RU2728641C1 - Flavobacterium sp_ 72 associative bacteria strain, active nitrogen fixator and producer of physiologically active substances used to increase rice yield - Google Patents

Abstract

FIELD: agriculture.SUBSTANCE: invention relates to agricultural microbiology and agriculture, namely to microbiological agents used for optimization of mineral nutrition of plants. Presented is Flavobacterium sp. 72, extracted from epiphytic microbocenosis of apical part of rice root and deposited in Departmental collection of useful microorganisms of agricultural purpose (Federal State Budgetary Scientific Institution the All-Russian Research Institute of Agricultural Meteorology) under registration number RCAM 04328. Strain is an active nitrogen fixator, a producer of physiologically active substances, can be used by pre-sowing treatment of seeds and get used in cereal plants rhizosphere, in particular rice, forming associative symbiosis.EFFECT: strain Flavobacterium sp_ 72 can be effectively used to increase rice yield.1 cl, 8 tbl, 1 dwg

Description

The invention relates to agricultural microbiology, biotechnology, microbiological industry and agriculture, namely to microbiological means for optimizing the mineral nutrition of plants by pre-sowing seed treatment with effective growth-stimulating bacteria capable of taking root in the rhizosphere of cereal plants, in particular rice, forming an associative symbiosis.

To reduce the anthropogenic load on the flooded soils of rice fields, it is necessary to introduce biological elements into agricultural technology for growing rice crops. Pre-sowing treatment of seeds with associative strains of microorganisms is an effective method for optimizing plant nutrition, ensuring an increase in yield and quality of the resulting product.

A number of strains isolated from rice rhizophera and rhizoplana are known, which are used in agriculture to stimulate the growth and increase the productivity of grain crops.

Known isolated from rice rhizoplana strain Enterobacter aerogenes 30F (AS SU 1356454 A1, C12N 1/20, C05F 11/08), which is a non-symbiotic nitrogen fixer for rice and allows you to increase its yield. The strain also showed high efficiency in crops of grain crops and is the basis of the microbial preparation Rhizoenterin, recommended for pre-sowing treatment of barley seeds (Litivintseva T.A. The effectiveness of using rhizoenterin on crops of malting barley // Agrochemical Bulletin, 2007. - No. 5. - P. 37 -38).

в умовах Степу/ Науково-практичний щорiчник. Посiбник

хлiбороба, 2013. - Т. 1. - С. 209-210).Known strain Agrobacterium (Rhizobium) radiobacter 204, isolated from the soil of the rhizosphere of rice and used for pre-sowing treatment of rice and wheat seeds in order to increase yields (AS 1621433, C05F 11/08, C12N 1/20, 15.10.1990). Strain Agrobacterium (Rhizobium) radiobacter 204 - nitrogen fixer, growth stimulator, is a bioagent of the microbial preparation Diazofit (rhizoagrin), recommended for the treatment of seeds of wheat, rice and other grain crops (Zavalin A.A. Biopreparations, fertilizers and harvest. M .: Publishing house VNIIA, 2005. - 302 p .; Melnichuk T., Kameneva I, Yaroshenko S. Stagnation of microbial preparations for viroshuvanny of wheat

in the minds of Stepu / Scientific and Practical Shorichnik. Posibnik

hliboroba, 2013. - T. 1. - S. 209-210).

Known strain of nitrogen-fixing bacteria Alcaligenes paradoxus 060207, which is isolated from the rhizosphere of rice grown in monoculture without mineral fertilizers, and is used for the production of bacterial fertilizers for rice and wheat (Patent UA No. 17694, C05F 11/08, C12N 1/20, publ. 31.10 .1997, bul. No. 5).

The reasons that impede the achievement of an increase in the productivity of the agrocenosis of a monoculture of rice is a decrease in the nitrogen-fixing activity of the known strains due to the high pesticide load on the soil. Agrochemicals prevent the survival of beneficial forms of microorganisms in the rhizosphere of plants, namely, strains that are the basis of microbial preparations, thereby reducing the potential functionality of the system "associative bacteria - rice plants".

Known strain of associative bacteria Phyllobacterium ifriqiyense 6, which was isolated from the apical part of the roots of rice varieties Khazar, grown in monoculture, and deposited in the Departmental collection of beneficial microorganisms for agricultural purposes (FGBNU VNIISHM, St. Petersburg) under registration number RCAM 04327. The strain is adapted to the conditions of the south of Russia, is an active nitrogen fixer, growth stimulator, increases the productivity of rice (Patent RU 2649362 C1, C12N 1/20, C01N 63/02, C12R 1/01, publ. 02.04.2018, bul. No. 10). However, despite the high degree of associativity, after 25 days of growing in the rhizosphere of bacterized plants, the number of Phyllobacterium ifriqiyense 6 was 97 thousand colony-forming units (CFU) in 1 year of soil, which may be due to the fact that Phyllobacterium ifriqiyense are not typical representatives of the rhizosphere of plants rice and soil intended for growing this crop.

Bacteria of the genus Flavobacterium are characteristic representatives of the microbocenosis of the rhizosphere and rhizoplana of various plants. (Orazova M.Kh., Polyanskaya L.M., Zvyagintsev D.G. Root heterogeneity as a habitat for microorganisms // Microbiology. - 1994. - T. 63. - No. 4. - S. 706-714.). The known microbial preparation Flavobacterin (based on the Flavobacterium sp. Strain) suppresses the development of phytopathogenic microorganisms both on the seed itself and when it enters the soil. (Zavalin A.A., Almetov N.S. The use of biological products and biological nitrogen in agriculture in the Non-Black Earth Region. - Moscow: VNIIA Publishing House, 2009. - 152 p .; Laktionov Yu.V. New technologies for the cultivation of winter crops based on the use of microbiological preparations of rhizoagrin and flavobacterin produced by the Federal State Unitary Enterprise "EKOS" of the Russian Agricultural Academy (St. Petersburg) // Chief Agronomist. - 2011. - No. 8. - P. 20-21). The disadvantage of the drug is its unstable efficiency in rice crops grown under conditions of prolonged flooding, which create specific conditions for soil microorganisms.

The basis of the invention is the task of obtaining a strain of soil bacteria with a complex of properties useful for plants, capable of forming an associative symbiosis with rice plants grown in monoculture and showing an adaptive potential to flooded conditions on dark chestnut soils of southern Russia, providing an increase in crop yield.

The problem was solved by isolating from the microbocenosis of the apical part of the roots of rice grown in monoculture, a strain of the associative bacteria Flavobacterium sp. 72, which has the ability to fix atmospheric nitrogen and produce physiologically active substances. The use of the Flavobacterium sp. 72 provides an increase in rice yield by 25-30%.

A significant advantage of the Flavobacterium sp. 72 is its ability to form an associative symbiosis with Oriza sativa L., since the selection of the strain was carried out according to the principle of associativity to a particular plant species (namely, rice grown under conditions of periodic flooding). The strain is a facultative anaerobe with a high adaptive potential for intensive cultivation of this crop. Also, the advantages of the strain include its prevalence (numerical representation). When isolates with a high degree of associativity with plants were isolated, the abundance of this species in the epiphytic microbocenosis of rice was 13.4%, and the frequency of occurrence was 30%.

The strain was isolated from the epiphytic microbocenosis of the apical part of the rice root using an improved technique (Sherstoboev M.K., Melnichuk T.M., Melnik L.I. Zb. - Chernigiv, 2006. - Vip. 3. - P. 34-42) on cabbage medium (Wednesday No. 19).

Composition of medium No. 19:

Cabbage broth - 1 l

Corn extract - 1.25 ml / l

beer wort - 25 ml / l

agar-agar - 20 g / l

pH 7.0-7.2

As a substrate for isolation, we used the meadow-chestnut soil of rice paddies, where the monoculture of rice was 10 years old (Crimea, Krasnoperekopsky district).

The identification of the strain was made on the basis of morphological and biochemical characteristics according to Bergi's determinant 1984 and methodological recommendations (Voznyakovskaya Yu.M., Popova Zh.P. Methodical recommendations for the identification of non-spore bacteria dominating in the rhizosphere of plants. Leningrad, 1985).

Bacterial cells look like rods. Spores do not form, motionless. Colonies on rhizoid agar media, up to 3 mm in diameter, flat, shiny, orange, smooth, uneven edges. The culture uses mineral and organic nitrogen, gelatin does not thin; poor growth on media with carbohydrates (lactose, arabinose) with acidification; able to hydrolyze starch; no growth at 37 ° C; catalase positive; optional anaerobic. The temperature range for the growth of the strain is 22-30 ° C. The optimum temperature for the growth and reproduction of this strain is 25-28 ° C, it is stably stored on cabbage agar medium at a temperature of + 4 ° C, pH 6.9-7.2. The frequency of reseeding is 1 time in 6 months.

Genetic identification was performed in 2016 at FGBNU VNIISKhM, St. Petersburg (with the support of grant No. 16-34-50060 Identification and characterization of the symbiotic properties of growth-stimulating bacteria in the rice rhizosphere).

Based on the analysis of the nucleotide sequence of the 16S rRNA gene, the newly isolated strain was identified as Flavobacterium sp. and deposited in the Departmental collection of beneficial microorganisms for agricultural purposes (FGBNU VNIISHM, St. Petersburg) under registration number RCAM 04328 dated October 21, 2016.The closest relatives of the claimed strain are: Flavobacterium pectinovorum strain NBRC 15945 (Identity - 98.52%), Flavobacterium pectinovorum strain DSM 6368 (Identity - 98.45%), Flavobacterium saccharophilum strain NBRC 15944 (Identity - 98.17%).

The associative symbiont has a nucleotide sequence of SEQIDNO: 1.

Flavobacterium sp. 72 is characterized by high manufacturability, because grows well in environments regulated for the production of biological products, gains a high titer, which lasts for a long time. With submerged cultivation for three days in corn molasses medium at 220 rpm, the bacterial titer reached 18 billion CFU / ml - sufficient for the treatment of rice seeds and other grain crops based on the optimal load per seed.

Studies carried out to study the mechanism of action of the strain Flavobacterium sp. 72, confirmed its ability to fix atmospheric nitrogen and produce physiologically active substances of auxin nature.

The potential nitrogen-fixing activity of the culture was determined by the acithelen reduction method on a Chrom-5 gas chromatograph with a flame ionization detector and was 22.1 C ₂ H ₄ nMol / ml / hour.

Flavobacterium sp. 72 at a concentration of 1:10 and 1: 100 stimulated an increase in the length of wheat healers per day by 44 and 32%, respectively, compared with the control and by 33 and 22%, respectively, in comparison with an authentic solution (IAA 10 ^-5 ), which indicates phytostimulating properties of this strain (Table 1)

Quantitative and qualitative analysis of auxins in the culture liquid of Flavobacterium sp. 72, carried out by high performance liquid chromatography, revealed the production of indolyl-3-lactic acid (IMA), indolyl-3-carboxylic acid (ICA), and indoleacetic acid (IAA) (Table 2).

The effect of stimulation was established on rice plants under the conditions of the growing experience in 2014 (Table 3). It has been shown that bacterization of seeds promotes plant growth and development of rice roots. Moreover, the Flavobacterium sp. 72 increases the shoot length by 40.5% relative to the control and by 15.6% relative to the reference strain A. radiobacter 204, root length by 103.5% and 12.3%, air-dry mass by 76.2% and 68, 2% respectively. This increases the content of chlorophylls in the leaves of plants by 25.7 and 9.1%, respectively, in comparison with the control and the reference A. radiobacter 204 strain. FIG. 1 shows a diagram of the chlorophyll content in the leaves of rice plants in the flowering phase.

Flavobacterium sp. 72 forms active associations with rice plants and, when introduced into the soil with seeds, takes root in the plant rhizosphere. So, after 25 days of rice vegetation, the amount of Flavobacterium sp. 72 was 687 thousand colony forming units (CFU) in 1 gram of rhizosphere soil, which exceeded the number of Phyllobacterium ifriqiyense 6 by 7 times.

Seed treatment before sowing with the Flavobacterium sp. 72 promoted an increase in the number of diazotrophs in the rhizosphere of rice in the tillering, flowering and ripening phases, respectively, by 46.9%, 42.9% and 26.5% relative to controls (Table 4).

An increase in biological activity in the rhizosphere of rice was found. Emissions of CO ₂ in the embodiment with the processing of seeds Flavobacterium sp. 72 in the tillering phase increased by 53% compared to the control and by 76.9% and 64.3%, respectively, to the reference strains A. radiobacter 204 and P. ifriqiyense 6 (Table 5). In the maturation phase, this indicator exceeded the control by 40% and the reference strains by 16.7% and 27.3%, respectively.

Seed treatment before sowing provided an increase in rice yield by an average of 40% over three years and exceeded the reference strain by 9.6% (Table 6).

When studying the ability of Flavobacterium sp. 72 to mobilize nutrients in the soil, it was noted that the strain led to a decrease in the content of water-soluble forms of Al, but increased the content of Fe, Cu, and Mn in the meadow chestnut soil. An increase in the content of water-soluble Al and Mn was found upon introduction into the dark chestnut weakly solonetsous soil.

Taking into account the ability of the strain to produce auxins, and also with the aim of expanding the range of crops for the introduction of the strain, we studied its influence on the sowing properties of wheat and a promising for Crimea crop of camelina. The stimulating effect of Flavobacterium sp. 72 was installed on seedlings of different varieties of winter wheat. Treatment of wheat seeds with a suspension of the Flavobacterium sp. 72 helps to increase their germination in the Stanichnaya variety by 2%, Victoria - 4.3%, Vassa - 1%.

The investigated strain of associative bacteria increases the length of the roots of winter wheat varieties Stanichnaya, Victoria and Krasnodarskaya 99 by 15.8, 46.0 and 13.2%, respectively. In variants with seed treatment with the Flavobacterium sp. 72 also noted the stimulation of the length of wheat seedlings by 10-33%, depending on the variety (table 7).

The stimulation of the growth of roots and seedlings of the oilseed crop camelina when treated with an associative strain of bacteria Flavobacterium sp. 72 by 29.4% and 4.1%, respectively (table 8).

Thus, as a result of solving a technical problem, a strain of associative bacteria Flavobacterium sp. 72, capable of fixing atmospheric nitrogen and producing phytohormones of auxin nature. Presowing seed treatment with Flavobacterium sp. 72 promotes growth stimulation, an increase in the amount of chlorophylls in leaves and an increase in rice grain yield, as well as an increase in biological activity in the rhizosphere of plants. Presowing seed treatment with a suspension of a strain of associative bacteria Flavobacterium sp. 72 promotes the stimulation of the growth of not only rice, but also wheat, as well as the oilseed camelina, which makes it possible to expand the range of crops for the use of the strain.

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Sequence listing

<110> Federal State Budgetary Institution of Science "Research Institute of Agriculture of Crimea" (FGBUN "Research Institute of Agriculture of Crimea")

<120> Strain of associative bacteria Flavobacterium sp. 72, an active nitrogen fixer and producer of physiologically active substances used to increase rice yields

<140> RU 2019127586

<141> 2019-08-30

<160> 1

<210> 1

<211> 1424

<212> DNA

<213> Flavobacterium sp.

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gcttaacaca tgcaagtcga ggggtatggt tcttcggaac cagagaccgg cgcacgggtg 60

cgtaacgcgt atgcaatcta ccttttacag agggatagcc cagagaaatt tggattaata 120

cctcatagta taacatgacc gcatggtcga gttattaaag tcacaacggt aaaagatgag 180

catgcgtccc attagctagt tggtaaggta acggcttacc aaggctacga tgggtagggg 240

tcctgagagg gagatccccc acactggtac tgagacacgg accagactcc tacgggaggc 300

agcagtgagg aatattggac aatgggcgca agcctgatcc agccatgccg cgtgcaggat 360

gacggtccta tggattgtaa actgctttta tacgagaaga aacactccga cgtgtcggag 420

cttgacggta tcgtaagaat aaggatcggc taactccgtg ccagcagccg cggtaatacg 480

gaggatccaa gcgttatccg gaatcattgg gtttaaaggg tccgtaggcg gtttggtaag 540

tcagtggtga aagcccatcg ctcaacggtg gaacggccat tgatactgcc agacttgaat 600

tattaggaag taactagaat atgtagtgta gcggtgaaat gcttagagat tacatggaat 660

accaattgcg aaggcaggtt actactaatt gattgacgct gatggacgaa agcgtgggta 720

gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatggatact agctgttgga 780

agcaatttca gtggctaagc gaaagtgata agtatcccac ctggggagta cgttcgcaag 840

aatgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 900

gatgatacgc gaggaacctt accaaggctt aaatgtagat tgaccggttt ggaaacagat 960

ctttcgcaag acaatttaca aggtgctgca tggttgtcgt cagctcgtgc cgtgaggtgt 1020

caggttaagt cctataacga gcgcaacccc tgttgttagt tgccagcgag tcaagtcggg 1080

aactctaaca agactgccag tgcaaactgt gaggaaggtg gggatgacgt caaatcatca 1140

cggcccttac gcccttgggc tacacacgtg ctacaatggc cggtacagag agcagccact 1200

gggcgaccag gagcgaatct ataaaaccgg tcacagttcg gatcggagtc tgcaactcga 1260

ctccgtgaag ctggaatcgc tagtaatcgg atatcagcca tgatccggtg aatacgttcc 1320

cgggccttgt acacaccgcc cgtcaagcca tggaagctgg gggtgcctga agtcggtgac 1380

cgcaaggagc tgcctagggt aaaactggta actagggcta agtc 1424

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Claims (1)

The strain of associative bacteria Flavobacterium sp. 72, deposited in the Departmental collection of beneficial microorganisms for agricultural purposes (VKSM) FGBNU VNIISHM under registration number RCAM 04328, is an active nitrogen fixer and producer of physiologically active substances used to increase the yield of rice.

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