Yanomama sphingolipids and uses thereof

Abstract

The application belongs to the technical field of microbial application, and provides a strain of sphingomonas yanoikuyae with a preservation number of CGMCC No.28289.The strain can effectively degrade peanut phenolic acid allelochemicals, alleviate autotoxicity, and antagonize peanut soil-borne pathogenic bacteria, and can alleviate peanut continuous cropping obstacles.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a strain of *Sphingosine monocytogenes* and its application in peanut cultivation. Background Technology

[0002] peanut( Arachis Hypogaea Peanuts (Linn.) are a major oilseed and cash crop in my country, playing a crucial role in ensuring the country's edible oil security and improving the health of its population. In recent years, due to increased demand and limited planting area, many major peanut-producing areas have engaged in large-scale, continuous peanut cultivation for several years in pursuit of economic benefits, resulting in severe continuous cropping. Long-term continuous cropping leads to stunted peanut plants, reduced photosynthesis, deterioration of soil physical and chemical properties, imbalance in soil microbial community structure, and decreased yield and quality.

[0003] Continuous cropping obstacle refers to the phenomenon that, even under normal management conditions, the same plant or closely related plants will experience poor growth and development, severe pests and diseases, reduced yield, and deteriorated quality after being continuously cropped together. The main causes of continuous cropping obstacle include: changes in soil physicochemical properties, such as poor soil physical characteristics, secondary salinization and acidification, and nutrient imbalance; deterioration of the soil biological environment, such as changes in soil enzyme activity, alterations in the structure and quantity of microbial communities; and allelopathic autotoxicity, such as the autotoxicity of plant water extracts, the toxicity of decomposing plant residues, and the allelopathic effects of root exudates. Continuous cropping obstacle can have varying degrees of impact on plant morphology, leaf photosynthetic rate, root vigor, yield, and quality.

[0004] Peanut continuous cropping obstacles are the result of the combined effects of many factors in the peanut plant-soil system. Currently, the main mechanisms of peanut continuous cropping obstacles are as follows: (1) Deterioration of soil physicochemical properties. Long-term continuous cropping of peanuts will cause a deficiency of elements such as phosphorus, potassium, calcium, boron, and iron in the soil, resulting in the plant's inability to absorb the required nutrients from the soil, thus affecting its normal growth and development. In addition, the concentration of organic acids secreted by peanut roots during long-term continuous cropping will continuously increase in the soil, which can cause soil acidification and leaching of soil nutrients. (2) Autotoxic effects of allelochemicals. During the growth process, peanut plants secrete and release chemical substances (i.e., allelochemicals) into the soil and aboveground environment during long-term continuous cropping, which have a direct or indirect impact on the growth and development of surrounding plants or microorganisms. Currently, research on peanut allelochemicals mainly focuses on the identification of root exudates, specifically including phenolic acids, fatty acids, alcohols, aldehydes, and ketones. When these allelochemicals accumulate to a certain concentration in the soil, they can disrupt the soil microbial community structure, soil enzyme activity, plant cell membranes, and photosynthesis, affecting the normal growth of peanut plants and increasing the incidence of disease. (3) Imbalance in the soil microbial community structure. In the rhizosphere soil of peanut plants that are continuously cropped, the microbial community structure is disrupted, and the number of pathogenic fungi among the fungi increases significantly, such as Fusarium oxysporum Meanwhile, the diversity and abundance of beneficial fungi decrease accordingly. Beneficial bacteria in continuously cropped soils undergo directional selection, causing soil microorganisms to shift from bacterial to fungal. This disrupts the soil microbial balance, hindering plant growth and development, reducing yield and quality, and severely impacting farmers' economic income.

[0005] To address the mechanism of peanut continuous cropping obstacles, the following methods can be used: (1) Reasonable crop rotation, intercropping, and relay cropping. Practice has shown that the simplest and most convenient measure to alleviate peanut continuous cropping obstacles is crop rotation, but this is limited in major peanut producing areas because it reduces the total peanut yield of the year. (2) Selecting high-yield and continuous cropping tolerant varieties. Currently, varieties tolerant to continuous cropping have a small yield reduction under continuous cropping conditions, but the yield of these varieties is very low and they are not favored by growers. (3) Using organic fertilizer and continuous cropping-specific fertilizer. Reasonable fertilization measures can alleviate peanut continuous cropping obstacles to a certain extent. (4) Improving soil tillage technology. Deep tillage can break the microbial community structure of the topsoil, allowing pathogens to be buried deep in the soil layer, but this technology is limited by tillage machinery. (5) Using disinfectants and fungicides. Disinfectants and fungicides kill beneficial bacteria while killing pathogens, which is not conducive to the healthy development of the soil. (6) Applying microbial inoculants. Applying microbial inoculants to alleviate peanut continuous cropping obstacles is currently a research hotspot and will gradually become a major means of controlling peanut diseases and pests caused by continuous cropping. Applying microbial inoculants is expected to be an important measure to alleviate or eliminate peanut continuous cropping obstacles, and screening for native antagonistic bacteria will yield even better results. Summary of the Invention

[0006] To address issues such as peanut continuous cropping obstacles, this invention provides a strain of *Sphingosine monocytogenes* (Yano). Sphingobium yanoikuyae It can effectively degrade peanut allelochemicals, alleviate autotoxicity, and antagonize peanut soil-borne pathogens, thus alleviating peanut continuous cropping obstacles.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A strain of Sphingosine monocytogenes ( Sphingobium yanoikuyae PN83, with accession number CGMCCNo.28289.

[0009] This strain can be used to degrade plant allelochemicals and reduce peanut continuous cropping obstacles.

[0010] The allelochemical is selected from at least one of cinnamic acid, p-hydroxybenzoic acid, and phthalic acid. Preferably, the allelochemical is cinnamic acid.

[0011] This strain can be used to antagonize Fusarium and to control peanut root rot caused by Fusarium.

[0012] This strain can be used to prepare pesticides or fertilizers, such as biocontrol agents or microbial fertilizers for peanut cultivation. Preferably, the concentration of this strain in the aforementioned biocontrol agents or biofertilizers is not less than 10%. 5 cfu / mL.

[0013] The present invention has the following advantages:

[0014] The *Sphingosine monocytogenes* provided by this invention ( Sphingobium yanoikuyae This strain can degrade phytoallergenic substances such as cinnamic acid, p-hydroxybenzoic acid, and phthalic acid, alleviate the autotoxic effects caused by peanut allelochemicals, and reduce peanut continuous cropping obstacles. This strain can also antagonize Fusarium oxysporum and prevent peanut root rot caused by Fusarium. Moreover, this bacterium was isolated from peanut planting soil, has no pathogenic effect on peanuts, and has little impact on the environment.

[0015] Biological Preservation Information

[0016] Yano Sphingosine mononitrate ( Sphingobium yanoikuyae PN83 was deposited on August 29, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 28289. Attached Figure Description

[0017] Figure 1 Phylogenetic tree of the target strain;

[0018] Figure 2 The variation of cinnamic acid content in soil at different locations over time;

[0019] Figure 3 The change of phthalic acid content in soils under different treatments over time;

[0020] Figure 4 The change in p-hydroxybenzoic acid content in soils under different treatments over time;

[0021] Figure 5 A diagram showing the confrontation and growth of *Sphingosine monocytogenes* and *Fusarium oxysporum*, the pathogen causing peanut root rot.

[0022] Figure 6 The effect of *Sphingosine monocytogenes* on the incidence of peanut root rot. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0024] Example 1: Screening and Identification of Target Strains

[0025] Weigh 1g of peanut rhizosphere soil sample from 5 consecutive years of peanut cropping, add it to 100mL of sterile water, shake on a shaker for half an hour, then transfer 1mL of the suspension to 99mL of sterile water to prepare a 10% concentration. -2 The suspension was shaken on a shaker for half an hour, and 1 mL of the suspension was then transferred to 99 mL of sterile water to prepare a concentration of 10. -3 The suspension was prepared sequentially in 10... -4 10 -5 Suspension. Take 10... -3 10 -4 and 10 -5 200 μL of the suspension was evenly spread on R2A plates, with each concentration repeated three times. After incubation at 28°C for 3–7 days, colony selection and purification were initiated. Each single colony was purified at least three times, numbered, and then sent to Shanghai Sangon Biotech Co., Ltd. for 16 rRNA sequencing using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACATTGTTACGACTT-3').

[0026] Based on sequencing results, among the more than 200 strains isolated, PN83 belongs to *Sphingosine monocytogenes* (Yano). Sphingobium yanoikuyae Its phylogenetic tree is as follows: Figure 1 As shown in the figure. Based on the results of previous studies, this strain was selected as the target strain for further research. The isolated *Sphingosine monocytogenes* colonies on R2A medium were round, mucous, and milky yellow; the cells were short rods, arranged singly or in pairs. It was deposited on August 29, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 28289.

[0027] Example 2: Degradation of peanut allelochemicals by *Sphingosine monocytogenes* PN83

[0028] 1. Experimental treatment

[0029] Weigh out a certain amount of analytical grade cinnamic acid, phthalic acid and p-hydroxybenzoic acid, dissolve them separately in a small amount of ethanol (5 mL of ethanol per liter of solution), and then dilute with distilled water to prepare a phenolic acid treatment solution of 400 mg / L for later use.

[0030] The *Sphingosine monocytogenes* isolated in Example 1 was inoculated into R2A liquid medium and cultured in a shaker at 200 r / min at 28°C. When OD... 600 When the value is 0.6, centrifuge at 5000 r / min for 10 min, collect the bacterial cells, and then resuspend the precipitate in sterile distilled water until the OD value is reached. 600 The value was 0.6, and the bacterial suspension was obtained for later use.

[0031] The soil used in the experiment was taken from a peanut field in Ju County, Rizhao City, Shandong Province, where peanuts had been continuously cropped for 10 years. The soil was collected after peanut harvest. The specific method was as follows: soil from the 0-20 cm topsoil layer was collected, and impurities such as gravel and plant / animal remains were removed. The soil was then sieved through a 2 mm sieve and mixed thoroughly. The soil type was brown soil, with a sandy loam texture. The pH value of the tested soil was 5.24, the organic matter content was 11.7 g / kg, and the total nitrogen, total phosphorus, and total potassium were 0.11 g / kg, 1.90 g / kg, and 22.84 g / kg, respectively. The contents of available nitrogen, available phosphorus, and available potassium were 45.92 g / kg, 30.76 g / kg, and 94.67 g / kg, respectively.

[0032] The experiment was conducted as follows: After drying, the soil was divided into plastic bottles, with 100 g of soil in each bottle (5 cm in diameter and 8.5 cm in height). The divided soil was treated with a prepared 400 mg / L phenolic acid solution, with 20 mL of the solution added to each bottle, so that the initial content of the mixture of the three phenolic acids reached 80 mg / kg. 干土 Add 5 mL of bacterial suspension to each soil sample, and add an equal volume of autoclaved bacterial suspension to the control. After treatment, seal each sample with sealing film, leaving small holes for ventilation, and maintain a moisture content of 20% (by weight adjustment). Nine samples were used for each treatment and incubated in the dark at 25°C. Samples were taken at 1, 3, 5, 7, 9, and 15 days after treatment, with three samples randomly selected each time, i.e., three replicates.

[0033] 2. Extraction and detection of allelochemicals

[0034] After mixing the soil thoroughly for each replicate, weigh 20 g of fresh soil into a centrifuge tube, add 25 mL of 2 mol / L NaOH solution, shake and extract for 24 h, centrifuge at 5000 r / min for 15 min, filter the supernatant with filter paper, acidify the filtrate with 5 mol / L hydrochloric acid to pH 2.5, centrifuge for 2 h to remove humic acid, then extract the supernatant five times with 20 mL of ethyl acetate, collect the extract and evaporate to dryness under reduced pressure at 40℃, dissolve the residue in 2 mL of 80% chromatographic methanol solution, and store the extract at 4℃ for later use.

[0035] The contents of three phenolic acids—cinnamic acid, p-hydroxybenzoic acid, and phthalic acid—in the extract were determined by HPLC. 0.01 g of each of the three phenolic acids were accurately weighed into a 10 mL amber volumetric flask, dissolved in an appropriate amount of 80% methanol solution, and diluted to the mark to prepare a 1 mg / mL standard for each phenolic acid. 0.1 mL of each standard was then pipetteed into another 10 mL amber volumetric flask, and diluted to the mark with 80% methanol solution to obtain a 10 μg / mL mixed standard.

[0036] Qualitative analysis of samples was performed using the chromatographic retention time of standards, and quantitative analysis was performed using peak area. The content of phenolic acids in the extract was calculated based on the ratio of the peak area of ​​each phenolic acid in the standards to the peak area of ​​the sample. The content of phenolic acids in the extract was calculated based on the dried weight of the soil. The high-performance liquid chromatograph (HPLC) was a Waters 2695 (USA), the detection column was an Agilent EC-C18 (4.6 mm × 100 mm), the mobile phase was methanol-1% phosphoric acid aqueous solution (2:5 v / v), the flow rate was 1 mL / min, the detection wavelength for phthalic acid was 254 nm, and the column temperature was 30℃; the detection wavelength for the other two phenolic acids was 280 nm, and the column temperature was 25℃.

[0037] The changes in the content of cinnamic acid, phthalic acid, and p-hydroxybenzoic acid over time are as follows: Figure 2-4 As shown in the three figures above, from day 1 to day 15 after treatment, the cinnamic acid content in both the treatment and control groups decreased, but the cinnamic acid content in the treatment group was significantly lower than that in the control group. The phthalic acid content also decreased significantly after treatment, but there was no significant difference between the treatment and control groups on days 7 and 15. The p-hydroxybenzoic acid content showed the same trend as phthalic acid, indicating that this strain of bacteria has a good degradation effect on cinnamic acid.

[0038] Example 3: Antagonistic effect of Yano Sphingosine monocytogenes PN83 on flower pathogens

[0039] 1. Flat-panel standoff

[0040] The plate confrontation method was used to compare the *Sphingosine monocytogenes* isolated in Example 1 with the pathogen *Fusarium oxysporum* isolated from peanut root rot. Fusarium oxysporum A confrontation experiment was conducted. An 8mm diameter *Fusarium oxysporum* mycelial disc was inoculated in the center of PDA medium. An 8mm diameter test strain mycelial disc was vertically inoculated 2.5cm away. Six replicates were set up, with a blank control without test strain mycelial discs. After incubation at 28℃ for 7 days, the strength of the resistance to the pathogen was determined by measuring the diameter of the inhibition zone and calculating the inhibition rate. The inhibition rate was calculated using the following formula:

[0041] Antibacterial rate = (radius of control experiment - radius of treatment experiment) / radius of control experiment.

[0042] The results are as follows Figure 5 As shown, the average diameter of the Fusarium oxysporum mycelium ring in the control group was 4.35 cm, while the average diameter of the inhibition ring in the treatment group was 2.84 cm, with an inhibition rate of 34.71%. This demonstrates that this strain has a significant inhibitory effect on Fusarium oxysporum.

[0043] 2. Pot experiment

[0044] Using a suspension of *Sphingosine monocytogenes* (concentration 1×10⁻⁶) 5The roots of peanut plants grown in vermiculite for 15 days were treated with 10 mL of a sterile bacterial suspension of the same concentration (CFU / mL). The control was an equal volume of sterile bacterial suspension of the same concentration. Five days later, the roots were treated with a bacterial suspension of Fusarium oxysporum (spore concentration 1×10⁻⁶ CFU / mL). 5 Water the peanut roots with 50 mL of water and observe the plant disease situation two weeks later.

[0045] The results of the pot experiment are as follows Figure 6 As shown, Fusarium oxysporum isolated from peanut root rot was inoculated after treatment with Sphingosine monocytogenes. Fusarium oxysporum The disease index of peanut plants in the control group was 11%, while the disease index of the control group was 52%, indicating that *Sphingomonas yannoides* can reduce the incidence of root rot in continuously cropped peanuts.

Claims

1. A strain of *Sphingosine monocytogenes* ( Sphingobium yanoikuyae PN83, with accession number CGMCC No.28289.

2. The application of *Sphingosine monocytogenes* PN83 as described in claim 1 in the degradation of plant allelochemicals, characterized in that... The allelochemical is selected from at least one of cinnamic acid, p-hydroxybenzoic acid, and phthalic acid.

3. The application according to claim 2, characterized in that, The allelochemical is cinnamic acid.

4. The application of Yano Sphingosine monocytogenes PN83 as described in claim 1 in the prevention and control of peanut root rot.

5. The application of *Sphingomonas PN83* as described in claim 1 in reducing peanut continuous cropping obstacles, characterized in that, Continuous cropping obstacles are caused by increased levels of plant allelochemicals and / or peanut root rot caused by Fusarium.

6. A pesticide containing *Sphingosine monocytogenes* PN83 as described in claim 1.

7. The pesticide according to claim 6, characterized in that, The pesticide is a biocontrol agent.

8. The pesticide according to claim 7, characterized in that, The concentration of PN83 of *Sphingosine monocytogenes* Yano is not less than 10. 5 cfu / mL.

9. A fertilizer containing the Yano sphingosine monocytogenes PN83 as described in claim 1.

10. The fertilizer according to claim 9, characterized in that, The fertilizer is a microbial fertilizer.

11. The fertilizer according to claim 10, characterized in that, The concentration of PN83 of *Sphingosine monocytogenes* Yano is not less than 10. 5 cfu / mL.

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