RU2654580C1 - Strain bradyrhizobium diazoefficiens ccm gs-4 (vkpm v-12660) - rhizobial component of microbial biopreparation for soy pretreatment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Strain of nodule bacteria Bradyrhizobium diazoefficiens CCM GS-4, which has the ability to form nodules with soybean plants. Strain Bradyrhizobium diazoefficiens is deposited in the Russian National Collection of Industrial Microorganisms under the registration number VKPM V-12660 and can be used to obtain microbiological fertilizer.

EFFECT: invention makes it possible to increase the productivity of soy.

1 cl, 1 dwg, 5 tbl

Description

The invention relates to biotechnology and agriculture and relates to a new strain of nodule bacteria Bradyrhizobium diazoefficiens SSM GS-4 to obtain microbiological fertilizers that increase soybean productivity.

Currently, to increase the yield of legumes using bacterial fertilizers made on the basis of nitrogen-fixing nodule bacteria. There are a number of previously patented strains of nodule bacteria that were used as monocomponent biological products for soybean, according to copyright certificates and invention patents from 1979 to 2013, namely: Rhizobium japonicum 626a, Rhizobium japonicum 646, Rhizobium japonicum 614a, Rhizobium japonicum 614a, 634b, Rhizobium japonicum 24108 (71 m), Bradyrhizobium japonicum M-8, Bradyrhizobium japonicum KH 10, Bradyrhizobium japonicum T66, Bradyrhizobium japonicum 36, Bradyrhizobium japonicum 46, Bradyrhizobium japonicum 206, Bradyrhizobium japonicum VKM B-2629D (MM-117), Bradyrhizobium japonicum BM-85k, Bradyrhizobium japonicum 859, Bradyrhizobium japonicum Cm-42k.

However, the question of the stability and effectiveness of such biological products in practice remains unresolved. This is mainly due to the creation of the so-called "universal biological products", the development of which does not take into account the dependence of plant-microbial symbiosis on a number of parameters: cultivar strain specificity, the influence of soil microbiota, agroclimatic conditions of cultivation of bean culture and agricultural background, on which the varieties used were selected plants. And most importantly, often the impact of the components of such biological products on soil biocenosis is not assessed, the residual effect of the introduced inoculum strains on the local populations of nodule bacteria is not evaluated, including their safety after the first year of vegetation of the host plants. Therefore, to create effective symbiosis, the principle of “complementarity of couples” is so important, based on knowledge of the biodiversity of partners.

Brief Description of the Drawing

The soybean microsymbionts biodiversity is mainly represented by 6 genetically removed groups of strains: 3 groups of B. japonicum : USDA 123 - this group includes competitive but ineffective strains of nodule bacteria; USDA 6T - a typical group of strains of this species, effective in symbiosis with soy, production strains 626a, 46 and KN-10 fall into it; USDA 4 - a group of effective strains, which include production strains 36 and M-8; 1 group of the species B. diazoefficiens USDA 110, to which the production strain 634b and the strain CCM GS-4 proposed in this application belong; and strains of species B. liaoningense, B. elkanii . The drawing shows a phylogenetic tree, built on the basis of data from multilocus sequencing (MLSA) of the nucleotide sequences of nodule bacteria of the genus Bradyrhizobium sp. using the Neighbor-Joining algorithm. The scale corresponds to 1 substitution per 100 base pairs (evolutionary distances). The numbers show the statistical certainty of the branching order (in%), determined using the “bootstrap” analysis of 1000 alternative trees.

The principle of “complementarity of pairs” is based on the co-evolution of strains of certain groups with host plant varieties, due to the agricultural background on which the used plant varieties were selected and the agro-climatic conditions of cultivation. The proposed strain B. diazoefficiens CCM GS-4 is a slow-growing nodule bacteria that are more effective on modern soybean varieties of domestic selection compared to previously used strains, including 634b.

The bacterial strain B. diazoefficiens CCM GS-4 was obtained C. Didovich and N.Z. Tolkachev in 2003 by the method of analytical selection. The strain is characterized by the following morphological-cultural and physiological-biochemical characteristics: the cells of the 7-day-old culture of the strain are in the form of rods, 0.4-1.2 μm wide and 1.5-2.0 μm long, mobile due to the polar flagellum (monotrich). The strain is gram-negative, does not form a spore, obligate aerobic. The growth of bacteria when streaking on an agarized pea medium with glucose or mannitol is weak, the stroke is whitish, mucous. They belong to slow-growing nodule bacteria, colonies appear on the surface of agar on days 7-8, have a round shape, up to 1 mm in diameter, convex, shiny, mucous. The strain actively absorb glucose, fructose, arabinose, raffinose, xylose, rhamnose, maltose, mannitol, sorbitol, dulcite. The lack of an invertase enzyme does not allow the absorption of sucrose. As a source of nitrogen, it uses ammonium, nitrate, amide nitrogen. On media with organic nitrogen (MPA), the strain does not grow. Milk with litmus alkalizes, does not peptone. Gelatin does not thin, does not have a proteolytic enzyme, gelatinase, does not hydrolyze starch, casein, agar, does not have amylase and oxidase activity, is characterized by denitrifying ability, urease and catalase activity. The strain has an active nitrate reductase and reduces nitrates. Prototroph, sensitive to antibiotics. It does not form fluorescent and phenazine pigments. The strain synthesizes ammonia from air nitrogen in symbiosis with the host plant, synthesizes growth-stimulating substances such as phytohormones. The temperature range of strain growth on solid and in liquid media is 18-37 ° C, and the temperature optimum is 25-28 ° C, the strains are attributed to mesophiles. Bacterial growth is observed in the pH range 4.5–8.5; the optimal reaction for the development of the cells of the strain is a medium pH of 6.5–7.5, which is close to neutral; the strain is assigned to neutrophils. Signs of the strain are persistent.

Strain B. diazoefficiens SSM GS-4 forms active nodules with modern soybean varieties ( Glycine max (L.) Merr.) Of domestic selection. On beans, chickpea, lupine, peas, beans, sainfoin, alfalfa, goatskin, clover, lamb, do not form nodules.

For storage and cultivation of strain B. diazoefficiens SSM GS-4 in the laboratory, mannitol-yeast agar (MDA) with the addition of glucose at pH 6.8 ± 0.2 of the following composition, g / l:

mannitol - 10 g

glucose - 10 g

yeast extract - 1 g

K ₂ NRA ₄ × 4H ₂ 0 - 0.5 g

MgSO ₄ × 7H ₂ O - 0.2 g

NaCl - 1 g, CaCO ₃ - 3 g

agar - 20 g

distilled water - 1 l

Strain the strain at 4-6 ° C. Reseeding 1 time in half a year.

For industrial cultivation of strain B. diazoefficiens SSM GS-4, medium is used at pH 6.8 ± 0.2 of the following composition, per liter: corn extract - 7 g, baker's yeast - 0.5 g, glucose - 12 g, (NH ₄ ) ₂ SO ₄ - 1 g, K ₃ PO ₄ × 3H ₂ O - 0.35 g, MgSO ₄ - 0.2 g, CaCO ₃ - 1 g. Sowing dose - 5% of the volume of the nutrient medium. Cultivation is carried out at 28 ° C for 3 days.

The effectiveness of the bacterial strain B. diazoefficiens CCMGS-4 was determined:

- in vegetation experiments on symbiotic indicators (number, weight and nitrogen-fixing activity of nodules - acetylene method for determining nitrogen-fixing activity / Hardy et al., 1968 /) on varieties of Ukrainian selection used as test for primary analysis;

- in field experiments on the productivity of the host plant (seed yield; the amount of crude protein in the seeds - Kjeldahl method / GOST 10846-91 “Grain and its processed products” /)) on production varieties of Russian selection.

The scheme of the experiments involved a comparison of the effect of the proposed strain B. diazoefficiens SSM GS-4 with the applied technical strains that showed the best results for the studied parameters in previous breeding on different soybean varieties (Tables 1, 2). The effectiveness of bean-rhizobial symbiosis is associated with the specificity of plant-microbial interactions. This strain of B. diazoefficiens shows great specificity with respect to soy, which allows it to be used as an active component for a microbiological preparation.

Note: N.A. - nitrogenase activity

In a small-plot experiment, tests were conducted on production varieties of the Russian selection Zusha and Beautiful Sword, 2 strains from each genetically removed group of soybean symbionts were used as inoculums (Table 4). A negative control was a variant of the experiment without microbial treatment. As can be seen from table 4, there were no local populations of soybean nodule bacteria in the soil; therefore, the data obtained are a consequence of the different efficacy of bean-rhizobial symbiosis with introduced strains.

The strain B. diazoefficiens SSM GS-4 was selected for the subsequent stage of field trials in the production varieties of the Russian selection of Zusha and Krasnyaya Mecha, and the strain A. diazoefficiens 634b and B. japonicum 36 were the largest number of active (pink) nodules and production comparison strains were tested. The tests were carried out in the Oryol region on the basis of the All-Russian Research Institute of Leguminous and Cereal Crops Federal State Budgetary Scientific Institution (Tables 5 and 6).

As a result of field tests, it was shown that the strain B. diazoefficiens SSM GS-4 proposed in this application provides a maximum increase in the yield of Zusha and Krasnyaya Mecha varieties - 3.5 c / ha (16.5%) and 3.9 c / ha (18.4%) compared with control, respectively. At the same time, there is an increase in protein content in soybean seeds of the Zusha variety - by 1.2% and the Beautiful Mecha variety - by 1.4%. Thus, in the processing of soybean seeds of modern varieties of domestic selection, the efficiency of biofertilizers obtained on the basis of the indicated strain B. diazoefficiens CCM GS-4 exceeded 7.5-8% in terms of seed yield, and in terms of crude protein content in seeds - by 0 , 6-1.2% similar indicators of the production strain of B. japonicum 634b.

All of the above indicates the possibility of using a strain of nodule bacteria B. diazoefficiens SSM GS-4 for the manufacture of fertilizer preparations for presowing treatment of soybean seeds in the form of a bacterial suspension concentrate with a titer of at least 10 ⁹ CFU / ml.

Claims (1)

The bacterial strain Bradyrhizobium diazoefficiens VKPM B-12660, increasing soybean yield.

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