Sphingolipid as well as microbial preparation and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to sphingobium sp., a microbial preparation of the sphingobium sp. And application of the sphingobium sp. The new sphingolipid strain D-491 is separated and screened from the desert environment, has the functions of nitrogen fixation, IAA secretion, alkali stress resistance and the like, enriches microbial strain resources and genome information, and broadens the application scene of the sphingolipid strain.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, this invention relates to a sphingobium sp., its microbial preparations, and applications. Background Technology

[0002] With the development of the human economy and the growth of the population, the demand for agricultural production is also increasing. Chemical fertilizers, as an important agricultural production tool, are widely used in farmland. However, while the use of chemical fertilizers has brought great convenience to agricultural production, it has also had a series of impacts on soil health and the ecological environment. Long-term, excessive use of chemical fertilizers has brought many adverse effects to the farmland environment, including soil structure deterioration, low nutrient utilization efficiency, and imbalance of the soil microbial environment.

[0003] With the development of modern agriculture, researchers have discovered that soil contains many beneficial microorganisms with functions such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and secretion of the plant hormone indoleacetic acid (IAA). Developing and utilizing these microorganisms can help improve nutrient absorption efficiency in crops, promote plant growth, regulate soil structure, and thus improve fertilizer utilization efficiency. Currently, the main soil microbial strains developed and applied in agriculture include Bacillus, Paenibacillus, Pseudomonas, Arthrobacter, and Rhizobium. However, reports on Sphingobium, especially its research and application in promoting plant growth, are very limited.

[0004] Therefore, there is an urgent need in this field to discover a new sphingolipid bacterium that can promote seed germination and plant growth, and after cultivation, can be used as a microbial agent in unconventional environmental conditions, providing a new method for the green development of agricultural production. Summary of the Invention

[0005] As mentioned above, existing research lacks practical applications of sphingolipids on plants or is limited to the effects of strains under conventional environmental conditions. Therefore, there is an urgent need in this field to discover new sphingolipid species to enrich the strain resources of agricultural microorganisms and broaden the application scenarios of sphingolipids.

[0006] In view of this, in a first aspect, the present invention provides a sphingobium sp. having the 16S rDNA sequence shown in SEQ ID NO. 1.

[0007] In a second aspect, the present invention provides a microbial preparation comprising the sphingolipids and / or cultures thereof of the first aspect of the present invention.

[0008] In a third aspect, the present invention provides the use of the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention in biological nitrogen fixation.

[0009] In a fourth aspect, the present invention provides the use of the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention in the production of indoleacetic acid (IAA).

[0010] In a fifth aspect, the present invention provides the use of the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention in improving soil nutrition.

[0011] In a sixth aspect, the present invention provides a method for promoting the germination of plant seeds, wherein the method includes contacting the plant seeds with a sphingolipid bacteria of the first aspect of the present invention or a microbial preparation of the second aspect of the present invention.

[0012] In a seventh aspect, the present invention provides a method for promoting plant growth, wherein the method comprises applying to the plant either a sphingolipid of the first aspect of the present invention or a microbial preparation of the second aspect of the present invention.

[0013] The beneficial effects of this invention include one or more of the following: It provides a novel sphingolipid bacterium that has nitrogen-fixing, IAA-secreting, and certain alkaline stress resistance capabilities, can promote seed germination and plant growth, and after cultivation, can be used as a microbial agent in unconventional environmental conditions (such as sandy soil environments), effectively increasing plant height, root length, and weight, enriching agricultural microbial strain resources, and providing a new method for the green development of agricultural production. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other implementation schemes can be obtained based on these drawings without creative effort.

[0015] Figure 1 A phylogenetic tree for Sphingolipid D-491 constructed based on the 16S rRNA sequence.

[0016] Figure 2 This is the genome map of Sphingolipid D-491.

[0017] Figure 3 The colony morphology of Sphingolipid D-491 on LB medium.

[0018] Figure 4 This is a Gram staining image of Sphingolipid D-491.

[0019] Figure 5 The results show the nitrogen-fixing capacity of Sphingolipid D-491.

[0020] Figure 6 The results show the assay results of the ability of Sphingolipid D-491 to secrete IAA.

[0021] Figure 7 The growth of Sphingolipid D-491 was shown at pH 9.0.

[0022] Figure 8 The effect of Sphingobacterium D-491 on seed germination was shown.

[0023] Figure 9 The effects of Sphingolipid D-491 on the growth of potted alfalfa and soybean grown in substrate soil are shown. The plants in the first row of photos are soybeans, and the plants in the second row are alfalfa.

[0024] Figure 10 The effects of Sphingolipid D-491 on the growth of potted alfalfa grown in sandy soil were shown. Detailed Implementation

[0025] The present invention will now be clearly and completely described in conjunction with its embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0026] Unless otherwise stated, all terms used herein should be understood to have their ordinary meaning as known in the art. Further, more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and the scope of the invention application, unless otherwise expressly listed definitions provide for a broader meaning.

[0027] Throughout the specification and the scope of the invention claims, the terms "comprising," "including," and variations thereof are open-ended and are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprising" covers situations where it means "consisting of." Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon reading the specification, or may be learned by practicing the invention. Moreover, the invention covers all possible combinations of the specific and particular embodiments described herein.

[0028] As mentioned above, existing research lacks practical applications of sphingolipids on plants or is limited to their effects under conventional environmental conditions.

[0029] Therefore, the purpose of this invention is to provide a novel sphingolipid bacteria that can promote seed germination and plant growth, and after cultivation, can be used as a microbial agent in unconventional environmental conditions (such as sandy soil environments), and effectively increase plant height, root length and weight.

[0030] The inventors isolated and screened a *Sphingobium* strain from the desert environment of Xinjiang and sequenced its 16S rDNA gene sequence. The nucleotide sequence of the 16S rDNA is shown in SEQ ID NO:1. Similarity searches were performed on the sequencing results using the BLAST program in the EZBiocloud database, and multiple sequence alignment and phylogenetic tree construction were conducted using MEGA. The strain showed the highest homology with *Sphingobium lignivorans* (ON306316) and *Sphingobium xanthum* (KF437579), with similarities of 96.84% and 96.76%, respectively. Since its similarity to known species was less than 98.5%, and the calculated genome ANI with the highest homology of the known species was 82.56% (less than 95%), this strain was identified as a new species of *Sphingobium* and named *Sphingobium sp. D-491*. The strain was deposited on October 21, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with the accession number GDMCC 65304.

[0031] Therefore, in a first aspect, the present invention provides a sphingobium sp. having the 16S rDNA sequence shown in SEQ ID NO.1.

[0032] As described above, the inventors found that the 16S rDNA sequence of this strain has a similarity of less than 98.5% with the 16S rDNA sequence of known sphingolipid species, and its genome ANI is less than 95% with the known species with the highest homology. Therefore, it can be identified that this strain comes from a new species of the genus Sphingolipid.

[0033] In some embodiments, the strain number of the sphingolipid is D-491, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 21, 2024, with accession number GDMCCNo.65304.

[0034] In a second aspect, the present invention provides a microbial preparation comprising the sphingolipids and / or cultures thereof of the first aspect of the present invention.

[0035] In this invention, "microbial preparation" refers to a type of biological product prepared directly using microorganisms, such as bacteria, or prepared using microbial cultures, wherein the microorganism is the main bioactive component. The microbial preparation can be in solid, liquid, or lyophilized form. The microbial preparation can be widely used in agriculture, horticulture, environmental protection, and other fields. For example, it can replace some chemical fertilizers and pesticides, reducing environmental pollution; it can improve soil quality, enhance soil biological activity, and promote vigorous soil development; and / or increase the efficiency of plant nutrient absorption and improve plant stress resistance.

[0036] The term "culture" refers to a specific group of microorganisms that has been cultured and propagated under artificially defined conditions. This can be a population of microbial cells or a growth on a liquid or solid culture medium. The main components of a culture include the microbial cells themselves, and optionally, the metabolic products of the microbial cells, as well as a conditioned medium or water. In this document, "conditioned medium" refers to a culture medium in which microbial cells have been cultured under specified conditions. Therefore, it is understood that, in addition to fresh culture medium, conditioned medium additionally contains the metabolic products of the microbial cells. In some cases, the conditioned medium may contain components that are undesirable for subsequent applications. Therefore, the cells and supernatant can be separated by centrifugation, and the cells can be resuspended in an appropriate volume of water for subsequent applications.

[0037] In some embodiments, the microbial preparation comprises Sphingolipid D-491 and / or its culture as an active ingredient.

[0038] In some embodiments, the OD of the sphingolipids in the microbial preparation 600 Value ≥ 0.5.

[0039] Those skilled in the art will know that, through the OD of bacterial cells 600 The density or concentration of the bacteria can then be calculated. Therefore, in some embodiments, the content of sphingolipids in the microbial preparation is ≥1.0 × 10⁻⁶. 8 CFU / mL.

[0040] In analyzing the genomic characteristics of Sphingolipid D-491, the inventors discovered that the strain's genome map contains the nitrogen-fixing gene nifH and the major carbon-fixing pathway map01200. Furthermore, in further functional verification analysis, the inventors found that this strain can indeed grow on nitrogen-free medium and possesses nitrogen-fixing capabilities.

[0041] Therefore, in a third aspect, the present invention provides the use of the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention in biological nitrogen fixation.

[0042] While analyzing the genomic characteristics of *Sphingolipidus* D-491, the inventors also discovered that the strain's genome map contains a key gene for the production of indoleacetic acid (IAA). Indoleacetic acid is one of the earliest discovered plant growth-promoting hormones, regulating stem growth rate, inhibiting lateral buds, and promoting rooting. It is used in agriculture to significantly promote the rooting of cuttings. In functional verification analysis, the inventors found that this strain indeed exhibits the ability to secrete IAA.

[0043] Therefore, in a fourth aspect, the present invention provides the use of the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention in the production of indoleacetic acid (IAA).

[0044] In a fifth aspect, the present invention provides the use of the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention in improving soil nutrition. The improvement of soil nutrition includes both direct application of sphingolipids D-491 or its microbial preparations to the soil and application of sphingolipids D-491 or its microbial preparations as bio-fertilizers or soil nutrient improvers to the soil.

[0045] Sandy soils, such as those in the Hotan Desert of Xinjiang, are characterized by high sand content, loose texture, low soil moisture and nutrient content, poor water and fertilizer retention capacity, and are typically alkaline (e.g., pH 9.0). These conditions create stress for microorganisms, hindering their growth and reproduction, resulting in low abundance and diversity. However, the inventors have discovered that when Sphingolipid D-491 or its microbial preparations are applied to plants grown in sandy soil (e.g., sandy soil with a pH of 9.0), the plants thrive despite the poor soil conditions. Therefore, the discovery of Sphingolipid D-491 broadens the application scenarios for Sphingolipid strains, enabling their use in sandy soil environments and demonstrating alkaline tolerance.

[0046] Therefore, in some implementations, the soil is sandy soil.

[0047] Furthermore, the inventors verified the practical application of Sphingolipid D-491 on plants, including its effects on seed germination and its growth-promoting effects on plants.

[0048] First, the experimental results showed that the germination rate of seeds soaked in Sphingobacterium D-491 solution was significantly higher than that of untreated seeds, and the root tip elongation was also more obvious, indicating that Sphingobacterium D-491 can significantly improve the germination rate of plant seeds.

[0049] Therefore, in a sixth aspect, the present invention provides a method for promoting the germination of plant seeds, wherein the method includes contacting the plant seeds with a sphingolipid of the first aspect of the present invention or a microbial preparation of the second aspect of the present invention.

[0050] The contact can be achieved in several ways. For example, a bacterial solution containing Sphingomyelin D-491 can be sprayed onto the surface of plant seeds. This spraying can be performed once or multiple times, such as at least two, three, or four times, with appropriate intervals between each spray, such as 5 minutes, 10 minutes, or 15 minutes, to ensure that Sphingomyelin D-491 can sufficiently stimulate seed germination. Alternatively, plant seeds can be soaked in a bacterial solution containing Sphingomyelin D-491 for a period of time, such as at least 30 minutes, to stimulate seed germination. In one embodiment, the contact is described as soaking plant seeds in a bacterial solution containing Sphingomyelin D-491 for a period of time, such as at least 30 minutes.

[0051] Furthermore, the plant seeds can be derived from the seeds of field crops and forage crops. "Field crops" refer to major crops cultivated in open fields on arable land, primarily including, for example, wheat, rice, sorghum, corn, cotton, rapeseed, and legumes such as soybeans. "Forage crops" refer to grasses or other herbaceous plants used for livestock feed, primarily including alfalfa, ryegrass, Mexican corn grass, Napier grass, sweet elephant grass, Sudan grass, foxtail grass, and elephant grass. In some embodiments, the plant seeds are derived from the seeds of field crops and forage crops. In a preferred embodiment, the field crop is soybean, and the forage crop is alfalfa.

[0052] Secondly, the experimental results showed that alfalfa and soybeans treated with Sphingobacterium D-491 bacterial solution exhibited significantly greater plant height, more abundant foliage, and more developed root systems compared to the control group without treatment, indicating that Sphingobacterium D-491 has a significant growth-promoting effect on alfalfa and soybean plants. Furthermore, the experiment also verified that Sphingobacterium D-491 has a significant growth-promoting effect on alfalfa and soybean plants in both conventional and sandy soils.

[0053] Therefore, in a seventh aspect, the present invention provides a method for promoting plant growth, wherein the method comprises applying to the plant either the sphingolipids of the first aspect of the present invention or the microbial preparations of the second aspect of the present invention.

[0054] In some implementations, the plants are grown in sandy soil.

[0055] In some implementations, the plants include field crops and forage crops. The field crops may include, for example, wheat, rice, sorghum, corn, cotton, rapeseed, and legumes such as soybeans, but are not limited thereto; the forage crops may include, but are not limited thereto, alfalfa, ryegrass, Mexican corn grass, Napier grass, sweet elephant grass, Sudan grass, foxtail grass, elephant grass, etc.

[0056] In some implementations, promoting plant growth includes: increasing plant weight; promoting stem growth to increase plant height; promoting leaf growth to increase the number and size of leaves; and / or promoting root development to increase taproot length.

[0057] In some embodiments, applying the sphingolipid or microbial agent to the plant includes applying it directly to the plant, for example, its roots, or to the plant's growing environment, which includes any environment suitable for plant growth, such as soil, water, or plant nutrient solution.

[0058] The present invention will be further described below through the following embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0059] Example

[0060] Example 1: Isolation of new strains of the genus *Sphingolipidae*

[0061] Rhizosphere soil from *Pennisetum affine* in the Hotan desert of Xinjiang was collected as experimental material. 1.0 g of sand was weighed for bacterial isolation. Using a serial dilution method, the sand was poured into a sterile centrifuge tube, 10 ml of sterile water was added, the mixture was shaken thoroughly, and allowed to stand for half an hour. The supernatant was then used for serial dilution until a 10⁻⁶ solution was obtained. -6 Select an appropriate dilution gradient, and evenly spread 100 μL of the sample dilution onto BHI medium. Perform three replicates for each dilution. Incubate the cultures at 28°C for 3 days. Select single colonies with different morphological characteristics for repeated purification; the resulting strains are the test strains. Mix 60% sterile glycerol with the bacterial culture at a 1:2 ratio and store at -80°C.

[0062] Example 2: Species identification of new strains of the genus *Sphingolipidae*

[0063] 2.116S rDNA sequence

[0064] The bacterial culture of the test strain obtained in Example 1 was subjected to bacterial PCR to amplify the 16S rDNA sequence of the strain. The primers used were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplified product was sequenced. The total length of the 16S rDNA sequence of Sphingobium sp. D-491 was 1387 bp. Homology comparison of the sequence results with the EZBiocloud database (https: / / www.ezbiocloud.net / ) revealed the highest homology with Sphingobium lignivorans (ON306316) and Sphingobium xanthum (KF437579), with similarities of 96.84% and 96.76%, respectively, but both were below 98.65%. Meanwhile, as... Figure 1 Phylogenetic analysis showed that *Sphingobium* sp. D-491 did not cluster with other strains. Since the 16S rDNA of *Sphingobium* sp. D-491 showed less than 98.65% similarity to the 16S rDNA of the most homologous known species, and its calculated genome ANI with the most homologous known species was 82.56%, this strain can be considered a new species of *Sphingobium*. This strain was deposited on October 21, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCCNo. 65304, and classified as *Sphingobium* sp. D-491.

[0065] The 1387bp 16S rDNA sequence was obtained by sequencing, as shown in SEQ ID No. 1.

[0066] SEQ ID NO:1:

[0067] CTGTGGTCGCCTGCCTCCTTGCGGTTAGCTCAACGCCTTCGAGTGAATC

[0068] CAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCTGGGAACGTAT

[0069] TCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGCCTTCATGCT

[0070] CTCGAGTTGCAGAGAACAATCCGAACTGAGACGACTTTTGGAGATTAG

[0071] CTCACCCTCGCGAGTTCGCTGCCCACTGTAGTCGCCATTGTAGCACGTG

[0072] TGTAGCCCAACGCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCT

[0073] TCCTCCGGCTTATCACCGGCGGTTCCTTTAGAGTGCCCAACTAAATGAT

[0074] GGCAACTAAAGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACAT

[0075] CTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCACCTATCCA

[0076] GCCGAACTGAAGGAAACCATCTCTGGTAACCGCGATAGGGATGTCAAA

[0077] CGTTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACC

[0078] GCTTGTGCAGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGT

[0079] ACTCCCCAGGCGGATAACTTAATGCGTTAGCTGCGCCACCCGAATTCCA

[0080] TGAACCCGGACAGCTAGTTATCATCGTTTACGGCGTGGACTACCAGGGT

[0081] ATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCAATACTT

[0082] GTCCAGCGAGCCGCCTTCGCCACTGGTGTTCTTCCGAATATCTACGAAT

[0083] TTCACCTCTACACTCGGAATTCCACTCGCCTCTCCAAGATTCTAGCCAT

[0084] CCAGTCTCAAAGGCAGTTCTGGAGTTGAGCTCCAGGCTTTCACCTCTG

[0085] ACTTAAATGGCCGCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAAC

[0086] GCTAGCCCCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGG

[0087] GCTTATTCTCCCGGTACTGTCATTATCATCCCGGGTAAAAGAGCTTTACA

[0088] ACCCTAAGGCCTTCATCGCTCACGCGGCATTGCTGGATCAGGCTTTCGC

[0089] CCATTGTCCAATATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGT

[0090] GTCTCAGTCCCAGTGTGGCTGATCATCCTCTCAGACCAGCTAAGGATC

[0091] GTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCTACGCGGGC

[0092] TCATCCAAAGGCGATAAATCTTTGGTCTTGCGACATCATCCGGTATTAG

[0093] CACCCCTTTCGAGGAGTTATTCCGAACCTTTGGGCAGATTCCCACGCGT

[0094] TACGCACCCGTGCGCCACTAGATCCGAAGATCTCGTTCGACTTG

[0095] 2.2 Genome characteristics of Sphingolipid D-491

[0096] Sequencing of *Sphingolipidae* D-491 yielded a 4,834,448 bp chromosome. The GC content was 65.52%, and 4502 coding genes were predicted. The genome map of this strain can be found at [link to genome map]. Figure 2 3111 functional genes were predicted in the KEGG database, including the nitrogen fixation gene nifH, nodulation factors nodA, nodB, nodc, and nodD, the main carbon fixation gene pathway map01200, the flavonoid gene, key genes for IAA production, and key genes for alkaline phosphatase production. The potassium solubilization pathway is also relatively complete.

[0097] Example 3: Colony morphology of Sphingolipid D-491

[0098] Using the streak plating method, *Sphingolipidella esculenta* D-491 was inoculated onto LB solid medium and then placed in a 28°C incubator. After 3 days of growth, round, yellow colonies appeared, with smooth surfaces, regular edges, and slightly moist texture (see [link to article]). Figure 3 Gram staining of the strain revealed that Sphingobium sp. D-491 appeared red under the microscope, indicating it is a Gram-negative bacterium (see [link to article]). Figure 4 ).

[0099] Example 4: Functional Verification Analysis of Sphingolipid D-491

[0100] 4.1 Nitrogen fixation capacity

[0101] Prepare a nitrogen-free Ashby medium with the following composition ( / L): 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 5g calcium carbonate, 10g mannitol, 0.1g calcium sulfate, and 15g agar; pH 7.0.

[0102] Sphingolipid D-491 was inoculated at a 10% inoculum in nitrogen-free Ashby liquid medium and cultured at 28°C on a shaker at 180 rpm for 48 h. A blank Ashby liquid medium without inoculation was used as a control group. After culture, the medium inoculated with Sphingolipid D-491 (treatment group) was found to be significantly more turbid than the control group. The OD values ​​of the experimental group and the control group (CK) were measured separately. 600 Value, OD of the control group 600 The value is 0.257, processing group OD 600 The value is 0.617, such as Figure 5 As shown in the figure. Experimental results indicate that Sphingolipid D-491 can grow on nitrogen-free medium and has nitrogen-fixing ability.

[0103] 4.2 IAA secretion capacity

[0104] Sphingolipid D-491 was inoculated into LB liquid medium (containing 0.5 mg / mL L-tryptophan) and cultured at 30℃ with shaking at 150 rpm for 3 days. The bacterial culture was then centrifuged at 12000 rpm for 15 min, and 800 μL of the supernatant was collected and an equal volume of Salkowski's reaction solution (1 mL 0.5 mol / L ferric chloride, 49 mL 35% perchloric acid) was added. The mixture was inverted and incubated in the dark for 30 min. The results showed that the mixture inoculated with Sphingolipid D-491 (treatment group) developed a color reaction and turned red, while the control showed no significant change. The absorbance was measured at 530 nm using a microplate reader. LB medium without the strain but treated in the same way served as the control group (CK). The OD of the control group... 530 The value is 0.257, OD of the treatment group 600 The value is 0.617, indicating that Sphingolipid D-491 has the ability to secrete IAA.

[0105] 4.3 Alkali stress resistance

[0106] Prepare LB solid culture medium with a pH of 9.0, and use the streak plate method to... Sphingolipids The bacterial suspension of D-491 was inoculated onto the culture medium and then placed in an incubator at 28°C to observe the growth of the strain. Figure 7 As shown, after 2 days of cultivation, the strain was found to grow under pH 9.0 conditions, indicating that it has alkali tolerance.

[0107] Example 5: Effects of Sphingolipid strain D-491 on seed germination

[0108] The cultured Sphingolipid D-491 bacterial suspension was centrifuged, and the bacterial cells were resuspended in sterile water to adjust the OD. 600 Adjust to 0.5. Disinfect alfalfa seeds (Algonquin) with 75% alcohol for 2 minutes, then rinse 5-6 times with sterile water until all alcohol residue is removed. 20 alfalfa seeds were allocated to each of the control and treatment groups and placed in blank petri dishes. The control group seeds were soaked in sterile water to promote germination, while the treatment group seeds were soaked in a resuspended bacterial solution to promote germination. After 24 hours, the germination rate was calculated based on the germination status of the seeds. The calculated germination rate was 75% for the control group and 90% for the treatment group. Furthermore, if... Figure 8 As shown, the germination rate of the treatment group inoculated with Sphingobacterium D-491 bacterial solution was higher and the root tip elongation was more obvious than that of the control group, indicating that Sphingobacterium D-491 can significantly improve the germination rate of alfalfa.

[0109] Example 6: Growth-promoting effect of Sphingolipid D-491 on potted alfalfa and soybean in conventional soil environment. Sphingolipid D-491 was inoculated at a 10% inoculum on LB liquid medium and cultured at 28°C for 3 days in a shaker at 180 rpm. The bacterial culture was centrifuged to collect the cells, and the cells were resuspended in sterile water to obtain OD. 600 Adjust the value to 0.5 (at which point the concentration of sphingolipids should be at least 1.0 × 10⁻⁵). 8 (CFU / mL) was used to treat potted alfalfa and soybeans.

[0110] The test materials for the pot experiment were alfalfa (Algonquin) and soybean (Longken 306). The seeds of both plants were disinfected with 75% alcohol for 2 minutes, followed by rinsing with sterile water 5-6 times. The pots used for the pot experiment were disinfected with 75% alcohol, and the soil was autoclaved substrate.

[0111] Alfalfa and soybean seeds were planted in the autoclaved substrate. At the same time as sowing, the seeds were inoculated with bacterial solution for the first time at a rate of 5 ml per seed. One week after the alfalfa and soybeans germinated, they were inoculated with bacterial solution again at a rate of 5 ml per plant. The control group was inoculated with an equal amount of sterile water.

[0112] Photos of alfalfa and soybean seedlings grown in substrate soil 15 days after emergence are shown. Figure 9 .Depend on Figure 9 It is evident that the germination rates of alfalfa and soybean seeds in the treatment group were significantly higher than those in the control group. These results indicate that Sphingolipid D-491 has a significant growth-promoting effect on alfalfa and soybean in conventional soil environments.

[0113] Example 7: Growth-promoting effect of Sphingolipid D-491 on potted alfalfa under sandy soil conditions. Sphingolipid D-491 was inoculated at a 10% inoculum on LB liquid medium and cultured at 28°C for 3 days in a shaker at 180 rpm. The bacterial culture was centrifuged to collect the cells, and the cells were resuspended in sterile water to obtain OD. 600 Adjust the value to 0.5 (at which point the concentration of sphingolipids should be at least 1.0 × 10⁻⁵). 8 (CFU / mL) was used to treat potted alfalfa.

[0114] The test material for the pot experiment was alfalfa (Algonquin). Alfalfa seeds were disinfected with 75% alcohol for 2 minutes, followed by rinsing with sterile water 5-6 times. The pots used for the pot experiment were disinfected with 75% alcohol, and the soil was high-pressure sterilized sand collected from the Xinjiang desert.

[0115] Alfalfa seeds were planted in autoclaved sand. At the same time as sowing, the seeds were inoculated with bacterial solution for the first time at a rate of 5 ml per seed. One week after the alfalfa germinated, the seeds were inoculated with bacterial solution again at a rate of 5 ml per plant. The control group was inoculated with an equal amount of sterile water.

[0116] Thirty days after alfalfa emergence, plant growth was observed and physiological traits were measured. The results are shown in Table 1. Compared with the control group, the treatment group inoculated with Sphingolipid D-491 showed an increase in plant height and root length of 32.01% and 2.75%, respectively, an increase in aboveground fresh weight and underground fresh weight of 90.29% and 10.21%, respectively, and an increase in the number of aboveground branches.

[0117] Table 1. Effects of inoculation with Sphingolipid D-491 on the growth of potted alfalfa

[0118]

[0119] also, Figure 10 A photograph of alfalfa seedlings 30 days after emergence is shown. Figure 10 As shown, compared with the control group, the plant height of the treatment group was significantly increased, the leaves were more lush, and the root system was more developed. These results indicate that even in nutrient-poor sandy soil environments, alfalfa plants treated with Sphingobacterium D-491 have a significant growth-promoting effect compared with the control group that was not treated with Sphingobacterium D-491. This provides the possibility for large-scale planting of alfalfa and other plants in sandy soil in the future.

Claims

1. Sphingobium sp., which has the 16S rDNA sequence shown in SEQ ID NO.

1.

2. The sphingolipid of claim 1, strain number D-491, is deposited at Guangdong Provincial Microbial Culture Collection Center on October 21, 2024, with accession number GDMCC No. 65304.

3. A microbial preparation, wherein the microbial preparation comprises the sphingolipids of claim 1 or 2 and / or their cultures.

4. The microbial preparation according to claim 3, wherein the OD of the sphingolipids in the microbial preparation is... 600 Value ≥ 0.

5.

5. The microbial preparation according to claim 3, wherein the content of sphingolipids in the microbial preparation is ≥1.0 × 10⁻⁶. 8 CFU / mL.

6. Use of the sphingolipids of claim 1 or 2 or the microbial preparation of any one of claims 3-5 in biological nitrogen fixation.

7. Use of the sphingolipids of claim 1 or 2 or the microbial preparation of any one of claims 3-5 in the production of indoleacetic acid (IAA).

8. Use of the sphingolipids of claim 1 or 2 or the microbial preparation of any one of claims 3-5 in improving the nutrition of soil, such as sandy soil.

9. A method for promoting the germination of plant seeds, wherein the method comprises contacting the plant seeds with the sphingolipids of claim 1 or 2 or a microbial preparation of any one of claims 3-5.

10. The method of claim 9, wherein the plant seeds are derived from field crops and forage crops such as alfalfa and soybeans.

11. A method for promoting plant growth, wherein the method comprises applying to the plant the sphingolipids of claim 1 or 2 or the microbial preparation of any one of claims 3-5.

12. The method according to claim 11, wherein, The plants are grown in sandy soil.

13. The method of claim 11, wherein the plants include field crops and forage crops such as alfalfa and soybeans.

14. The method of claim 11, wherein promoting plant growth comprises: Increase the weight of the plant; Promotes stem growth and increases plant height; Promotes leaf growth, increasing the number and size of leaves; and / or It promotes root development and increases the length of the taproot.

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