CN113604399B - Sphingolipid bacteria with growth promoting function of garden plants and application thereof - Google Patents

Abstract

The invention discloses sphingolipid bacteria with a growth promoting function of garden plants and application thereof. The sphingolipid bacteria is sphingolipid bacteria (Sphingobium jiangnanensis) 3R8, and the registration number of the sphingolipid bacteria in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.21506. The unit IAA content of the sphingolipid bacteria 3R8 in south China is 458.66mg/L fermentation liquor, and the ratio of the phosphorus-dissolving ring to the colony diameter on an inorganic phosphorus plate is 1.34 +/-0.14. Compared with a negative control group which is not inoculated, the germination rate of marigold seeds and the growth of rice seedlings can be obviously improved, and the germination rate of marigold seeds is improved by 2 times. This indicates that sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 can secrete auxin IAA, has a phosphorus dissolving function, and can be used as a microbial organic fertilizer to improve soil fertility.

Description

Sphingolipid bacteria with growth promoting function of garden plants and application thereof

Technical Field

The invention relates to sphingolipid bacteria with a growth promoting function of garden plants and application thereof.

Background

The plant rhizosphere harbors a wide variety of microbial communities that play a key role in the growth and reproduction of plants. Some rhizosphere microorganisms can regulate the growth of plants and the flowering time of the plants by regulating nitrogen circulation and plant hormones (such as auxin and the like); the biological fertilizer has the functions of degrading organic pollutants, enriching heavy metals, reducing the toxicity of the heavy metals in soil, improving the soil pore condition, improving the soil fertility and improving the soil biological properties, can biologically control pathogenic bacteria, and improves the growth adaptability of landscape plants in saline-alkali and barren soil environments and special urban spaces. The development and utilization of microbial resources can promote the growth of landscape plants, improve the health quality of urban green land soil and have wide development prospect.

Sphingobacteriaceae (Sphingobacteriaceae) has been isolated from various environments such as soil, fresh water, groundwater, the ocean, plant surfaces, rhizosphere, and even extreme environments such as contaminated areas, arctic and antarctic soils, deeply buried sediments, and soil surfaces in arid areas. In previous studies, members of the genus sphingolipid (Sphingobium) have been reported to degrade a variety of natural and synthetic aromatic compounds, such as Polycyclic Aromatic Hydrocarbons (PAHs), lignin-derived aromatic compounds, heterocyclic aromatic hydrocarbons, chlorinated aromatic sulfonated aromatic hydrocarbons, azo compounds, and pesticides and herbicides, among others. And sphingolipid bacteria have been less studied for their growth-promoting function.

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.

In order to solve the technical problems, the invention firstly provides sphingolipid bacteria.

The sphingolipid bacteria provided by the invention are sphingolipid bacteria (Sphingobium jiangnanensis) in south of the Yangtze river.

The sphingolipid bacteria (Sphingobium jiangnanensis) provided by the invention can be sphingolipid bacteria (Sphingobium jiangnanensis) 3R8, the registration number of which in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.21506, and hereinafter referred to as sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 for short.

Sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 is a gram-negative bacterium, rod-shaped and no spore formation. Sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 has 16S rRNA gene shown in sequence 1 in the sequence table.

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

The culture of sphingolipid bacteria provided by the invention is a substance obtained by culturing sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 in a microorganism culture medium (namely, a fermentation product, such as a fermentation broth containing sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 and a substance secreted into a liquid culture medium, or a solid fermentation product containing sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 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 sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 or/and a metabolite of sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 or/and the culture.

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

m1) promoting plant growth;

m2) promoting the germination of plant seeds;

m3) producing auxin IAA;

m4) dissolving phosphorus.

Herein, the metabolite of the sphingolipid of south China (Sphingobium jiangnanensis) 3R8 can be obtained from a fermentation broth of the sphingolipid of south China (Sphingobium jiangnanensis) 3R8. The metabolite of the sphingolipid of south China (Sphingobium jiangnanensis) 3R8 can be a sterile metabolite of the sphingolipid of south China (Sphingobium jiangnanensis) 3R8 or a bacteria-containing metabolite of the sphingolipid of south China (Sphingobium jiangnanensis) 3R8. Specifically, the sterile metabolite (sterile fermentation filtrate) of the sphingolipid bacteria Jiangnanensis (Sphingobium jiangnanensis) 3R8 can be prepared by culturing the sphingolipid bacteria Jiangnanensis (Sphingobium jiangnanensis) 3R8 in a liquid culture medium, and filtering to remove the sphingolipid bacteria Jiangnanensis (Sphingobium jiangnanensis) 3R8 in the liquid culture medium (fermentation liquid) to obtain the sterile metabolite of the sphingolipid bacteria Jiangnanensis (Sphingobium jiangnanensis) 3R8. Specifically, the bacterial-containing metabolite of sphingolipid of south China (Sphingobium jiangnanensis) 3R8 can be prepared by culturing sphingolipid of south China (Sphingobium jiangnanensis) 3R8 in a liquid fermentation medium, and collecting a fermentation broth (containing sphingolipid of south China (Sphingobium jiangnanensis) 3R8 and a substance secreted into the liquid culture medium), i.e., the bacterial-containing metabolite of sphingolipid of south China (Sphingobium jiangnanensis) 3R8.

The active ingredient of the microbial inoculum can be sphingolipid of south China (Sphingobium jiangnanensis) 3R8 or/and sphingolipid of south China (Sphingobium jiangnanensis) 3R8 metabolite or/and the culture, and the active ingredient of the microbial inoculum can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by the technicians in the field according to the effects of the microbial inoculum.

In the above bacterial agent, the bacterial agent 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, sphingolipid (Sphingobium jiangnanensis) 3R8 and/or a metabolite of sphingolipid (Sphingobium jiangnanensis) 3R8 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 needs, 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 microecological preparation or the biological fertilizer provided by the invention contains the sphingolipid bacteria, the culture or/and the microbial inoculum, and the product can be a microbial inoculum, a microecological preparation or a biological fertilizer.

Any one of the following applications of the sphingolipid bacteria, the culture or the microbial inoculum or the biological fertilizer also belongs to the protection scope of the invention:

n1, application in promoting plant seed germination;

n2, and application in preparation of products for promoting plant seed germination;

n3, application in promoting plant growth;

n4, application in preparation of products for promoting plant growth;

n5, application in the production of auxin IAA;

n6, application in preparation of products for producing auxin IAA;

n7, application in dissolving phosphorus;

n8 and application in preparing a product for dissolving phosphorus.

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

p1) dicotyledonous plants or monocotyledonous plants,

p2) plants of the subclass Chrysoideae,

p3) plants of the order Chrysanthemum or Cyperales,

p4) Compositae plants or Gramineae plants,

p5) a plant of the Pooideae family,

p6) plants of the genus Tagetes or plants of the genus 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 the sphingolipid Jiangnathus (Sphingobium jiangnanensis) 3R8 or/and the sphingolipid Jiangnathus (Sphingobium jiangnanensis) 3R8 metabolite or/and the culture as the components of the microbial inoculum to obtain the microbial inoculum.

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

Through IAA secretion test, phosphorus dissolving test and seed germination test, the unit IAA content of sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 is 458.66mg/L fermentation liquor, and the ratio of phosphorus dissolving ring to colony diameter on an inorganic phosphorus plate is 1.34 +/-0.14. Compared with a negative control group which is not inoculated, the germination rate of marigold seeds and the growth of rice seedlings can be obviously improved, and the germination rate of marigold seeds is improved by 2 times. This indicates that sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 can secrete auxin IAA, has a phosphorus dissolving function, and can be used as a microbial organic fertilizer to improve soil fertility.

Deposit description

The strain name is as follows: sphingolipid bacteria of south China

Latin name: sphingobium jiangnanensis

The strain number is as follows: 3R8

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.21506

Drawings

FIG. 1 is a photograph showing the colonies of Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 cultured on an R2A plate for 2 days.

FIG. 2 is a phylogenetic tree of sphingolipid of south China (Sphingobium jiangnanensis) 3R8 and related model bacteria constructed based on 16S rRNA gene sequences. Wherein 3R8 is sphingolipid bacteria (Sphingobium jiangnanensis) 3R8.

FIG. 3 is a phylogenetic tree of sphingolipid of south China (Sphingobium jiangnanensis) 3R8 and the genome of related model strain of Sphingobium. Wherein 3R8 is sphingolipid bacteria (Sphingobium jiangnanensis) 3R8.

FIG. 4 is a photograph showing qualitative detection of auxin secretion from Sphingobacterium jiang (Sphingobium jiangnanensis) 3R8. In the figure, the first row represents a positive control, the second row represents a negative control, and the third row represents a sphingolipid (Sphingobium jiangnanensis) 3R8 treatment.

FIG. 5 is a photograph showing the generation of phosphate solubilizing circles by growing 3d of Sphingobacterium jiang (Sphingobium jiangnanensis) 3R8 on an inorganic phosphate detection plate (inorganic phosphate medium).

FIG. 6 is a photograph of one month after transplanting of rice seedlings. The left flowerpot was the control group and the right flowerpot was the test group.

FIG. 7 is a photograph of marigold seedlings after 30d of transplantation. The left flowerpot is a test group, and the right flowerpot is a control group.

Detailed Description

The research adopts a multiphase classification method to determine the classification status of the 3R8 rhizosphere bacteria of the agapanthus, researches the functions of the agapanthus on IAA secretion and phosphorus dissolution, and detects the growth promoting function of the agapanthus through a seed germination test. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

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.

The media configuration used in the following examples was as follows:

1/50R2A solid culture medium comprises 0.01g of yeast extract powder, 0.01g of peptone, 0.01g of casein hydrolysate, 0.01g of glucose, 0.01g of soluble starch, 0.0006g of potassium dihydrogen phosphate, 0.00048g of magnesium sulfate heptahydrate, 0.006g of sodium pyruvate and 15g of agar, distilled water is used for diluting to 1000mL, the pH value is adjusted to 7.2 +/-0.2, and sterilization is carried out at 121 ℃ for 15min.

R2A liquid 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, 0.3g of monopotassium phosphate, 0.024g of magnesium sulfate heptahydrate and 0.3g of sodium pyruvate, wherein the volume is adjusted to 1000mL by using distilled water, the pH value is adjusted to 7.2 +/-0.2, and the mixture is sterilized at 121 ℃ for 15min.

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, 0.3g of monopotassium phosphate, 0.024g of magnesium sulfate heptahydrate, 0.3g of sodium pyruvate and 0.2g of L-tryptophan, wherein the volume is set to 1000mL by using distilled water, the pH value is adjusted to 7.2 +/-0.2, and the mixture is sterilized at 121 ℃ for 15min.

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 15g of agar, wherein the volume is fixed to 1000mL by using distilled water, the pH value is adjusted to 7.0-7.2, and the sterilization is carried out for 30min at 121 ℃.

Example 1 isolation and identification of Strain 3R8

1. Isolation of Strain 3R8

African agapanthus rhizosphere soil samples were collected from Shanghai city garden science planning institute Wu bridge foundation ground (30 ° 58'N,121 ° 25' E) and brought back to 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 a sterile mortar, fully grinding the plant roots, transferring the ground plant roots into a 150mL conical flask filled with glass beads and 50mL of sterile water, oscillating the plant roots at room temperature of 150R/min for 30min, taking 1mL of diluent, performing serial dilution by using the sterile water, taking 100 mu L of serial diluent, coating the serial diluent on a 1/50R2A flat plate, performing inversion culture at 30 ℃ for 1 week, picking a single colony by using a bamboo stick according to the characteristics of physiological morphology, purifying the single colony on the flat plate, transferring the single colony on a slope after determining that the single colony is pure bacteria, storing the single colony at 4 ℃ for a short time, transferring the single colony to a 25% glycerol tube, and storing the single colony at-80 ℃ for a long time. One of the isolated and purified strains was named strain 3R8.

2. Identification of Strain 3R8

2.1 morphological identification of the Strain

And (3) performing single colony state description on the strain 3R8 which is in a logarithmic growth phase and has stable colony size and is obtained by separation and purification in the first step, wherein the single colony state description mainly comprises the size, color, transparency, colony surface state and colony edge state of the colony. Bacterial strain 3R8 was subjected to smear gram staining using a gras staining kit of solibao according to the manufacturer's instructions, and the morphology of the cells was observed with an optical microscope.

The results show that after 48 hours of R2A plate culture, the colony of the strain 3R8 is yellow, 2-3mm in diameter, convex, round and smooth, and complete in edge (FIG. 1); the strain 3R8 cells are gram-negative, rod-shaped and have no sporulation.

2.2 molecular characterization

Operating according to the specification, usingGenomic DNA was extracted using a Tiangen company TIANAmp bacterial genomic DNA extraction kit, and 16S rRNA gene amplification was performed using bacterial universal primers 27F (5. 25 μ L PCR amplification system: 2 × Taq PCR Mix 12.5 μ L,27F (10 μmol/L) 1 μ L,1492R (10 μmol/L) 1 μ L, ddH ₂ O8.5. Mu.L, 2. Mu.L of 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, and 30 cycles; final extension at 72 ℃ for 10min. 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 sequencing length of the 16S rRNA gene of strain 3R8 was 1,387bp, and the sequence alignment with EzBioCloud showed that strain 3R8 was a member of the genus Sphingobium, and that the sequences of Sphingobium limnetium DSM25076 (T) (97.84%), sphingobium vermicompostis DSM21299 (T) (97.04%) were highly similar, and < 97% to the sequence of other species of the genus Sphingobium. The strain 3R8 can be preliminarily determined to be a new species in the Sphingobium genus. 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 Bootstrap value was 1000 times, and the developed tree was shown in FIG. 2. The strain 3R8 and Sphingobium limneticum are gathered in the same branch.

2.3 genomic analysis

Genomic DNA was sent to the anovulda gene technology ltd and the draft genome of strain 3R8 was sequenced using the Illumina NovaSeq 6000 sequencing system. Assembly using Unicycler v0.4.7 yielded 81 contigs with a coverage of about 232 x and an N50 length of 157538bp. The genome 4.73Mb, G + C content was 64.0mol%, and the Average Nucleotide Identity (ANI) was calculated on the http:// jspecies. Ribohost. Com/jspecies sws/# analysis website. Digital DNA-DNA hybridization (dDDH) values between strain 3R8 and the reference strain were calculated by genome-to-genome sequence comparison using a genome-to-genome distance calculator (GGDC) 2.0 server (http:// ggdc.dsmz.de/distcalac.php). Based on the similarity of the 16S rRNA gene sequences of strain 3R8 and phylogenetic analyses, sphingobium limneticum DSM25076 (T) and Sphingobium vermicompositi DSM21299 (T) were selected as reference strains for physiological testing and chemical taxonomic analyses.

The ANI values ranged from 77.08-88.79% compared to other strains of the Sphingobium species with published genomic sequences, below the 95-96% cutoff previously proposed for species demarcation, and the dDDH values for 3R8 and its model strains ranged between 21.3-41.1%, well below the 70% species demarcation threshold. Both ANI and dDDH results support strain 3R8 as a new species of Sphingobium.

TABLE 1 Strain 3R8 and its cognate strains ANI and dDDH values

3R8	ANI(％)	dDDH(％)
			Sphingobium cupriresistens	88.79	41.1
Sphingobium paulinellae	83.51	28.9
			Sphingobium limneticum	83.51	28.9
Sphingobium algicola	83.50	28.9
			Sphingobium terrigena	80.74	24.9
Sphingobium xenophagum	80.01	24.3
			Sphingobium hydrophobicum	79.97	24.2
Sphingobium yanoikuyae	79.69	24.2
			Sphingobium amiense	79.34	24.1
Sphingobium indicum	78.72	23.3
			Sphingobium francense	78.54	23.3
Sphingobium chlorophenolicum	78.27	23.1
			Sphingobium aromaticivastans	78.15	22.4
Sphingobium vermicomposti	77.08	21.3

To further investigate the relationship of strain 3R8 to related species of the genus Sphingobium, phylogenetic trees were reconstructed using the latest bacterial core gene set (UBCG) pipeline. A phylogenetic tree based on the 92 bacterial core gene sequences showed that 3R8 forms a unique phylogenetic lineage in the genus Sphingobium (fig. 3), indicating that strain 3R8 should belong to a new species of the genus Sphingobium.

2.4 physiological and chemical Classification characterization

Oxidase and catalase activities were measured by French Merrier (API) oxidase assay kit and H at 3% (v/v), respectively ₂ O ₂ The generation of bubbles in the solution. The physiological properties of strain 3R8 and related model strains were determined using the API 20NE and API ZYM kit (bioMerieux) of Meriella France. GEN III MicroPlates from bilog corporation, usa were used to determine the oxidation of carbon sources and the sensitivity to inhibitory compounds.

The results showed that the strain 3R8 was catalase negative and oxidase positive. The 20NE test results showed that the nitrate reduction reaction of the strain 3R8 was negative, indole-producing was not performed, escin was hydrolyzed, galactosidase-positive, glucose, arabinose, maltose, malic acid and citric acid assimilation reaction was positive, glucose fermentation was negative, arginine-di-hydrolase was negative, urea and gelatin were not hydrolyzed, mannose, mannitol, N-acetyl-glucosamine, sodium gluconate, capric acid, adipic acid and phenylacetic acid assimilation reaction was negative, the enzyme-like enzymes (C4), lipase-like enzymes (C8), leucine arylamine, valine arylamine, chymotrypsin, beta-galactosidase, beta-uronidase, acid and alkaline phosphatase, naphthol-AS-BI phosphohydrolase, alpha-glucosidase and beta-glucosidase-positive, lipase-like enzymes (C14), cystine arylamine, trypsin, alpha-galactosidase, alpha-mannosidase and beta-fucosidase were negative. Glucan, D-cellobiose, D-turanose, beta-formyl-D-glucoside, D-salicin, alpha-D-glucose, D-galactose, D-trehalose, L-rhamnose, D-sorbitol, D-glucose-6-phosphate, aminoacetyl-L-proline, L-aspartic acid, L-glutamic acid, L-galactonic acid, glucuronic acid, quinic acid, alpha-keto-glutaric acid, L-hydroxysuccinic acid, bromo-succinic acid, beta-hydroxy-D, L-butyric acid, acetoacetic acid, acetic acid and formic acid can be utilized as carbon sources and can be grown in the presence of 1% sodium lactate, rifamycin SV, lincomycin, tetrazolium blue, naphthyridine, potassium tellurite and aztreonam. The difference between the strain 3R8 and the related model strain is shown in Table 2.

The results show that the strain 3R8 and the model strain Sphingobium vermicompositi DSM21299 have obvious differences in the following 33 physiological and biochemical characteristics:

1) The strain 3R8 can assimilate arabinose, maltose, malic acid and citric acid, and the model strain Sphingobium vermicompostis DSM21299 cannot;

2) The extracellular zymogram of the strain 3R8 contains chymotrypsin, beta-uronic acid glycosidase and alpha-glucosidase, does not contain trypsin, and the extracellular zymogram of the model strain Sphingobium vermicompostis DSM21299 does not contain chymotrypsin, beta-uronic acid glycosidase and alpha-glucosidase and contains trypsin;

3) Strain 3R8 can utilize glucan, D-cellobiose, D-glucose-6-phosphate, aminoacetyl-L-proline, L-aspartic acid, quinic acid, β -formyl-D-glucoside, D-salicin, D-sorbitol, α -keto-glutaric acid, L-galactonic acid, L-hydroxysuccinic acid, formic acid as carbon sources, the model strain Sphingobium vermicompositi DSM21299 cannot utilize these substances, strain 3R8 cannot utilize α -D-lactose, L-pyroglutamic acid, D-fructose, 3-formyl-glucose, D-fructose-6-phosphate, galacturonic acid and glucuronamide as carbon sources, the model strain Sphingobium vermicompostis DSM21299 can utilize;

4) In the presence of 1% sodium lactate, blue tetrazolium, aztreonam and potassium tellurite, the strain 3R8 was able to grow, while the model strain Sphingobium vermicompositi DSM21299 was not able to grow, and the tetrazole violet was able to inhibit the growth of the strain 3R8 and was not able to inhibit the growth of the model strain Sphingobium vermicompositi DSM 21299.

The strain 3R8 and the model strain Sphingobium limneticum DSM25076 (T) have obvious differences in the following 21 physiological and biochemical characteristics:

1) The strain 3R8 can assimilate citric acid and maltose, and the model strain Sphingobium limneticum DSM25076 (T) can not assimilate citric acid and maltose;

2) The extracellular zymogram of the strain 3R8 contains chymotrypsin, beta-uronic acid glycosidase and beta-galactosidase, and does not contain trypsin, and the extracellular zymogram of the model strain Sphingobacterium limneticum DSM25076 (T) contains trypsin, and does not contain chymotrypsin, beta-uronic acid glycosidase or beta-galactosidase;

3) Strain 3R8 cannot utilize gentiobiose, D-trehalose, D-melibiose, L-serine, L-alanine, galacturonic acid and glucuronamide as carbon sources, the model strain Sphingobium limneticum DSM25076 (T) can utilize these substances, strain 3R8 can utilize D-sorbitol and α -keto-glutaric acid as carbon sources, and the model strain Sphingobium limnetium DSM25076 (T) cannot;

4) In the presence of rifamycin SV and potassium tellurite, strain 3R8 grew, whereas the model strain Sphingobium limneticum DSM25076 (T) did not, oleandomycin, minocycline, D-serine and tetrazole violet inhibited strain 3R8 growth and did not inhibit the model strain Sphingobium limneticum DSM25076 (T) growth.

TABLE 2 Difference characteristics between Strain 3R8 and related model strains

Note: +, positive or available; negative or not available; w, weak positive.

The fatty acid profile of strain 3R8 and 2 closely related bacteria was determined using cells grown on R2A agar at 30 ℃ for 2 days, and the fatty acid methyl esters obtained from cells grown on R2A at 30 ℃ for 2 days were analyzed using Sherlock microbial identification System (MIDI). The results of MIDI analysis are shown in Table 3, the main fatty acids of strain 3R 8: (A), (B)>5%) is the 8 th feature type (C) _18:1 Omega 6C and/or C _18:1 ω 7C), type 3 feature (C) _16:1 Omega 6C and/or C _16:1 ω7c)、C _16:0 、C _17:1 Omega 6C and C _14:0 2OH, the results show that the fatty acid components of the strain 3R8 and the closely related bacteria Sphingobium limneticum DSM25076 (T) and Sphingobium vermicompositi DSM21299 (T) are basically the same, but the content is greatly different.

TABLE 3 cellular fatty acid composition of Strain 3R8 and its type strains

Note: the values shown are the percentage of total fatty acids. tr, trace of (<1%). The characteristic type is a mixture of two or three fatty acids which cannot be separated by GLC in MIDI system, and the 3 rd characteristic type comprises C _16:1 Omega 6C and/or C _16:1 ω 7C, characteristic type 8 includes C _18:1 Omega 6C and/or C _18:1 ω7c。

In conclusion, the similarity of the strain 3R8 to the 16S rRNA of Sphingobium limneticum 301 (T) is 97.84%, the similarity to the 16S rRNA of Sphingobium vermicompostin VC-230 (T) is 97.04%, and the sequence similarity to the 16S rRNA of other species of Sphingobium is less than 97%; compared with the strains of Sphingobium species, the ANI value of the strain 3R8 is 77.08-88.79%, and is lower than the critical value which is previously proposed for species demarcation by 95-96%, and the dDDH value of the strain 3R8 and the model strain thereof is 21.3-41.1%, and is far lower than the species demarcation threshold of 70%; and the physiological and biochemical characteristics and the fatty acid content are greatly different, so that the strain 3R8 can be determined to be a new species of Sphingobium, named Sphingobium jiang nanensis, wherein the name is sphingolipid bacteria in south China. The sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 has been preserved in China general microbiological culture Collection center (CGMCC for short, no. 3 of West Lu 1 of Beijing Kogyo area of Chaoyang) within 18 days 12.2020, and the preservation number is CGMCC No.21506. Hereinafter referred to as sphingolipid bacteria (Sphingobium jiangnanensis) 3R8.

Example 2 qualitative detection and quantitative analysis of secreted auxin of sphingolipid of south Jiangnan (Sphingobium jiangnanensis) 3R8

1. Qualitative detection

After being activated, the sphingolipid of Jiangnan (Sphingobium jiang sphingoensis) 3R8 is inoculated in an IAA liquid fermentation culture medium, shaking culture is carried out at 35 ℃ and 180R/min for 2d, and centrifugation is carried out at 12 000r/min for 10min, 100 mu L of supernatant is dripped on a white porcelain plate, and equivalent Salkowski colorimetric fluid is added for color development reaction. 100 μ L of IAA aqueous solution (100 mg/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 fermentation liquor of the sphingolipid bacteria (Sphingobium jiangnanensis) 3R8, the color of the mixed liquor turns red (figure 4), which indicates that the sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 can secrete plant growth hormone IAA.

2. Quantitative analysis

Preparation of 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 _{530 nm} Drawing a graph for the ordinate to obtain an IAA standard curve.

Measurement of IAA concentration in bacterial solution: after the activation of 3R8 of sphingolipid of Jiangnan (Sphingobium jiangnanensis) in IAA-producing liquidMeasuring OD of fermentation liquor after shaking culture for 72h at 35 ℃ and 180r/min in fermentation medium ₆₀₀ And the fermentation liquid is centrifuged at 12 000rpm for 10min, and the supernatant is mixed with an equal volume of Salkowski colorimetric solution. Standing at room temperature in dark for 30min, and determining OD _{530 nm} Value (control of mixed solution of IAA-producing liquid fermentation medium without inoculation and Salkowski colorimetric solution in the same volume). By IAA concentration and OD _{530 nm} The corresponding IAA concentration was calculated from the standard relationship curve of (a). And according to OD _{600 nm} Value, unit IAA yield was calculated according to the formula:

unit IAA yield (mg/L) = IAA content in fermentation liquor/OD of fermentation liquor _{600 nm} The value is obtained.

The regression equation of the standard curve of the detected IAA content is y =0.013x +0.0672, and the correlation coefficient R ² =0.9934, OD of Sphingobium jiang sphingolipid (Sphingobium jiangnanensis) 3R8 after culturing for 72h _{600 nm} The value was 0.46, and the supernatant was mixed with Salkowski colorimetric solution to obtain OD _{530 nm} The strain is 2.81, and the unit IAA content of the suspension of sphingolipid of Jiangnan (Sphingobium jiangnanensis) 3R8 is 458.66mg/L.

Example 3 phosphorus solubilizing Properties of sphingolipid bacteria (Sphingobium jiangnanensis) 3R8

And (3) determining the phosphorus dissolution property: inoculating sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 on an inorganic phosphorus culture medium, culturing at 30 ℃ for 4-6d, observing whether a phosphorus-dissolving ring is generated, measuring the diameter (d 2) of the phosphorus-dissolving ring and the diameter (d 1) of a bacterial colony, and calculating the ratio (d 2/d 1) of the phosphorus-dissolving ring to the bacterial colony. 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 sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 was 1.34 + -0.14 for 3d growth on inorganic phosphorus assay plates (inorganic phosphorus medium) (FIG. 5). It is shown that sphingolipid bacteria (Sphingobium jiangnanensis) 3R8 can dissolve inorganic phosphorus.

Example 4 growth promotion of plants by Sphingobacterium jiang 3R8

1. Promotion of marigold seed germination

Selecting sphingolipid from south of Yangtze river (Sphingobium jiangnanensis) 3R8 single colony, inoculating to 250mL cone containing 100mL R2A liquid culture mediumThe cells were cultured in a flask at 30 ℃ for 48 hours on a shaker at 150 rpm. Centrifuging at 10000r/min for 5min, collecting thallus, and making OD with sterile water _600nm Bacterial suspension of =0.1 (with sterile water as blank). The marigold seeds with relatively consistent growth vigor are selected and placed in a 9cm plate with filter paper laid at the bottom, 10 seeds are placed in each plate, 10ml of bacterial suspension is added (test group), 10ml of sterile water is added to negative control, and the plate is placed in a 30 ℃ constant temperature incubator. Each group was replicated three times, 3 plates each time. Tracking and observing the germination condition of marigold, recording the germination number, and analyzing the promoting effect of sphingolipid (Sphingobium jiangnanensis) 3R8 on marigold seed germination.

Germination% = (number of germinated seeds/number of test seeds on specified days) × 100%.

The result shows that after the cultivation for 7 days, the germination rate of the negative control seeds is 20%, the germination rate of the test group seeds is 60%, and the result shows that the germination rate of the marigold seeds can be obviously improved by 3R8 of sphingolipid bacteria (Sphingobium jiangnanensis) and is increased by 2 times.

2. Promoting the growth of rice and marigold seedlings

A single colony of sphingolipid (Sphingobium jiangnanensis) 3R8 was picked and inoculated into a 250mL Erlenmeyer flask containing 100mL R2A liquid medium, and cultured for 48h at 30 ℃ in a shaker at 150 rpm. Centrifuging at 10000r/min for 5min, collecting thallus, and making OD with sterile water _600nm Bacterial suspension of =0.1 (sterile water as blank control).

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. Each of the CK wells of the test group and the control group was set to 3 wells. The root irrigation treatment of the bacterial suspension is carried out, and the bacterial suspension (test group) or sterile water 25mL (control group) is irrigated 1 time 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 14h. And after 30d of transplanting, observing the growth vigor of the plants.

The rice seedling potting results are shown in table 4 and fig. 6, the fresh weight of rhizomes, the root length, the dry weight of roots and stems of the experimental group are increased by 125.0%, 25.7%, 66.6%, 77.8% and 38.3% respectively compared with the control group, the experimental result shows that the 3R8 has obvious growth promoting effect on rice seedlings.

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

Group of	Root weight	Weight of the stem	Root length	Length of stem	Dry weight of root	Dry weight of stem
							Experimental group 1	0.57	1.03	24.3	33.4	0.34	0.55
Experimental group 2	0.78	1.57	25.2	33.9	0.29	0.72
							Experimental group 3	0.66	1.52	28	33.5	0.31	0.69
Experimental group 4	0.54	1.16	30	32.9	0.33	0.64
							Experimental group	0.63±0.11a	1.32±0.27a	26.88±2.61a	33.42±0.41a	0.32±0.02a	0.65±0.07a
Control group 1	0.27	0.94	17.2	27	0.19	0.47
							Control group 2	0.27	1.32	16.1	30.3	0.17	0.45
Control group 3	0.24	0.78	15.6	29.5	0.17	0.45
							Control group 4	0.35	1.16	15.6	31.8	0.17	0.52
Mean value of	0.28±0.05b	1.05±0.24b	16.13±0.75b	29.65±2.01a	0.18±0.01b	0.47±0.03b

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

The potted marigold seedlings result is shown in table 5 and fig. 7, and other indexes of the test group are obviously superior to those of the control group except for the root length. Test results show that the 3R8 has obvious growth promotion effect on marigold seedlings.

TABLE 5 determination results of marigold seedlings potted plant experiments

Group of	Root weight/g	Weight per gram of stem	Root length/cm	Stem length/cm	Root dry weight/g	Dry weight of stem/g
							Test group 1	0.74	1.47	17.43	16.00	0.35	0.92
Test group 2	1.47	1.45	15.63	16.50	0.46	1.03
							Test group 3	1.07	1.50	22.00	18.55	0.44	1.16
Test group	1.09±0.36a	1.47±0.02a	18.35±3.29a	17.02±1.35a	0.42±0.06a	1.04±0.12a
							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±1.38a	12.54±0.92b	0.2±0.06b	0.55±0.09b

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 within 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 examples, 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 use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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

<120> sphingolipid bacteria having growth promoting function of landscape plants and use thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1387

<212> DNA

<213> sphingolipid bacteria of Jiangnan (Sphingobium jiangnanensis)

<400> 1

aacggggggg acgctatact gcagtcgaac gagaccttcg ggtctagtgg cgcacgggtg 60

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agttggtggg gtaaaggctc accaaggcta cgatccttag ctggtctgag aggatgatca 240

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aaagctcttt tacccgagat gataatgaca gtatcgggag aataagctcc ggctaactcc 420

gtgccagcag ccgcggtaat acggagggag ctagcgttgt tcggaattac tgggcgtaaa 480

gcgcacgtag gcggcgattt aagtcagagg tgaaagcccg gggctcaacc ccggaactgc 540

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aattcgtaga tattcggaag aacaccagtg gcgaaggcgg cccgctggac aagtattgac 660

gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta 720

aacgatgata actagctgct ggggcacatg gtgtttcagt ggcgcagcta acgcattaag 780

ttatccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac gggggcctgc 840

acaagcggtg gagcatgtgg tttaattcga agcaacgcgc agaaccttac caacgtttga 900

catccctatc gcggatcgtg gagacacttt ccttcagttc ggctggatag gtgacaggtg 960

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ccggaggaag gtggggatga cgtcaagtcc tcatggccct tacgcgttgg gctacacacg 1140

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atgggagttg gattcactcg aaggcgttga gctaaccgca aggaggcagg cgaccacagg 1380

catggcg 1387

Claims (7)

1. Sphingolipid bacteria, characterized in that: the sphingolipid bacteria are sphingolipid bacteria of south China (A)Sphingobium jiangnanensis) The strain number is 3R8, and the preservation number of the strain in the China general microbiological culture Collection center is CGMCC No.21506.

2. A culture characterized by: the culture is obtained by culturing the sphingolipid according to claim 1 in a microbial culture medium.

3. The microbial inoculum is characterized in that: the microbial agent comprising the sphingolipid according to claim 1 or/and the culture according to claim 2.

4. A microbial ecological agent or a biological fertilizer, characterized in that: the microbial preparation or biofertilizer comprising the sphingolipid according to claim 1, the culture according to claim 2 or/and the microbial agent according to claim 3.

5. The sphingolipid according to claim 1, the culture according to claim 2, the microbial agent according to claim 3 or the probiotic or biofertilizer according to claim 4 for any one of the following uses:

n1, application in promoting plant seed germination;

n2, and application in preparation of products for promoting plant seed germination;

n3, application in promoting plant growth;

n4, application in preparation of products for promoting plant growth;

n5, application in the production of auxin IAA;

n6, application in preparation of products for producing auxin IAA;

n7, application in dissolving phosphorus;

n8 and application in preparing a product for dissolving phosphorus.

6. Use according to claim 5, characterized in that: the plant is marigold or rice.

7. A method for producing the microbial preparation according to claim 3, comprising a step of obtaining the microbial preparation by using the sphingolipid according to claim 1 or/and the culture according to claim 2 as a component of the microbial preparation.

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