Sphingosine box bacteria for preventing and controlling plants from absorbing heavy metals, fungicide and preparation method and application of spingosine box bacteria and fungicide

Abstract

The invention belongs to the technical field of agriculture and environmental microorganisms, and particularly relates to sphingosine box bacteria for preventing and controlling plants from absorbing heavy metals, a fungicide and a preparation method and application of the sphingosine box bacteria and the fungicide. The sphingomyza sp. Is named as YHY20-d, the sphingomyza sp. Is preserved in the China General Microbiological Culture Collection Center on December 30, 2024, and the preservation number of the sphingomyza sp. Is CGMCC (China General Microbiological Culture Collection Center) NO.33226. The sphingomyza sp. The sphingomyza sp. YHY20-d is inoculated to the heavy metal contaminated soil, the content of available heavy metal in the soil can be reduced, plants can be effectively prevented and controlled from absorbing heavy metal cadmium, lead and copper, and the YHY20-d can also increase the content of organic matter and available phosphorus in rhizosphere soil of the plants, promote conversion of ammonium nitrogen in the soil to nitrate nitrogen and improve the ecological environment of the soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural and environmental microorganisms, and particularly relates to a sphingosine box bacterium for preventing and controlling plants from absorbing heavy metals, a bacterial agent, and a preparation method and application thereof. Background Art

[0002] Due to overexploitation of mineral resources, improper use of fertilizers and pesticides, and the advancement of industrialization, heavy metal contamination in soil is becoming an increasingly serious problem. Heavy metal pollution does not naturally degrade over time, but instead accumulates in the environment. Plants, especially crops such as wheat and rice, are extremely susceptible to the effects of heavy metals in the soil during their growth. For fruit trees or crops like wheat and rice, excessive absorption of heavy metals not only reduces yield and quality but also enters the human body through the food chain, harming human health and causing various diseases such as neurological damage and kidney disease. Currently, numerous methods are available to control heavy metal contamination in soil and reduce plant uptake of heavy metals, including physical remediation, chemical remediation, and bioremediation. Physical remediation is costly and can damage soil structure; while chemical remediation is effective, it can introduce secondary pollution. Using microbial agents to inhibit the absorption and accumulation of heavy metals in wheat is an efficient and environmentally friendly bioremediation method that has attracted the attention of researchers.

[0003] Currently reported microbial species that inhibit plant absorption and accumulation of heavy metals primarily include Bacillus, Enterobacter, Serratia, Sphingomonas, and Pseudomonas. The inventor's previous patent, CN116463245A, discloses an Agrobacterium strain that inhibits wheat absorption of heavy metals and its applications. The Agrobacterium strain NJ20 (Agrobacterium sp.) can inhibit wheat from absorbing the heavy metals lead and cadmium. Patent CN114317373A also discloses a Sphingomonas sp. PAH02, a microbial preparation, and its application as a functional conditioner for crop cadmium reduction and selenium enrichment. The Sphingomonas sp. PAH02 can reduce cadmium absorption by rice roots.

[0004] However, most microbial strains or agents discovered so far can only fix or inactivate a single heavy metal, making it difficult to simultaneously prevent plant uptake of multiple heavy metals, limiting their application in complex contaminated soils. Furthermore, the interactions between microorganisms in the complex soil-microbe-plant system are influenced by multiple factors, including soil type and heavy metal concentrations. Therefore, it is crucial to provide a wider variety of microorganisms that can prevent plant uptake and accumulation of heavy metals, enrich the resource of microorganisms with inactivation / inactivation capabilities, and offer alternative strategies for different plant and environmental conditions.

[0005] Therefore, screening out new multi-metal-fixing bacteria that can stably adapt and have a wide range of functions, while reducing the accumulation of multiple heavy metals in plants, is of great significance for ensuring the safe production of crops in complex contaminated soils. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, one of the objects of the present invention is to provide a sphingopyxis sp., which is named YHY20-d and deposited in the China General Microbiological Culture Collection Center on December 30, 2024, with a deposit number of CGMCC NO.33226.

[0007] A second object of the present invention is to provide a bacterial agent, which includes the sphingomyelinase box bacteria YHY20-d described above.

[0008] Preferably, the bacterial agent is a liquid bacterial agent, which is fermented by Sphingobacterium sphingosine YHY20-d.

[0009] Preferably, the number of viable bacteria in the liquid bacterial agent is 2×10 8 -9×10 9 CFU·mL -1 within the range.

[0010] The third object of the present invention is to provide a method for preparing the above-mentioned bacterial agent, wherein the preparation method of the bacterial agent is to inoculate the sphingomyelinase YHY20-d into LB liquid culture medium, and culture it in a shaking incubator at 28°C and 150-180 r / min for 20-30 hours until the effective viable bacteria count reaches 2×10 8 ~9×10 9 The desired bacterial agent is obtained when the CFU / mL range is reached. Before inoculation, the sphingomyelinase box bacteria YHY20-d is first cultured on LB solid medium at 28°C for 72 hours for activation.

[0011] A fourth object of the present invention is to provide a use of the above-mentioned Sphingosine Box Bacteria YHY20-d or the above-mentioned bacterial agent in preventing and controlling the absorption of heavy metals by plants.

[0012] Preferably, the heavy metal is cadmium and / or lead and / or copper.

[0013] A fifth object of the present invention is to provide a use of the above-mentioned Sphingosine Box Bacteria YHY20-d or the above-mentioned bacterial agent in increasing the content of organic matter and / or available phosphorus in soil and improving soil fertility.

[0014] A sixth object of the present invention is to provide a method for preventing plants from absorbing heavy metals, which comprises applying an effective amount of the above-mentioned bacterial agent to plants or seeds.

[0015] Preferably, the above-mentioned bacterial agent is used to water the roots of plants, or to soak or coat seeds.

[0016] The beneficial effects of the present invention are:

[0017] 1. The sphingomyelinase box bacteria YHY20-d provided by the present invention can tolerate cadmium, lead and copper, grow well in culture solutions containing cadmium, lead or copper, and can adsorb soluble cadmium, lead or copper in the culture solution. The adsorption and removal rates of cadmium, lead and copper reach 55.72%, 49.50% and 66.69% respectively, providing a new bacterial strain resource for the remediation of heavy metal contaminated soil and ensuring the safe production of plants, especially crops.

[0018] 2. The sphingomyelinase YHY20-d provided by the present invention can produce indoleacetic acid, has a certain dissolving effect on insoluble tricalcium phosphate, and can be used to increase the content of available phosphorus in the soil.

[0019] 3. Wheat experiments showed that the sphingosine box bacteria YHY20-d provided by the present invention can increase the organic matter and available phosphorus content in the wheat rhizosphere soil, promote the conversion of ammonium nitrogen in the soil to nitrate nitrogen, increase soil fertility, and at the same time reduce the available cadmium, lead and copper content in the wheat rhizosphere soil, thereby improving the soil ecological environment.

[0020] 4. Experiments have shown that the sphingomyelinase YHY20-d provided by the present invention can simultaneously reduce the cadmium, lead and copper contents in wheat grains, straw and roots; the bacterial agent prepared by the sphingomyelinase YHY20-d can be used to prevent and control the absorption and accumulation of cadmium, lead and copper in wheat in complex contaminated soils, which is beneficial to the safe production of wheat in complex contaminated soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of Sphingobacterium YHY20-d.

[0022] Figure 2 This is the phylogenetic tree of the 16S rDNA sequence of Sphingobacterium YHY20-d;

[0023] Figure 3The growth of Sphingosine box bacteria YHY20-d in Example 3 is compared. Figure A shows the normal growth colony without adding any heavy metals; Figure B shows the colony grown in 50 mg / L Cd 2+ Colony map of culture medium; C is grown in 600mg / L Pb 2+ Colony diagram of culture medium; D is the colony grown in 200 mg / L Cu 2+ Colony image of culture medium.

[0024] Figure 4 3 is a comparison of the OD values ​​of Sphingosine box bacteria YHY20-d in Example 3. In the figure, A is a curve showing the change of OD value with different cadmium concentrations; B is a curve showing the change of OD value with different lead concentrations; and C is a curve showing the change of OD value with different copper concentrations.

[0025] Figure 5 This is the growth curve of strain YHY20-d in Example 4 under different conditions.

[0026] Figure 6 This is the standard curve of the phosphate solubilization ability of strain YHY20-d in Example 6.

[0027] Figure 7 This is the standard curve of the IAA production ability of strain YHY20-d in Example 6. DETAILED DESCRIPTION

[0028] The present invention will be further described below by way of examples.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0030] Example 1

[0031] Isolation of strains

[0032] The isolated and preserved strain Sphingobacterium sphingosine YHY20-d was isolated and purified from wheat rhizosphere soil. The specific isolation steps are as follows:

[0033] Dig out the vigorously growing wheat plants with their roots and soil, remove the non-rhizosphere soil away from the roots, and use a sterile brush to collect the rhizosphere soil within 1-2 mm of the root surface. Weigh 1 g of wheat rhizosphere soil, add it to 100 mL of sterile water, place it on a shaker, and shake it at 28°C and 180 rpm for 30 minutes to obtain 10 -2 Soil suspension. Pipette 1mL 10 -2 The soil suspension was added to 9 mL of sterile water and mixed well to obtain 10 -3 The soil dilution solution was diluted 10 times in sequence, and the operation was the same as above to obtain 10 -4 and 10-5 Soil dilution. Pipette 100 μl of the dilution and apply it on the soil containing Cd. 2+ 25mg / L LB solid medium (peptone 10.0g, yeast powder 5.0g, sodium chloride 10.0g, agar 20.0g, distilled water 1000mL, pH 7.0-7.2) was placed in a biochemical incubator at 28-30℃ for 48h. After 48h, the actively growing colonies were picked and streaked on a new plate containing Cd. 2+ Culture on a 25 mg / L LB solid medium plate at 28°C for 48 h. Repeat the operation three times. Pick a single colony and add it to LB liquid medium. Place it on a shaker and culture it at 28°C and 180 r / min for 28-36 h. Use 40% glycerol suspension to preserve the strain at -80°C for later use.

[0034] Example 2

[0035] Identification of strain YHY20-d

[0036] Strain YHY20-d was tested according to the Manual of Systematic Identification of Common Bacteria.

[0037] See also Figure 1 When grown on LB solid medium, strain YHY20-d produces yellow colonies with smooth surfaces, regular edges, opaque ridges, and no halos. Its physiological and biochemical characteristics include Gram-negative staining, amylase-negative, VP-negative, MR-negative, catalase-positive, and hydrogen sulfide-negative. It tolerates 7% sodium chloride and grows well at 30°C. It can use glucose, lactose, sucrose, starch, maltose, and mannose as carbon sources, and ammonium and nitrate nitrogen as nitrogen sources.

[0038] DNA from strain YHY20-d was amplified by PCR using primers 27F and 1492R, universal primers for the bacterial 16S rRNA gene, and the resulting PCR product was sequenced. The 16S rDNA sequence was analyzed using BLAST against the NCBI nucleic acid database. Figure 2 The results showed that strain YHY20-d belongs to the genus Sphingopyxis sp. and has a maximum similarity of 99.38% with the model strain Sphingopyxis chilensis strain S37. A phylogenetic tree based on 16S rRNA revealed that strain YHY20-d and Sphingopyxis chilensis strain S37 clustered on the same branch.

[0039] The DNA sequence of strain YHY20-d is shown in the sequence listing.

[0040] To further explore the taxonomic status of strain YHY20-d, whole genome sequencing was performed, and ANIb and ANIm were calculated using the online website JspeciesWS (http: / / jspecies.ribohost.com / jspeciesws), and the Genome-to-Genome Distance Calculator was used.

[0041] The DDH value was calculated using the GGDC (http: / / ggdc.dsmz.de / ggdc.php). The ANI analysis and dDDH results are shown in Table 1. The thresholds for identifying a new species using ANI analysis and dDDH analysis are ANI values ​​<95%-96% and dDDH values ​​<70%, respectively. The results showed that, based on the whole genome sequence, the ANI values ​​of strain YHY20-d and its highly similar model strains were both less than 95%, and the dDDH values ​​were both less than 70%, both below the thresholds for species identification. Therefore, strain YHY20-d is considered a new species of the genus Sphingopyxis, distinct from previously reported species.

[0042] Table 1 ANI and dDDH analysis results of closely related model strains of YHY20-d strain

[0043]

[0044] Example 3

[0045] Tolerance of strain YHY20-d to heavy metals cadmium, lead and copper

[0046] Preparation of Cd containing different concentration gradients 2+ (5, 25, 50, 75, 100mg / L), Pb 2+ (200, 400, 600, 800, 900, 1000 mg / L) and Cu 2+ The strains were streaked onto solid medium containing and without heavy metals and incubated at 28°C for 72 hours to observe their growth.

[0047] The strain YHY20-d was inoculated into liquid LB medium and cultured in a constant temperature shaker at 160 r / min and 28°C for 24 h. 2+ The concentrations were 5, 25, 50, 75, and 100 mg / L; Pb 2+ The concentrations are 200, 400, 600, 800, 900, and 1000 respectively; Cu 2+The concentrations were 25, 50, 75, 100, 120, 150, and 200 mg / L in liquid LB culture medium, and each concentration treatment was repeated 3 times. The cells were cultured in a shaking incubator at 160 r / min and 28°C for 48 h, and the OD value at 600 nm was measured using a spectrophotometer.

[0048] like Figure 3 As shown in A, strain YHY20-d can grow well on LB solid medium without heavy metals. 2+ Colonies or bacterial mosses appeared on the culture medium with concentrations of 5, 25, 50, and 75 mg / L, but with the increase of Cd 2+ The growth rate of strain YHY20-d gradually weakened with the increase of Cd 2+ The growth condition on the 50mg / L plate is as follows Figure 3 As shown in B. In Cd 2+ When the concentration reached 100 mg / L, no more colonies appeared on the culture medium, indicating that Cd 2+ The concentration of 100 mg / L was lethal to strain YHY20-d. 2+ Colonies or bacterial mosses appeared on the culture medium with concentrations of 200, 400, 600, and 800 mg / L, and the growth rate gradually weakened with the increase of concentration. 2+ The growth condition on the 600mg / L plate is as follows Figure 3 As shown in C. When the concentration reaches 900 mg / L, no more colonies appear on the culture medium, indicating that Pb 2+ The concentration of 900 mg / L was lethal to strain YHY20-d. 2+ Colonies or bacterial mosses appeared on the culture medium at 25, 50, 75, 100, 120, 150, and 200 mg / L. When the concentration reached 200 mg / L, the growth of the strain weakened but it was still able to grow ( Figure 3 (D), indicating that the strain has good copper tolerance.

[0049] As Cd 2+ With the increase of concentration, the OD of the strain 600 The value gradually decreases. When Cd 2+ When the concentration reaches 100 mg / L, OD 600 Values ​​close to 0 ( Figure 4 A). With the increase of Pb in the culture medium 2+ With the increase of concentration, the OD 600 The value gradually decreases. 2+ When the concentration is 900 mg / L, OD 600 The value tends to 0 ( Figure 4 B). With Cu 2+When the concentration increased from 25 mg / L to 200 mg / L, the OD 600 The value gradually decreases ( Figure 4 Middle C).

[0050] In conclusion, the sphingomyelinase box bacteria YHY20-d has a strong tolerance to cadmium, lead and copper.

[0051] Example 4

[0052] Growth curves of strain YHY20-d under different conditions

[0053] The strain YHY20-d was inoculated into liquid LB medium and cultured in a constant temperature shaker at 160 r / min and 28°C for 24 h; the strain was inoculated into a medium free of heavy metals and Cd at a 1% inoculum. 2+ The concentration is 5mg / L, Pb 2+ The concentration is 100mg / L, Cu 2+ Each treatment was repeated three times in 100 mL of liquid LB medium with a concentration of 10 mg / L. The culture was shaken at 160 rpm and 28°C for 48 h. The OD value at 600 nm was measured using a spectrophotometer every 3 h starting from 0 h, and the measurement process continued until the end of 48 h.

[0054] See also Figure 5 The results showed that the strain was free of heavy metals and Cd 2+ The concentration is 5mg / L, Pb 2+ The concentration is 100mg / L, Cu 2 + Under the condition of 10mg / L concentration, all of them can grow normally, and enter the logarithmic growth phase after about 9h of culture, and enter the stable phase after 30h. The growth performance is as follows: no heavy metal group>Cu 2+ 10mg / L group>Cd 2+ 5mg / L group>Pb 2+ 100mg / L group.

[0055] Example 5

[0056] Removal capacity of strain YHY20-d for heavy metals cadmium, lead and copper

[0057] A single colony of strain YHY20-d was picked and inoculated into LB liquid medium, and cultured at 28℃ with a shaker at 180r / min for 24h. The bacterial solution was inoculated into 5mL of Cd-containing liquid at a 3% inoculum volume. 2+ The concentration is 5mg / L, Pb 2+ Concentration 100mg / L, Cu 2+The cells were cultured in LB liquid medium with a concentration of 10 mg / L at 28°C and shaken at 180 rpm for 72 hours. After the culture, the supernatant was collected at 6000 rpm and the heavy metal content was determined by atomic absorption spectroscopy. The results are shown in Table 2.

[0058] Table 2 Removal of cadmium and lead in culture medium by strain YHY20-d

[0059]

[0060] It can be seen that the average removal rate of cadmium by strain YHY20-d is 55.73%, the average removal rate of lead is 49.50%, and the average removal rate of copper is 66.69%.

[0061] Example 6

[0062] Determination of phosphate solubilization ability and indoleacetic acid (IAA) production of strain YHY20-d

[0063] 1. Phosphate solubilization ability of strain YHY20-d

[0064] The bacterial strain is inoculated in a 5mL test tube, and after 28 ℃, 160r / min shaking culture for 30h on a shaking table, it is connected to 10mL phosphate-dissolving medium (glucose 10.0g, ammonium sulfate 0.1g, potassium chloride 0.2g, magnesium sulfate heptahydrate 0.25g, magnesium chloride hexahydrate 5.0g, calcium phosphate 5.0g, distilled water 1000mL, pH 7.0, 115 ℃, 30min) by 5% inoculum size, cultivated for 72h at 28 ℃ of shaking tables 160r / min, and the culture fluid is centrifugal at 8000r / min, and 20 microlitre supernatants are added to 1mL of molybdenum antimony anti-developer, and distilled water is settled to 10mL, develops the colour for 15~20min, and surveys the OD value at 700nm place. It is 0.00, 0.25, 0.50, 1.00, 1.25, 1.50mg / L to make a standard curve with the gradient dilution of 5mg / L phosphorus standard solution to phosphorus content. Add molybdenum antimony anti-coloring agent at room temperature, color for 20 minutes, measure its absorbance and make a standard curve. The standard curve is y = 0.3717x-0.0055, and the correlation coefficient R 2 =0.9997( Figure 6 The phosphorus content in the culture medium of strain YHY20-d was calculated based on the standard curve to be 29.93 mg / L. This indicates that the strain has a certain ability to solubilize phosphate and can convert insoluble calcium phosphate into soluble phosphorus.

[0065] 2. Determination of IAA secretion of strain YHY20-d

[0066] The standard curve was prepared using analytically pure IAA. A 0.2 mg / mL IAA standard solution was serially diluted to concentrations of 0, 5, 10, 15, 20, 25, and 30 mg / L. The absorbance at 530 nm was measured using the Salkowski colorimetric method. The resulting standard curve was y = 0.0192x - 0.0133, with a correlation coefficient of R. 2 =0.9992.

[0067] Nitrogen-containing medium (sucrose 10.0 g, ammonium sulfate 1.0 g, potassium hydrogen phosphate 2.0 g, magnesium sulfate heptahydrate 0.5 g, sodium chloride 0.1 g, yeast extract 0.5 g, calcium carbonate 0.5 g, pH 7.2, distilled water 1000 mL, 115°C for 30 min) was dispensed into test tubes, 4 mL per tube. After sterilization, 1 mL of filter-sterilized tryptophan was added to make the tryptophan concentration in the medium 0.5 mg / mL. The target strain was inoculated into the medium and cultured at 28°C in a shaker at 160 rpm for 48 h. After the incubation period, the culture was centrifuged at 6000 rpm for 10 min. 1 mL of the supernatant was added with 50 μL of 10 mmol / L orthophosphoric acid and 2 mL of Sackowski's colorimetric reagent. The mixture was thoroughly mixed and color developed at 25°C in the dark for 30 min. The absorbance at 530 nm was measured and substituted into the standard curve ( Figure 7 ) The results showed that the IAA content in the culture medium of strain YHY20-d was 5.72 mg / L.

[0068] In summary, strain YHY20-d has certain abilities of solubilizing phosphate and producing IAA, indicating that the strain has certain potential to promote plant growth.

[0069] Example 7

[0070] The inhibitory effect of strain YHY20-d on heavy metal absorption by wheat

[0071] 1. Preparation of liquid bacterial agent of strain YHY20-d

[0072] Use an inoculation loop to pick a loop from the YHY20-d glycerol tube, streak it onto LB solid medium aseptically, and culture it at 28°C for 72 hours. Then, select a single colony of the YHY20-d strain with vigorous growth and inoculate it into LB liquid medium. Incubate it at 28°C in a shaker at 150-180 rpm for 20-30 hours until the effective viable count reaches 2×10 8- 9×10 9 CFU / ml range, that is, the liquid bacterial agent of strain YHY20-d is obtained.

[0073] 2. This example was conducted using a field experiment in heavy metal-contaminated farmland in Anhui Province. The soil's physical and chemical properties were as follows: total cadmium 1.62 mg / kg, total lead 241.60 mg / kg, total copper 136.60 mg / kg, available cadmium 0.87 mg / kg, available lead 65.48 mg / kg, and available copper 32.89 mg / kg. The wheat varieties used in the experiment were Zhenmai 15 and Yangmai 28, with seeds of plump, uniform grain size.

[0074] The experiment set up 4 groups of treatments, each group of treatments was repeated 4 times, and the details of the 4 groups of treatments are as follows:

[0075] Control group 1: Zhenmai 15 was not inoculated;

[0076] Experimental group 1: Zhenmai 15 inoculated with YHY20-d treatment;

[0077] Control group 2: Yangmai 28 was not inoculated;

[0078] Experimental group 2: Yangmai 28 was inoculated with YHY20-d.

[0079] Field plots of 1 square meter each were set up, with each treatment group located within one plot. A randomized complete block design was employed, with protected rows 20-40 cm wide between the plots. 200-300 mL of liquid inoculant strain YHY20-d was applied to each plot. When the wheat reached the jointing stage, 100-240 mL of liquid inoculant strain YHY20-d was added via root irrigation. After the wheat matured, the kernels, straw, and roots were collected and dried, and then cadmium, lead, and copper contents were determined. The determination method involved grinding the kernels, straw, and roots into powder, and then analyzing the contents using nitric acid-perchloric acid-atomic absorption spectrometry. The results are shown in Tables 3, 4, and 5.

[0080] Table 3 Effects of strain YHY20-d on cadmium content in various parts of wheat

[0081]

[0082] Table 4 Effects of strain YHY20-d on lead content in various parts of wheat

[0083]

[0084]

[0085] Table 5 Effects of strain YHY20-d on copper content in various parts of wheat

[0086]

[0087] Note: * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and there is a significant difference between treatments

[0088] It can be seen from Tables 3, 4 and 5 that compared with control group 1, the cadmium content in the grains of experimental group 1 was significantly reduced by 26.88%, the lead content was significantly reduced by 43.47%, and the copper content was significantly reduced by 48% and 57% respectively; the cadmium content in the straw was significantly reduced by 17.41%, the lead content was significantly reduced by 19.15%, and the copper content was significantly reduced by 7.94%; the cadmium content in the roots was significantly reduced by 10.17%, the lead content was significantly reduced by 7.05%, and the copper content was significantly reduced by 14.15%.

[0089] Compared with control group 2, the cadmium content in the grains of experimental group 2 was significantly reduced by 15.98%, the lead content was significantly reduced by 23.72%, and the copper content was significantly reduced by 21.16%; the cadmium content in the straw was significantly reduced by 18.42%, the lead content was significantly reduced by 10.65%, and the copper content was significantly reduced by 10.86%; the cadmium content in the roots was significantly reduced by 11.24%, the lead content was significantly reduced by 9.52%, and the copper content was significantly reduced by 15.77%.

[0090] Based on this, the inoculation with YHY20-d could significantly reduce the cadmium and copper contents in the grains, straw and roots of the two wheat varieties, and significantly reduce the lead content in the grains.

[0091] Example 8

[0092] Effects of strain YHY20-d on soil organic matter and available phosphorus content

[0093] The rhizosphere soil of the test plants was obtained from Example 7: After the experimental wheat matured, the shaking-off method was used. The plant roots were first gently shaken by hand to remove the loose non-rhizosphere soil around the roots. Then, the rhizosphere soil attached to the roots was carefully scraped off with sterile tweezers or a brush and collected in a ziplock bag or centrifuge tube for later use.

[0094] The soil organic matter determination method is as follows: air-dry and sieve the soil sample to be tested, weigh 0.2 g of rhizosphere soil, add 10 mL of a 0.136 mol / L K2Cr2O7-H2SO4 standard solution, and gently shake to mix. Heat at 170-190°C, counting for 7 minutes when condensation appears in the tube. After cooling, add 3-4 drops of o-phenanthroline indicator and titrate with 0.2 mol / L ferrous sulfate standard solution. The endpoint is when the solution changes color from yellow to green and then suddenly to reddish-brown.

[0095] The available phosphorus content in the soil was determined using the molybdenum-antimony colorimetric method. The results are shown in Table 6.

[0096] Table 6 Effects of strain YHY20-d on soil organic matter and available phosphorus content

[0097]

[0098] Note: * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and there is a significant difference between treatments

[0099] Soil organic matter content and available phosphorus content are important indicators for evaluating soil fertility. Table 6 shows that compared with control group 1, the organic matter and available phosphorus contents in experimental group 1 increased significantly, by 59.52% and 25.30%, respectively. Compared with control group 2, the organic matter and available phosphorus contents in experimental group 2 also increased significantly, by 42.00% and 32.35%, respectively. This indicates that inoculation with the bacterial strain increased the organic matter and available phosphorus contents in the soil, thereby improving soil fertility.

[0100] Example 9

[0101] Effects of strain YHY20-d on soil nitrate and ammonium nitrogen contents

[0102] The rhizosphere soil of the test plants was obtained from Example 7: After the experimental wheat matured, the shaking-off method was used. The plant roots were first gently shaken by hand to remove the loose non-rhizosphere soil around the roots. Then, the rhizosphere soil attached to the roots was carefully scraped off with sterile tweezers or a brush and collected in a ziplock bag or centrifuge tube for later use.

[0103] The method for determining the nitrate nitrogen content in soil is as follows: take 5.0 g of soil sample, add 50 mL of 2 mol / L KCl solution, shake for 1 hour, let the suspension stand for 3 to 5 minutes and then filter, measure the absorbance of the extract at 220 nm and 275 nm, and calculate the corrected absorbance A according to the following formula: A = A220-2*A275.

[0104] The method for determining the ammonium nitrogen content in soil is as follows: weigh 10.0 g of soil sample, place it in a 250 mL conical flask, add 50 mL of KCl solution, plug the flask, shake for 30 minutes, filter, take 2.5 mL of the extract into a 25 mL colorimetric tube, add 2.5 mL of KCl, then add 2.5 mL of phenol solution and 2.5 mL of NaClO alkaline solution, shake well, place at room temperature of 20°C for 1 hour, add 0.5 mL of masking agent to dissolve any precipitate, then dilute to 25 mL with water, and measure the absorbance at 625 nm.

[0105] Soil ammonium nitrogen primarily originates from the decomposition of nitrogen-containing organic matter by microorganisms. Under good aeration, ammonium nitrogen can be converted into nitrate nitrogen, which promotes nutrient absorption by plant roots and promotes crop growth. Changes in soil nitrate and ammonium nitrogen levels are shown in Table 7.

[0106] Table 7 Effects of strain YHY20-d on soil nitrate nitrogen and ammonium nitrogen contents

[0107]

[0108] Note: * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and there is a significant difference between treatments

[0109] As shown, compared with control group 1, the nitrate nitrogen content in experimental group 1 increased significantly by 41.95%, while the ammonium nitrogen content decreased significantly by 49.97%. Compared with control group 2, the nitrate nitrogen content in experimental group 2 increased significantly by 56.70%, while the ammonium nitrogen content decreased by 34.37%. Therefore, after inoculation with the bacterial strain, the less mobile ammonium nitrogen is converted into nitrate nitrogen, which is highly mobile with water. This helps to replenish nitrogen deficiency in the root zone, facilitates nitrogen absorption by plants, and improves soil fertility.

[0110] Example 10

[0111] Effects of strain YHY20-d on the contents of available cadmium, available lead and available copper in soil

[0112] The rhizosphere soil of the test plants was obtained from Example 7: After the experimental wheat matured, the shaking-off method was used. The plant roots were first gently shaken by hand to remove the loose non-rhizosphere soil around the roots. Then, the rhizosphere soil attached to the roots was carefully scraped off with sterile tweezers or a brush and collected in a ziplock bag or centrifuge tube for later use.

[0113] The method for determining the available heavy metal content in soil is as follows: extraction is performed according to a solid-liquid ratio of 1:5, that is, 2 g of soil sample is weighed, 10 mL of DTPA extractant is added and mixed, and the mixture is fully shaken at 20°C and 180 r / min for 2 hours, then allowed to stand for 30 minutes, filtered and fixed to volume, and then determined by atomic absorption spectrometer.

[0114] See Table 8 for the results.

[0115] Table 8 Effects of strain YHY20-d on the contents of available cadmium, available lead and available copper in soil

[0116]

[0117] Note: * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and there is a significant difference between treatments

[0118] As shown in Table 8, the available cadmium in the soil of experimental group 1 was significantly reduced by 15.16%, the available lead in the soil was significantly reduced by 15.67%, and the available copper in the soil was significantly reduced by 10.25% compared with the control group 1. The available cadmium in the soil of experimental group 2 was significantly reduced by 16.52%, the available lead in the soil was significantly reduced by 14.81%, and the available copper in the soil was significantly reduced by 9.94%.

[0119] Based on this, inoculation can reduce the content of effective heavy metals in the soil, alleviate soil heavy metal pollution, and thus improve the soil ecological environment.

[0120] In summary, the strain YHY20-d obtained by the present invention can grow well under the stress of heavy metals cadmium, lead, and copper, and can adsorb and remove soluble cadmium, lead, and copper in the culture medium. At the same time, strain YHY20-d can produce indoleacetic acid, which has a certain dissolving effect on insoluble tricalcium phosphate, and can improve soil phosphorus nutrition. Inoculating the strain of the present invention into heavy metal-contaminated soil can effectively prevent wheat from absorbing heavy metals cadmium, lead, and copper, reducing the accumulation of cadmium, lead, and copper in wheat grains, straw, and roots; increasing the organic matter and available phosphorus content in the wheat rhizosphere soil, promoting the conversion of ammonium nitrogen to nitrate nitrogen in the soil, increasing soil fertility, and at the same time reducing the content of available heavy metals in the soil and improving the soil ecological environment.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A Sphingopyxis sp., named YHY20-d, deposited in the China General Microbiological Culture Collection on December 30, 2024, with a deposit number of CGMCC NO.33226.

2. A bacterial agent, characterized in that The bacterial agent includes the sphingomyelinase YHY20-d as claimed in claim 1.

3. The bacterial agent according to claim 2, wherein The bacterial agent is a liquid bacterial agent, which is fermented by Sphingosine Box Bacteria YHY20-d.

4. The microbial agent according to claim 3, characterized in that The number of viable bacteria in the liquid bacterial agent is 2×10 8 -9×10 9 CFU·mL -1 within the range.

5. A method for preparing the bacterial agent according to claim 3 or 4, characterized in that: The preparation method of the bacterial agent is as follows: Sphingosine box bacteria YHY20-d is inoculated into LB liquid culture medium, and cultured in a shaking incubator at 28°C and 150-180 r / min for 20-30 hours until the effective viable bacteria count reaches 2×10 8 ~9×10 9 The required bacterial agent is obtained within the CFU / mL range.

6. Use of the sphingomyelinase YHY20-d according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in preventing and controlling the absorption of heavy metals by plants.

7. The use according to claim 6, characterized in that The heavy metal is cadmium and / or lead and / or copper.

8. Use of the Sphingobacterium sphingosine box bacteria YHY20-d according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in increasing the content of organic matter and / or available phosphorus in soil.

9. A method for preventing plants from absorbing heavy metals, characterized in that: Apply an effective amount of the bacterial agent according to any one of claims 2 to 4 to the plants or seeds.

10. The method according to claim 9, wherein The microbial agent according to any one of claims 2 to 4 is used to water the roots of plants, or to soak or coat seeds.

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