RU2426778C2 - Strain of legume bacteria bradyrhizobium japonicum 206 vkpm v-9505, virulent to recognised varieties of soya - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a strain of legume bacteria Bradyrhizobium japonicum 206 VKPM V- 9505 which is virulent to recognised varieties of soya. The Bradyrhizobium japonicum 206 strain is extracted from Belgorodskaya-48 soya nodules recognised in Belgorod region, and selected using a cross-inoculation method in order to achieve virulence to other soya varieties grown in Belgorod region and other regions in the Central-European part of Russia. The Bradyrhizobium japonicum 206 strain is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under number VKPM V-9505 and has virulence to soya varieties grown in Russia, Ukraine and Belarus. The obtained strain is meant for preplant inoculation of plants by treating seeds or adding preparations based on the strain directly into the soil.

EFFECT: invention increases yield and quality of soya grain grown in soils which contain or do not contain virulent types of legume bacteria previously introduced in any way.

1 tbl, 7 ex

Description

The strain Bradyrhizobium japonicum 206 VKPM B-9505 is intended for pre-sowing inoculation of seeds or pre-sowing application in the soil in order to provide nitrogen-fixing symbiosis of soybean plants and nodule bacteria through the formation of nodules. The strain is grown either by the traditional liquid-phase method, or by the method of solid-phase fermentation on highly dispersed, moisture-intensive, non-swelling mineral carriers containing solutions of carbon sources, nitrogen, mineral salts and growth-promoting additives.

It is used in the form of aqueous suspensions with the addition of film-forming substances for pre-sowing inoculation of soybean seeds, or in the form of aqueous suspensions, or neutral granular mixtures for application to the soil in the spring before sowing soybeans in a uniformly distributed dose of about 500 · 10 ⁹ CFU / ha.

The strain is virulent to all tested soybean varieties zoned in the central black earth regions of Russia and its neighboring regions. Provides increased yield, oil content and protein content in soybean grain. The relative increase in these indicators due to the use of the strain lies in the range of 15-40%, depending on the conditions of use.

Description of the invention.

The invention relates to the fields of biotechnology and agricultural microbiology and is a new strain of nodule bacteria specific for zoned soybean varieties, providing an effective symbiosis of soybean plants with this strain of nodule bacteria.

The symbiosis of legumes and nodule bacteria has been known since the end of the 19th century [1 - p. 293, 2 - p. 454, 4 - p. 395-398]. It consists in the fact that nodule bacteria of the genus Rhizobium and Bradyrhizobium are able to infect young roots of leguminous plants. Intruding into the root cell, they stimulate its growth and multiply in it, eating carbohydrates entering the cell. In this case, a supraorganic symbiotic system is formed in which the plant and bacterial cells together synthesize a new substance called leghemoglobin, which accumulates in the root nodules formed at the site of infection and performs a threefold function: it supplies oxygen and molecular nitrogen to aerobic bacteria, protects the nitrogen-fixing apparatus of bacteria (enzyme nitraginase) from the toxic effects of oxygen and, thus, provides effective fixation and biotransformation of molecular nitrogen into Available for the assimilation of it as microorganisms and plants. Moreover, most (up to 90%) of fixed atmospheric nitrogen is absorbed by the plant, and the rest by microorganisms. Legumes are able to absorb nitrogen from the soil, but in the presence of symbiosis with nodule bacteria, the yield of legume seeds is significantly higher by 10-40% than in the original, even well-fertilized soil. In addition, plants and their seeds, grown in symbiosis with nodule bacteria, contain more protein, which is an important indicator of their quality. This is especially important for mass legumes, in particular for soybeans.

Since the discovery and study of symbiosis, the direction of artificial inoculation of leguminous plants with nodule bacteria grown in laboratory and industrial conditions has begun to develop [3 - p. 3-9]. It turned out that artificial inoculation provides increased yield and quality of legumes not only in soils that do not contain natural virulent cultures of nodule bacteria, but also in soils where such bacteria are present, and there is a natural symbiosis [1 - pp. 294-298 ]. This is because nodule bacteria of the genus Rhizobium and Bradyrhizobium are capable of multiplying either in the root nodules of leguminous plants, or in artificial conditions. In the soil, they can only survive for a while in suitable local niches. That is, one of the factors of the economic efficiency of symbiosis is the concentration of virulent species of nodule bacteria in the soil. In addition, more virulent strains with increased nitrogen-fixing activity can be selected under artificial conditions. It has been established that effective symbiosis requires the introduction of about 500 billion virulent cells of nodule bacteria into the soil during sowing per 1 ha of sown area [3 - p. 7].

According to virulence (the ability to infect legumes of legumes with the formation of nodules), there is a species specificity of bacteria of the genera Rhizobium and Bradyrhizobium, i.e. Certain types of bacteria of the genus Rhizobium form a symbiosis with individual species or with a group of legume plants. In particular, for soybean there is one symbiont type species Bradyrhizobium japonicum [4 - p. 396, 3 - p. 5]. In addition, for soybean there is varietal specificity of the strains of Bradyrhizobium japonicum, i.e. Bradyrhizobium japonicum strains exist, virulent only with respect to certain groups of soybean varieties. A fairly common occurrence is the need for breeding of new strains of Bradizhizobium japonicum to new or zoned soybean varieties in connection with the expansion of the cultivation of this crop in new regions for it. In particular, this is the case for central black earth and central Russia, Belarus, Volga regions, i.e. with the spread of soybean crops from the south to the more northern and northeastern regions.

At present, many strains of nodule bacteria for soybean have been isolated, selected and maintained under artificial conditions in the world (genera: Rhizobium and Bradyrhizobium, species - Bradyrhizobium japonicum); 3 - p. 7, VKM and VKPM catalogs].

Some of them were tested on soybean varieties of the central chernozem region, in particular in the Belgorod region. Some of them showed virulence to local and zoned varieties. This phenomenon became the basis for the selection of a highly effective strain of nodule bacteria for soybean varieties zoned in the Belgorod region, central black earth regions, as well as the purpose of the present invention.

The selection was based on the selected culture of nodules found on the site of long-term cultivation of soybean at the experimental hospital of the Belgorod State Agricultural Academy (BSAA) on the soybean “Belgorodskaya-48” of the BSAHA selection. The isolated pure culture was propagated on an artificial medium and again used to inoculate soybean Belgorod-48, Belgorod-6. As a result of this operation, a rather high virulence was observed on the Belgorodskaya-48 variety and some slight virulence on the Belgorodskaya-6 soybean. Nodule bacteria isolated from Belgorodskaya-6 soybean nodules were used to inoculate Belgorodskaya-48, Belgorodskaya-6, Lanceolate, Glazastaya, Yaselda, Armavirskaya soybeans and others. virulence and efficacy were observed on the varieties Belgorodskaya-48 and Belgorodskaya-6, while the remaining varieties were low and medium. Isolation, propagation and subsequent re-inoculation of soybean seeds with the selected cultures increased the virulence and symbiosis of the strain with the maternal variety Belgorodskaya-48 and other varieties. The subsequent stages of breeding were reduced to selecting a culture of nodule bacteria from the most effective symbioses of the studied series of soybean varieties and spread to other soybean varieties. The accelerated work in stages was provided by a modified technique [3 - p. 437]. The modification of the technique consisted in the fact that not only soybean seeds, but their seedlings were placed in test tubes with an agarized transparent medium, and the seedlings were inoculated using a micropipette with a diluted suspension of a pure strain culture after introducing the root of the growth into the agar medium. The following composition of the medium was used: MgSO ₄ - 0.1%, K ₂ HPO ₄ - 0.1%, Ca ₃ (PO ₄ ) ₂ - 0.002%, FeSO ₄ - traces, agar - 1%. Transparent agar medium made it possible to estimate the time of nodule appearance, their number and size over a certain period of time. Selected options were compared in experiments on growing soybeans on soil in a phytotron under laboratory conditions. Further, the effectiveness of symbioses was evaluated in the field on the fields of the first soybean sowing and on the fields of long-term soybean cultivation, where the constant presence of Bradyrhizobium japonicum was already detected in the soil.

The virulence of the strains was evaluated by the number of nodules formed on average per plant, and the effectiveness by the average weight of one nodule, yield growth and protein content in soybean grain.

As an illustration of the properties of the selected and selected strain, we present a table of the results of field trials of a drug based on it in 2006 (table 1).

The genetic stability of the strain is evidenced by the constancy of the indicators of virulence and the effectiveness of symbiosis with the Belgorodskaya-48 soybean base variety under numerous culture passages on mannitol-yeast (DSM) and pea agarized media both during sowing through a liquid suspension and during direct passaging with a loop.

The strain was identified both by the results of the operations described above (characteristic properties) and by microscopic, biochemical and physiological characteristics in accordance with the determinant of Bergey bacteria (Moscow, Mir, 1977, p. 80-81).

The strain turned out to be slow-growing and was named Bradyrhizobium japonicum 206. This strain is donated to VKPM as Bradyrhizobium japonicum 206 VKPM B-9505.

Cultural and morphological characters.

Strain Bradyrhizobium japonicum 206 - rod-shaped straight, curved cells. Gram-negative, do not form a dispute.

Temperature - (26-30) ° С, pH = 6-7.

The colonies are round, translucent, whitish, more or less granular, with a diameter of not more than 0.5 mm after 5-7 days of incubation on an agar medium with yeast extract, mannitol and soil extract. Growth on the medium is accompanied by the formation of extracellular mucus of a polysaccharide nature.

A characteristic feature of these organisms is the ability to penetrate into the root hairs of soybean (family Glycine) varieties Belgorod-6, Belgorod-48, Lanceolate, Glazastaya.

Physiological signs.

The strain is an aerobic, grows in the temperature range of 15-35 ° C, the temperature optimum is 26-28 ° C at pH = 6-7. Optimum pH = 7.0-7.2 (start of cultivation). It grows on rich nutrient media based on carbohydrates, extracts and yeast autolysates.

Biochemical signs.

The strain is a chemoorotroph.

In liquid media, it absorbs D-mannitol, glucose, and also does not metabolize mixtures of these carbohydrates with sucrose, cellulose and starch. Sources of nitrogen are ammonia salts, nitrates, amino acids. The MPA is not growing. When grown on media with carbohydrates, it secrets mucus of a polysaccharide nature. When growing on a liquid medium, DSM causes it to alkalize to pH = 8, starting from the second half-growth period.

A characteristic property (feature) is the ability to penetrate into the root hairs of soybeans (a number of varieties zoned in the Central Chernozem Region and regions close to climatic conditions).

Depending on the used compositions of the liquid media and cultivation conditions, a culture fluid (QL) with a titer of 10 ⁸ -10 ¹⁰ CFU / cm ^{3 is} obtained within 96 hours.

When grown on a loose solid-phase carrier (sterile powder fraction of a moisture-intensive neutral inorganic material, filled with liquid nutrient medium to a moisture content of 70-75% and seeded with culture) with the initial inoculation of 10 ⁶ -10 ⁷ CFU / g after exposure to thermostat at a temperature of 28-30 ° With no mixing and additional aeration for seven days, the finished product with a titer of at least 8 · 10 ⁹ CFU / g is obtained.

Storage medium.

A solid DSM storage medium containing D-mannitol, yeast extract, soil extract is seeded with culture and cultivated at a temperature of (26-28) ° C for 5-7 days, then stored in the refrigerator.

Frequency of reseeding - 1 time in 3 months.

The invention is illustrated by the following examples.

Example 1. Obtaining seed and growing culture on DSM-agar.

From a test tube storage loop under sterile conditions, the culture is streaked onto the surface of the mowed D8M (or bean) agar seed tube and grown in an incubator at a temperature of (26-28) ° C under aerobic conditions for 5-7 days. The grown layer of culture is washed off the surface with a nutrient medium (composition: molasses, glucose, ammonium sulfate, potassium phosphate, magnesium sulfate, manganese sulfate, ammonium molybdenum acid) and poured into a seed flask. The flush volume in the seed flask was adjusted with nutrient medium to 100 ml and stirred on a rocking chair at 190-220 rpm. 100 ml of seed are obtained with a titer of about 10 ⁸ CFU / ml. Thus, the seed material is produced in the required volume for use both for liquid-phase deep cultivation by aeration and for solid-phase cultivation in breathable bags.

Example 2. Liquid-phase cultivation in flasks.

90 ml of liquid nutrient medium is placed in rocking flasks (750 ml each), sterilized at 120-125 ° С in an autoclave, cooled and inoculated under a burner flame. 10 ml of seed obtained in Example 1. The flasks are mounted on a rocking chair with a vibration frequency of 190 -220 rpm in a temperature-controlled room at a temperature of 28-30 ° C and stirred continuously for 96 hours.

A culture fluid is obtained with a titer of about 10 ⁹ CFU / ml.

Example 3. Liquid-phase cultivation in a fermenter.

In the Bior-250 fermenter (200 liter capacity) equipped with a stirrer and a bubbler type air aerator, 150 liters of culture medium are prepared, sterilized with deaf steam (through the wall of the shirt) is carried out at a temperature of 120-125 ° C, then the medium is cooled and seeded 5 liters of seed obtained in example 1 or 2. Cultivation of the culture is carried out at a temperature of 28-30 ° C, barbation of air 0.5-1 volume per 1 volume of medium with a working stirrer with a frequency of 60 rpm for 96 hours.

At the end of the process, 150 L of culture fluid (QOL) is obtained with a titer of about 10 ⁹ CFU / ml.

Example 4. Solid phase growth.

200 g of neutral moisture-intensive, non-swelling mineral powder is loaded into a 2 L package, hermetically sealed and sterilized in an autoclave at a temperature of 132 ° C for 60 minutes. Then, under sterile conditions, a culture-seeded liquid medium is introduced into the bag with sterilized powder, the puncture is sealed with a piece of adhesive tape or other similar material. By rotating and kneading the contents of the package, it is homogenized, the package is placed in an air thermostat at a temperature of 28-30 ° C and a culture is grown, periodically ventilating the air space of the thermostat for seven days. Get the finished preparation of nodule bacteria with a titer of at least 8 · 10 ⁹ CFU / g A hectare dose of this drug when applied to the soil by pre-sowing seed treatment of 80 g, which is 640 billion living cells per 1 ha.

Example 5. Assessment of virulence of the strain in the laboratory.

An environment is prepared containing: MgSO ₄ - 0.1%, K ₂ HPO ₄ - 0.1%, Ca ₃ (PO ₄ ) ₂ - 0.002%, FeSO ₄ - traces, agar - 1% and hot it is poured into 15 ml glass tubes. After some time, agar gel forms in test tubes when cooled.

10 seeds of soybean varieties are selected: Belgorodskaya-48, Belgorodskaya-6, Lanceolate, Glazastaya, Yaselda. The seeds are thoroughly washed with water, immersed in ethyl alcohol for 3-4 seconds, then for 10 minutes in a 0.1% mercuric chloride solution, again thoroughly washed with distilled water and placed on a napkin moistened with distilled water in a Petri dish. Petri dishes with seeds set in a thermostat with a temperature of 30 ° C, germinated for 1.5-2 days (until the emergence of a root size of 0.5-0.8 cm).

Seeds with seedlings are placed on the surface of the agar gel in test tubes, pushing the roots deep into the gel and leaving the seeds on the surface, close the tubes with cotton-gauze plugs and in this form they are kept at a temperature of 25-27 ° С for three days until the root system is formed in a transparent gel and stems with buds of leaves above the surface. Then, on the basis of seeds (example 1,2) or culture liquid (example 3) or preparation (example 4), an aqueous suspension of nodule bacteria cells with a titer of 10 CFU / ml is prepared and 0.1 ml of these suspensions are added using a micropipette to plant roots in test tubes so that this volume of suspension is absorbed into the microcracks formed around the root in an agar gel.

Distilled water was added to control tubes in the same way. Test tubes are installed in a phytotron with illumination at a temperature of 25-27 ° C for growing plants. After 7-9 days, with the help of a magnifier, one can observe the formation of nodules, which are clearly visible to the naked eye on the 21st day after inoculation, and their number can be easily calculated and sizes measured using, for example, a catheter.

In control tubes, nodules do not form, and in tubes with the introduction of a suspension of strain in the root systems of the above soybean varieties, 7-10 nodule buds were detected after 7 days, and after 20 days, the number of root nodules was found to be from 15 to 20 pieces with sizes 1-2 mm Due to unnatural conditions for soybean growth in the subsequent period, the number and size of nodules did not increase significantly.

Example 6. Evaluation of virulence in the phytotron on vessels with soil.

The experiment used soil (ordinary chernozem) that did not contain nodule bacteria virulent to soybean, which was established by control experiments with pure soybean seeds.

2 kg of soil was placed in ceramic vessels, moistened to a moisture content of 30% and sowed seeds of different sorts, prepared and sterilized as in Example 5. In the control crops, soybean seeds were not inoculated, but treated with distilled water in an amount of 1% by weight of seeds. In experimental variants, soybean seeds were treated with aqueous suspensions of the Bradyrhizobium japonicum culture strain containing 6 × 10 ⁸ active cells in 1 ml. In this case, liquid and granular forms of the preparation were used, obtained as in examples 1-4. Seeds (4 seeds per vessel) were sown in vessels with soil to a depth of 2 cm and placed in a phytotron for growing at a temperature of 20-25 ° C with intense uniform illumination for 12 hours and for the remaining 12 hours in a day in the dark. After germination, only one best-looking plant was left in the vessel and continued to grow. Growth was completed 7 days after the formation of the first ternary leaf for each soybean variety. The soil with the plant was carefully removed from the vessels, mounted on a pallet, and the soil was carefully washed off with a stream of water. The root system was examined, the resulting root nodules were collected, counted and weighed. In breeding experiments, nodules were selected for subsequent work from variants with the largest number and mass of these formations.

Thus, for the selected strain, the virulence and symbiosis efficiency of the strain with various soybean varieties were evaluated (this work is ongoing to assess the limitations of varietal and regional-climatic applicability of the strain and the possibility of its further selection).

Example 7. Evaluation of virulence and the effectiveness of the symbiosis of drugs based on the strain in the field.

In the field (plot and production experiments), the virulence and efficiency of the strain were evaluated under conditions close to agricultural technologies for soybean cultivation. The plot experiments were carried out at the BSHA field experimental hospital in areas with soil with the presence of nodule bacteria and in remote areas where soybean was sown for the first time, as well as in country plots where nodule bacteria virulent to soybeans were also absent. In experiments, soybean seeds were inoculated with working solutions of liquid and granular preparations in doses ensuring the content of 500 · 10 ⁹ living cells of the strain in the hectare norm of seed sowing. To exclude swelling of seeds and uniformity of treatment, the working solution was formulated so that its volume (weight) containing a hectare dose of the preparation was about 1% of the weight of the seeds (water for working solutions should not contain disinfectants). For example, with a hectare norm of sowing seeds of 100 kg, 1 l of a working solution containing 200 ml of liquid or 80 g of a loose preparation containing a culture of Bradyrhizobium japonicum 206 and 100 ml of adhesive (organomineral complex) is used. The working solution is supplied with a small stream or in portions to constantly stirred seeds in a tilted vessel, for example, a rotating concrete mixer, thoroughly tedding (pouring) a half-filled bag with seeds with a plastic liner or using special devices with a dispenser, a solution sprayer and mixing the seeds.

Inoculation and storage of the treated seeds is carried out in places protected from direct sunlight and other active radiation, harmful to microorganisms. Without additional protection against drying, storage of treated seeds is permissible for no more than one day. Sowing seeds is carried out using one of the accepted methods (continuous narrow-row, wide-row or periodically ordinary sowing). In control variants, the seeds were treated with water. During the growing season, phenological observations of crops were carried out, nodule formation was monitored, and after ripening, yield and quality (protein content) in soya grains were evaluated. The results of part of the experiments are shown in table 1.

References.

1. Mishustin E.N. Microorganisms and agricultural productivity. M .: Nauka, 1972

2. Workshop on microbiology, edited by A.I. Netrusov. M .: ACADEMA, 2005.

3. Methods of cultivation of nitrogen-fixing bacteria, methods for the preparation and use of drugs based on them (guidelines). Compiled by A.V. Khotyanovich. VASKHNIL, VNIISKHM, Len., 1991

4. Schlegel G. General Microbiology (translated from German). M .: Mir, 1987

Claims (1)

The strain of nodule bacteria Bradyrhizobium japonicum 206 VKPM B-9505, virulent to zoned soybean varieties.