (0.2 l / t), in the control, with water. The flow rate of the working fluid is 3 l / t. A sample weight of potato tubers was 5 kg. The repetition of the experiment is 10 times. The total mass of tubers of each variant is 50 kg. Placement of samples: in an embankment of potatoes at a depth of 20-30 from the surface.

The analysis of the results of the experiment conducted in the potato storage facility of GNU VNIIIKH confirmed the data of laboratory experiments on the effectiveness of the selected strain for the protection of potato tubers. At the end of the storage period in March 2013, a tuber analysis was carried out and the prevalence of diseases and weight loss of potatoes during storage were determined (tables 3-5).

Table 3 The effect of bacillus strains on the defeat of potato tubers by diseases during storage (September 6, 2012 - March 26, 2013) Variants of the experiment, the titer of cells in dilutions of the culture fluid of the strain, CFU / ml The number of tubers,% healthy pathogens Total including common scab (S. scabies) ring rot (C. michiganensis var. sepedonicum) late blight (P. infestans) dry rot (Phomaexigua) Control, water 13.3 86.7 52.3 7.4 11.8 15.2 B. subtilis B-10 VIZR, 10 ⁹ (prototype) 30.4 69.6 47.3 4.8 8.5 9.0 B. subtilis B 93 VIZR, 10 ⁹ 32.4 67.6 53.8 2.6 8.2 3.0 B.subtilis B 93 VIZR, 10 ⁸ 35.5 64.5 55.7 2.9 4.4 1.5 Maxim, KS, standard, 0.2 l / t 30.0 70.0 54.1 3.5 8.3 4.1 NDS _0.5 2.3 5.4 6.6 0.4 1.4 0.2

A significant decrease in the development of diseases on seed potato tubers after treatment with B. subtilis strain B 93 VIZR and the standard Maxim, KS was noted compared with the control. The number of healthy tubers after application of the strain reached 32.4-35.5%, while in the prototype this indicator was 30.4%, in the control it did not exceed 13.3%. Thus, in comparison with the control, the yield of healthy products increased 2.7 times. The treatment with the strain effectively reduced tuber damage by all the diseases that appeared, with the exception of common scab. The strain showed the greatest effect on pathogens of ring and dry rot of potatoes. In the variant using strain B. subtilis B 93 VIZR, the prevalence of ring rot was 2.6–2.9%, dry rot — 1.5–3.0%, which is significantly lower than those for strain B. subtilis B-10 (prototype) and the standard fungicide Maxim, KS (4.8 and 3.5%; 9.0 and 4.1%, respectively).

Thus, an analysis of the results of the experiment showed that the treatment of tubers with B. subtilis strain B 93 VIZR before being stored in the potato storage reduced the number of tubers infected with fungal and bacterial diseases and increased the yield of healthy potatoes 2.6–2.7 times. The biological effectiveness of strain B. subtilis B 93 VIZR with respect to ring rot, dry rot and late blight reached 80-90% and was significantly higher than the efficiency of the prototype (28.0-40.8%) and standard fungicide Maxim, KS (29.7-73.0%) (table. four).

Table 4 The biological effectiveness of strain B. subtilis B 93 VIZR against pathogens of potato tubers during storage (09/06/2012-26.03.2013) Variants of the experiment, the titer of cells in dilutions of the culture fluid of the strain, CFU / ml Biological effectiveness (%) against pathogens ring rot (Clavibacter michiganensis var. sepedonicum) late blight (Phytophthora infestans) dry rot (Phoma exigua) Control, water - - - B. subtilis B-10 VIZR, 10 ⁹ (prototype) 35.1 28.0 40.8 B. subtilis B 93 VIZR, 10 ⁹ 64.9 30.5 80.3 B. subtilis B 93 VIZR, 10 ⁸ 60.8 62.7 90.1 Maxim, KS, standard, 0.2 l / t 52.7 29.7 73.0

Table 5 The influence of bacillus strains on the safety of potato tubers during storage (September 6, 2012 - March 26, 2013) Variants of the experiment, the titer of the cells in dilutions of the culture fluid, CFU / ml Mass of tubers Economic efficiency% healthy mass loss,% kg % Total including technical waste absolute rot natural decline Control, water 3.66 73.2 26.8 8.0 8.4 10.4 one hundred B. subtilis B-10, 10 ⁹ (prototype) 3.04 60.7 39.3 6.2 18.3 14.8 83.1 B. subtilis B 93 VIZR, 10 ⁹ 3.92 78.5 21.5 7.3 4.2 10.0 107.1 B. subtilis B 93 VIZR, 108 4.06 81.3 18.7 4.6 7.7 6.4 110.9 Maxim, KS, standard, 0.2 l / t 3.69 73.9 26.1 5.3 7.2 13.6 one hundred NDS _0.5 0.02 1.3 1.1 2.1 0.9

The safety of tubers after their treatment with strain B. subtilis B 93 VIZR was significantly higher compared to the prototype and standard - fungicide Maxim, KS (78.5-81.3%, 60.7% and 73.9%, respectively). Economic efficiency in variants using the strain B. subtilis B 93 VIZR in both concentrations of culture fluid reached 107.1-110.9%, which significantly exceeded this indicator in the prototype. These test variants were characterized by the lowest weight loss of tubers during storage (18.7-21.5%). In the variant with the prototype, the weight loss of tubers reached 39.3%. The natural decrease in the mass of tubers was 1.5-2.3 times lower than that of the prototype, 1.4-2 times lower than in the standard, and 1.5 times lower than in the control (table 5).

Thus, the studies showed the high efficiency of the selected strain of B. subtilis B 93 VIZR and the prospects of its use as a producer of a biological product to protect potato tubers from a complex of diseases during storage.

Literature

1. N.V. Samusenko. The scientific rationale for the use of antagonist bacteria during long-term refrigerated storage of carrot root crops. Dis. Ph.D., St. Petersburg, 2001.

2. The state catalog of pesticides and agrochemicals approved for use on the territory of the Russian Federation, 2013. http://www.mcx.ru.

3. Methods of research on potato culture. M., VASKHNIP, NIIKH, 1967, 189 p.

4. B.A. Armor. The methodology of field experience (with the basics of statistical processing of research results). - 5th ed., Ext. and revised .: Agropromizdat, 1985 .-- 351 p.

5. Guidelines for state testing of fungicides, antibiotics and seed dressers for crops. M, 1985.130 s.

6. Research methods for the protection of potatoes from diseases, pests, weeds and immunity. M., VNIIKKH, 1995 .-- 105 p.

7. Guidelines for registration testing of fungicides in agriculture. - St. Petersburg. - 2009 .-- 377 p.

8. Workshop on Microbiology / Ed. Egorova N.S. - M .: Moscow State University, 1976 .-- 308 p.

9. M.N. Pimenova, N.N. Grechushkina, L.G. Azova, A.I. Netrusov, E.V. Semenova, N.N. Kolotilova, L.M. Zakharchuk, V.V. Zinchenko, S.I. Mylnikova, M.V. Nefedova, I.V. Botvinko. Guide to practical exercises in microbiology. Textbook allowance / Ed. N.S. Egorova. - 3rd ed., Revised. and add. - M.: Publishing House of Moscow State University, 1995 .-- 224 p.

10. Hoult D., Krieg N. et al. Identifier of the bacteria Bergey. In 2 volumes. 9th edition. Publishing house "Mir". 1997.

Claims (1)

The bacterial strain Bacillus subtilis B 93 VIZR to protect potatoes from diseases during storage.