RU2464308C2 - BACTERIAL STRAIN AZOTOBACTER CHROOCOCCUM 5 V(e) USED FOR OBTAINING NITROGEN FIXING FERTILISER FOR GRAIN AND FORAGE CROPS - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: bacterial strain Azotobacter chroococcum 5 V(e) is selected from light-grey forest soil of village Raifa of Zelenodolsk district of the Republic of Tatarstan and is deposited in RNCIM under the number B-10387. The strain synthesizes vitamins of group B and indolyl-3-acetic acid. The strain has nitrogen-fixing activity 515.7 nM N₂/ml·h.

EFFECT: invention enables to increase yield of grain and forage crop.

13 tbl, 4 ex

Description

The invention relates to microbiology and can be used in agriculture, namely in the production of bacterial fertilizers to improve the nitrogen nutrition of plants.

To improve the nitrogen nutrition of plants, it is not enough to introduce nitrogen compounds into the soil. It is necessary to increase the efficiency of nitrogen consumption by the plant. For this, the interaction of a microorganism producing nitrogen in a form acceptable for plants and cells of plant roots has long been used.

So, the known strains of bacteria Azotobacter chroococcum, allowing to improve the nitrogen nutrition of tomatoes (patent No. 2076088 RU), cucumbers (patent No. 2074157 RU), barley (copyright certificate No. 1316189 SU). These strains are isolated from the rhizosphere or rhizoplans of the object of increasing nitrogen nutrition, and their disadvantage is a narrow effect.

A known bacterial strain Azotobacter chroococcum B35 (patent No. 2074159 RU), which can be used to obtain bacterial fertilizers for green crops, such as lettuce, celery, parsley, etc. When used in fertilizer, the level of nitrates in the soil is reduced. The disadvantage is that the strain is also isolated from the rhizosphere of the tomato Lycopersicon esculentum and may be poorly adapted to survive in climatic and biological conditions other than the region of origin of the tomato Lycopersicon esculentum.

The closest analogue is the bacterial strain Azotobacter chroococcum for the preparation used in crop production (patent No. 1703634 SU), which has a wide spectrum of inhibition of phytopathogenic fungi, as well as the ability to synthesize indolyl-3-acetic acid (IAA). This strain is isolated from serozem.

The aim of the invention is to obtain a bacterial strain for nitrogen-fixing fertilizer, which has an effect on a wide range of cereals and fodder plants, and also has a high resistance to external conditions.

The goal is achieved through the use of the bacterial strain Azotobacter chroococcum 5 V (e). The strain was found in vivo, isolated from the light gray forest soil of the village of Raif, Zelenodolsky district of the Republic of Tatarstan.

The culture was identified to the species by analysis of 16S RNA in the All-Russian Collection of Industrial Microorganisms (FSUE GosNIIgenetiki), where it was registered under No. B-10387.

Cultural and morphological features of the strain: The size of the cells of a 5-day-old culture on Ashby's medium is about (0.7-1.7) × (3.0-3.6) microns. The cells are large, round, single or paired, motionless, gram-negative. On medium Ashby form colonies of medium size (5 mm). On the 5th day, the colonies are brilliant, light beige, round with scalloped edges and a smooth surface, convex, homogeneous in structure.

The activity (productivity) of the strain, other production indicators: Nitrogenase activity of the strain 515.7 nm N ₂ / ml · h; able to synthesize and secrete B vitamins (mg / l) into the environment: biotin (B ₇ ) - 1.2, pyridoxine (B ₆ ) - 1.31, thiamine (B ₁ ) - 2.1, pantothenic acid (B ₃ ) - 1.2, as well as indolyl-3-acetic acid (IAA) - 77.5 (The maximum accumulation occurs on the 4th day).

The optimal growth temperature of the strain Azotobacter chroococcum 5 V (e) is 25-30 ° C. The pH range for its growth is 5.5-8.5, the optimum is 7.0-7.5.

For inoculation, a 4-day old culture was grown on Ashby liquid medium (Ashby medium (g / l): K ₂ HPO ₄ 0.2; MgSO ₄ 0.2; NaCl 0.2; KH ₂ PO ₄ (or K ₂ SO ₄ ) - 0.1; CaCO ₃ - 5.0; sucrose - 20.0; tap water - 1000 ml [Koleshko OI Ecology of soil microorganisms. Laboratory workshop. - Minsk: Higher school, 1981. - 175 pp.]. After 4 days using a light microscope, cells were counted in the Goryaev chamber — the titer was determined, which should be 2.0 · 10 ⁹ -8.0 · 10 ⁹ cells / ml.

Method, conditions and composition of media for long-term storage of the strain: The strain can be stored without loss of useful properties at a temperature of + 4-6 ° C (in the refrigerator) on jambs with Ashby agar medium.

Method, conditions and composition of media for the propagation of the strain: Ashby medium - 0.02% K ₂ HPO ₄ , 0.02% MgSO ₄ , 0.02% NaCl, 0.01% KH ₂ PO ₄ , 0.5% CaCO ₃ , 2% mannitol, 2% agar.

Optimum conditions and composition of the fermentation medium: Burk's medium - 0.064% K ₂ HPO ₄ , 0.02% MgSO ₄ , 0.02% NaCl, 0.016% KH ₂ PO ₄ , 2% sucrose, 0.005% CaSO ₄ · 2H ₂ O , 0.0001% NaMoO ₄ , 0.0003% FeSO ₄ .

The strain Azotobacter chroococcum 5 V (e) has the ability to attach to the root surface of various crops, where it multiplies and functions for 5-6 months after inoculation.

The use of the claimed strain of Azotobacter chroococcum 5 V (e) can significantly increase the yield of various crops. In addition, the life time of this strain (and, consequently, the duration of exposure to bacterial fertilizers) is at least 5 months.

Nitrogen-fixing activity (515.7 nM N ₂ / ml · h) was determined in the isolated Azotobacter chroococcum 5 V (e) strain. The isolated strain can be classified as highly active, since its acetylene reduction is higher than 150 nM N ₂ / ml · h (Sadykov B.F., Umarov M.M. Productivity of nitrogen fixation in the plant philosphere // Microbiology. - 1980. - T. 49. - Issue 6. - S.998-1002).

The proposed strain is able to synthesize B vitamins and indolyl-3-acetic acid (IAA).

The greatest ability to produce physiologically active compounds was established for him on the 4th day ((mg / l): biotin (B ₇ ) - 1.2, pyridoxine (B ₆ ) - 1.9, thiamine (B ₁ ) - 2.1, pantothenic acid (B ₃ ) - 1.2, as well as indolyl-3-acetic acid (IAA) - 107.5). These vitamins play an important role in the metabolism of nitrogenous substances. It is known that heteroauxin has a stimulating effect in the range of concentrations of 17.5-140.0 mg / l (Guskov A.V., Zemskaya V.A. Changes in auxin oxidase activity in rooted bean cuttings under the action of IAA and 2,4-D // Plant Physiology. - 1985.- T.32, issue 6. - S.1137-1143). Therefore, the proposed strain can be used to produce a growth-promoting drug.

Also, the strain is well cultivated in low-cost nutrient media, which reduces the cost of producing fertilizers based on this strain.

It is known that good quality of seed, high energy of seed germination and friendly growth of seedlings are important conditions that ensure high yields in the future. The results of tests showing the effectiveness of the proposed strain are shown in the examples.

For all examples, the seeds of the test cultures were treated with a suspension of Azotobacter chroococcum 5 V (e) for 1 h at 28 ° C (cell titer 10 ⁹ ).

When studying the effect of a suspension of Azotobacter chroococcum 5 V (e) on the development of seedlings of test cultures, their length and biomass, its growth-promoting effect was established (Table 1-5).

Example 1

Suspension of Azotobacter chroococcum 5 V (e) processed seeds of wheat, rye, millet, and corn. An increase in the energy of seed germination by 5-30% was obtained depending on the crop compared to untreated seeds. The data are presented in table 1.

Example 2

Suspension of Azotobacter chroococcum 5 V (e) processed seeds of wheat, rye, millet, and corn. A significant increase in the length of the roots was obtained (from 20% to 80% on the 10th day), which is very important, since the roots provide the plant with various nutrients, including mineral ones (Table 2-3).

The development of roots contributed to the active growth of seedlings of test plants (Table 4-5).

Thus, the data obtained allow us to recommend the strain Azotobacter chroococcum 5 V (e), which has a multifunctional positive effect on plants, for pre-sowing treatment of seeds of grain and forage crops.

Example 3

The studies were continued in vegetative experiments with spring wheat (Tables 6–8), where the test plants were highly responsive to seed treatment with a suspension of Azotobacter chroococcum 5 V (e).

Example 4

Vegetation experiments were conducted with corn, which also showed high efficiency in seed treatment with a suspension of Azotobacter chroococcum 5 V (e) (Table 9-13).

Table 1 Effect of Azotobacter chroococcum 5 V (e) on seed germination energy of various crops Culture The control Azotobacter chroococcum 5V (e) % to control Cereal Wheat 72 88 +22.2 Rye 82 86 +4.9 Millet 90 96 + 6.7 Feed Corn 72 94 +30.6

table 2 The effect of Azotobacter chroococcum 5 V (e) on the length of the roots of various crops Culture The control Azotobacter chroococcum 5V (e) % to control Cereal Wheat 9.2 16.7 +81.5 Rye 10.3 17.4 +68.9 Millet 4.3 6.4 +48.8 Feed Corn 17.8 21,4 +20.2

Table 3 The effect of Azotobacter chroococcum 5 V (e) on the biomass of roots of various crops Culture The control Azotobacter chroococcum 5V (e) % to control Cereal Wheat 0.0055 0.0075 +36.4 Rye 0.0078 0.0103 +32.1 Millet 0,0003 0,0004 +33.3 Feed Corn 0.0232 0,0297 +28.0

Table 4 Effect of Azotobacter chroococcum 5 V (e) on the length of seedlings of various crops Culture The control Azotobacter chroococcum 5V (e) % to control Cereal Wheat 10.6 12,4 +17.0 Rye 12.0 14.9 +24.2 Millet 7.8 8.9 +14.1 Feed Corn 9.0 13.9 +54.4

Table 5 Effect of Azotobacter chroococcum 5 V (e) on the biomass of seedlings of various crops Culture The control Azotobacter chroococcum 5V (e) % to control Cereal Wheat 0.0103 0.0127 +23.3 Rye 0.0118 0.0130 +10.2 Millet 0.0014 0.0018 +28.6 Feed Corn 0,0574 0,0593 +3.3

Table 6 Effect of Azotobacter chroococcum 5 V (e) on spring wheat grain yield (growing experiment) No. p / p Option Productivity, g / vessel % to control one Fertilizer-free control 7.6 - 2 Azotobacter chroococcum 5V (e) 8.2 +7.9

Table 7 Effect of Azotobacter chroococcum 5 V (e) on the weight of spring wheat straw (growing experiment) No. p / p Option Weight of straw, g / vessel % to control one Fertilizer-free control 16.1 - 2 Azotobacter chroococcum 5V (e) 18.5 +14.9

Table 8 The effect of Azotobacter chroococcum 5 V (e) on the quality of spring wheat grain (vegetative experience) No. p / p Option The protein content,% % to control one Fertilizer-free control 11.3 - 2 Azotobacter chroococcum 5V (e) 12.5 + 10.6

Table 9 Effect of Azotobacler chroococcum 5 V (c) on soil phosphorus, potassium, nitrate (N-NO ₃ ) and ammonia (N-NH ₄ ) nitrogen in the phase of seven corn leaves (vegetative experiment) No. p / p Option Content, mg / kg of soil P ₂ O ₅ K ₂ O N-NO ₃ N-NH ₄ N-NO ₃ + N-NH ₄ one Fertilizer-free control 1065 915 386 6.9 392.9 2 Azotobacler chroococcum 5 V (e) 1658
(+ 55.7%) 920
(+ 0.6%) 397
(+ 2.8%) 7.1
(+ 2.9%) 404.1
(+ 2.9%) Note: (+ 55.7%) -% of control

Table 10 Effect of Azotobacter chroococcum 5 V (e) on the leaf surface area of corn plants No. p / p Option The leaf surface area of one plant, cm ² % to control one Fertilizer-free control 1180 - 2 Azotobacter chroococcum 5 V (e) 1493 +26.5

Table 11 Effect of Azotobacter chroococcum 5 V (e) on the yield of green maize No. p / p Option Productivity, g / vessel % to control one Fertilizer-free control 200,0 - 2 Azotobacter chroococcum 5 V (e) 232.3 +16.2

Table 12 Effect of Azotobacter chroococcum 5 V (e) on the structure of the corn crop No. Option The weight of the green mass of corn, g / vessel Including grain weight, g / vessel stem weight leaf weight ear weight one Fertilizer-free control 118 29.0 53.0 6.8 2 Azotobacter chroococcum 5V (e) 127
(+ 7.6%) 32,0
(+ 10.3%) 72.6
(+ 37.0%) 7.1
(+ 4.4%) Note: (+ 7.6%) -% of control

Table 13 Effect of Azotobacter chroococcum 5 V (e) on microbial biomass in the rhizosphere of maize during harvest No. p / p Option C _mic , mg / 100 g % to control one Fertilizer-free control 30.6 - 2 Azotobacter chroococcum 5 V (e) 56.8 +85.6

Claims (1)

The bacterial strain Azotobacter chroococcum 5 V (e), deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) FSUE "GosNIIGenetika" under No. B-10387, used to obtain nitrogen-fixing fertilizers for grain and forage crops.