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

Abstract

The invention discloses a sphingomycete with the function of promoting the growth of garden plants and its application. The sphingomycete is Sphingobium jiangnanensis 3R8, and its registration number in the General Microbiology Center of China Committee for Culture Collection of Microorganisms is CGMCC No. 21506. The unit IAA content of Jiangnan Sphingomyces 3R8 was 458.66mg/L fermentation broth, and the ratio of phosphorus-dissolving circle to colony diameter on the inorganic phosphorus plate was 1.34±0.14. Compared with the non-inoculated negative control group, it can significantly improve the germination rate of marigold seeds and the growth of rice seedlings, and increase the germination rate of marigold seeds by 2 times. This indicates that Sphingobium jiangnanensis 3R8 can secrete auxin IAA and has the function of dissolving phosphorus, which can be used as 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 a sphingomycete with the function of promoting growth of garden plants and its use.

Background technique

The plant rhizosphere hosts a diverse microbial community that plays key roles in plant growth and reproduction. Some rhizosphere microorganisms can regulate the growth of plants and the flowering time of plants by regulating nitrogen cycle and plant hormones (such as auxin, etc.); they can degrade organic pollutants, enrich heavy metals, reduce the toxicity of heavy metals in soil, improve soil pore conditions, increase Soil fertility, the function of improving soil biological properties, can carry out biological control of pathogenic bacteria, and improve the growth adaptability of garden plants in saline-alkali, barren soil environment and special urban space. The development and utilization of microbial resources can promote the growth of garden plants and improve the health and quality of urban green space soil, which has broad development prospects.

Sphingobacteriaceae (Sphingobacteriaceae) have been isolated from soil, freshwater, groundwater, ocean, plant surface, rhizosphere, and even extreme environments such as polluted areas, Arctic and Antarctic soils, deeply buried sediments, and soil surfaces in arid regions. environment separation. In previous studies, members of the genus Sphingobium were reported to be able to degrade a variety of natural and synthetic aromatic compounds, such as polycyclic aromatic hydrocarbons (PAHs), lignin-derived aromatic compounds, heterocyclic aromatic hydrocarbons, chlorine Substituted aromatic hydrocarbons, sulfonated aromatic hydrocarbons, azo compounds, pesticides and herbicides, etc. However, there are few studies on the growth-promoting function of Sphingomyces.

Contents of the invention

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

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

A sphingomycete provided by the present invention is Sphingobium jiangnanensis.

The Sphingobium jiangnanensis provided by the present invention can specifically be Sphingobium jiangnanensis 3R8, and its registration number in the General Microorganism Center of China Microbiological Culture Collection Management Committee is CGMCC No.21506, hereinafter It is referred to as Sphingobium jiangnanensis 3R8.

Sphingobium jiangnanensis 3R8 is a Gram-negative bacterium, rod-shaped and non-spore-forming. Sphingobium jiangnanensis 3R8 has a 16S rRNA gene shown in sequence 1 in the sequence listing.

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

The culture of Sphingobium jiangnanensis provided by the present invention is the material obtained by cultivating Sphingobium jiangnanensis 3R8 in a microbial culture medium (i.e. a fermentation product, such as containing Sphingobium jiangnanensis 3R8 and secreted into The substance in the liquid medium is the fermentation broth, or the substance containing Sphingobium jiangnanensis 3R8 and secreted into the solid medium is the solid fermentation product).

In order to solve the above technical problems, the present invention also provides bacterial agents.

The microbial agent provided by the present invention contains Sphingobium jiangnanensis 3R8 or/and the metabolites of Sphingobium jiangnanensis 3R8 or/and the above culture.

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

M1) promote plant growth;

M2) promote plant seed germination;

M3) produce auxin IAA;

M4) Dissolving phosphorus.

Herein, the metabolite of Sphingobium jiangnanensis 3R8 can be obtained from the fermentation broth of Sphingobium jiangnanensis 3R8. The metabolite of Sphingobium jiangnanensis 3R8 may be a sterile metabolite of Sphingobium jiangnanensis 3R8 or a bacterium-containing metabolite of Sphingobium jiangnanensis 3R8. The aseptic metabolite (sterile fermentation filtrate) of Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 specifically can be prepared according to the following method, cultivate Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 in a liquid medium, and remove the liquid culture (fermentation filtrate) by filtration. Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 in liquid) to obtain the sterile metabolite of Sphingobium jiangnanensis 3R8. The bacterium-containing metabolite of Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 can be specifically prepared according to the following method, cultivate Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 in a liquid fermentation medium, collect the fermentation broth (containing Sphingobium jiangnanensis) 3R8 and substances secreted into the liquid medium), the fermentation broth is the bacterial metabolite of Sphingobium jiangnanensis 3R8.

The active ingredient of the above bacterial agent can be Sphingobium jiangnanensis 3R8 or/and the metabolite of Sphingobium jiangnanensis 3R8 or/and the above-mentioned culture, and the active ingredient of the above bacterial agent can also contain other biological Components or non-biological components, other active ingredients of the above bacterial agents can be determined by those skilled in the art according to the effect of the bacterial agents.

In the bacterial agent mentioned above, the bacterial agent may also include a carrier. The carrier can be a solid carrier or a liquid carrier.

The solid carrier can be a mineral material, a biological material; the mineral material can be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomite; the biological material It can be at least one of straw, pine shell, straw, peanut shell, corn flour, soybean flour, starch, peat and animal manure of various crops; the liquid carrier can be water; in the bacterial agent, Jiangnan Sphingobium jiangnanensis 3R8 or/and metabolites of Sphingobium jiangnanensis 3R8 may be in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or mixture of cells and filtrate exist. The dosage form of the bacterial agent can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the bacterial agent.

In order to solve the above-mentioned technical problems, the present invention also provides probiotics or biological fertilizers.

The probiotics or biological fertilizer provided by the present invention contains the sphingolipid bacteria, the culture or/and the bacterial agent. In the above, the product can be a bacterial agent, a probiotic agent or a biological fertilizer.

Any of the following applications of the sphingolipid bacteria, the culture or the bacterial agent or the biological fertilizer also belongs to the protection scope of the present invention:

N1, application in promoting plant seed germination;

N2, the application in the preparation of the product that promotes plant seed germination;

N3. Application in promoting plant growth;

N4. Application in the preparation of products for promoting plant growth;

N5. Application in the production of auxin IAA;

N6. Application in the preparation of products producing auxin IAA;

N7. Application in dissolved phosphorus;

N8. Application in the preparation of dissolved phosphorus products.

Herein, the plant can be any of the following plants:

P1) dicots or monocots,

P2) Chrysanthemum plants,

P3) Plants of the order Asteraceae or Cyperaceae,

P4) Compositae or grasses,

P5) Gramineae plants,

P6) plants of the genus Marigold or Oryza,

P7) Marigold or rice.

The invention also provides a method for preparing the bacterial agent.

The method for preparing the bacterial agent provided by the present invention comprises using Sphingobium jiangnanensis 3R8 or/and metabolites of Sphingobium jiangnanensis 3R8 or/and the above-mentioned cultures as components of the bacterial agent, The step of obtaining the bacterial agent.

Herein, the dissolved phosphorus may be dissolved inorganic phosphorus. The plant growth promotion can be promoting the growth of rice seedlings and/or promoting the growth of marigold seedlings. The promoting plant seed germination can be improving the germination rate of marigold seeds.

Through secretion IAA test, phosphorus dissolution test and seed germination test, it was found that the unit IAA content of Sphingobium jiangnanensis 3R8 was 458.66mg/L fermentation broth, and the ratio of phosphorus-dissolving circle to colony diameter on the inorganic phosphorus plate was 1.34±0.14 . Compared with the non-inoculated negative control group, it can significantly increase the germination rate of marigold seeds and the growth of rice seedlings, and increase the germination rate of marigold seeds by 2 times. This indicates that Sphingobium jiangnanensis 3R8 can secrete auxin IAA and has the function of dissolving phosphorus, which can be used as microbial organic fertilizer to improve soil fertility.

Preservation instructions

Species name: Sphingomyces jiangnan

Latin name: Sphingobium jiangnanensis

Strain number: 3R8

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Depository institution abbreviation: CGMCC

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: December 18, 2020

Registration number of the collection center: CGMCC No.21506

Description of drawings

Figure 1 is a picture of the colonies of Sphingobium jiangnanensis 3R8 cultured on the R2A plate for 2 days.

Figure 2 is a phylogenetic tree of Sphingobium jiangnanensis 3R8 and related model bacteria constructed based on the 16S rRNA gene sequence. Among them, 3R8 is Sphingobium jiangnanensis 3R8.

Fig. 3 is a phylogenetic tree constructed from the genomes of Sphingobium jiangnanensis 3R8 and related model strains of the genus Sphingobium. Among them, 3R8 is Sphingobium jiangnanensis 3R8.

Fig. 4 is a photo of qualitative detection of auxin secreted by Sphingobium jiangnanensis 3R8. In the figure, the first row is a positive control, the second row is a negative control, and the third row is Sphingobium jiangnanensis 3R8 treatment.

Fig. 5 is a photograph of phosphate-dissolving circles produced by Sphingobium jiangnanensis 3R8 grown on an inorganic phosphorus detection plate (inorganic phosphorus medium) for 3 days.

Figure 6 is a photo of rice seedlings one month after transplanting. The flowerpot on the left is the control group, and the flowerpot on the right is the experimental group.

Fig. 7 is a photo of marigold seedlings 30 days after transplanting. The flowerpot on the left is the test group, and the flowerpot on the right is the control group.

detailed description

In this study, a multiphase classification method is used to determine the taxonomic status of Agapanthus rhizosphere bacteria 3R8, to study its secretion of IAA and phosphorus-dissolving functions, and to detect its growth-promoting function through a seed germination test. The present invention will be further described in conjunction with specific examples below. . It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The medium configuration method used in the following examples is as follows:

1/50R2A solid medium: yeast extract powder 0.01g, peptone 0.01g, casein hydrolyzate 0.01g, glucose 0.01g, soluble starch 0.01g, potassium dihydrogen phosphate 0.0006g, magnesium sulfate heptahydrate 0.00048g, sodium pyruvate 0.006g, 15g agar, dilute to 1000mL with distilled water, adjust the pH to 7.2±0.2, and sterilize at 121°C for 15min.

R2A liquid medium: yeast extract powder 0.5g, peptone 0.5g, casein hydrolyzate 0.5g, glucose 0.5g, soluble starch 0.5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate heptahydrate 0.024g, sodium pyruvate 0.3g , dilute to 1000mL with distilled water, adjust the pH to 7.2±0.2, and sterilize at 121°C for 15min.

IAA-producing liquid fermentation medium: yeast extract powder 0.5g, peptone 0.5g, casein hydrolyzate 0.5g, glucose 0.5g, soluble starch 0.5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate heptahydrate 0.024g, sodium pyruvate 0.3g, L-tryptophan 0.2g, dilute to 1000mL with distilled water, adjust the pH to 7.2±0.2, and sterilize at 121°C for 15min.

Inorganic phosphorus medium: sodium chloride 0.3g, glucose 10g, potassium chloride 0.3g, tricalcium phosphate 5g, ammonium sulfate 0.5g, magnesium sulfate heptahydrate 0.3g, manganese sulfate 0.03g, ferrous sulfate heptahydrate 0.03g, Agar 15g, dilute to 1000mL with distilled water, adjust the pH to 7.0-7.2, and sterilize at 121°C for 30min.

Embodiment 1, isolation and identification of bacterial strain 3R8

1. Isolation of strain 3R8

Agapanthus rhizosphere soil samples were collected at Wuqiao Base (30°58'N, 121°25'E) of Shanghai Garden Science Planning Research Institute, and brought back to the laboratory for storage at 4°C. Shake off the soil attached to the plant roots, and only keep the rhizosphere soil closely adhered to the root surface, weigh 2g of plant roots with rhizosphere soil and grind them in a sterile mortar. Put 50ml of sterile water in a 150ml Erlenmeyer flask, shake at room temperature at 150r/min for 30min, take 1mL of the diluted solution for serial dilution with sterile water, take 100μL of the serial diluted solution and spread it on a 1/50R2A plate, and incubate it upside down at 30°C for 1 week. According to the physiological morphological characteristics, a single colony was picked with a bamboo stick and purified on a plate. After it was confirmed as pure bacteria, it was transferred to an inclined plane at 4°C for short-term storage, and then transferred to a 25% glycerol tube for long-term storage at -80°C. One of the isolated and purified strains was named strain 3R8.

2. Identification of strain 3R8

2.1 Morphological identification of strains

The strain 3R8, which is in the logarithmic growth phase and has a stable colony size, and is isolated and purified in the above step 1, is described for the state of a single colony, mainly including the size, color, transparency, surface state of the colony, and edge state of the colony. According to the manufacturer's instructions, the smears of strain 3R8 were stained with Gram staining kits using the Gram staining kit from Solebo, and the morphology of the bacteria was observed with an optical microscope.

The results showed that after 48 hours of culture on the R2A plate, the colony of bacterial strain 3R8 was yellow, 2-3mm in diameter, raised, round and smooth, with complete edges (Fig. 1); bacterial strain 3R8 cells were Gram-negative, rod-shaped, without sporulation.

2.2 Molecular identification

According to the instructions, Genomic DNA was extracted with TIANamp Bacterial Genomic DNA Extraction Kit from Tiangen Company, and 16S rRNA gene amplification was carried out using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') increase. 25 μL PCR amplification system: 2×Taq PCR Mix 12.5 μL, 27F (10 μmol/L) 1 μL, 1492R (10 μmol/L) 1 μL, ddH ₂ O 8.5 μL, 2 μL DNA template. The PCR amplification program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 1 min, 72°C extension for 90 s, 30 cycles; 72°C final extension for 10 min. The PCR amplification product was detected by 1% agarose gel electrophoresis, and the amplified fragment was about 1400bp. After electrophoresis verification, the positive result PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequence obtained by sequencing was uploaded to Ezbiocloud (www.ezbiocloud.net/eztaxon) for sequence comparison.

The sequence length of the 16S rRNA gene of strain 3R8 is 1,387bp, and the sequence comparison result with EzBioCloud shows that strain 3R8 is a member of the Sphingobium genus, and Sphingobium limneticum DSM25076 (T) (97.84%), Sphingobium vermicomposti DSM 21299 (T) (97.04%) %) has a high degree of similarity, and the similarity with other species of the Sphingobium genus is less than 97%. The strain 3R8 can be preliminarily determined to be a new species in the genus Sphingobium. Those closely related 16S rRNA gene sequences were retrieved from the EzBioCloud server and compared using the clustal W program. The phylogenetic tree was constructed by Neighbor-Joining method using MEGA 7 software. Using the Kimura two-parameter model to calculate the evolutionary distance of the NJ method, when the Bootstrap value is 1000 times, the resulting developmental tree is shown in Figure 2. Strain 3R8 and Sphingobium limneticum clustered in the same clade.

2.3 Genome analysis

Genomic DNA was sent to Annoroad Gene Technology Co., Ltd., and the draft genome of strain 3R8 was sequenced using the Illumina NovaSeq 6000 sequencing system. Assembly was performed using Unicycler v0.4.7, resulting in 81 contigs with a coverage rate of approximately 232× and an N50 length of 157538bp. Genome 4.73 Mb, G+C content 64.0 mol%, average nucleotide identity (ANI) calculated on http://jspecies.ribohost.com/jspeciesws/#analyse website. Digital DNA-DNA hybrids (dDDH) between strain 3R8 and a reference strain were calculated by genome-to-genome sequence comparison using the Genome-to-Genome Distance Calculator (GGDC) 2.0 server (http://ggdc.dsmz.de/distcalc.php) )value. Based on the similarity and phylogenetic analysis of the 16S rRNA gene sequence of strain 3R8, Sphingobium limneticum DSM25076(T) and Sphingobium vermicomposti DSM 21299(T) were selected as reference strains for physiological tests and chemical taxonomic analyses.

Compared with other strains of Sphingobium species with published genome sequences, the ANI values were 77.08-88.79%, lower than the 95-96% cut-off value previously proposed for species delineation, and the dDDH values of 3R8 and its type strains were in 21.3-41.1%, far below the 70% threshold for species division. Both the results of ANI and dDDH supported that strain 3R8 was a new species of Sphingobium.

Table 1. ANI and dDDH values of strain 3R8 and its close relatives

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 francesse 78.54 23.3 Sphingobium chlorophenolicum 78.27 23.1 Sphingobium aromaticivastans 78.15 22.4 Sphingobium vermicomposti 77.08 21.3

To further explore the relationship of strain 3R8 to related species of the Sphingobium genus, a phylogenetic tree was reconstructed using the latest Bacterial Core Gene Set (UBCG) pipeline. According to the phylogenetic tree of 92 bacterial core gene sequences, 3R8 formed a unique phylogenetic lineage in Sphingobium (Fig. 3), which indicated that strain 3R8 should belong to a new species of Sphingobium.

2.4 Physiological and chemical taxonomic identification

Oxidase and catalase activities were determined by French Mérieux (API) oxidase assay kit and bubbling in 3% (v/v) H ₂ O ₂ solution, respectively. Physiological characteristics of strain 3R8 and related model strains were determined using API 20NE and API ZYM kits (bioMérieux) from France Mérieux. GEN III MicroPlates from BIOLOG, USA, were used to determine oxidation of carbon sources and sensitivity to inhibitory compounds.

The results showed that strain 3R8 was negative for catalase and positive for oxidase. The results of the 20NE test showed that the nitrate reduction reaction of the strain 3R8 was negative, could not produce indole, could hydrolyze escin, was positive for galactosidase, was positive for assimilation of glucose, arabinose, maltose, malic acid and citric acid, and negative for glucose fermentation , Negative for arginine dihydrolase, unable to hydrolyze urea and gelatin, negative assimilation reactions of mannose, mannitol, N-acetyl-glucosamine, sodium gluconate, capric acid, adipic acid and phenylacetic acid, ZYM enzyme activity identification In the test, lipase (C4), lipase (C8), leucine aromatase, valine aromatase, chymotrypsin, β-galactosidase, β-glucuronidase , acid and alkaline phosphatase, naphthol-AS-BI phosphohydrolase, α-glucosidase and β-glucosidase positive, lipase (C14), cystine arylase, trypsin, α- Galactosidase, α-mannosidase, and β-fucosidase were negative. Glucan, D-cellobiose, D-turanose, β-formyl-D-glucoside, D-salicin, α-D-glucose, D-galactose, D-trehalose, L -Trehalose, L-rhamnose, D-sorbitol, D-glucose-6-phosphate, aminoacetyl-L-proline, L-aspartic acid, L-glutamic acid, L-galactobionic acid , glucuronic acid, quinic acid, α-keto-glutaric acid, L-hydroxysuccinic acid, bromo-succinic acid, β-hydroxy-D,L-butyric acid, acetoacetic acid, acetic acid and formic acid as carbon sources, It can grow in the presence of 1% sodium lactate, rifamycin SV, lincomycin, tetrazolium blue, nalidixic acid, potassium tellurite and aztreonam. The differences between strain 3R8 and related model species are shown in Table 2.

The results showed that the strain 3R8 was significantly different from the type strain Sphingobium vermicomposti DSM 21299 in the following 33 physiological and biochemical characteristics:

1) The strain 3R8 can assimilate arabinose, maltose, malic acid and citric acid, but the type strain Sphingobium vermicomposti DSM 21299 cannot;

2) The extracellular zymogram of strain 3R8 contains chymotrypsin, β-glucuronidase and α-glucosidase, but does not contain trypsin, and the extracellular zymogram of the type strain Sphingobium vermicomposti DSM 21299 does not contain chymotrypsin protease, beta-glucuronidase and alpha-glucosidase, including 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-galactobionic acid, L-hydroxysuccinic acid, and formic acid were used as carbon sources, and the type strain Sphingobium vermicomposti DSM21299 could not utilize these substances, Strain 3R8 is unable to utilize α-D-lactose, L-pyroglutamate, D-fructose, 3-formyl-glucose, D-fructose-6-phosphate, galacturonic acid and glucuronamide as carbon sources, pattern Strain Sphingobium vermicomposti DSM 21299 can utilize;

4) In the presence of 1% sodium lactate, tetrazolium blue, aztreonam and potassium tellurite, the strain 3R8 can grow, but the type strain Sphingobium vermicomposti DSM 21299 cannot grow, tetrazolium violet can inhibit the growth of the strain 3R8, but cannot inhibit the growth of the model strain The strain Sphingobium vermicomposti DSM 21299 was grown.

There are obvious differences between the strain 3R8 and the type strain Sphingobium limneticum DSM25076(T) in the following 21 physiological and biochemical characteristics:

1) Strain 3R8 can assimilate citric acid and maltose, but the type strain Sphingobium limneticum DSM25076(T) cannot assimilate citric acid and maltose;

2) The extracellular zymogram of strain 3R8 contains chymotrypsin, β-glucuronidase and β-galactosidase, but does not contain trypsin. The extracellular zymogram of the type strain Sphingobium limneticum DSM25076(T) contains pancreatic Protease, without chymotrypsin, beta-glucuronidase 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 type strain Sphingobium limneticum DSM25076 (T) can utilize these substances, strain 3R8 can utilize D-sorbitol and α-keto-glutaric acid as carbon sources, but the type strain Sphingobiumlimneticum DSM25076 (T) cannot utilize;

4) In the presence of rifamycin SV and potassium tellurite, the strain 3R8 could grow, but the type strain Sphingobium limneticum DSM25076 (T) could not grow, troleandomycin, minocycline, D-serine and tetracycline Azopurin can inhibit the growth of strain 3R8, but cannot inhibit the growth of model strain Sphingobium limneticum DSM25076(T).

Table 2. Differences between strain 3R8 and related model strains

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

The fatty acid profiles of strain 3R8 and two closely related bacteria were determined using cells grown on R2A agar at 30°C for 2 days. Obtained fatty acid methyl esters. The results of MIDI analysis are shown in Table 3. The main fatty acids (>5%) of strain 3R8 are the eighth characteristic type (C _18:1 ω6c and/or C _18:1 ω7c), the third characteristic type (C _16:1 ω6c and/or or C _16:1 ω7c), C _16:0 , C _17:1 ω6c and C _14:0 2OH, the results showed that the fatty acid composition of strain 3R8 and the closely related bacteria Sphingobium limneticum DSM25076(T) and Sphingobium vermicompostiDSM21299(T) were basically the same , but there is a large difference in the content.

Table 3. Cellular fatty acid composition of strain 3R8 and its type strains

Note: Values shown are percentages of total fatty acids. tr, trace (<1%). Signature types are mixtures of two or three fatty acids that cannot be separated by GLC with the MIDI system, 3rd feature type includes C _16:1 ω6c and/or C _16:1 ω7c, 8th feature type includes C _18:1 ω6c and/or C _18:1 ω7c.

In summary, the 16S rRNA similarity between strain 3R8 and Sphingobium limneticum 301(T) was 97.84%, and that of Sphingobium vermicomposti VC-230(T) was 97.04%. 16S rRNA sequence similarity <97%; ANI values of 77.08-88.79% for strain 3R8 compared with strains of Sphingobium genus, lower than 95-96% cut-off value previously proposed for species demarcation, strain 3R8 and its pattern The dDDH value of the strain is between 21.3-41.1%, which is far below the threshold value of 70% for species classification; and the physiological and biochemical characteristics and fatty acid content are quite different, so it can be determined that the strain 3R8 is a new species of the genus Sphingobium, named Sphingobium jiangnanensis, Its Chinese name is Jiangnan Sphingomyces. The Sphingobium jiangnanensis 3R8 has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (CGMCC for short, address: No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing) on December 18, 2020. The deposit number is CGMCCNo.21506. Hereinafter referred to as Sphingobium jiangnanensis 3R8 for short.

Example 2, Qualitative detection and quantitative analysis of auxin secreted by Sphingobium jiangnanensis 3R8

1. Qualitative detection

Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 was activated and inoculated in the IAA-producing liquid fermentation medium, cultured with shaking at 35°C and 180r/min for 2 days, centrifuged at 12 000r/min for 10min, and 100μL of the supernatant was dropped on a white porcelain plate , adding an equal volume of Salkowski colorimetric solution for color reaction. 100 μL IAA aqueous solution (100 mg/L) and IAA-producing liquid fermentation medium without bacteria were used as positive control and negative control, respectively. Place the white porcelain plate at room temperature and avoid light for 30 minutes and then observe it. If it turns pink, it is positive, indicating that the strain can secrete IAA. The experiment was repeated three times.

Qualitative test results show that after adding Salkowski colorimetric solution dropwise to the supernatant of Sphingobium jiangnanensis 3R8 fermentation broth, the color of the mixture turns red (Fig. 4), indicating that Sphingobium jiangnanensis 3R8 can Secretes plant growth hormone IAA.

2. Quantitative analysis

Preparation of the standard curve: After dissolving an appropriate amount of IAA with a small amount of ethanol, dilute with distilled water, prepare IAA standard solutions with concentrations of 0, 25, 50, 100, 200, and 250 mg/L, and compare the color with Salkowski at a volume ratio of 1:1 solution was mixed, placed at room temperature in the dark for 30 min, and then the OD _530nm of each concentration was measured respectively (the 1:1 mixed solution of distilled water and Salkowski colorimetric solution was used as the blank control). Finally, the IAA standard curve was obtained by plotting the IAA concentration as the abscissa and OD _{530 nm} as the ordinate.

Determination of the concentration of IAA in the bacterial liquid: after the activation of Sphingobium jiangnanensis 3R8, the OD ₆₀₀ value of the fermented liquid was measured in the IAA-producing liquid fermentation medium at 35°C and 180r/min for 72 hours, and the fermented liquid was 12 Centrifuge at 000 rpm for 10 min, and mix the supernatant with an equal volume of Salkowski colorimetric solution. Place it in the dark at room temperature for 30 minutes, and measure its OD _{530 nm} value (using the mixed solution of uninoculated IAA-producing liquid fermentation medium and equal volume of Salkowski colorimetric solution as a control). The corresponding IAA concentration was calculated by the standard relationship curve of IAA concentration and OD _{530 nm} . And according to the _OD600nm value, calculate the unit _IAA output according to the formula:

Unit IAA yield (mg/L) = IAA content in fermentation broth/OD _{600 nm} value of fermentation broth.

The regression equation of the standard curve of the detected IAA content is: y=0.013x+0.0672, the correlation coefficient R ² =0.9934, the OD _{600 nm} value of Sphingobium jiangnanensis 3R8 after 72 hours of cultivation is 0.46, and its supernatant and Salkowski The mixed OD _{530 nm} of the colorimetric solution was 2.81, and the unit IAA content of the Sphingobium jiangnanensis 3R8 suspension was 458.66 mg/L.

Embodiment 3, the phosphorus-dissolving characteristic of Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8

Determination of phosphorus-dissolving characteristics: Inoculate Sphingobium jiangnanensis 3R8 on inorganic phosphorus medium, culture at 30°C for 4-6 days, observe whether there is a phosphorus-dissolving circle, measure the diameter of the phosphorus-dissolving circle (d2) and the diameter of the colony ( d1), calculate the ratio of phosphorus-dissolving circle to colony diameter (d2/d1). The larger the ratio, the stronger the phosphorus-dissolving ability. The results showed that the ratio of phosphorus-dissolving circle to colony diameter of Sphingobium jiangnanensis 3R8 was 1.34±0.14 after growing on the inorganic phosphorus detection plate (inorganic phosphorus medium) for 3 days ( FIG. 5 ). It shows that Sphingobium jiangnanensis 3R8 can dissolve inorganic phosphorus.

Embodiment 4, Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 promotes plant growth

1. Promote the germination of marigold seeds

A single colony of Sphingobium jiangnanensis 3R8 was picked and inoculated into a 250mL Erlenmeyer flask containing 100mL R2A liquid medium, and cultured in a shaker at 30°C and 150rpm for 48h. Centrifuge at 10000 r/min for 5 min, collect the bacterial cells, and prepare a bacterial suspension with OD _600nm =0.1 with sterile water (use sterile water as a blank control). Select marigold seeds with relatively consistent growth and place them in a 9cm flat plate with filter paper on the bottom. Put 10 seeds on each flat plate, add 10ml of bacterial suspension (test group), and add 10ml of sterile water to the negative control. 30°C constant temperature incubator. Each group was replicated three times, with 3 plates per replicate. Follow up and observe the germination of marigolds, record the number of germinations, and analyze the effect of Sphingobium jiangnanensis 3R8 on the germination of marigold seeds.

Germination rate%=(the number of germinated seeds in the specified days/the number of tested seeds)×100%.

The result shows that after cultivating for 7 days, the negative control seed germination rate is 20%, and the test group seed germination rate is 60%, showing that Sphingobium jiangnanensis (Sphingobium jiangnanensis) 3R8 can significantly improve the germination rate of marigold seeds, and improve the germination rate of marigold seeds 2 times.

2. Promote the growth of rice and marigold seedlings

A single colony of Sphingobium jiangnanensis 3R8 was picked and inoculated into a 250mL Erlenmeyer flask containing 100mL R2A liquid medium, and cultured in a shaker at 30°C and 150rpm for 48h. Centrifuge at 10000 r/min for 5 min, collect the bacterial cells, and prepare a bacterial suspension with OD _600nm =0.1 with sterile water (use sterile water as a blank control).

Select rice and marigold seedlings with similar growth and transplant them into flower pots, with 4-5 plants per pot. Three pots of CK were set up for the test group and the control group respectively. Root irrigation with bacterial suspension was carried out, and each pot was irrigated with bacterial suspension (test group) or 25 mL of sterile water (control group) once a week. Water every 2-3 days during plant growth. The experiments were carried out in a smart greenhouse. The day and night temperatures are 25°C/20°C respectively, and the sunshine time is 14h. After 30 days of transplanting, the growth of the plants was observed.

The potted results of rice seedlings are shown in Table 4 and Figure 6. Compared with the control group, the fresh weight of rhizome, root length, dry weight of root and stem in the experimental group increased by 125.0%, 25.7%, 66.6%, 77.8% and 38.3%, respectively. Significantly better than the control group, the test results show that 3R8 has an obvious growth-promoting effect on rice seedlings.

Table 4. Measuring results of rice seedling pot experiments

group root weight stem weight root length stem length root dry weight stem dry weight 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 test 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 average value 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<0.05, different lowercase numbers in the same column indicate significant difference.

The potted results of marigold seedlings are shown in Table 5 and Figure 7. Except for root length, other indicators of the test group were significantly better than those of the control group. The test results showed that 3R8 had obvious growth-promoting effect on marigold seedlings.

Table 5. Marigold seedling pot experiment measurement results

group root weight/g Stem weight/g root length/cm stem length/cm root dry weight/g Stem dry weight/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<0.05, different lowercase numbers in the same column indicate significant difference.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

sequence listing

<110> Shanghai Academy of Garden Science Planning; Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences

<120> Sphingomycetes with garden plant growth-promoting function and use thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1387

<212>DNA

<213> Sphingobium jiangnanensis

<400> 1

aacggggggg acgctatact gcagtcgaac gagaccttcg ggtctagtgg cgcacgggtg 60

cgtaacgcgt gggaatctac ccttgggttc ggaataacgt cgggaaactg acgctaatac 120

cggatgatga cgaaagtcca aagattttc gcccagggat gagcccgcgt aggattagct 180

agttggtggg gtaaaggctc accaaggcta cgatccttag ctggtctgag aggatgatca 240

gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatattg 300

gacaatgggg gcaaccctga tccagcaatg ccgcgtgagt gatgaaggcc ttagggttgt 360

aaagctcttt tacccgagat gataatgaca gtatcgggag aataagctcc ggctaactcc 420

gtgccagcag ccgcggtaat acggagggag ctagcgttgt tcggaattac tgggcgtaaa 480

gcgcacgtag gcggcgattt aagtcagagg tgaaagcccg gggctcaacc ccggaactgc 540

ctttgagact ggattgctag aatcttggag aggcgggtgg aattccgagt gtagaggtga 600

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 gagaacacttt ccttcagttc ggctggatag gtgacaggtg 960

ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1020

accctcgcct ttagttgcca tcatttagtt gggtactcta aaggaaccgc cggtgataag 1080

ccggaggaag gtggggatga cgtcaagtcc tcatggccct tacgcgttgg gctacacacg 1140

tgctacaatg gcgactacag tgggcagcca ctccgcgagg aggagctaat ctccaaaagt 1200

cgtctcagtt cggatcgttc tctgcaactc gagagcgtga aggcggaatc gctagtaatc 1260

gcggatcagc atgccgcggt gaatacgttc ccaggccttg tacacaccgc ccgtcacacc 1320

atgggagttg gattcactcg aaggcgttga gctaaccgca aggaggcagg cgaccacagg 1380

catggcg 1387

Claims (7)

1. Sphingolipid bacterium, characterized in that: the sphingolipid bacterium is Sphingobium jiangnanensis ( Sphingobium jiangnanensis ), its strain number is 3R8, and its preservation number in the General Microbiology Center of China Microbiological Culture Collection Management Committee is CGMCCNo.21506 . 2. Culture, it is characterized in that: described culture is the material obtained by cultivating the sphingolipid bacteria described in claim 1 in a microbial culture medium. 3. Bacteria agent, characterized in that: said bacterium agent contains the sphingolipid bacteria described in claim 1 or/and the culture described in claim 2. 4. Micro-ecological preparation or biological fertilizer, characterized in that: said micro-ecological preparation or biological fertilizer contains the sphingolipid bacterium according to claim 1, the culture according to claim 2 or/and the bacterium according to claim 3 agent. 5. the following any application of the sphingomycetes described in claim 1, the culture described in claim 2, the bacterial agent described in claim 3 or the probiotics described in claim 4 or biological fertilizer: N1, application in promoting plant seed germination; N2, the application in the preparation of the product that promotes plant seed germination; N3. Application in promoting plant growth; N4. Application in the preparation of products for promoting plant growth; N5. Application in the production of auxin IAA; N6. Application in the preparation of products producing auxin IAA; N7. Application in dissolved phosphorus; N8. Application in the preparation of products for dissolving phosphorus. 6. The application according to claim 5, characterized in that: the plant is marigold or rice. 7. The method for preparing the bacterial agent according to claim 3, comprising using the sphingomycetes according to claim 1 or/and the culture according to claim 2 as a component of the bacterial agent to obtain the step of the bacterial agent.

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