CN113564086A - Rhizobium with phosphorus dissolving function and garden plant growth promoting function and application thereof - Google Patents

Abstract

The invention discloses rhizobium with a phosphorus dissolving function and a garden plant growth promoting function and application thereof. The Rhizobium is 3T7 of lotus Rhizobium (Rhizobium rhizobia) and the registration number of the Rhizobium in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21507. The agapanthus Rhizobium (Rhizobium rhizobia) 3T7 can secrete IAA, has an inorganic phosphorus dissolving function, and can be used as a microbial organic fertilizer for improving soil fertility.

Description

Rhizobium with phosphorus dissolving function and garden plant growth promoting function and application thereof

Technical Field

The invention relates to rhizobia with a phosphorus dissolving function and a garden plant growth promoting function in the technical field of biology and application thereof.

Background

Traditionally, rhizobia species have been considered to be leguminous endophytes and most have been isolated from root nodules of leguminous plants. Some studies have also shown that non-symbiotic rhizobia can also be isolated from other sources (e.g. soil, plant rhizosphere). Rhizobia is of great importance in agricultural practice, particularly for legume crops. In addition to having symbiotic nitrogen fixation capacity, rhizobia strains can promote the growth of non-legume plants.

Phosphorus is one of essential nutrient elements for plants, and phosphorus in soil mainly exists in the form of insoluble phosphate, so that the utilization rate of the phosphorus by the plants is usually extremely low. The phosphate solubilizing bacteria can dissolve inorganic phosphorus compounds which are not easy to absorb by plants in soil by producing organic acids and the like, and have the advantages of improving the effective phosphorus content of the soil, reducing the usage amount of phosphate fertilizer and promoting the growth of plants. The plant rhizosphere microorganisms can also secrete plant hormone substances, promote the growth of plant root systems and improve the absorption and utilization of plants on nutrients.

Disclosure of Invention

The technical problem to be solved by the invention is how to promote the growth of plants and/or promote the germination of plant seeds and/or dissolve phosphorus.

In order to solve the technical problems, the invention provides rhizobium.

The Rhizobium provided by the invention is Rhizobium (Rhizobium rhizobia).

The agapanthus Rhizobium (Rhizobium rhizobiaum rhizobia) provided by the invention can be specifically agapanthus Rhizobium (rhizobiaum rhizobia) 3T7, the registration number of the agapanthus Rhizobium (Rhizobium rhizobia) 3T7 is CGMCC No.21507 and is hereinafter referred to as agapanthus Rhizobium (rhizobiaum rhizobia) 3T 7.

Lotus Rhizobium rhizogenes (Rhizobium rhizobia) 3T7 is a gram-negative bacterium, rod-shaped, and has no spore formation. Colonies of 3T7 on the YMA plate were white, round, convex, translucent and sticky. The 16S rRNA gene of 3T7 of lotus Rhizobium (Rhizobium rhizobia) has a DNA fragment shown in sequence 1 in a sequence table.

In order to solve the above technical problems, the present invention also provides a culture of rhizobia.

The Rhizobium culture provided by the invention is a substance obtained by culturing Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 in a microbial culture medium (namely a fermentation product, such as a fermentation liquid containing Rhizobium japonicum Rhizobium 3T7 and a substance secreted into a liquid culture medium, or a solid fermentation product containing Rhizobium japonicum (Rhizobium rhizobia) 3T7 and a substance secreted into a solid culture medium).

In order to solve the technical problems, the invention also provides a microbial inoculum.

The microbial inoculum provided by the invention contains the metabolites of lotus Rhizobium (Rhizobium rhizobia) 3T7 or/and lotus Rhizobium (Rhizobium rhizobia) 3T7 or/and the cultures.

In the microbial inoculum, the microbial inoculum has at least one of the following functions:

m1) promoting plant growth;

m2) promoting plant seed germination;

m3) secretes IAA;

m4) dissolving inorganic phosphorus.

The active ingredients of the microbial inoculum can be metabolites of Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 or/and Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 or/and the culture, and can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by the skilled person according to the effect of the microbial inoculum.

In the above microbial inoculum, the microbial inoculum may further comprise a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier can be mineral material and biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material can be at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal excrement of various crops; the liquid carrier can be water; among the microbial agents, metabolites of Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 or/and Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture, or a mixture of cells and filtrate. The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

In order to solve the technical problems, the invention also provides a microbial ecological agent or a biological fertilizer.

The microbial ecological agent or the biological fertilizer provided by the invention contains the rhizobium, the culture or/and the microbial inoculum, and the product can be a microbial inoculum, a microbial ecological agent or a biological fertilizer.

Any of the following applications of the rhizobia, the cultures, the microbial inoculum or the biological fertilizer are also within the scope of the invention:

n1, application in promoting plant seed germination;

n2, application in preparing products for promoting plant seed germination;

n3, application in promoting plant growth;

n4, application in preparing products for promoting plant growth;

n5, use in the secretion of IAA;

n6, application in preparing IAA-secreting products;

n7, application in dissolving phosphorus;

n8, and application in preparing phosphorus-dissolved products.

Herein, the plant may be any one of the following plants:

p1) dicotyledonous or monocotyledonous plants,

p2) plant of the subclass Chrysanthemum,

p3) plants of the order Chrysanthemum or Cyperales,

p4) Compositae plant or Gramineae plant,

p5) of a plant of the Pooideae family,

p6) Tagetes or Oryza,

p7) marigold or rice.

The invention also provides a method for preparing the microbial inoculum.

The method for preparing the microbial inoculum comprises the step of taking metabolites of Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 or/and Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 or/and the culture as components of the microbial inoculum to obtain the microbial inoculum.

Herein, the metabolite of the lotus Rhizobium radiculosum (Rhizobium rhizobia) 3T7 can be obtained from a fermentation broth of lotus Rhizobium (Rhizobium rhizobia) 3T 7. The metabolite of the lotus Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 can be a sterile metabolite of the lotus Rhizobium japonicum Rhizobium (Rhizobium rhizobia) 3T7 or a bacteria-containing metabolite of the lotus Rhizobium (Rhizobium rhizobia) 3T 7. Specifically, the sterile metabolite (sterile fermentation filtrate) of the lotus Rhizobium (Rhizobium rhizobia) 3T7 can be prepared by culturing the lotus Rhizobium (Rhizobium rhizobia) 3T7 in a liquid culture medium, and filtering to remove the lotus Rhizobium (rhizobiam rhizobia) 3T7 in the liquid culture medium (fermentation broth) to obtain the sterile metabolite of the lotus Rhizobium (rhizobiam rhizobia) 3T 7. Specifically, the fungus-containing metabolites of lotus Rhizobium japonicum (Rhizobium) 3T7 can be prepared by culturing lotus Rhizobium japonicum (Rhizobium) 3T7 in a liquid fermentation medium, and collecting a fermentation broth (containing the Rhizobium (Rhizobium rhizobia) 3T7 and substances secreted into the liquid culture medium), wherein the fermentation broth is the fungus-containing metabolites of lotus Rhizobium (Rhizobium) 3T 7.

Herein, the dissolved phosphorus may be dissolved inorganic phosphorus.

Herein, the promoting plant growth may be promoting rice seedling growth and/or promoting marigold seedling growth. The promotion of plant seed germination can be the improvement of marigold seed germination rate.

The IAA secretion test, the phosphorus dissolving test and the seed germination test prove that the ratio of the phosphorus dissolving ring to the colony diameter of the agapanthus rhizobia (Rhizobium rhizobia) 3T7 on an inorganic phosphorus plate is 1.34 +/-0.06. Compared with a negative control group which is not inoculated, the germination rate of marigold seeds and the growth of rice and marigold seedlings can be obviously improved, and the germination rate of the marigold seeds is improved by more than 1 time. The agapanthus Rhizobium (Rhizobium rhizobia) 3T7 can secrete IAA, the unit IAA content of the agapanthus Rhizobium (Rhizobium rhizobia) 3T7 is 38.88mg/L fermentation liquor, and the agapanthus Rhizobium (Rhizobiaum rhizobia) 3T7 has an inorganic phosphorus dissolving function, can be used as a microbial organic fertilizer and is used for improving soil fertility.

Deposit description

The strain name is as follows: agapanthus rhizobia rhizobium

Latin name: rhizobium rhizolagapanthis

The strain number is as follows: 3T7

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 12 and 18 months in 2020

Registration number of the preservation center: CGMCC No.21507

Drawings

FIG. 1 is a photograph of colony obtained after 3 days of culturing Rhizobium Rhizobium rhizogenes (Rhizobium rhizozaapanthis) 3T7 on YMA plates.

FIG. 2 is a phylogenetic tree of Rhizobium japonicum (Rhizobium rhizobia) 3T7 and related model bacteria constructed based on 16S rRNA gene sequences. GenBan of the strain in bracketsk sequence number; in the figure, all reference strains are standard strains of all species. Neorhizobium huautlense S02^TAs an outer group.

FIG. 3 is a genome BLAST distance phylogenetic tree (GBDP) tree constructed from the whole genome of Rhizobium japonicum (Rhizobium rhizobia) 3T7 and related species within the genus Rhizobium. Wherein 3T7 is Lotus Rhizobium (Rhizobium rhizobia) 3T 7.

FIG. 4 is a photograph showing the formation of a phosphate solubilizing ring after 3 days of culturing Rhizobium japonicum (Rhizobium rhizozaapanthis) 3T7 on an inorganic phosphate medium.

FIG. 5 is a photograph showing qualitative detection of auxin secretion by Rhizobium Rhizobium (Rhizobium rhizozaapanthis) 3T 7. In the figure, IAA is a positive control, R2A is a negative control, and 3T7 is 3T7 treatment of Rhizobium japonicum Rhizobium rhizogenes (Rhizobium rhizobia apantanthus).

FIG. 6 is a photograph of rice seedlings at 1 month after transplantation. The left side 4 flowerpots are control groups, and the right side 4 flowerpots are test groups.

FIG. 7 is a photograph of marigold seedlings at 1 month after transplantation. The left 3 flowerpots are control groups, and the right 3 flowerpots are test groups.

Detailed Description

The research adopts a multiphase classification method to determine the classification status of the agapanthus rhizosphere bacteria 3T7, researches the IAA production and phosphorus dissolving functions of the agapanthus rhizosphere bacteria, and detects the growth promoting function of the agapanthus rhizosphere bacteria through a seed germination test and a pot culture test.

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

1/100TSA solid culture medium comprises tryptone 0.15g, soytone 0.05g, sodium chloride 0.05g, agar 15.0g, and distilled water 1000mL, and is sterilized at 121 deg.C for 20min, with pH adjusted to 7.3 + -0.2.

YMA medium: 1.0g of yeast extract, 10.0g of mannitol, 0.5g of dipotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 15.0g of agar and 1000mL of distilled water, adjusting the pH value to 6.8-7.0, and sterilizing at 121 ℃ for 20 min.

YMA liquid medium: 1.0g of yeast extract, 10.0g of mannitol, 0.5g of dipotassium phosphate, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water, adjusting the pH value to 6.8-7.0, and sterilizing at 121 ℃ for 20 min.

Inorganic phosphorus culture medium: 0.3g of sodium chloride, 10g of glucose, 0.3g of potassium chloride, 5g of tricalcium phosphate, 0.5g of ammonium sulfate, 0.3g of magnesium sulfate heptahydrate, 0.03g of manganese sulfate, 0.03g of ferrous sulfate heptahydrate and 1000mL of distilled water, adjusting the pH value to 7.0-7.2 and sterilizing at 121 ℃ for 30 min.

Producing an IAA liquid fermentation medium: 0.5g of yeast extract powder, 0.5g of peptone, 0.5g of casein hydrolysate, 0.5g of glucose, 0.5g of soluble starch, 0.03g of monopotassium phosphate, 0.024g of magnesium sulfate heptahydrate, 0.3g of sodium pyruvate and 0.2g of L-tryptophan, wherein the volume is fixed to 1000mL by using distilled water, the pH value is adjusted to 7.2 +/-0.2, and the mixture is sterilized at 121 ℃ for 15 min.

Example 1 isolation and characterization of Lotus Rhizobium rhizogenes (Rhizobium rhizobia) 3T7

1. Isolation of Lotus Rhizobium rhizogenes (Rhizobium rhizobia) 3T7

A rhizosphere soil sample of the African agapanthus was collected at Wu bridge base (30 degrees 58'N, 121 degrees 25' E) of the institute of garden science and planning in Shanghai city, and was brought back to the laboratory for storage at 4 ℃. Shaking off soil attached to plant roots, only retaining rhizosphere soil tightly adhered to the surfaces of the roots, weighing 2g of plant roots with the rhizosphere soil, grinding the plant roots in an aseptic mortar, fully grinding the plant roots, transferring the ground plant roots into a 150mL conical flask filled with glass beads and 50mL of aseptic water, oscillating the plant roots at room temperature of 150r/min for 30min, taking 1mL of diluent, performing serial dilution by using the aseptic water, taking 100 mu L of serial diluent, coating the serial diluent on an 1/100TSA flat plate, performing inverted culture at 30 ℃ for 1 week, picking single bacterial colonies by using a bamboo stick according to the characteristics of physiological morphology, purifying the single bacterial colonies on the flat plate, transferring the single bacterial colonies after determining to be pure bacteria, performing short-term storage at an inclined plane of 4 ℃, transferring the single bacterial colonies into a 25% glycerol tube, and performing long-term storage at-80 ℃. One of the isolated and purified strains was named 3T 7.

2. Identification of Lotus Rhizobium (Rhizobium rhizobia) 3T7

2.1 morphological characterization of the Strain

The strain 3T7 which is in the logarithmic growth phase and has stable colony size and is obtained by the separation and purification of the first step is subjected to single colony state description, and the single colony state description mainly comprises the size, color, transparency, colony surface state and colony edge state of colonies. 3T7 was stained with gram stain using a gram stain kit from Solebao according to the manufacturer's instructions, and the form of the cells was observed with an optical microscope.

Colonies of 3T7 on the YMA plate were white, round, convex, translucent and sticky (fig. 1). The cells were gram-negative, rod-shaped, and free of sporulation.

2.2 molecular characterization

The 16S rRNA gene amplification was performed using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') by extracting genomic DNA using a TIANMAP bacterial genomic DNA extraction kit (Gentiangen). 25 μ L PCR amplification system: 2 XTaq PCR Mix 12.5. mu.L, 27F (10. mu. mol/L) 1. mu.L, 1492R (10. mu. mol/L) 1. mu.L, ddH2O8.5. mu.L, 2. mu.L DNA template. The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 90s, and 30 cycles; final extension at 72 ℃ for 10 min. Detecting the PCR amplification product by using 1% agarose gel electrophoresis, wherein the amplification fragment is about 1400bp, and after electrophoresis verification, sending the positive result PCR product to Beijing Optimalaceae Biotechnology Limited for sequencing. The sequenced sequences were uploaded to Ezbiocloud (www.ezbiocloud.net/eztaxon) for sequence alignment.

The 16S rRNA gene of strain 3T7 was sequenced 1,403bp in length, and alignment with the EzBioCloud database showed that strain 3T7 is a member of the genus Rhizobium, and Rhizobium altiplani Hambi3664^T(99.43％)，Rhizobium tibeticum CGMCC 1.7071^T(99.36％)、Rhizobium favelukesii LPU83^T(99.29%) and Rhizobium mesometricum Hambi 3151^T(98.89%) has a heightSimilarity, with Rhizobium other species model bacterial sequence similarity < 98.7%. Those closely related 16S rRNA gene sequences were retrieved from the ezbiocoud server and aligned using the clustal _ W program. The phylogenetic tree was constructed by the neighbor method (neighbor-Joining) using MEGA 7 software. The evolutionary distance of the NJ method was calculated using the Kimura two-parameter model, and the development tree formed when Bootstrap was 1000 times as shown in FIG. 2. The strain 3T7 is clustered in the same branch as Rhizobium mesomericum.

2.3 genomic analysis

Genomic DNA was sent to Annuodda Gene technology (Beijing) Ltd and draft genomic sequencing was performed on strain 3T7 using Illumina NovaSeq 6000 sequencing System. Assembly using Unicycler v0.4.7 software yielded 80 contigs with approximately 185 x coverage and a length of 232080bp for N50. Genome 5.94Mb, with a G + C content of 59.9%, genome wide average nucleotide identity (OrthoANI) analysis of strain 3T7 with a strain of the genus Rhizobium was performed using the orthologous average nucleotide identification tool (OAT) v0.93.1 software of ChunLab. The digital DNA-DNA hybridization (dDDH) values between strain 3T7 and the reference strain were calculated using a genome-to-genome distance calculator (GGDC)2.0 server (http:// GGDC. dsmzz. de/distcalac. php) comparison.

As shown in Table 1, the OrthoANI values ranged from 78.6 to 88.5% compared to other strains of Rhizobium species with published genomic sequences, which were 95 to 96% below the critical values previously proposed for species demarcation, the dDDH values for 3T7 and its model strains ranged between 22.5 to 37.5%, and were well below the 70% species demarcation threshold. Both ANI and dDDH results support strain 3T7 as a new species of Rhizobium.

TABLE 1 Strain 3T7 and its related strains ANI and dDDH values

3T7	OrthoANI(％)	dDDH(％)
			R.tibeticum GCA_900110205	88.5	37.5
R.favelukesii GCA_000577275	88.3	37.2
			R.altiplaRi GCA_001542405	86.7	33.4
R.mesoamericanum IMG_2738541345	86.5	32.6
			R.grahamii GCA_000298315.2	85.4	31.3
R.metallidurans GCA_014196505	79.9	23.2
			R.phaseoli GCA_003985125	79.0	22.7
R.sophoriradicis GCA_003939025	78.8	22.8
			R.aethiopicum GCA_900094625	78.6	22.5

The whole genome sequence of 3T7 was submitted to the TYGS website of the model bacterial genome server for alignment, and the results showed that strain 3T7 did not belong to any species found in the TYGS database, and was a potential new species. Genome-wide based taxonomic analysis showed that 3T7 was closely related to Rhizobium tibeticum and Rhizobium favelukesii and clustered with these two model strains in a phylogenetic tree, as shown in fig. 3.

2.4 physiological and chemical Classification characterization

The physiological and biochemical properties of strain 3T7 and related model strains were determined using the API 20NE and API ZYM kit (bioMerieux) of Meriella, France. GEN III MicroPlates from BIOLOG USA were used to determine the oxidation of carbon sources and the sensitivity to inhibitory compounds.

The 20NE test result shows that the nitrate reduction reaction of the strain 3T7 is positive, indole can be generated, esculin and urea can be hydrolyzed, and gelatin cannot be hydrolyzed. The assimilation reaction of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose and gluconate is positive, the assimilation reaction of malic acid is weak positive, galactosidase is positive, gluconation is negative, arginine double hydrolase is negative, and the assimilation reaction of capric acid, adipic acid, citric acid and phenylacetic acid is negative. In the ZYM enzyme activity identification test, the leucine arylamine enzyme, the acid phosphatase, the naphthol-AS-BI phosphohydrolase and the beta-glucosidase are positive. BIOLOG results showed that 3T7 can utilize L-alanine, L-serine, sugar acid, D-glucose-6-phosphate D-fructose-6-phosphate, aminoacetyl-L-proline, L-arginine, L-glutamic acid, quinic acid, L-lactic acid, D-malic acid, bromo-succinic acid, acetoacetate, pectin, L-histamine, mucic acid, methyl pyruvate, formic acid, gentiobiose, raffinose, alpha-D-lactose, melibiose, beta-formyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-beta-D-mannosamine, N-acetyl-D-galactosamine, alpha-D-glucose, beta-formyl-D-glucoside, etc, D-mannose, D-fructose, D-galactose, D-fructose, L-rhamnose, D-sorbitol, D-mannitol, D-arabitol, inositol, glycerol, L-aspartic acid, D-galacturonic acid, D-glucuronic acid, glucuronamide, L-malic acid, acetic acid, D-maltose, D-cellobiose and sucrose as carbon sources can be grown in the presence of tetrazolium blue, nalidixic acid, potassium tellurite and aztreonam. The difference between the strain 3T7 and the related model strain is shown in Table 2.

The strain 3T7 is obviously different from a model strain Tibet Rhizobium (Rhizobium tibetimicum) CGMCC 1.7071 (LMG 24453) in the following 32 physiological and biochemical characteristics: 1) the strain 3T7 can assimilate N-acetyl-glucosamine and gluconate, and the model strain CGMCC 1.7071^TFail to work; 2) the extracellular zymogram of the strain 3T7 does not contain beta-galactosidase and alpha-glucosidase, but CGMCC 1.7071^TThe extracellular enzyme spectrum contains the two enzymes; 3) the strain 3T7 can take 18 substances such as L-alanine, L-serine, sugar acid, D-glucose-6-phosphate, D-fructose-6-phosphate, aminoacetyl-L-proline, L-arginine, L-glutamic acid, quinic acid, L-lactic acid, D-malic acid, bromo-succinic acid, acetoacetic acid, pectin, L-histamine, mucic acid, methyl pyruvate and formic acid as carbon sources, and the model strain CGMCC 1.7071^TThese substances could not be utilized, and strain 3T7 could not utilize D-turanose, stachyose, N-acetylneuraminic acid, inosine and D-gluconic acid as carbon sources, while CGMCC 1.7071^TThese 5 substances can be utilized as a carbon source; 4) 3T7 can grow in the presence of sodium bromate, 1% sodium lactate, acearundomycin, rifamycin SV and lincomycin, and CGMCC 1.7071^TAnd growth was not possible.

Strain 3T7 and the model strain Rhizobium mesomerinum Hambi 3151^TThere are significant differences in the following 19 physiological and biochemical characteristics: 1) strain 3T7 can assimilate N-acetyl-glucosamine and gluconate, cannot assimilate citric acid, is negative to arginine double hydrolase, and has 3151^TCan not assimilate N-acetyl-glucosamine and gluconate, can assimilate citric acid, and is positive to arginine double hydrolase; 2) strain 3T7 extracellular zymogramContaining alkaline phosphatase and alpha-glucosidase, and HAMBI 3151^TThe extracellular enzyme spectrum contains the two enzymes; 3) strain 3T7 was unable to use citric acid and D-turanose as carbon sources, HAMBI 3151^TCitric acid and D-turanose can be used, and strain 3T7 can be made from L-alanine, L-serine, glyconic acid, L-histamine, mucic acid, methyl pyruvate, formic acid and gentiobioseIs a carbon source and is used as a carbon source, HAMBI 3151^Tis not available; 4) 3T7 can grow in the presence of lithium chloride and 1% sodium lactate, while HAMBI 3151^TCan not grow, and sodium butyrate can inhibit the growth of 3T7 but can not inhibit HAMBI 3151^TAnd (5) growing.

Strain 3T7 and model strain Rhizobium altiplani Hambi3664^TThere are significant differences in the following 17 physiological and biochemical characteristics: 1) strain 3T7 can hydrolyze urea, HAMBI3664^TFail to work;

2) the N-acetyl-glucosamine assimilation reaction of the strain 3T7 is positive, and the model strain HAMBI3664^TThe assimilation reaction of N-acetyl-glucosamine is negative;

3) the extracellular zymogram of the strain 3T7 does not contain esterase (C4) and trypsin, while HAMBI3664^TThe extracellular enzyme spectrum contains the two enzymes;

4) strain 3T7 can not use L-galacturonolactone, propionic acid, citric acid, D-turanose, stachyose and N-acetylneuraminic acid as carbon source, model strain HAMBI3664^TIn addition, the strain 3T7 can use L-alanine, L-serine, sugar acid and D-glucose-6-phosphate as carbon source, HAMBI3664^TIs not available;

5) 3T7 can grow in the presence of lithium chloride and sodium bromate, while HAMBI3664^TCannot grow, vancomycin can inhibit 3T7 growth, but cannot inhibit HAMBI3664^TAnd (5) growing.

TABLE 2 differential characterization of Strain 3T7 with related model species

Note: +, positive or available; negative or unavailable; w, weak positive.

Cells from strain 3T7 and 3 closely related bacteria were assayed using the Sherlock microbial identification system (MIDI). The results of the MIDI analysis are shown in Table 2, and the major fatty acids (> 5%) of strain 3T7 are of type 8 (C)_18：1Omega 6C and/or C_18：1ω 7C), type 2 feature (C)_12：0Aldehyde and/or unknown fatty acid with equivalent carbon chain length of 10.9525), C_16：0、C_18：0And C_19：0cyclo ω 8 c. The results supported the strain 3T7 to be Rhizobium. The subtle differences in fatty acid profiles between the four strains are shown in table 3.

TABLE 3 cellular fatty acid composition of Strain 3T7 and its type strains

Fatty acids	3T7	CGMCC 1.7071	HAMBI 3664	HAMBI 3151
					C16：0	7.63	9.33	7.47	4.84
C15：0-2OH	0.79	1.35	1.59	1.66
					C17：0 cyclo	0.66	1.04	0.50	0.54
C16：0-3OH	1.74	1.70	1.38	1.19
					C18：0	5.64	6.51	8.83	6.97
11-methyl C18：1ω7c	0.22	1.15	0.56	0.74
					C19：0 cycloω8c	14.60	20.03	18.52	16.37
C18：0 3-OH	-	-	2.57	-
					Type 2 feature	6.81	4.66	4.93	4.32
Type 3 feature	1.15	1.69	0.87	0.69
					Type 8 feature	58.62	49.95	51.46	60.87

Note: the values shown are the percentage of total fatty acids. -: no detection was made, the profile was a mixture of two or three fatty acid components which could not be separated by GLC by the MIDI system, and profile 2 included C_14：03OH and/or C_16：1iso I, feature type 3 includes C_16：1Omega 6C and/or C_16：1ω 7C, characteristic type 8 includes C_18：1Omega 6C and/or C_18：1ω7c。

According to homology comparison, construction of phylogenetic trees, and combination of morphological, physiological and biochemical identification, fatty acid composition and genome analysis results, the strain 3T7 can be determined to be a new species of Rhizobium, and the proposed classification is named as Rhizobium Rhizobium rhizogenes, wherein the Chinese name is agapanthus rhizogenes. Baizilian Rhizobium (Rhizobium rhizobia) 3T7 has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 1 Xilu-1 of Beijing Kogyang district, Beijing) at 12 months and 18 days in 2020, and the deposition number is CGMCC No. 21507. Hereinafter, abbreviated as "Baizilian Rhizobium (Rhizobium rhizozaapanthis) 3T 7".

Example 2 examination of phosphorus solubilizing ability of Lotus rhizobia (Rhizobium rhizobia) 3T7

Inoculating 10 μ L of 3T7 bacterial solution of agapanthus Rhizobium rhizosphere (Rhizobium rhizobia) on inorganic phosphorus culture medium, culturing at 30 deg.C for 4d, observing whether there is phosphorus dissolving ring, and measuring the diameter of phosphorus dissolving ring (d)₂) And colony diameter (d)₁) Calculating the ratio of the phosphorus-dissolving ring to the colony diameter (d)₂/d₁). The larger the ratio, the stronger the phosphorus dissolving ability. The results showed that the ratio of the phosphorus-solubilizing circle to the colony diameter of 3T7 of Rhizobium japonicum Rhizobium (Rhizobium rhizozaapaanthus) was 1.34. + -. 0.06 after 4 days of growth on inorganic phosphorus medium (FIG. 4). Indicating that the lotus Rhizobium (Rhizobium rhizozaapaanthus) 3T7 can dissolve inorganic phosphorus.

Example 3 qualitative detection and quantitative analysis of secreted auxin of Rhizobium Rhizobium japonicum (Rhizobium rhizozaapanthis) 3T7

1. Qualitative detection

Activating 3T7 of agapanthus radicans rhizobia (Rhizobium rhizobia) and inoculating the activated agapanthus radicans rhizobia (3T 7) to an IAA-producing liquid fermentation medium, carrying out shake culture at 35 ℃ and 180r/min for 2d, centrifuging at 12000 rpm for 10min, then dripping 100 mu L of supernatant of the agapanthus radicans rhizobia (Rhizobium rhizobia) 3T7 fermentation liquid on a white porcelain plate, and adding an equal volume of Salkowski colorimetric solution to carry out a color reaction (the agapanthus radicans rhizobia (Rhizobium rhizobia) 3T7 treatment). 100 μ L of IAA aqueous solution (100mg/L) and an IAA-producing liquid fermentation medium without addition of bacteria were used as a positive control and a negative control, respectively. And (3) the white porcelain plate is placed for 30min at room temperature in a dark condition and then observed, and if pink appears, the strain is positive, which indicates that the strain can secrete IAA. The experiment was performed in triplicate.

The qualitative detection result shows that after Salkowski colorimetric solution is dripped into the supernatant of the agapanthus Rhizobium (Rhizobium rhizobia) 3T7 fermentation liquor, the color of the bacterial solution turns pink (figure 5), which indicates that the agapanthus Rhizobium (Rhizobium rhizobia) 3T7 can secrete plant growth hormone (IAA).

2. Quantitative analysis

And (3) preparing a standard curve: dissolving appropriate amount of IAA with small amount of ethanol, diluting with distilled water, preparing IAA standard solution with concentration of 0, 25, 50, 100, 200, 250mg/L, mixing with Salkowski colorimetric solution at volume ratio of 1: 1, standing at room temperature in dark place for 30min, and measuring OD of each concentration_530nm(A1: 1 mixed solution of distilled water and Salkowski colorimetric solution was used as a blank). Finally, the IAA concentration is taken as the abscissa, OD_530nmDrawing a graph for the ordinate to obtain an IAA standard curve.

Measuring the IAA concentration in the bacterial liquid: after 3T7 of the agapanthus radicans Rhizobium (rhizobiaum rhizobia) is activated, the OD of the fermentation liquor is measured after shaking culture for 48 hours at 35 ℃ and 180r/min in an IAA liquid fermentation culture medium_600nmThe mixture was centrifuged at 12000 rpm for 10min, and the supernatant was mixed with an equal volume of Salkowski colorimetric solution. Standing at room temperature in dark for 30min, and determining OD_530nmValue (the mixed solution of the IAA-producing liquid fermentation medium without bacteria and the Salkowski colorimetric solution with the same volume is used as a control). By IAA concentration and OD_530nmThe corresponding IAA concentration was calculated from the standard relationship curve of (a). And according to OD₆₀₀Value, unit IAA yield was calculated according to the formula:

unit IAA yield (mg/L) is IAA content in fermentation liquor/OD of fermentation liquor_600nmThe value is obtained.

The regression equation of the standard curve of the detected IAA content is that y is 0.013x +0.0672, the correlation coefficient R2 is 0.9934, and the OD of 3T7 of the lotus Rhizobium (Rhizobium rhizobia) after 72h culture_600nmThe value was 0.52, and the supernatant was mixed with Salkowski colorimetric solution to obtain OD_530nm0.33, the unit IAA content of 3T7 fermentation liquor of agapanthus Rhizobium (Rhizobium rhizobia) is 38.88 mg/L.

Example 4 growth promotion of plants by Lotus Rhizobium (Rhizobium rhizozaapanthis) 3T7

1. Promotion of marigold seed germination

Single colonies of Rhizobium rhizogenes (Rhizobium rhizobia) 3T7 were picked and inoculated into 250mL Erlenmeyer flasks containing 100mL of YMA liquid medium, and cultured at 30 ℃ for 48 hours. Centrifuging at 10000r/min for 5min, collecting thallus, and making OD with sterile water_600nmBacterial suspension (sterile water as blank) 0.1. Selecting plump marigold seeds, placing the seeds in a 9cm plate with filter paper laid at the bottom, placing 10 seeds in each plate, adding 10ml of bacterial suspension, adding 10ml of sterile water in negative control, and treating 3 plates each. It was placed in a 30 ℃ incubator. Tracking and observing the germination condition of marigold, recording the germination number, and analyzing the promoting effect of the test bacteria on the germination of marigold seeds.

The germination percentage is ═ 100% (number of germinated seeds on specified days/number of seeds tested).

After the cultivation for 7 days, the germination rate of the negative control seeds is 20 percent, and the germination rate of the test group seeds is 45 percent, which shows that the germination rate of the marigold seeds can be obviously improved by using the lotus Rhizobium rhizogenes (Rhizobium rhizobia apaanthus) 3T 7.

2. Promoting the growth of rice and marigold seedlings

Single colonies of Rhizobium rhizogenes (Rhizobium rhizobia) 3T7 were picked and inoculated into 250mL Erlenmeyer flasks containing 100mL of YMA liquid medium, and cultured at 30 ℃ for 48 hours. Centrifuging at 10000r/min for 5min, collecting thallus, and making OD with sterile water_600nmBacterial suspension (sterile water as blank) 0.1. Selecting rice and marigold seedlings with similar growth vigor, and transplanting the rice and marigold seedlings into flowerpots, wherein 4-5 seedlings are planted in each flowerpot. Test and control CK groups were set up with 4 wells (referred to as test groups 1-4 and control groups 1-4, respectively). The root irrigation treatment of the bacterial suspension is carried out, and 25mL of the bacterial suspension (test group) or sterile water (control group) is irrigated in each basin every week. Watering every 2-3d during the growth period of the plants. The experiments were performed in a smart greenhouse. The temperature of day and night is respectively 25 ℃/20 ℃, and the sunshine duration is 14 h.

The fresh weight and dry weight of the overground part, the plant height (the height from the stem base of the plant to the top end of the leaf), the root length, the root weight and the like were measured after 1 month of transplantation. The final experimental data are the average per pot of plants.

The rice seedling potting results are shown in table 4 and fig. 6, the fresh stem weight, the root length, the dry root and stem weight of the experimental group are respectively increased by 80.0%, 17.8%, 33.3% and 53.2% compared with the control group, the experimental result shows that the 3T7 has obvious growth promotion effect on rice seedlings.

TABLE 4 determination results of the experiment for potting of rice seedlings

Group of	Fresh weight of root/g	Fresh weight of stem/g	Root length/cm	Plant height/cm	Dry underground part weight/g	Dry weight of aerial parts/g
							Control group 1	0.27	0.94	17.20	37.00	0.19	0.47
Control group 2	0.27	1.32	16.10	40.30	0.17	0.45
							Control group 3	0.24	0.78	15.60	39.50	0.17	0.45
Control group 4	0.35	1.16	15.60	41.80	0.17	0.52
							Control group	0.28±0.05a	1.05±0.24b	16.13±0.75b	39.65±2.01a	0.18±0.01b	0.47±0.03b
Test group 1	0.36	2.27	19.30	44.60	0.27	0.89
							Test group 2	0.38	2.04	18.50	43.90	0.28	0.79
Test group 3	0.35	1.53	20.10	45.20	0.22	0.66
							Test group 4	0.31	1.71	18.10	39.20	0.19	0.55
Test group	0.35±0.03a	1.89±0.33a	19.00±0.89a	43.23±2.74a	0.24±0.04a	0.72±0.15a

Note: p is less than 0.05, and the difference of lower case numbers in the same column indicates that the difference is obvious.

As shown in Table 5 and FIG. 7, the fresh weight of roots and stems of the test group was increased by 94.7% and 34.2% respectively, compared with the fresh weight of the control group. The test result shows that the 3T7 has obvious growth promoting effect on marigold seedlings.

TABLE 5 determination results of marigold seedlings potted plant experiments

Group of	Fresh weight of root/g	Fresh weight of stem/g	Root length/cm	Stem length/cm	Root Dry weight/g	Dry weight of stem/g
							Control group 1	0.49	0.88	20.75	13.25	0.26	0.65
Control group 2	0.37	0.70	22.00	11.50	0.21	0.47
							Control group 3	0.30	0.69	23.50	12.88	0.14	0.52
Control group	0.38±0.09b	0.76±0.11b	22.08±1.38b	12.54±0.92a	0.2±0.06a	0.55±0.09a
							Test group 1	0.63	1.15	15.43	17.00	0.27	0.56
Test group 2	0.82	0.97	16.25	15.25	0.25	0.71
							Test group 3	0.77	0.94	19.33	13.83	0.31	0.72
Test group	0.74±0.10a	1.02±0.11a	17.00±2.06a	15.36±1.59a	0.28±0.03a	0.66±0.09a

Note: p is less than 0.05, and the difference of lower case numbers in the same column indicates that the difference is obvious.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences; shanghai city garden science planning research institute

<120> rhizobium with phosphorus dissolving function and garden plant growth promoting function and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1403

<212> DNA

<213> Lotus rhizosphere nodule bacteria (Rhizobium rhizobia)

<400> 1

aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gccccgcaag gggagcggca 60

gacgggtgag taacgcgtgg gaacgtaccc tttactacgg aataacccag ggaaacttgg 120

actaataccg tatgtgccct tcgggggaaa gatttatcgg taaaggatcg gcccgcgttg 180

gattagctag ttggtggggt aaaggcctac caaggcgacg atccatagct ggtctgagag 240

gatgatcagc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg 300

gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360

agggttgtaa agctctttca ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420

ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggatttactg 480

ggcgtaaagc gcacgtaggc ggatcgatca gtcaggggtg aaatcccaga gctcaactct 540

ggaactgcct ttgatactgt cgatctggag tatggaagag gtgagtggaa ttccgagtgt 600

agaggtgaaa ttcgtagata ttcggaggaa caccagtggc gaaggcggct cactggtcca 660

ttactgacgc tgaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 720

acgccgtaaa cgatgaatgt tagccgtcgg gcagtatact gttcggtggc gcagctaacg 780

cattaaacat tccgcctggg gagtacggtc gcaagattaa aactcaaagg aattgacggg 840

ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgcaga accttaccag 900

cccttgacat gcccggccag ctacagagat gtagtgttcc cttcggggac cgggacacag 960

gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1020

gcaaccctcg cccttagttg ccagcattta gttgggcact ctaaggggac tgccggtgat 1080

aagccgagag gaaggtgggg atgacgtcaa gtcctcatgg cccttacggg ctgggctaca 1140

cacgtgctac aatggtggtg acagtgggca gcgagaccgc gaggtcgagc taatctccaa 1200

aagccatctc agttcggatt gcactctgca actcgagtgc atgaagttgg aatcgctagt 1260

aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320

caccatggga gttggtttta cccgaaggta gtgtgctaac cgcaaggagg cagctaacca 1380

cggtagggtc agcgactggg gtg 1403

Claims (10)

1. Rhizobia, which is characterized in that: the rhizobia is Rhizobium rhizogenes (Rhizobium rhizobia).

2. A rhizobia bacterium according to claim 1, wherein: the Rhizobium is 3T7 of lotus Rhizobium (Rhizobium rhizobia) and the registration number of the Rhizobium in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 21507.

3. The culture of the rhizobia bacterium according to claim 1 or 2, which is obtained by culturing the rhizobia bacterium according to claim 1 in a microbial culture medium.

4. The microbial inoculum is characterized in that: the microbial inoculum comprises the rhizobia of claim 1 or 2 or/and a metabolite of the rhizobia of claim 1 or 2 or/and a culture of claim 3.

5. The microbial inoculum according to claim 4, characterized in that: the microbial inoculum has at least one of the following functions:

m1) promoting plant growth;

m2) promoting plant seed germination;

m3) secretes IAA;

m4) dissolving inorganic phosphorus.

6. The microbial inoculum of claim 5, wherein: the plant is any one of the following plants:

p1) dicotyledonous or monocotyledonous plants,

p2) plant of the subclass Chrysanthemum,

p3) plants of the order Chrysanthemum or Cyperales,

p4) Compositae plant or Gramineae plant,

p5) of a plant of the Pooideae family,

p6) Tagetes or Oryza,

p7) marigold or rice.

7. A method for producing the microbial agent according to claim 4, 5 or 6, comprising the step of obtaining the microbial agent by using the rhizobia according to claim 1 or 2 or/and the metabolite of the rhizobia according to claim 1 or 2 or/and the culture according to claim 3 as a component of the microbial agent.

8. A microbial ecological agent or biofertilizer characterized in that: the probiotic preparation or biofertilizer comprising the rhizobium of claim 1 or 2, the culture of claim 3 or/and the microbial inoculum of claim 4, 5 or 6.

9. Use of any one of the rhizobia of claim 1 or 2, the culture of claim 3, or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for:

use of N1, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for promoting germination of plant seeds;

use of N2, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for the preparation of a product for promoting germination of plant seeds;

use of N3, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for promoting plant growth;

use of N4, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for the preparation of a product for promoting plant growth;

use of N5, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 in secreting siderophores;

use of N6, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for the preparation of an IAA secreting product;

use of N7, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for solubilizing phosphorus;

use of N8, the rhizobia of claim 1 or 2, the culture of claim 3 or the inoculant of claim 4, 5 or 6 or the biofertilizer of claim 7 for the preparation of a product for solubilizing phosphorus.

10. Use according to claim 9, characterized in that: the plant is any one of the following plants:

p1) dicotyledonous or monocotyledonous plants,

p2) plant of the subclass Chrysanthemum,

p3) plants of the order Chrysanthemum or Cyperales,

p4) Compositae plant or Gramineae plant,

p5) of a plant of the Pooideae family,

p6) Tagetes or Oryza,

p7) marigold or rice.

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