RU2487932C1 - STRAIN OF NODULE BACTERIA Bradyrhizobium japonicum 859 FOR PRODUCTION OF FERTILISER FOR SOYA - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain of nodule bacteria Bradyrhizobium japonicum 859 (VNIISHM No.24117) is proposed, used as a facility to produce a fertiliser for soya. When soya seeds are treated with the fertiliser produced on the basis of the specified strain Bradyrhizobium japonicum, the index of germination increases by more than 8%, and crop capacity increases by 17-30%.

EFFECT: higher germination of plants, improved crop capacity.

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Description

The invention relates to biotechnology and agriculture and relates to a new strain of nodule bacteria Bradyrhizobium japonicum 859 to obtain microbiological fertilizers that increase soybean yield.

Currently, to increase the yield of legumes using bacterial fertilizers made on the basis of nitrogen-fixing nodule bacteria.

So, from the description of the USSR Author Certificate No. 491695, the strain Rhizobium japonicum 646 is known, which is used for the manufacture of nitragin. Its disadvantage is the relatively low nitrogen-fixing activity and competitive ability.

From the description of the USSR Author's Certificate No. 640989 (published on January 8, 1979), a strain of nodule bacteria of soybean Rhizobium japonicum 614a is known, which is a highly active symbiotic nitrogen fixator. Its disadvantage is sensitivity to chemical seed dressers, therefore, the treatment of seeds of legumes must be carried out immediately before sowing, without combining it with chemical dressing.

From the description of the USSR Author's Certificate No. 1034358 (priority date 04.01.1982, published 07.04.1984), a strain of soybean nodule bacteria Rhizobium japonicum 634 B is also known, which is used in the industrial manufacture of the bacterial drug nitragin for soybeans. Moreover, the indicated strain 634 B actively fixes atmospheric nitrogen in symbiosis with soy, and contributes to the active accumulation of protein by plants. Its disadvantage is the relatively low efficiency.

A known bacterial strain Rhizobium japonicum 24108 (71t) is described in the materials for the Author's certificate No. 939547 (priority from 07/31/1990, published on 06/30/1993), which allows to obtain an increased yield of green mass and soybean compared to other known at the priority date similar strains. Its disadvantage is low competitiveness with local strains of soybean nodule bacteria.

A number of strains of nodule bacteria are known for the industrial production of bacterial preparations for soybeans described in Ukrainian patents, for example:

- Bradyrhizobium japonicum M-8 (declaration patent (UA) for the invention 39545 A, published June 15, 2001);

- Bradyrhizobium japonicum KH 10 (declaration patent (UA) for the invention 69993 A, published September 15, 2004);

- Bradyrhizobium japonicum T66 (patent (UA) for the invention 83298 C2, published June 25, 2008);

- Bradyrhizobium japonicum 36 (patent (UA) for utility model 36700 U, published November 10, 2008);

- Bradyrhizobium japonicum 46 (patent (UA) for the invention 85943 C2, published March 10, 2009).

Their disadvantage is that these strains were selected and tested on soybean varieties cultivated in Ukraine, in relation to local soil and climatic conditions. However, there is no data on the effectiveness of these strains in the soil and climatic conditions of the main regions of soybean cultivation in Russia: in the Krasnodar, Stavropol Territories, in the Rostov Region, the Far East, and Siberia.

Also known is the microbiological preparation Agrostim B containing a consortium of strains of Pseudomonas fluorescens and Azotobacter spp. (see, for example, the article “Increasing the yield of leguminous crops through the use of the drug Agrostim B”, authors Kirsanova EV, Daryuga K.V. / FSEI HPE Orel GAU, Musalatova NN, Tsukanova Z.R., Borzenkova G.A. / GNU VNIIZBK /, published in the journal Vestnik Orel GAU No. 4 for 2008, UDC 635. 656: 633.353: 635.655). The specified preparation can be used both for pre-sowing seed treatment, and for spraying the soil and vegetative plants. It has a growth-promoting and inhibiting harmful microflora effect. The disadvantage is that when the indicated preparation is applied on the plant roots, nodules do not form, fixing the atmospheric nitrogen necessary for plant nutrition, which does not significantly increase soybean productivity and leads to the need to introduce additional nitrogenous fertilizers into the soil.

Also known is a microbiological fertilizer for soybean Rizotorfin based on a strain of soybean nodule bacteria Rhizobium japonicum 629 ^a (prototype), described in the materials for the Certificate of Authorship No. 939547 (priority from 01/05/1981, published on 06/30/1982), which allows to obtain a soybean crop with a high protein content in the grain. The specified strain 629 ^a (87) is suitable for the industrial manufacture of preparations of nodule bacteria (Rizotorfina). The disadvantage is the relatively low efficiency on the main production varieties of soybeans grown on the soils of the Far East.

The task of the invention is to identify a new strain of bacteria with high nitrogen-fixing activity, suitable for use in agriculture in order to increase soybean yield.

This problem is solved due to the fact that a strain of nodule bacteria Bradyrhizobium japonicum 859 is used for fertilizing under soy.

The bacterial strain was isolated from nodules growing on powerful chernozem of the Gulkevichsky district of the Krasnodar Territory of soybeans and was originally named as a strain of Bradyrhizobium japonicum M-8, which was deposited in the collection of the GNU VNIISM ROSSELHOZAKADEMII on December 05, 2008 under registration number 24117 in the nodule group. Then, as a result of a patent search, it was revealed that in Ukraine another strain was named Bradyrhizobium japonicum M-8 Kircyner (patent application (UA) for invention 39545 A, published on June 15, 2001), after which, in order to avoid problems with intellectual property infringement , the strain isolated by the Applicant was renamed 02/11/2010 in the strain Bradyrhizobium japonicum 859.

The strain Bradyrhizobium japonicum 859 is characterized by the following morphological-cultural and physiological-biochemical characteristics.

The cells have the shape of regular sticks with rounded ends and a polar arrangement of the flagellum. Cell size (0.6-0.8) × (1.4-1.9) microns.

The strain Bradyrhizobium japonicum 859 does not form a spore, negatively stained by Gram. Growth in a liquid and semi-liquid nutrient medium is aerobic, respiratory metabolism. On mannitol-yeast agar forms mucous, convex, round, matte colonies with smooth edges, with a diameter of 2-5 mm. The optimum temperature for growth is 28 ° C. At temperatures less than 20 ° C and more than 35 ° C, growth is slow. The optimal pH of the medium is 6.8; growth also occurs at pH from 5.0 to 8.5. Aerob, demanding on oxygen.

The strain Bradyrhizobium japonicum 859 does not hydrolyze casein, gelatin. When the strain grows in litmus milk, mild acid formation is observed with a change in the shade of the medium when compared with the control. The strain is able to use arabinose, xylose, glucose, galactose and fructose as the sole carbon source. The strain is not able to use mannitol, sucrose and lactose.

For storage and cultivation of the strain Bradyrhizobium japonicum 859 in laboratory conditions, mannitol-yeast agar (MDA) is used at pH = 6.8 ± 0.2 of the following composition, g / l:

Mannitol - 10.0 Yeast extract - 0.5 K ₂ HPO ₄ - 0.5 MgSO ₄ × 7H ₂ O - 0.2 NaCl - 0.1 Agar - 15.0

As you know, nitrogen is required for plant development. Nitrogen fixation is the biological assimilation of air nitrogen molecules by nitrogen-fixing bacteria to form nitrogen compounds available for use by other organisms; is one of the most important processes of the nitrogen cycle in nature and an indicator of soil fertility. Nitrogen fixation is carried out by both free-living nitrogen-fixing bacteria - azotobacter, cyanobacteria, azospirillum and others (non-symbiotic nitrogen fixation), and symbiotic nitrogen fixers living in symbiosis with higher plants (e.g., nodule bacteria). In this regard, the nitrogen-fixing activity of the strain is an important characteristic that affects the stimulation of plant growth and productivity.

It was revealed and experimentally established that the bacteria of the claimed strain Bradyrhizobium japonicum 859 form nodules on the roots of plants, have good nitrogen-fixing properties and positively affect the field germination of soybean, its growth and yield. In this case, the concentration of bacteria of the strain for its effective use in various biological products should be at least 10 ⁸ CFU / ml. It was also experimentally established that the optimal concentration of bacteria in the range of values (10 ⁸ -10 ⁹ ) CFU / ml, because the use of the strain at a concentration of less than 10 ⁸ CFU / ml leads to a decrease in the indicated effect, and an increase in concentration above 10 ⁹ CFU / ml does not increase it.

The ability of the bacteria of the claimed strain Bradyrhizobium japonicum 859 to form nodules on the roots of plants is confirmed by vegetative experiments conducted on the experimental basis of GNU VNIISKHM. First, they prepared the soil by mixing 80% of the soil from the experimental field (sod-podzolic) with 20% of the peat soil of the Terravita brand. Then, the resulting soil was decomposed into 5 kg of vegetation vessels of 3 l each and poured 700 ml of tap water into the vessel. After this, the seeds were laid for germination: first, the seeds were surface-sterilized with 70% ethanol for 2 minutes, then they were washed with sterile water three times for 2 minutes, after which the sterile seeds were germinated in Petri dishes in physiological saline (0.85%) NaCl ) at a temperature of 28 ° C for 3 days, and the seedlings were soaked in a bacterial suspension of Bradyrhizobium japonicum 629 ^a and Bradyrhizobium japonicum 859 with a cell titer of 5 × 10 ⁷ CFU / ml for 15 min and planted in the soil. At the end of the experiment, the plants were dug up along with the roots, the roots were washed, dried and weighed separately aboveground and underground parts of the plants, and the number of nodules was counted. The results of the described vegetative experiments with soybeans are presented in table 1.

Table 1. The number of soybean nodules and the weight of plants in the growing experiment with soybean Experience option Weight of plants, g Number of nodules Control - no processing 2,55 0 Bacterial treatment of strain Bradyrhizobium japonicum 629 ^a 3.87 18.6 Bacterial treatment of Bradyrhizobium japonicum 859 strain 4,55 27.3

From the data presented in table 1, it follows that when soybean seeds are treated with bacteria of the claimed strain of Bradyrhizobium japonicum 859, soybean plants are larger in weight and nodules are formed on their roots than in the case of bacteria treated with the Bradyrhizobium prototype strain japonicum 629 ^a.

The effectiveness of the bacterial strain Bradyrhizobium japonicum 859 was tested in vegetation and field experiments using microbiological fertilizer based on it, which the applicant named BisolbiRiz. When studying the effectiveness of the bacterial strain Bradyrhizobium japonicum 859, it was found that presowing treatment of soybean seeds with BisolbiRiz fertilizer affects their germination and soybean productivity. With this fertilizer, immediately before sowing, soybean seeds were processed at the rate of 0.3-0.7 liters of BisolbiRiz per hectare rate of soybean seeds, which depends on the soybean variety and ranges from 70 to 120 kg / ha.

Table 2 presents the results of experiments to determine the field germination of seeds and yields of soybean varieties Lancet, depending on the processing of microbiological fertilizers.

Table 2. The effect of microbiological fertilizers, including Bisolbi Rhys, on field seed germination and soybean yield Experience option Field germination,% Harvest
t / ha The mass of 1000 seeds, g The protein content in the seeds,% 1. Control (no processing) 74 2.07 114 36.6 2. Microbiological preparation Agrostim B (Pseudomonas fluorescens + Azotobacter spp.). Seed treatment + seedling spraying on the 7th day after sowing 79 2.47 117 37.0 3. Risotorfin (Bradyrhizobium japonicum 629 ^a ). Seed treatment 77 2.42 115 37,2 4. Bisolbi Rhys (Bradyrhizobium japonicum 859). Seed treatment 80 2.48 118 37.5

From the data presented in table 2, it follows that during the processing of soybean seeds with BisolbiRiz fertilizer, the germination rate increased by 8.1% compared with the control, and by 3.9% compared to the Rizotorfin fertilizer.

Due to the use of BisolbiRiz fertilizer, the excess along the length of seedlings compared to the control reached 14%, roots up to 18%, and compared to Rizotorfin fertilizer - up to 4% and 7%, respectively.

From the data presented in table 2, it also follows that as a result of processing soybean seeds with BisolbiRiz fertilizer, its yield increased by 0.41 t / ha or 19.8% compared with the control, and by 0.06 t compared with the Rizotorfin fertilizer / ha or 2.5%.

The increase in soybean productivity due to the use of BisolbiRiz fertilizer is mainly due to an increase in plant productivity, i.e. due to an increase in the number of beans compared to the control - up to 21%, bean grains - up to 12%, the number of seeds per plant - up to 24%, the weight of seeds per plant - up to 25%.

In addition, when treating soybean seeds with BisolbiRiz fertilizer, there was an increase in the quality of soybean seeds in the form of an increase in the protein content in them by an average of 0.9% compared with the control, by 0.5% compared to Agrostim B and by 0.3% Compared to Rizotorfin fertilizer.

At the same time, to achieve similar agrotechnical indicators (field germination and yield) of soybeans with the help of Agrostim B, they needed to process not only seeds, but also to spray seedlings on the 7th day after sowing, which confirms the great effectiveness of BisolbiRiz fertilizer based on the Bradyrhizobium nodule bacteria strain japonicum 859 compared with the microbiological preparation Agrostim B.

Thus, the data presented in table 2 confirm the greater efficiency of the BisolbiRiz fertilizer obtained on the basis of the claimed strain of nodule bacteria Bradyrhizobium japonicum 859, not only in comparison with the preparation Agrostim B containing a consortium of strains of Pseudomonas fluorescens and Azotobacter spp., But also in comparison with fertilizer Risotorfin obtained on the basis of the prototype strain of nodule bacteria Bradyrhizobium japonicum 629 ^a .

That is, from table 2 it follows that the claimed strain of nodule bacteria Bradyrhizobium japonicum 859, has a higher nitrogen-fixing activity compared to the known strain of nodule bacteria Bradyrhizobium japonicum 629 ^a , which has a positive effect on stimulating plant growth and soybean productivity, as well as improving the quality soybean seeds as an increase in their protein content.

All of the above indicates the possibility of using a strain of nodule bacteria Bradyrhizobium japonicum 859 for the manufacture of fertilizer preparations for soybeans.

The following is an example of a liquid form of fertilizer based on the inventive strain of nodule bacteria Bradyrhizobium japonicum 859.

Uterine culture

To obtain a uterine culture of a strain of nodule bacteria Bradyrhizobium japonicum 859, liquid mannitol-yeast medium was used, which was poured 100-150 ml into 750 ml rocking flasks. Autoclaving was carried out at a temperature of at least 121 ° C for 20-30 minutes. After this, the medium in each flask was seeded by flushing from one tube with a beveled MDA with a pure culture of bacteria from the refrigerator, after which the flasks were placed on a shaker (220 ± 10 rpm) for 72 ± 3 hours at a temperature of 28-30 ° С. Thus, a uterine culture with a bacterium titer of (3-5) · 10 ⁹ CFU / ml was obtained in the flasks, which was stored in the refrigerator for subsequent inoculation with bottles or fermenters.

Work culture

For industrial cultivation of the strain Bradyrhizobium japonicum 859, a nutrient medium was used at pH = 6.8 ± 0.2 of the following composition (with a possible deviation of ± 5% of the indicated amount), g / l: corn extract - 7.0; molasses - 5.0; (NH) ₂ SO ₄ - 1.0; K ₂ HPO ₄ 0.35; KH ₂ PO ₄ 0.35; MgSO ₄ × 7H ₂ O 0.2; CaCO ₃ - 1.0, which was autoclaved at a temperature of at least 131 ° C for at least 60 minutes to ensure sterility. The sterility of the culture medium was checked by plating a sterile sample from the fermenter on standard prepared media: nutrient agar (manufacturer Biocompas-S LLC, Russia) or tryptic soy agar (TSA, manufacturer Sigma, USA) and judged on it by the absence of bacterial growth within 48 hours at a temperature of 28 ± 1 ° C. The sterile culture medium prepared in this way was seeded in the fermenter with a uterine culture of Bradyrhizobium japonicum 859 strain with a bacterial titer of (3-5) × 10 ⁹ CFU / ml, the seed volume being 5-10% of the seed culture medium and cultured for 72 ± 3 hours at a temperature of 28 ± 1 ° С and purging with sterile air at the rate of: air volume per minute per volume of nutrient medium (with a possible deviation from this ratio of ± 5%). Sterility of the air was ensured by blowing it through a bacterial filter installed at the inlet of the fermenter, for example, Sartorius Stedim Biotech brand, manufactured by SARTOGOSM CJSC. In this case, before culturing the Bradyrhizobium japonicum 859 strain, the degree of sterility of the supplied air was checked by blowing it through this bacterial filter at the inlet of the fermenter for 24 hours, followed by inoculation of the culture medium taken sterile from the fermenter onto standard prepared media: nutrient agar (manufacturer Biocompas LLC -С ”, Russia) or tryptic soy agar (TSA, manufacturer Sigma, USA) and judged on it by the absence of bacterial growth for 48 hours at a temperature of 28 ± 1 ° С.

The resulting culture fluid of Bradyrhizobium japonicum 859 strain was microscopic and plated on mannitol-yeast agar (MDA) to count the number of nodule bacteria and tryptic soy agar (TSA) to count the number of extraneous microorganisms.

Thus, a concentrate of a bacterial suspension of fertilizer based on the bacterial strain Bradyrhizobium japonicum 859 with a titer of at least 10 ⁹ CFU / ml was obtained.

The liquid form of Bisolbi Rize fertilizer based on the Bradyrhizobium japonicum 859 strain of nodule bacteria

A liquid microbiological fertilizer based on the Bradyrhizobium japonicum 859 strain of nodule bacteria was obtained by diluting the bacterial suspension concentrate with water, using sterile, distilled water obtained, for example, using a Geyser fine chemical, mechanical treatment and disinfection unit (manufacturer ZAO Eco Geyser ", Russia). The acceptable degree of sterility of water was determined by seeding it on nutrient agar and the absence of bacterial growth on it for 48 hours at a temperature of 28 ± 1 ° C.

In order to obtain fertilizer, the following ingredients were added to sterile water based on 1 liter of water (with a possible deviation of ± 5% of the indicated amount for the ingredients), g / l:

bacterial suspension concentrate -100.0 K ₂ HPO ₄ (potassium hydrosulfate) - 1.5 KH ₂ PO ₄ (potassium hydrogen phosphate) - 1.5 MgSO ₄ · 7H ₂ O (potassium sulfate heptohydrate) - 1.5 lignohumates (humic fertilizers) - 2.0

Then, the pH of the resulting fertilizer was determined and adjusted to a level of 6.8-7.2.

The obtained liquid fertilizer was kept for 3-5 days at a temperature of 25-28 ° C until a bacterium titer of Bradyrhizobium japonicum 859 was obtained in the obtained fertilizer of at least 10 ⁸ CFU / ml.

After that, a liquid microbiological fertilizer based on the bacterial strain Bradyrhizobium japonicum 859, which the applicant named BisolbiRiz, was ready for processing crops, namely soybeans.

Strict control and proper management of the liquid form preparation process predetermine the high quality of BisolbiRiz fertilizer and its corresponding activity in the processing of soybean seeds.

Testing the effectiveness of BisolbiRiz fertilizer based on a strain of nodule bacteria Bradyrhizobium japonicum 859

The effectiveness of the BisolbiRiz microbiological fertilizer, obtained on the basis of the Bradyrhizobium japonicum 859 strain of nodule bacteria, was tested in production experiments in the Ulyanovsk and Amur Regions.

Immediately before sowing one part soybean seeds treated with nothing (control), another part of the seeds treated with the liquid form of fertilizers based rizotorfina nodule bacteria strains Bradyrhizobium japonicum 629 ^a, and the last portion was treated with the fertilizer obtained by the method described above, the rate of 0.5 liters BisolbiRiz per hectare rate of soybean seeds.

The results of the experiments are shown in Tables 3 and 4.

From the data given in tables 3 and 4, it is seen that the use of liquid fertilizer BisolbiRiz, obtained on the basis of a strain of nodule bacteria Bradyrhizobium japonicum 859, increased soybean yield compared to control (without fertilizer treatment) by an average of 17-30%, which is more effective than using the well-known fertilizer Rizotorfin based on the prototype strain of nodule bacteria Bradyrhizobium japonicum 629 ^a .

Moreover, from table 4 it follows that when applied in the Far East - in the farms of the Amur region - the claimed strain of nodule bacteria Bradyrhizobium japonicum 859 is most effective in comparison with the strain of nodule bacteria Bradyrhizobium japonicum 629 ^a - prototype.

Claims (1)

The bacterial strain Bradyrhizobium japonicum 859, deposited in the collection of the GNU VNISKH RUSSIAN ECONOMY on December 05, 2008 under registration number 24117 in the group of nodule bacteria, as a means of obtaining fertilizer for soybeans.