CN113564086B - Rhizobium with phosphorus dissolving function and capable of promoting garden plant growth and application thereof - Google Patents

Abstract

The invention discloses rhizobia with a phosphorus dissolving function and capable of promoting the growth of garden plants and application thereof. The rhizobia is agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7, and the registration number of the rhizobia in the common microorganism center of the China general microbiological culture collection center is CGMCC No.21507. The agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 can secrete IAA, has the function of dissolving inorganic phosphorus, and can be used as a microbial organic fertilizer for improving soil fertility.

Description

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

Technical Field

The invention relates to rhizobium with a phosphorus dissolving function and a function of promoting the growth of garden plants and application thereof in the field of biotechnology.

Background

Traditionally, rhizobium species are considered leguminous endophytes and most are isolated from leguminous plant nodules. 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, especially for legume crops. In addition to having symbiotic nitrogen fixation capability, rhizobia strains can promote the growth of non-leguminous plants.

Phosphorus is one of the essential nutrient elements for plants, and phosphorus in soil exists mainly in the form of insoluble phosphates, and the utilization rate of the phosphorus by plants is usually extremely low. The phosphate-dissolving bacteria can dissolve inorganic phosphorus compounds which are not easy to be absorbed by plants in soil by generating organic acids and other substances, and has the advantages of improving the effective phosphorus content of the soil, reducing the using amount of phosphate fertilizer and promoting the growth of plants. The plant rhizosphere microorganism can also secrete plant hormone substances, promote plant root growth and improve nutrient absorption and utilization of plants.

Disclosure of Invention

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

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

The rhizobia provided by the invention is agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus).

The agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) provided by the invention can be specifically agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7, and the registration number of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) in the common microorganism center of the China general microbiological culture collection center is CGMCC No.21507, and hereinafter, the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 is called.

The agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 is gram-negative bacteria, rod-shaped and sporefree. Colonies of 3T7 on YMA plates were white, round, convex, translucent and mucinous. The 16S rRNA gene of the 3T7 rhizobia (Rhizobium rhizagapanthus) of the agapanthus rhizosphere has a DNA fragment shown as a sequence 1 in a sequence table.

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

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

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

The microbial inoculum provided by the invention contains the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 or/and the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 metabolites or/and the culture.

Among the above-mentioned bacterial agents, the bacterial agent has at least one of the following functions:

m1) promoting plant growth;

m2) promoting germination of plant seeds;

m3) IAA secretion;

m4) dissolving inorganic phosphorus.

The active ingredient of the microbial inoculum can be a metabolite of 3T7 of rhizosphere rhizobia (Rhizobium rhizagapanthus) or/and 3T7 of rhizosphere rhizobia (Rhizobium rhizagapanthus) or/and the culture, and the active ingredient of the microbial inoculum can also contain other biological ingredients or non-biological ingredients, and other active ingredients of the microbial inoculum can be determined by a person skilled in the art according to the effects of the microbial inoculum.

In the microbial inoculum, the microbial inoculum can further comprise a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier can be mineral material or biological material; the mineral material may be at least one of turf, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material can be at least one of straws, pine shells, straws, peanut shells, corn flour, bean flour, starch, turf and animal excrement of various crops; the liquid carrier may be water; in the microbial inoculum, the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 or/and the metabolite of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 can exist in the form of cultured living cells, fermentation liquor of the living cells, filtrate of cell culture or mixture of cells and filtrate. The dosage form of the microbial inoculum can be various dosage forms, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.

In order to solve the technical problems, the invention also provides a microecological preparation or a biological fertilizer.

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

Any of the following applications of the rhizobia, the culture, the microbial inoculum or the biofertilizer also fall within the scope of the invention:

n1, application in promoting plant seed germination;

n2, application in preparing a product for promoting germination of plant seeds;

n3, use in promoting plant growth;

n4, use in the preparation of a product for promoting plant growth;

n5, use in secreting IAA;

n6, use in the preparation of IAA-secreting products;

n7, use in dissolving phosphorus;

n8, in the preparation of phosphorus-dissolving products.

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

p1) dicotyledonous plants or monocotyledonous plants,

p2) a plant of the chrysanthemi subclass,

p3) a plant of the order Chrysanthemum or Cyperales,

p4) a plant of the Compositae family or of the Gramineae family,

p5) a plant of the subfamily gramineae,

p6) plants of the genus Tagetes or Oryza,

p7) Tagetes erecta or rice.

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

The method for preparing the microbial inoculum provided by the invention comprises the step of taking 3T7 of the rhizosphere rhizobia of agapanthus (Rhizobium rhizagapanthus) or/and a metabolite of 3T7 of the rhizosphere rhizobia of agapanthus (Rhizobium rhizagapanthus) or/and the culture as components of the microbial inoculum to obtain the microbial inoculum.

Herein, the metabolite of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 can be obtained from the fermentation broth of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7. The metabolite of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 can be a sterile metabolite of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 or a bacteria-containing metabolite of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7. Sterile metabolite (sterile fermentation filtrate) of the 3T7 rhizosphere bacteria (Rhizobium rhizagapanthus) of the agapanthus can be specifically prepared by culturing the 3T7 rhizosphere bacteria (Rhizobium rhizagapanthus) of the agapanthus in a liquid culture medium, filtering to remove 3T7 of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) in the liquid culture (fermentation broth) to obtain the sterile metabolite of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7. The bacterial-containing metabolite of the rhizosphere bacteria (Rhizobium rhizagapanthus) 3T7 of the agapanthus can be specifically prepared by culturing the rhizosphere bacteria (rhizosphere bacteria) 3T7 of the agapanthus in a liquid fermentation medium, collecting fermentation broth (containing rhizosphere rhizobium (Rhizobium rhizogenes) 3T7 and substances secreted into liquid culture medium), the fermentation broth is a bacteria-containing metabolite of the rhizobia (Rhizobium rhizagapanthus) 3T7 of the agapanthus.

Herein, the dissolved phosphorus may be dissolved inorganic phosphorus.

Herein, the promotion of plant growth may be promotion of rice seedling growth and/or promotion of growth of marigold seedlings. The promotion of plant seed germination may be an increase in the germination rate of marigold seeds.

The invention proves that the ratio of the diameter of the phosphate-dissolving ring to the diameter of the colony of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 on the inorganic phosphorus plate is 1.34+/-0.06 through the IAA secretion test and the phosphate-dissolving test and the seed germination test. Compared with the non-inoculated negative control group, the germination rate of the 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 rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 can secrete IAA, the unit IAA content of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 is 38.88mg/L fermentation liquid, and has the function of dissolving inorganic phosphorus, and can be used as a microbial organic fertilizer for improving soil fertility.

Preservation description

Strain name: barbate rhizosphere rhizobium

Latin name: rhizobium rhizagapanthus

Strain number: 3T7

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3

Preservation date: 2020, 12 months and 18 days

Accession numbers of the preservation center: CGMCC No.21507

Drawings

FIG. 1 is a photograph of colonies of the plant African agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7 after 3d cultivation on YMA plates.

FIG. 2 is a phylogenetic tree of agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 and related model bacteria constructed from the 16S rRNA gene sequence. The GenBank serial numbers of the strains are in brackets; in the figure, the reference strains are all standard strains of the species. Neorhizobium huautlense S02 ^T As an outer group.

FIG. 3 shows the rhizosphere Rhizobium (Rhizobium rhizagapanthus) 3T7 and the inner kindred of Rhizobium the whole genome of a species constructs a genomic BLAST distance phylogenetic tree (GBDP) tree. Wherein 3T7 is agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7.

FIG. 4 is a photograph of a 3T7. Hundred percent rhizosphere rhizobia (Rhizobium rhizagapanthus) cultured for 3 days on inorganic phosphorus medium to form a phosphate solubilizing circle.

FIG. 5 is a photograph of a qualitative measurement of the secretion of auxin by the plant rhizobia (Rhizobium rhizagapanthus) 3T7. In the figure, IAA is a positive control, R2A is a negative control, and 3T7 is treated with agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7.

FIG. 6 is a photograph of rice seedlings 1 month after transplanting. The left 4 flowerpots are used as a control group, and the right 4 flowerpots are used as a test group.

FIG. 7 is a photograph of a marigold seedling 1 month after transplanting. The left 3 flowerpots are used as a control group, and the right 3 flowerpots are used as a test group.

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 dissolution functions of the agapanthus rhizosphere bacteria, and detects the growth promotion function of the agapanthus rhizosphere bacteria through a seed germination test and a potting test.

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

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

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

1/100TSA solid medium, tryptone 0.15g, soytone 0.05g, sodium chloride 0.05g, agar 15.0g, distilled water 1.000 mL, pH 7.3+ -0.2, sterilization at 121℃for 20min.

YMA medium: 1.0g of yeast extract, 10.0g of mannitol, 0.5g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 15.0g of agar, 1.000 mL of distilled water, adjusting the pH value to 6.8-7.0 and sterilizing for 20min at 121 ℃.

YMA liquid medium: 1.0g of yeast extract, 10.0g of mannitol, 0.5g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 1.000 mL of distilled water, adjusting the pH value to 6.8-7.0 and sterilizing for 20min at 121 ℃.

Inorganic phosphorus 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, 1.000 mL of distilled water, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 30min.

IAA-producing liquid fermentation medium: yeast extract powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, potassium dihydrogen phosphate 0.03g, magnesium sulfate heptahydrate 0.024g, sodium pyruvate 0.3g, L-tryptophan 0.2g, distilled water to 1000mL, pH 7.2+ -0.2, and sterilizing at 121deg.C for 15min.

Example 1 isolation and identification of African agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7

1. Isolation of agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7

The root soil sample of agapanthus was collected at the bridge base of Shanghai city garden science planning institute (30 ° 58'n,121 ° 25' e), and was returned to the laboratory for storage at 4 ℃. Shaking off soil attached to plant roots, only reserving rhizosphere soil tightly adhered to root surfaces, weighing 2g of plant roots with rhizosphere soil, grinding in a sterile mortar, fully grinding, transferring into a 150mL conical flask filled with glass beads and 50mL of sterile water, oscillating for 30min at room temperature of 150r/min, taking 1mL of diluent, serially diluting with sterile water, taking 100 mu L of serial diluent, coating on a 1/100TSA plate, inversely culturing at 30 ℃ for 1 week, picking single colony with bamboo sticks according to physiological morphological characteristics, purifying on the plate, transferring into an inclined plane for short-term preservation at 4 ℃, transferring into a 25% glycerol tube, and storing at-80 ℃ for long term. One of the isolated and purified strains was designated 3T7.

2. Identification of agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7

2.1 morphological identification of strains

The bacterial strain 3T7 which is in the logarithmic growth phase and has stable bacterial colony size and is obtained by separation and purification in the step one is subjected to single bacterial colony state description, and the single bacterial colony state description mainly comprises bacterial colony size, color, transparency, bacterial colony surface state and bacterial colony edge state. The 3T7 was subjected to smear gram staining using a gram staining kit of soribao company according to the manufacturer's instructions, and the morphology of the cells was observed using an optical microscope.

Colonies of 3T7 on YMA plates were white, round, convex, translucent and mucinous (fig. 1). Cells were gram negative, rod-shaped, and sporulation free.

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 following the procedure described, using TIANASE TIANAMP bacterial genomic DNA extraction kit. 25. Mu.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, ddH2O 8.5. Mu.L, 2. Mu.L DNA template. The PCR amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 1min, extension at 72℃for 90s,30 cycles; final extension at 72℃for 10min. The PCR amplified product is detected by 1% agarose gel electrophoresis, the amplified fragment is about 1400bp, and after electrophoresis verification, the positive result PCR product is sent to Beijing qing biological science and technology Co., ltd for sequencing. The sequences obtained by sequencing were uploaded to Ezbiocloud (www.ezbiocloud.net/eztaxon) for sequence alignment.

The sequencing length of the 16S rRNA gene of strain 3T7 was 1,403bp, and the comparison with the EzBioCloud database revealed that strain 3T7 was 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 mesoamericanum HAMBI 3151 ^T (98.89%) has a high degree of similarity with the sequence of model bacteria of other species of the genus Rhizobium < 98.7%. Those closely related 16S rRNA gene sequences were retrieved from the EzBioCloud server and aligned using the Clustal_W program. Phylogenetic tree was constructed by proximity method (neighbor-Joining) using MEGA 7 software. The Kimura two-parameter model was used to calculate the evolutionary distance of the NJ method, and the formed developmental tree was shown in FIG. 2 when Bootstrap was 1000 times. Strains 3T7 and Rhizobium mesoamericanum are clustered in the same branch.

2.3 genomic analysis

Genomic DNA was sent to An Nuo you da gene technologies (beijing) limited and strain 3T7 was sequenced in a genomic sketch using Illumina NovaSeq 6000 sequencing system. Assembly using Unicycler v0.4.7 software gave 80 contigs with coverage of approximately 185X and N50 length of 232080bp. Genome 5.94Mb, 59.9% G+C content, and full genome average nucleotide identity (average nucleotide identity, orthoANI) analysis of strain 3T7 and Rhizobium close strains was performed using ChunLab orthologous average nucleotide identification tool (OAT) v0.93.1 software. 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. Dsmz. De/distcalc. Php) comparison.

As shown in Table 1, the OrthoANI values were 78.6-88.5% lower than the previously proposed threshold for species demarcation, and the dDDH values for 3T7 and its model strains were between 22.5-37.5% and well below the 70% species demarcation threshold, compared to other strains of Rhizobium species having the disclosed genomic sequence. The results of ANI and dDDH both support the strain 3T7 as a new species of Rhizobium.

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

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 model genome server TYGS website for alignment, and the results showed that strain 3T7 was not of any species found in TYGS database, and was a potential new species. The whole genome based taxonomic analysis showed that 3T7 was closely related to Rhizobium tibeticum and Rhizobium favelukesii and aggregated with both modes of bacteria in phylogenetic tree as shown in figure 3.

2.4 physiological and chemical Classification identification

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

The test result of 20NE shows that the nitrate reduction reaction of the strain 3T7 is positive, indole can be produced, escin and urea can be hydrolyzed, and gelatin cannot be hydrolyzed. Glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose and gluconate assimilation reaction are positive, malic acid assimilation reaction is weak positive, galactosidase is positive, gluconization is negative, arginine double hydrolase is negative, and decanoic acid, adipic acid, citric acid and phenylacetic acid assimilation reaction is negative. In the ZYM enzyme activity assay, leucine arylamidase, acid phosphatase, naphthol-AS-BI phosphohydrolase and beta-glucosidase were positive. BIOLOG results show that 3T7 can be grown using L-alanine, L-serine, glycosyl 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, formic acid, gentiobiose, melezitose, 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, 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-glucuronide, L-malic acid, acetic acid, D-glucose, D-naphthyridine, and D-glucose, and tebuxole as a source of sucrose, and potassium may be present as a source. The difference characteristics of the strain 3T7 and the related model species are shown in Table 2.

The strain 3T7 and the model strain Rhizobium ***s (Rhizobium tibeticum) CGMCC 1.7071 (=LMG 24453) have obvious differences in the following 32 physiological and biochemical characteristics: 1) Strain 3T7 is able to assimilate N-acetyl-glucosamine and gluconate,whereas the model strain CGMCC 1.7071 ^T Failure to do so; 2) The strain 3T7 extracellular zymogram does not contain beta-galactosidase and alpha-glucosidase, but CGMCC 1.7071 ^T The extracellular zymogram contains the two enzymes; 3) Strain 3T7 was able to use 18 substances of L-alanine, L-serine, glyconic 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 model strain CGMCC 1.7071 ^T The strain 3T7 cannot utilize D-melezitose, stachyose, N-acetylneuraminic acid, inosine and D-gluconic acid as carbon sources, but CGMCC 1.7071 ^T These 5 substances can be utilized as carbon sources; 4) 3T7 can grow in the presence of sodium bromate, 1% sodium lactate, vinegar bamboo peach mycin, rifamycin SV and lincomycin, and CGMCC 1.7071 ^T Cannot grow.

Strain 3T7 and model strain Rhizobium mesoamericanum HAMBI 3151 ^T There are significant differences in the physiological and biochemical characteristics of 19 items: 1) Strain 3T7 can assimilate N-acetyl-glucosamine and gluconate, can not assimilate citric acid, arginine double hydrolase is negative, HAMBI 3151 ^T Can not assimilate N-acetyl-glucosamine and gluconate, can assimilate citric acid, and has positive arginine double hydrolase; 2) The extracellular enzyme spectrum of the strain 3T7 does not contain alkaline phosphatase and alpha-glucosidase, but HAMBI 3151 ^T The extracellular zymogram contains the two enzymes; 3) Strain 3T7 was unable to use citric acid and D-melezitose as carbon, HAMBI 3151 ^T The strain 3T7 can be prepared from L-alanine, L-serine, glyconic acid, L-histamine, mucic acid, methyl pyruvate, formic acid and gentiobioseIs used as a carbon source and is used as a carbon source,HAMBI 3151 ^T cannot be utilized; 4) 3T7 can grow in the presence of lithium chloride and 1% sodium lactate, while HAMBI 3151 ^T Cannot grow, sodium butyrate inhibits 3T7 growth, but cannot inhibit HAMBI 3151 ^T And (5) growing.

Strain 3T7 and model strain Rhizobium altiplani HAMBI3664 ^T There are obvious differences in the physiological and biochemical characteristics of the following 17 items: 1) Strain 3T7 hydrolyzes urea, HAMBI3664 ^T Failure to do so;

2) N-acetyl-glucosamine assimilation reaction of Strain 3T7 was positive, model Strain HAMBI3664 ^T Is negative for the N-acetyl-glucosamine assimilation reaction;

3) Strain 3T7 has extracellular enzyme spectrum free of esterase (C4) and trypsin, while HAMBI3664 ^T The extracellular zymogram contains the two enzymes;

4) Strain 3T7 was unable to use L-galacturolactone, propionic acid, citric acid, D-melezitose, stachyose and N-acetylneuraminic acid as carbon source, model strain HAMBI3664 ^T Strain 3T7 may be HAMBI3664 using L-alanine, L-serine, glyconic acid and D-glucose-6-phosphate as carbon sources ^T Cannot be utilized;

5) 3T7 can grow in the presence of lithium chloride and sodium bromate, while HAMBI3664 ^T Failure to grow, vancomycin inhibits 3T7 growth but does not inhibit HAMBI3664 ^T And (5) growing.

TABLE 2 characterization of the differences between Strain 3T7 and the related model species

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

Cells from strain 3T7 and 3 closely related bacteria were assayed using the shelf microorganism identification system (MIDI). The MIDI analysis results are shown in Table 2, the main fatty acid (> 5%) of strain 3T7 is of the type 8 (C _18：1 Omega 6C and/or C _18：1 Omega 7C), type 2 feature (C _12：0 Aldehyde and/or unknown fatty acid of equivalent carbon chain length 10.9525), C _16：0 、C _18：0 And C _19：0 Cycloω8c. As a result, the support strain 3T7 was rhizobium. The subtle differences in fatty acid profile between the four strains are shown in table 3.

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

Fatty acid	3T7	CGMCC 1.7071	HAMBI 3664	HAMBI 3151
					C 16：0	7.63	9.33	7.47	4.84
C 15：0 -2OH	0.79	1.35	1.59	1.66
					C 17：0 cyclo	0.66	1.04	0.50	0.54
C 16：0 -3OH	1.74	1.70	1.38	1.19
					C 18：0	5.64	6.51	8.83	6.97
11-methyl C 18：1 ω7c	0.22	1.15	0.56	0.74
					C 19：0 cycloω8c	14.60	20.03	18.52	16.37
C 18：0 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 that: the values shown are percentages of total fatty acids. -: no detection was made, the type of feature was a mixture of two or three fatty acids which were not separable by GLC by the MIDI system, type 2 included C _14：0 3OH and/or C _16：1 iso I, type 3 features include C _16：1 Omega 6C and/or C _16：1 Omega 7C, type 8 feature includes C _18：1 Omega 6C and/or C _18：1 ω7c。

According to homology comparison, the construction of phylogenetic tree, morphological, physiological and biochemical identification, fatty acid composition and genome analysis results are combined to determine that the strain 3T7 is a new species of Rhizobium, the suggested classification is Rhizobium rhizagapanthus, and the Chinese name is agapanthus rhizobia. The 3T7 of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) is preserved in China general microbiological culture collection center (CGMCC) with the address of CGMCC No. 1 and 3 of North Xicilu No. 1 in the Korean region of Beijing city at the 12 th month and 18 days of 2020, and the preservation number is CGMCC No.21507. Hereinafter, it is abbreviated as agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7.

Example 2 detection of phosphorus-solubilizing Capacity of African agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7

Inoculating 10 μl of 3T7 strain solution of rhizosphere rhizobium (Rhizobium rhizagapanthus) of BAIZILIANZI onto inorganic phosphorus culture medium, culturing at 30deg.C for 4d, observing the presence or absence of phosphate-dissolving ring, and measuring diameter (d) ₂ ) And colony diameter (d) ₁ )，Calculating the diameter ratio (d) of the phosphate solubilizing ring and the colony ₂ /d ₁ ). The larger the ratio is, the stronger the phosphorus dissolving capability is. The results showed that the ratio of phosphate solubilizing circle to colony diameter of 3T7 of agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) was 1.34.+ -. 0.06 (FIG. 4) after 4d growth on inorganic phosphorus medium. Indicating that the agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7 can dissolve inorganic phosphorus.

EXAMPLE 3 qualitative detection and quantitative analysis of secreted auxin of the plant rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7

1. Qualitative detection

3T7 of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) is activated and then inoculated into an IAA-producing liquid fermentation medium, shaking culture at 35deg.C and 180r/min for 2d, centrifuging at 12,000 rpm for 10min, collecting 100 μl supernatant of 3T7 fermentation broth of BAIZILIANGENERATE (Rhizobium rhizagapanthus) on a white porcelain plate, an equal volume of Salkowski colorimetric solution was added for the color reaction (African agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 treatment). As positive and negative controls, 100. Mu.L of IAA aqueous solution (100 mg/L) and IAA-producing liquid fermentation medium without adding bacteria, respectively, were used. White porcelain plates are placed at room temperature for 30min under the dark condition and then observed, and if pink appears, the white porcelain plates are positive, which indicates that the strain can secrete IAA. The experiment was repeated three times.

Qualitative detection results show that after Salkowski colorimetric solution is dripped into supernatant of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 fermentation liquid, the bacterial liquid turns pink (fig. 5), which shows that the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) 3T7 can secrete plant growth hormone (IAA).

2. Quantitative analysis

And (3) manufacturing a standard curve: dissolving IAA in 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 for 30min, and measuring OD of each concentration _530nm (1:1 mixture of distilled water and Salkowski color solution is used as blank control). Finally, take IAA concentration as abscissa, OD _530nm In order to make a graph on the ordinate,obtaining the IAA standard curve.

Determination of IAA concentration in bacterial liquid: 3T7 activation of rhizobia (Rhizobium rhizagapanthus) of agapanthus, shake culture at 35deg.C and 180r/min in IAA production liquid fermentation medium for 48 hr, and measuring OD of fermentation liquid _600nm The values were centrifuged at 12 rpm for 10min and the supernatant was mixed with an equal volume of Salkowski broth. Standing at room temperature in dark place for 30min, and measuring OD _530nm Values (control with mixed solution of non-inoculated IAA-producing liquid fermentation medium and equal volume of Salkowski broth). By IAA concentration and OD _530nm The corresponding IAA concentration was calculated based on the standard relationship curve. And according to OD ₆₀₀ Value, unit IAA yield calculated according to the formula:

unit IAA yield (mg/L) =iaa content in broth/broth OD _600nm Values.

The regression equation of the standard curve of the IAA content is that y=0.0135x+0.0672, the correlation coefficient R2= 0.9934, and the OD of 3T7 of the agapanthus rhizobia (Rhizobium rhizagapanthus) after 72 hours of culture _600nm The value was 0.52, and the supernatant was mixed with Salkowski color solution OD _530nm The unit IAA content of the 3T7 fermentation broth of the agapanthus rhizobia (Rhizobium rhizagapanthus) is 38.88mg/L.

Example 4 plant growth promotion by African agapanthus rhizobia (Rhizobium rhizagapanthus) 3T7

1. Promoting germination of marigold seeds

A single colony of 3T7 rhizobia (Rhizobium rhizagapanthus) of agapanthus was inoculated into a 250mL conical flask containing 100mL YMA liquid medium and cultured at 30℃for 48h. Centrifuging at 10000r/min for 5min, collecting thallus, and preparing OD with sterile water _600nm Bacterial suspension=0.1 (sterile water as a blank). The large and full marigold seeds are selected and placed in 9cm plates with filter paper laid at the bottom, 10 seeds are placed on each plate, 10ml of bacterial suspension is added, 10ml of sterile water is added to negative control, and 3 plates are treated each. It was placed in a constant temperature incubator at 30 ℃. Tracking and observing the germination condition of the marigold, recording the germination number, and analyzing the promotion effect of the test bacteria on the germination of the marigold seeds.

Percent germination = (number of germinated seeds per number of tested seeds on specified days) ×100%.

After 7d of culture, the germination rate of the negative control seeds is 20%, and the germination rate of the test group seeds is 45%, which shows that 3T7 of the agapanthus rhizosphere rhizobia (Rhizobium rhizagapanthus) can obviously improve the germination rate of marigold seeds.

2. Promoting growth of rice and marigold seedlings

A single colony of 3T7 rhizobia (Rhizobium rhizagapanthus) of agapanthus was inoculated into a 250mL conical flask containing 100mL YMA liquid medium and cultured at 30℃for 48h. Centrifuging at 10000r/min for 5min, collecting thallus, and preparing OD with sterile water _600nm Bacterial suspension=0.1 (sterile water as a blank). Selecting rice and marigold seedlings with similar growth vigor, and transplanting the seedlings into flowerpots, wherein each pot is 4-5 plants. Test and control CK groups were each set with 4 pots (referred to as test groups 1-4 and control groups 1-4, respectively). The bacterial suspension was root irrigated with 25mL of bacterial suspension (test group) or sterile water (control group) per basin per week. The plants were watered every 2-3d during growth. Experiments were performed in a smart greenhouse. The day and night temperatures are 25 ℃/20 ℃ and the sunlight time is 14 hours respectively.

The fresh and dry weight of the aerial parts, plant height (height from the plant stem base to the leaf tip), root length, root weight, etc. were measured after 1 month of transplanting. The final experimental data are the average of each pot of plants.

The potting results of the rice seedlings are shown in table 4 and fig. 6, and compared with the control group, the fresh weight, root length, root and dry weight of the experimental group are respectively increased by 80.0%, 17.8%, 33.3% and 53.2%, which are obviously superior to those of the control group, and the experimental results show that the 3T7 has obvious growth promoting effect on the rice seedlings.

TABLE 4 determination of the potted rice seedlings

Group of	Fresh weight/g of root	Fresh weight of stem/g	Root length/cm	Plant height/cm	Dry weight/g of underground part	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 that: p is less than 0.05, and the difference is obvious in the same-column small-written number.

The pot culture results of marigold seedlings are shown in table 5 and fig. 7, and the fresh weight of roots and stems of the test group is improved by 94.7% and 34.2% respectively compared with that of the control group. The test result shows that 3T7 has obvious growth promoting effect on marigold seedlings.

TABLE 5 determination of pot experiments of marigold seedlings

Group of	Fresh weight/g of root	Fresh weight of stem/g	Root length/cm	Stem length/cm	Dry weight of root/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 that: p is less than 0.05, and the difference is obvious in the same-column small-written number.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of agricultural resource and agricultural division of national academy of agricultural science; shanghai city garden science planning institute

<120> rhizobium having phosphorus dissolving function and promoting growth of garden plants and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1403

<212> DNA

<213> African agapanthus rhizobia (Rhizobium rhizagapanthus)

<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 (9)

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

rhizagappanchus) 3T7 with accession number CGMCC No.21507 at the general microbiological center of the chinese microbiological bacterial culture management committee.

2. The microbial inoculum is characterized in that: the microbial agent comprises the rhizobia of claim 1.

3. The microbial agent of claim 2, wherein: the microbial inoculum has at least one of the following functions:

m1) promoting plant growth, said plant being marigold or rice;

m2) promoting germination of seeds of a plant, said plant being marigold;

m3) IAA secretion;

m4) dissolving inorganic phosphorus.

4. A process for preparing the microbial agent of claim 2 or 3, comprising the step of obtaining the microbial agent by using the rhizobia of claim 1 as a component of the microbial agent.

5. The microecological preparation is characterized in that: the microbial preparation contains the rhizobia of claim 1 and/or the microbial agent of claim 2 or 3.

6. The biological fertilizer is characterized in that: the biofertilizer contains the rhizobia of claim 1 and/or the microbial inoculum of claim 2 or 3.

7. Use of the rhizobia of claim 1 for any one of the following:

n1, the use in promoting germination of seeds of a plant, said plant being marigold;

n2, in the preparation of a product for promoting germination of seeds of a plant, said plant being marigold;

n3, application in promoting plant growth, wherein the plant is marigold or rice;

n4, application in preparing a product for promoting plant growth, wherein the plant is marigold or rice;

n5, use in dissolving phosphorus;

n6, in the preparation of phosphorus-dissolved products.

8. Use of the microbial agent of claim 2 or 3 for any of the following:

n1, the use in promoting germination of seeds of a plant, said plant being marigold;

n2, in the preparation of a product for promoting germination of seeds of a plant, said plant being marigold;

n3, application in promoting plant growth, wherein the plant is marigold or rice;

n4, application in preparing a product for promoting plant growth, wherein the plant is marigold or rice;

n5, use in dissolving phosphorus;

n6, in the preparation of phosphorus-dissolved products.

9. Use of the biofertilizer of claim 6 for any of the following:

n1, the use in promoting germination of seeds of a plant, said plant being marigold;

n2, in the preparation of a product for promoting germination of seeds of a plant, said plant being marigold;

n3, application in promoting plant growth, wherein the plant is marigold or rice;

n4, application in preparing a product for promoting plant growth, wherein the plant is marigold or rice;

n5, use in dissolving phosphorus;

n6, in the preparation of phosphorus-dissolved products.