RU2729576C1 - Bacterial strain bacillus subtilis b-94 vizr for protection of potatoes against diseases during vegetation and storage, increased yield and improved quality of tubers - Google Patents

Abstract

FIELD: biotechnology; agriculture.SUBSTANCE: invention relates to biotechnology and agriculture and concerns a new strain as a base for producing biopreparations for growing agricultural products. New strain B. subtilis B-94 VIZR, having high antagonist activity on phytopathogenic fungi and bacteria, is promising as a producer of a biopreparation for protection of potatoes against diseases during vegetation and storage of tubers, increased yield and improved quality of tubers. B. subtilis B-94 VIZR strain is deposited in the VIZR Collection, is active against alternaria, phytophthora and rhizoctonia. Biological efficiency of strain in vegetation period in relation to complex of diseases makes 60–70 %.EFFECT: use of the strain significantly improves consumer quality of tubers during storage.1 cl, 9 tbl, 4 ex, 1 dwg

Description

The invention relates to microbiological protection of plants and agricultural microbiology and is a strain that produces a complex of biologically active substances with fungicidal and bactericidal action, can be used to obtain a biological product for protection against fungal and bacterial diseases of potatoes during the growing season and storage of crops, as well as increasing productivity and improving the quality of tubers.

At present, the chemical method occupies a leading place in the protection of potatoes from phytopathogens during cultivation and storage. However, the widespread use of chemical remedies often leads to negative consequences - disruption of the structure of biocenoses, a decrease in their ability to self-regulation, the accumulation of toxic pesticide residues in soil, water and food. In addition, the resistance of populations of phytopathogenic fungi and bacteria to chemical fungicides increases, which reduces the effectiveness of their application.

Analysis of domestic and foreign scientific and technical literature has shown that microbiological control can successfully compete with chemical means of protection, without having their drawbacks. The microbiological method has significant economic benefits and does not use non-renewable natural resources. A relatively new direction in the development of biocontrol is the use of biological products based on active antagonist strains during storage of crop production and improvement of its quality.

Known strains of Bacillus subtilis are producers of a number of biological fungicides - Vitaplan, Fitosporin, Alirin-B, Gamair, Baktofit, Albit, etc. ("State catalog of pesticides and agrochemicals permitted for use on the territory of the Russian Federation", 2019). The strains used in them have low biological effectiveness in vegetable storage conditions and do not provide comprehensive effective protection of plant products from diseases during the growing season and during storage of the crop. The State Catalog of Pesticides and Agrochemicals Permitted for Use on the Territory of the Russian Federation does not contain biological products recommended to protect potatoes from diseases in storage conditions.

It is known to use Bacillus subtilis strain 413 to reduce the loss of carrots from infectious diseases and improve the quality of products during long-term refrigerated storage (Samusenko, 2001).

Known strains B. subtilis strain V-10 VIZR (RF patent No. 2081167, 1997) - producer of the drug Alirin-B and V. subtilis strain M-22 VIZR (RF patent №2084152, 1997) - producer of the drug Gamair to protect agricultural crops from fungal and bacterial diseases ("State catalog of pesticides and agrochemicals permitted for use in the Russian Federation", 2019). But these strains have a narrower spectrum of action and insufficient biological effectiveness against phytopathogenic fungi and bacteria under low temperature conditions during long-term storage of crop products. Alirin-B and Gamair are recommended for use on potatoes only during the growing season against "black leg", rhizoctonia, late blight and alternaria.

The closest analogue, taken as a prototype, is the Bacillus subtilis strain - B 93 VIZR (RF patent No. 2538157, 2015), which has a high antagonistic activity against phytopathogenic fungi and bacteria and is promising as a producer of a biological product for protecting potatoes from diseases during storage. The disadvantage of the known invention is the low biological efficiency of the strain in protecting potato plants during the growing season, as well as the lack of a positive effect on the quality of tubers.

The objective of the invention is to create a strain with a wide spectrum of action and high antagonistic activity against causative agents of fungal and bacterial diseases of potatoes and providing comprehensive protection against potato plant diseases during the growing season and during storage of tubers, increasing the yield of potatoes and improving the quality of tubers.

The task was accomplished by stepwise selection of active isolates from monoclonal sowing of the Bacillus subtilis strain B 93 VIZR, which produces a complex of biologically active substances with high antagonistic activity against pathogens of fungal and bacterial potato diseases. The strain was deposited in the State Collection of Microorganisms Pathogenic for Plants and Their Pests FGBNU VIZR. The original strain was isolated from the soils of India (Delhi) for the signs of antagonistic activity against phytopathogenic fungi and bacteria. Identification of the strain was carried out according to the Bergey bacteria guide (Hoult D., Krieg I. et al. Bergey bacteria guide. In 2 v. 9th edition. Publishing house Mir. 1997).

CHARACTERISTIC FEATURES

1. Morphology: Straight rods with rounded ends, often forming chains.

2. Size: 0.8-0.9x2.5-3.5 microns.

3. Cell wall and Gram staining: gram-positive, thick gram-positive cell wall (analysis method - staining with gentian dye and Gram iodine solution). Does not form capsules.

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

5. Type of division: transverse binary.

6. Prostash: absent.

7. Mobility: mobile.

8. Flagellation: peritrichous flagella.

9. Pigmentation of colonies: colonies are creamy, dull, flat, with an uneven edge, rapidly spreading over the surface of the nutrient medium, later the colonies become compacted, shiny and acquire a brownish-beige tint.

10. Pigment is not released on Wednesday.

11. There are no selective media.

12. Oxidase: positive.

13. Catalase: positive.

14. Attitude to oxygen: aerobic. Does not grow in anaerobic agar.

15. Chemoorganotroph, respiratory type metabolism. Used carbon sources for aerobic growth: glucose, sucrose, mannitol, maltose and lactose. When glucose, arabinose, xylose and mannitol are used, it forms acid and acetoin, does not form gas. Forms hydrolases. Hydrolyzes starch and casein. Reduces nitrate to nitrite.

16. Does not grow on sorbitol and inositol under aerobic conditions.

17. Non-oxidizing inorganic substrates.

18-20. Does not grow under anaerobic conditions.

21. Relationship to pH: the strain grows in the pH range from 4.5 to 8.5. Optimal pH = 6.5-7.5.

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

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

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

26. Cytochromes: not determined.

27. Menaquinones: not determined.

28.% HZ: not determined.

29. Requirements for growth factors or special supplements: not required.

30. Additional information: strain-producer of a biological product for the protection of growing plants and potato tubers from phytopathogenic fungi and bacteria - pathogens, increasing the yield and improving the quality of tubers. Method for determining the activity of the product: biological to suppress the growth of test cultures of phytopathogenic fungi and bacteria.

31. Method of storing the strain:

a) jambs with SPA or MPA environment.

b) in a lyophilized state.

32. Shelf life: reseeding after 6 months.

33. Attitude towards phages: no phage was isolated.

34. Genetic features: genotyping of the strain was carried out by the DREAM method (double splitting, selective labeling).

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

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

Fusarium sambucinum - 35.0

F. solani var. coeruleum - 22.0

F. avenaceum - 24.5

Fusarium oxysporum - 35.0

Rhizoctonia solani - 28.0

Alternaria brassicicola - 25.0

Colletotrichum lagenarium - 25.0

Verticillium dahliae - 25.5

Sclerotinia sclerotiorum - 25.0

Phoma exigua - 24.0

Bipolaris sorokiniana - 25.0

Helmintosporium solani - 30.0

Pythium debarianum - 17.0

Ascochytafabae - 22.0

Septoria nodorum - 25.0

Rhizoctonia solani - 28.0

Phytophthora infestans - 34.0

Pectobacterium carotovorum var. sepedonicum - 25.0

Clavibacter. michiganensis var. sepedonicum - 30.0.

Compared to the prototype, the proposed strain has a wider spectrum of action against causative agents of fungal diseases and high antagonistic activity against phytopathogenic bacteria (Table 1).

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

Example 1. The cultivation of the B. subtilis B-94 VIZR strain was carried out in submerged conditions in Erlenmeyer flasks for 72 hours on a circular shaker at 28 ° C until the end of the sporulation phase.

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

Sprat pancreatic hydrolyzate 10.05; sodium chloride 4.95; glucose 10.0; water - up to 1 liter; pH 7.1 ± 0.2 sterilization 0.5 atm / 30 min.

Determination of the antagonistic activity of a 3-day culture liquid obtained by deep cultivation of the strain was carried out by the standard microbiological method of wells (Workshop on Microbiology, ed. By N.S. Egorov, 1976; Guide to practical exercises in Microbiology, ed. By N.S. Egorov, 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 in comparison with the prototype are presented in table. 1.

As can be seen from table 1, the B. subtilis strain B-94 VIZR VIZR, in contrast to the prototype, has a higher antifungal and antibacterial activity. Significantly higher in the proposed strain antagonistic activity against phytopathogenic bacteria Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis var. sepedonicum, Pseudomonas corrugata and fungi Phytophthora infestans, Rhizoctonia solani, Phoma exigua. Bipolaris sorokiniana, Fusarium oxysporum, Ascochyta fabae.

Example 2.

The culture fluid of the B. subtilis strain B-94 VIZR VIZR was obtained in the same way as in example 1. The study of biological efficiency in model experiments and in a potato storage was carried out in accordance with the standard methods set forth in the following manuals: "Research methodology for potato culture", M ., 1967; "Methodology of field experience", Moscow, 1985; "Guidelines for state testing of fungicides, antibiotics and seed dressings for agricultural crops" / M., 1965 /; "Research methodology for the protection of potatoes from diseases, pests, weeds and immunity", M., 1995; "Methodological guidelines for registration tests of fungicides in agriculture", St. Petersburg, 2009.

The laying of samples of potato tubers for long-term storage in order to assess the biological effectiveness of the strain in relation to a complex of diseases was carried out in the potato storage of the State Scientific Institution VNIIKKH (Moscow region, Lyuberetsky district, Kraskovo village, Korenevo microdistrict).

The experiment used tubers of the Sante variety (medium early, universal use). The yield is high. The variety is resistant to potato cancer (the causative agent of Synchytrium endobioticum), golden potato cyst nematode (Globodera rostochiensis), viral diseases, susceptible to late blight by tops. Medium resistant to common scab, susceptible to rhizoctonia and phomosis. The set of material for the experiments was produced 3 days after the harvest. Before processing and storage, tuber analysis was carried out in order to determine the phytopathological state of potatoes. Before placing 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 the fungicide Maxim, KS (0.2 l / t), in the control - with water. Working fluid consumption - 3 l / t. The sample weight of potato tubers was 5 kg. The experiment was repeated 10 times. The total mass of tubers of each variant is 50 kg. Placement of samples: in a potato mound at a depth of 20-30 from the surface.

At the end of the storage period in March 2019, tuber analysis was carried out and the disease incidence and weight loss of potatoes during storage were determined (Tables 2-3).

A significant decrease in the development of diseases on seed potato tubers after treatment with the B. subtilis strain B-94 VIZR VIZR, and the standard Maxim, KS, compared with the control, was noted. The number of healthy tubers after application of the strain reached 35.4-40.5%, while in the prototype this figure was 25.4%, in the control did not exceed 13.5%. Thus, in comparison with the control, the yield of healthy products increased 3.0 times. The treatment with the strain effectively reduced the infestation of tubers with all the diseases that manifested itself, 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 the B. subtilis B-94 VIZR strain, the prevalence of ring rot was 3.6-3.9%, dry rot - 0.1-7.0%, which is significantly lower than these indicators in the B. subtilis B 93 VIZR strain (prototype) - 5.0% and 13.1 % and fungicide standard Maxim, KS - 9.5 and 12.2%, respectively.

Thus, the analysis of the results of the experiment showed that the treatment of tubers with the B. subtilis B-94 VIZR strain before placing them for storage in a potato storage reduced the number of tubers suffering from fungal and bacterial diseases and increased the yield of healthy potatoes by 3-3.5 times. The biological effectiveness of the B. subtilis B-94 VIZR strain against ring rot, dry rot and late blight was significantly higher than the effectiveness of the prototype and the standard fungicide Maxim, KS.

Example 3. In field experiments, the efficacy of the Bacillus subtilis B-94 VIZR strain against Rhizoctonia spp., Alternaria spp. And late blight (Phytophthora infestans) on potatoes of the Firefly 1 variety was studied. Cultural fluid of the B. subtilis B- strain. 94 VIZR was obtained in the same way as in example 1 and 2. During the growing season, the plants were treated with dilutions of the culture fluid of the strain with a titer of viable cells 10 ⁸ and 10 ⁹ CFU / ml (Table 4). Seeding rate: 3.0 t / ha. Planting date: 25.04, date of emergence of shoots 15.05. Plant development phase at the time of treatment: 0 (tubers), spraying: closing of rows, end of flowering. Tillage: disking, plowing, harrowing, continuous cultivation, manual planting, two-fold loosening of row spacings. Fertilizers: ammonium nitrate - 100 kg / ha when irrigated. Measures for the care of experimental plots: one watering during the growing season by sprinkling, hilling bushes, twice loosening in rows, treatment against pests.

Plot size and placement: 25 m ² , randomized. Number of repetitions: 4, processing time: 20.04; 20.05; 5.06. Multiplicity of processing: tubers 1 - multiple, spraying with a suspension of the drug during the growing season - 2 times. Apparatus used: backpack sprayer type ORI. Working fluid consumption: 10 l / t (pickling), 300 l / ha (spraying).

Registration dates: 6.06; 18.07. Date of appearance of diseases: 20.05 (rhizoctoniasis); 5.06 (alternaria); June 25 (late blight). Method of harvesting and accounting of the crop: manually from each option. Harvest date: July 20th.

The test results showed that the effectiveness of the B. subtilis B-94 VIZR strain against potato late blight at 2 concentrations of the suspension and the chemical standard was approximately the same (table 5). With a titer of 10 ⁹ CFU / ml, it was 62.5%, with a titer of 10 ⁸ CFU / ml - 57.5%; in the standard Maxim, KS - 42.9% with the development of the disease in the control of 28.0%. With an increase in the development of the disease in the control to 38.5%, the effectiveness of the strain at a titer of 10 ⁸ CFU / ml decreased and amounted to 42.9%, at a titer of 10 ⁹ CFU / ml it slightly increased (68.8%), the effectiveness of the standard decreased to 31, 2%. The effectiveness of the prototype was significantly inferior to that of the B. subtilis B-94 VIZR strain and did not exceed 15.6-26.8%.

Against Alternaria, the best results were obtained with a suspension titer of 10 ⁹ CFU / ml (54.5-66.7%), exceeding the efficiency of the standard (43.2-45.4%). The effectiveness of the strain at a titer of 10 ⁸ CFU / ml was 29.5-40.6%. The development of the disease in the control during the observation period increased from 22.0 to 42.1%. In relation to Alternaria, the effectiveness of the prototype was lower (18.2-38.2%)

The test results showed that 2-fold spraying of potato plants with a suspension of the strain at both concentrations against the background of treatment of tubers was effective against Rhizoctonia stems. The maximum efficiency against this disease was obtained in the variant with a titer of 10 ⁹ CFU / ml (70.9%) with the development of the disease in the control 31.6%. The efficiency at a titer of 10 ⁸ CFU / ml was below 47.8%. The efficiency of the Maxim, KS standard was 77.8%. The efficiency of the prototype was inferior to the proposed strain and amounted to 39.2%.

The effectiveness of the prototype (strain B. subtilis B 93 VIZR) against late blight, alternaria and rhizoctonia was inferior to the effectiveness of the proposed strain during the entire growing season (Table 5). The increase in yield was also lower than in the variants of the experiment using the B. subtilis B-94 VIZR strain and did not exceed 38%. The maximum yield increase was obtained in the experimental version (42-45%), it significantly exceeded the yield increase in the standard - 24%.

According to the results of the autumn tuber analysis of the obtained crop, the biological efficiency of the culture fluid of the strain, B. subtilis B-94 VIZR against pathogens of dry rot, ring rot and rhizoctoniae reached 82-100% and was at the level of the chemical standard (table 5). The efficiency of the prototype was 10% lower.

Thus, the studies carried out have shown the high efficiency of the selected B. subtilis B. subtilis B-94 VIZR strain and the prospects of its use as a producer of a biological product to protect potato tubers from a complex of diseases during the growing season and during storage of the crop by pre-planting tubers with a suspension with a titer of 10 ⁸ -10 ⁹ CFU / ml and spraying during the growing season at a rate of flow of the working fluid of 300 l / ha, 2 times.

Example 4. The yield of tubers obtained in the field experiment described in Example 3 was analyzed for quality indicators. The analyzes were carried out according to the following methods: Determination of dry matter in fresh material.

The thermostat-weight method is used to determine the dry matter content in tubers. The investigated sample of leaves or tubers is well crushed, thoroughly mixed and leveled with a thin layer. From different places of the prepared layer take 2 weighed portions of 25-50 g in cups dried to constant weight.

Preliminary drying of the material is carried out in a drying cabinet at a temperature of 50-60 ° C for 4 hours, after which the sample is continued to dry at a temperature of 100-105 ° C for another 3-4 hours. Then the cups with weighed portions are cooled in a desiccator and weighed. Re-drying takes 2 hours. The cups are then weighed again. Drying continues until constant weight. The amount of dry matter is determined by the formula:

где

Where

B is the weight of a sample of fresh material,

D is the weight of absolutely dry material.

Determination of starch content by specific gravity.

The specific gravity of tubers is determined by weighing them in air and in water and calculated by the formula:

где

Where

A is the weight of potato tubers in the air (g),

B - weight of potato tubers in water (g)

Before taking and weighing the sample, the tubers are thoroughly washed and dried in the air. Use only visually healthy, undamaged potatoes. For weighing, use a specially equipped laboratory balance with a mesh (or basket) for immersing tubers in water. When weighing in water, pay attention to its temperature and air bubbles formed on the surface of the tubers, which must be removed, which is achieved by moving the basket up and down. The water in which the tubers are immersed must be clean and have a temperature of 17-18 ° C. After calculating the formula according to a special table, the percentage of starch in the potato tubers is determined.

Determination of the content of ascorbic acid in plants (according to Murry).

The method is based on the transfer of ascorbic acid into a solution and on the ability to reduce it in an acidic medium, a blue indicator sodium 2,6-dichlorophenolindophenolate to leucoform, while ascorbic acid is oxidized to dehydroascorbic acid. The reaction is due to the enzyme ascorbate oxidase: the higher the content of ascorbic acid, the higher the activity of the enzyme ascorbate oxidase.

A weighed portion of plant material (potato tubers) 5 g is ground in a mortar with 20 ml of 1% hydrochloric acid solution for no more than 10 minutes. The resulting mass is transferred through a funnel into a 100 ml volumetric flask with a 1% solution of oxalic acid. Bring the solution in the flask to the mark with oxalic acid. The flask is vigorously shaken several times and left to stand for 15 minutes for the proteins to settle. The contents of the flask are filtered off, 10 ml of solution are taken from the filtrate in three glasses, the contents in the glasses are titrated from a microburette with 0.001 N sodium 2,6-dichlorophenolindophenolate solution (Tillmans paint) until a pink color that does not disappear within 1 minute. The calculation of the content of ascorbic acid is made according to the formula:

где

Where

X is the content of ascorbic acid in mg per 100 g of the substance;

a is the number of ink used for titration, ml;

T - Tillmans paint titer (0.14);

V is the volume of the volumetric flask with the extract;

c - the number of extract taken for titration, ml;

c - sample of the test material, g.

Determination of the content of reducing sugars is carried out according to the Sumner method.

The method is based on the ability of reducing sugars to give colored compounds with 3,5-dinitrosalicylic acid.

For analysis, 10-15 potato tubers are taken from an average sample. Tubers, washed and air-dried, are cut along the centerline into two or more pieces. Segments for analysis are selected from each part so that they reflect all zones of the analyzed tuber. Then the selected parts are crushed on a grater or homogenizer and mixed.

We put 5 g of potato pulp in a jar (or flask), add 50 ml of distilled water heated to a temperature of 60-70 ° C and set it to shake for 10 minutes. on the rocking chair. Then we filter the solution through a regular filter (or centrifuge). Transfer 1 ml of the filtered solution into a test tube, add 1 ml of Sumner's reagent and boil in a water bath for 5 minutes. Simultaneously with the test samples, we put a control on a tripod - a test tube with 1 ml of Sumner and 1 ml of distilled water. After 5 minutes of boiling, cool the rack with test tubes in cold water. Then add 15 ml of distilled water to each tube, mix well and look at the FEK at a wavelength of 540 nm.

The amount of sugars is determined by the formula:

reducing sugars,% = a × V × 100 / H × 1000, where

a - the amount of sugars found from the calibration curve, mg;

V is the total volume of the filtrate (50 ml);

H - weight (5 g);

1000 - coefficient for converting mg to g.

Building a graph for sugar. Dissolve 2.5 g of glucose in water (50 ml volumetric flask). 1 ml of the resulting solution contains 50 mg of glucose. In volumetric flasks of 100 ml, place the following amount of the original solution: 0.5; 1.0; 1.5; 2.0; 2.5 ml. Bring to the mark with distilled water. We mix. Next, we collect 1 ml of the working solution from the cones into test tubes, add 1 ml of Sumner's reagent. We boil in a water bath for 5 minutes. We cool in cold water. Add 15 ml of distilled water, mix and look at the FEK at a wavelength of 540 nm.

Sumner reagent preparation. To prepare 100 ml of Sumner's reagent, 1.6 g of NaOH, 30 g of K-Na-tartaric acid, 1 g of 3.5 dinitrosalicylic acid must be dissolved in distilled water and brought to the mark (for better dissolution, the prepared solution is heated in a water bath).

Ionometric method for the determination of nitrates in plant samples.

The method is based on the extraction of nitrates with a solution of potassium alum with subsequent measurement of the concentration of nitrates using an ion-selective nitrate electrode and is express.

Prepare the electrodes for work. For this, the membrane nitrate ion-selective electrode and the silver chloride electrode are prepared for operation in accordance with the instructions attached to the electrodes.

Before starting work, the membrane of the ion-selective electrode is soaked for 24 hours in a solution of potassium nitrate or sodium nitrate C _NO3- = 0.1 mol / dm ³ at a temperature of (20 ± 5) ° C.

Between measurements, the electrode is stored in a reference solution C _NO3- = 0.0001 mol / dm ³ . During long breaks in work, the electrode is stored dry; before measurement, the electrode is soaked for 1-2 hours in a reference solution C _NO3- = 0.1 mol / dm ³ . The auxiliary silver chloride electrode is stored in water.

The calibration graph is constructed as follows: a basic solution of potassium nitrate or sodium nitrate is prepared on the day of the test. To prepare a basic solution of potassium nitrate or sodium nitrate with (NO ₃ ) = 0.1 mol / dm ³ (pC _NO3 = - LgC = 1), 10.110 g of potassium nitrate or 8.500 g of sodium nitrate is dissolved in a solution of potassium alum and the volume is adjusted to 1000 cm ^{3 with} this solution. The solution is stored for no more than one year. When turbidity or sediment appears, the solution is replaced with a freshly prepared one.

Comparison solution with (NO ₃ ) = 0.01 mol / dm ³ (pC _NO3 = - LgC = 2): prepared from the stock solution with (NO ₃ ) = 0.1 mol / dm ³ dilution 10 times. To do this, take 10 cm ^{3 of the} basic solution with a pipette, add it to a volumetric flask with a capacity of 100 cm ³ and bring the volume to 100 cm ³ with a solution of potassium alum, mix. The potassium alum solution is used for all subsequent dilutions.

Comparison solution with (NO ₃ ^- ) = 0.001 mol / dm ³ (pC _NO3 = - LgC = 3): prepare on the day of the test by diluting 10 times the solution with (NO ₃ ) = 0.01 mol / dm ³ .

The reference solution c (NO ₃ ^- ) = 0.0001 mol / dm ³ (pC _NO3 ^- = - LgC = 4): prepare on the day of the test by diluting the solution 10 times with (NO ₃ ) = 0.001 mol / dm ³ .

Measurement of potentials E (mV) in reference solutions on an ionomer. The nitrate electrode is connected on the rear panel of the device to the "Measure" socket, and the silver chloride electrode - to the "Aux" socket. The electrodes are immersed in the test sample and the potential of the electrode pair E, mV is determined, while the "Type of work" button is set to the "mV" position, after the measurement, the network is turned off by pressing the "t" button.

Before each measurement of the reference solution or the test solution, the electrodes are washed several times with water, dried with filter paper, washed with the reference solution or the test solution, and only then immersed in the solution to be measured. The instrument readings are read not earlier than 1 min after the end of the drift of the instrument readings. The determination of the test samples is carried out simultaneously with the calibration of the electrodes.

Calibration of electrodes is carried out by measuring the potentials E, mV, in reference solutions prepared at room temperature. The measurement is carried out starting with solutions of low concentrations, each time rinsing the electrode with a solution of a higher concentration. The measurement results are recorded in table 7.

Based on the results obtained, a calibration graph is built. The abscissa axis represents values pC _NO3 ^-, respective comparison solutions of potassium nitrate or sodium nitrate:

- c (NO ₃ ) = 0.1 mol / dm ³ (pC _NO3 = 1);

- c (NO ₃ ) = 0.01 mol / dm ³ (pC _NO3 = 2);

- c (NO ₃ ) = 0.001 mol / dm ³ (pC _NO3 = 3);

- c (NO ₃ ) = 0.0001 mol / dm ³ (pC _NO3 = 4).

The ordinate is the corresponding value of the potential, E, mV.

The calibration of the electrodes is checked at least three times during the working day, using fresh portions of reference solutions each time.

The electrode has a linear function in the pC _NO3 range from 1 to 4 with a slope of (56 ± 3) mV per unit pC _NO3 at a temperature of 25 ± 5 ° C. If the characteristic of the electrode differs from the specified one, the electrode is not suitable for work.

FIG. 1 shows the graph of the dependence of the potential E (mV) on the concentration of NO ₃ ^- (pC _NO3 ).

Analysis process: the analyzed product (potato tubers) in an amount of 10 g is crushed, placed in a flat-bottomed or conical flask, 50 cm ^{3 of a} solution of potassium alum is poured in, closed with a stopper and shaken on a shaking apparatus for 5 minutes, placed in a glass cup, immersed electrodes into it and measure the potential of the electrode pair E, mV. From the obtained value of E according to the calibration curve, the value of pC _{NO3 is found} .

Processing of the results: the content of nitrates, mg / kg, or mass concentration, mg / dm ³ , is found by the pC value of _NO3 in special tables (6). For the final analysis result, the arithmetic mean of the results of two parallel determinations is taken, the permissible discrepancy between which in relation to the arithmetic mean at

P = 0.95 should not exceed:

30 - with nitrate content up to 200 mg / kg;

25 - with a nitrate content of 200 mg / kg and above.

To assess the taste and nutritional qualities of potato tubers, clean, unpeeled tubers are boiled without salt. The end of cooking is determined with a specially prepared wooden stick. The cooked tubers are laid out in plates and hot assessed for taste, pulp consistency, digestibility, smell, pulp color, darkening.

The taste is assessed according to a 5-point scheme: excellent - 5 points; very good - 4 points; good - 3 points; satisfactory - 2 points; bad - 1 point.

The darkening of the pulp of the tuber and the color of the pulp of the boiled tubers are assessed immediately after boiling and in cold form after about 1.5-2 hours: the surface and pulp of the tubers did not darken - 3 points; darkened slightly - 2 points; darkened strongly - 1 point.

The data from the analyzes are presented in Table 8. As can be seen from the table, the use of the B. subtilis B-94 VIZR strain for treating tubers and spraying plants during the growing season significantly influenced the consumer qualities of the crop during storage. The dry matter content in tubers was 14.6-33.5%, starch 8.2-10.5%, sugars - 25.0-90.0% more compared to the control. The nitrate content in tubers was 23.5-69.1% lower than in the control. The use of the strain improved the taste of potatoes. The treatment of tubers and potato plants with the culture liquid of the prototype strain was less effective: the dry matter content was only 1.9% higher and the sugar content 5% higher than in the control. The starch content remained at the control level, the nitrate concentration was only 10.2% lower. The use of chemical fungicides as a standard did not improve the quality of the tubers.

No darkening of the pulp of the tuber immediately after cooking and cold after 2 hours was noted.

Literature

1. N.V. Samusenko. Scientific substantiation of the use of antagonist bacteria during long-term refrigerated storage of carrots. Dis. Ph.D., St. Petersburg, 2001.

2. State catalog of pesticides and agrochemicals permitted for use on the territory of the Russian Federation, 2014. www.mcx.ru.

3. Research methodology for potato culture. M., VASKHNIP, NIIKH, 1967, 189 p.

4. B.A. Armor. Field experiment technique (with the basics of statistical processing of research results). - 5th ed., Add. and revised .: Agropromizdat, 1985 .-- 351 p.

5. Guidelines for state testing of fungicides, antibiotics and seed dressings for agricultural crops. M., 1985.130 p.

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

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

8. I.I. Novikov; A.I. A.I. Litvinenko, T.A. Nugmanova, G.V. Calco. Bacillus subtilis bacteria strain used to protect plants from phytopathogenic fungi. RF patent No. 2081167, filing date 04/08/1994, publication date 06/10/1997.

9.I.I. Novikova, G.A. Bykova, M.E. Sergeeva, V.A. Pavlyushin. Bacillus subtilis bacterial strain used to protect plants from phytopathogenic bacteria. RF patent No. 2084152, filing date 10.10.1994, publication date 20.07.1997.

10.I.I. Novikova, I. V. Boykova, V.A. Pavlyushin, V.N. Zeyruk. Bacillus subtilis bacterial strain B 93 VIZR to protect potatoes from diseases during storage. RF patent No. 2538157, filing date 11/27/2013, publication date 01/10/2015.

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

12. M.N. Pimenova, N.N. Grechushkina, L.G. Azov, A.I. Netrusov, E.V. Semenova, N.N. Kolotilova, L.M. Zakharchuk, V.V. Zinchenko, S.I. Mylnikova, M.V. Nefedova, I.V. Botvinko. A guide to practical exercises in microbiology. Textbook. allowance / Ed. N.S. Egorova. - 3rd ed., Rev. and add. - M .: Publishing house of Moscow State University, 1995 .-- 224 p. Research methodology on potato culture (ed. Andryushina N.A., Batsanov N.S.) - M. - 1967. - 264 p.

13. Guidelines for assessing potato varieties at test (test) sites - M., GNU VNIIKKH Rossel-khozakademiya - 2013. - 15 p.

14. Methodology of physiological and biochemical studies of potatoes (edited by V.P. Kiryukhan, E.A. Ladygina, M.M. Chegolina, A.V. Parfenova) - M. - 1989. - 144 p.

15. Hoult D., Krieg N. et al. Bergey's Keys to Bacteria. In 2 volumes. 9th edition. Publishing house Mir. 1997.

Claims (1)

Strain bacteriaB. subtilisB-94 VIZR for protecting potatoes from diseases during vegetation and storage, increasing yields and improving the consumer qualities of tubers.

Images