RU2654578C1 - Rhizobium leguminosarum ccm 32 (rncim b-12661) strain - risobial component of microbic biologic drug for presowing treatment of pea - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a strain of Rhizobium leguminosarum SSM 32 RNCIM B-12661 nodule bacteria, used as an active component for producing biofertilizers for peas.

EFFECT: invention allows to increase the yielding capacity of peas depending on its breed and agroclimatic growing conditions.

1 dwg, 2 tbl

Description

The invention relates to biotechnology and agriculture and relates to a new strain of nodule bacteria Rhizobium leguminosarum CCM 32 to obtain microbiological fertilizer that increases the productivity of peas.

To increase the yield of legumes, bacterial fertilizers are used, made on the basis of nitrogen-fixing nodule bacteria, both in the format of a monocomponent preparation and as part of consortia of useful soil microorganisms. Currently, there are only two RF patents for strains of the genus Rhizobium of various species. One of them involves the industrial use of culture as a producer of biopolymers (RU 2604495 C2), in the second, strains of the genus Rhizobium are proposed as a basis for a biological product for horned lamb (RU 2592677 C2). No patents for strains of nitrogen-fixing bacteria under peas have been identified.

The objective 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 the productivity of peas. This problem is solved due to the fact that for the biofertilizer under peas, a strain of nodule bacteria Rhizobium leguminosarum CCM 32 is used.

Until now, when developing microbial biological products for legumes, 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.

Legume-rhizobial interactions between plants of the tribe Vicieae: peas (Pisum), beans (Vicia fabae), rank (Lathyrus), lentils (Lens) - Rhizobium leguminosarum bv. viciae (Rlv) are considered one of the most specific. The strains within this biovar are capable of cross-inoculation of the host plants represented, the so-called cross-inoculation group (GUI). The effectiveness of strains of R. leguminosarum bv. viciae varies widely. This is due to genetic differences between the symbiotic genes and the “household” genes of the inoculum strains. The authors of this application previously developed a method for identifying and differentiating prokaryotic organisms (RF patent for the invention No. 2486251) by the chromosomal hin region marker, including nodule bacteria of the genus Rhizobium.

Thus, the biodiversity of microsymbionts of GUI plants for this marker is represented by 3 genetically removed groups of strains: R. leguminosarum I / A - strains that are more effective in interaction with peas (Pisum), R. leguminosarum I / B - strains that are more effective in interaction with fodder beans (Vicia fabae) and R. pisi.

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 of R. leguminosarum CCM 32 is a fast-growing nodule bacteria that are more effective on modern varieties of pea domestic selection compared to previously used strains, including R. leguminosarum 245a.

Brief Description of the Drawings

The drawing shows a phylogenetic tree, based on data from a comparative analysis of the sequences of the hin region of Rhizobium sp. - symbionts of peas and beans using the NJ algorithm. The scale corresponds to 1 substitution per 100 base pairs. The numbers show the statistical reliability of the branching order (in%), determined using the “bootstrap” analysis (1000 replicas). Bootstrap values below 70% are not shown. Rlv - Rhizobium leguminosarum bv. viciae.

This strain was obtained N.Z. Tolkachev in 2003 by the method of analytical selection. It is characterized by the following morphological-cultural and physiological-biochemical characteristics: 3-day-old cells have a rod-shaped shape 2.6–2.8 × 0.7–0.8 μm in size, motile. Strain SSM 32 - gram-negative, does not form a spore, obligate aerobic. Refers to fast-growing nodule bacteria. On a pea agar medium on the third day forms convex, shiny, mucous colonies up to 2-3 mm in diameter and actively accumulates polysaccharide mucus. The strain assimilates sucrose, glucose, fructose, arabinose, raffinose, xylose, rhamnose, maltose, mannitol, sorbitol, and dulcite. 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 acidifies, causes precipitation of casein and the formation of a transparent zone (1.0-1.5 cm) in the upper part of the medium. 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. It does not form fluorescent and phenazine pigments. The temperature range of the growth of the strain on solid and in liquid media is 18-37 ° C, and the temperature optimum is 25-28 ° C, the strain is attributed to mesophiles. Bacterial growth is observed in the pH range 4.5-8.5, optimal for the development of the cells of the strain is a close to neutral reaction of the medium pH 6.5-7.5, the strain is classified as neutrophils. The signs of the strain are stable. The culture is stored on mannitol-yeast or bean agar with sucrose at a temperature of 4-5 ° C, reseeded once every 6 months. Instead of sucrose, you can use a mixture of mannitol and glucose. The strain forms active nodules with varieties of peas (Pisum sativum L.), vetch (Vicia sativa L.), ranks (Lathyrus L.), lentils (Lens Mill.). In soy, beans, chickpeas, sainfoin, alfalfa, this strain of nodules does not form.

For storage and cultivation of strain CCM 32, an agar medium of the following composition is used: pea broth (100 g peas / 1 l water), sucrose 20 g / l, KH ₂ PO ₄ 0.5 g / l, MgSO ₄ 0.2 g / l, CaCO ₃ - 1 g / l, agar - 20 g / l, pH 6.8-7.0, a mixture of trace elements according to Fedorov - 1 ml / l. Strain the strain at 4-6 ° C. Reseeding 1 time in half a year.

For the industrial cultivation of strain CCM 32, the following composition is used: pea broth (100 g peas / 1 l water), sucrose 20 g / l, KH ₂ PO ₄ 0.5 g / l, MgSO ₄ 0.2 g / l, CaCO ₃ - 1 g / l, pH 6.8-7.0, a mixture of trace elements according to Fedorov - 1 ml / l. Sowing dose - 5% of the volume of the nutrient medium. Cultivation is carried out at 28 ° C for 3 days.

The ability of the bacteria of the claimed strain of R. leguminosarum SSM 32 to form active nodules on the roots of plants is confirmed by field experiments conducted at the experimental base of the Crimean Scientific Research Institute of Agriculture of the Crimea.

The scheme of the experiments involved a comparison of the effect of the proposed R. leguminosarum CCM 32 with the applied technological strain 245a, which showed the best results according to the studied parameters in previous breeding, on different varieties of peas. From table 1 it is seen that the proposed strain of R. leguminosarum CCM 32 actively forms nitrogen-fixing nodules on pea roots in the growing experiment and provides an increase in the efficiency of bean-rhizobial symbiosis, which leads to an increase in plant phytomass.

As a result of tests conducted in the field experiment, it was shown that the strain R. leguminosarum CCM 32 proposed in this application provides a maximum increase in yield on the Berkut variety - 38.2%, on the Vostok variety - 35.2%. At the same time, there is an increase in the protein content in the seeds of these pea varieties to 2.2% in comparison with the control (without microbial treatment). Thus, when processing pea seeds of modern production varieties, the efficiency of biofertilizers obtained on the basis of the indicated strain of R. leguminosarum CCM 32 exceeded 6.8-11.6% in terms of seed yield, and the increase in protein content in seeds reached 1.1 % relative to similar indicators of the production strain of R. leguminosarum 245a (table 2).

Note: * N.A. - nitrogenase activity

All of the above indicates the possibility of using a strain of nodule bacteria R. leguminosarum SSM 032 for the manufacture of fertilizer preparations for pre-sowing treatment of pea seeds in the form of a bacterial suspension concentrate with a titer of at least 10; CFU / ml.

Claims (1)

The bacterial strain Rhizobium leguminosarum CCM 32, deposited in the bacterial collection VKPM No. B-12661, as an active component for obtaining biofertilizer under peas.

Images