Sphingomonas for controlling plant absorption of heavy metals, bacterial agent, preparation method and application thereof

Abstract

The present application belongs to the field of agricultural and environmental microbial technology, and particularly relates to a sphingopyxis sp. for blocking and controlling plant absorption of heavy metals, a bacterial agent, a preparation method and application thereof. The sphingopyxis sp. is named YHY20-d, and was preserved in the China General Microbiological Culture Collection Center on December 30, 2024, with a preservation number of CGMCC NO.33226. Inoculation of the sphingopyxis sp. YHY20-d in heavy metal contaminated soil can reduce the content of available heavy metals in the soil, effectively block and control plant absorption of heavy metals such as cadmium, lead and copper, increase the content of organic matter and available phosphorus in the rhizosphere soil of the plant, promote the transformation of ammonium nitrogen to nitrate nitrogen in the soil, and improve the ecological environment of the soil.

Description

Technical Field

[0001] This invention belongs to the field of agricultural and environmental microbiology technology, specifically relating to a sphingosine box bacterium that inhibits the absorption of heavy metals by plants, its inoculum, preparation method, and application. Background Technology

[0002] Due to the overexploitation of mineral resources, the improper use of fertilizers and pesticides, and the advancement of industrialization, soil heavy metal pollution is becoming increasingly serious. Heavy metal pollution does not degrade naturally over time; instead, it accumulates continuously in the environment. Plants, especially crops (such as wheat and rice), are highly 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 the absorbed heavy metals can also enter the human body through the food chain, harming human health and causing various diseases, such as nervous system damage and kidney disease. Currently, there are many methods for remediating soil heavy metal pollution and reducing plant absorption of heavy metals, including physical remediation, chemical remediation, and bioremediation. Physical remediation is costly and easily damages soil structure; while chemical remediation is highly effective, it may introduce secondary pollution. Utilizing microbial agents to inhibit the absorption and accumulation of heavy metals in wheat is a highly efficient and environmentally friendly bioremediation method that has attracted the attention of researchers.

[0003] Currently reported microbial species that inhibit the absorption and accumulation of heavy metals in plants mainly include Bacillus, Enterobacter, Serratia, Sphingomonas, and Pseudomonas. The inventor's previous patent CN116463245A disclosed an Agrobacterium species that inhibits the absorption of heavy metals in wheat and its application; this Agrobacterium NJ20 can inhibit the absorption of lead and cadmium in wheat. Patent CN114317373A disclosed a Sphingomonas PAH02, a microbial preparation, and its application as a cadmium-reducing and selenium-enriching functional regulator for crops; this Sphingomonas PAH02 can reduce the absorption of cadmium by rice roots.

[0004] However, on the one hand, most of the currently discovered microbial strains or agents can only fix or passivate one type of heavy metal, making it difficult to simultaneously block the absorption of multiple heavy metals by plants, thus limiting their application in soils with complex pollution. On the other hand, the interactions between microorganisms in the complex soil-microorganism-plant system are also affected by multiple factors such as soil and heavy metal concentrations. Therefore, it is also essential to provide a wider variety of microorganisms that can block the absorption and accumulation of heavy metals by plants, enrich the resources of microorganisms with passivation / blocking functions, and provide selectable strategies for blocking heavy metals in different plants and environments.

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

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

[0007] A second objective of this invention is to provide a microbial agent comprising the sphingosine box bacterium YHY20-d as described above.

[0008] Preferably, the microbial agent is a liquid microbial agent, which is produced by fermentation of *Sphingosine Tetracycline* YHY20-d.

[0009] Preferably, the liquid bacterial agent contains 2 × 10⁻⁶ viable bacteria. 8 -9×10 9 CFU·mL -1 Within the range.

[0010] The third objective of this invention is to provide a method for preparing the bacterial agent as described above. The method involves inoculating *Sphingosine Tetracyclis* YHY20-d into LB liquid medium and culturing it at 28°C on a shaker at 150–180 r / min for 20–30 h to achieve an effective viable count of 2 × 10⁻⁶. 8 ~9×10 9 The desired bacterial agent can be obtained within the CFU / mL range. Before inoculation, the *Sphingosine Box Bacteria* YHY20-d was first activated by culturing on LB solid medium at 28°C for 72 hours.

[0011] The fourth objective of this invention is to provide an application of the sphingosine box bacterium YHY20-d as described above or the bacterial agent as described above in controlling the absorption of heavy metals by plants.

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

[0013] The fifth objective of this invention is to provide an application of the sphingosine sphingolipids YHY20-d or the bacterial agent described above in increasing the content of organic matter and / or available phosphorus in soil and improving soil fertility.

[0014] The sixth objective of this invention is to provide a method for controlling the absorption of heavy metals by plants, wherein the method involves applying an effective amount of the above-mentioned microbial agent to the plant or seeds.

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

[0016] The beneficial effects of this invention are as follows:

[0017] 1. The sphingosine-containing bacteria YHY20-d provided by this invention can tolerate cadmium, lead, and copper. It grows well in culture media containing cadmium, lead, or copper and can adsorb soluble cadmium, lead, or copper in the culture media. The adsorption and removal rates of cadmium, lead, and copper reach 55.72%, 49.50%, and 66.69%, respectively. This provides a new strain resource for the remediation of heavy metal contaminated soil and for ensuring the safe production of plants, especially crops.

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

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

[0020] 4. Experiments show that the sphingosine sphingolipids YHY20-d provided by this invention can simultaneously reduce the cadmium, lead and copper content in wheat grains, straw and roots; the bacterial agent prepared by sphingosine sphingolipids YHY20-d can be used to control the absorption and accumulation of cadmium, lead and copper in wheat in soils with multiple contamination, which is beneficial to the safe production of wheat in soils with multiple contamination. Attached Figure Description

[0021] Figure 1 The colony morphology of *Sphingosine Box Bacteria* YHY20-d.

[0022] Figure 2 Phylogenetic tree of 16S rDNA sequence of Sphingosine Box Bacterium YHY20-d;

[0023] Figure 3This is a comparison of the growth of *Sphingosine Box Bacteria YHY20-d* in Example 3. Figure A shows the normal growth colonies without the addition of any heavy metals; Figure B shows the colonies grown at 50 mg / L Cd. 2+ Colony diagram of the culture medium; C represents growth on 600 mg / L Pb. 2+ Colony diagram of the culture medium; D represents growth on 200 mg / L Cu 2+ Colony diagram of the culture medium.

[0024] Figure 4 The figure shows a comparison of the OD values ​​of *Sphingosine Tetracyclis* YHY20-d in Example 3. Figure A shows the OD value changing with different cadmium concentrations; Figure B shows the OD value changing with different lead concentrations; and Figure C shows the OD value changing with different copper concentrations.

[0025] Figure 5 The growth curves of strain YHY20-d in Example 4 under different conditions are shown.

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

[0027] Figure 7 This is a standard curve showing the IAA production capacity of strain YHY20-d in Example 6. Detailed Implementation

[0028] The present invention will be further described below through embodiments.

[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 *Sphingosine Tetranychus* strain YHY20-d isolated and preserved in this invention was obtained from wheat rhizosphere soil and purified. The specific isolation steps are as follows:

[0033] Dig up vigorous wheat plants with their roots and soil intact, remove non-rhizosphere soil far from the roots, and collect the rhizosphere soil adhering to the root surface within 1-2 mm using a sterile brush. Weigh 1g of wheat rhizosphere soil, add it to 100mL of sterile water, place it on a shaker, and shake for 30 minutes at 28℃ and 180r / min to obtain 10 -2 Soil suspension. Take 1 mL of 10... -2 The soil suspension was added to 9 mL of sterile water and mixed thoroughly to obtain 10 -3 The soil dilution solution was then diluted 10-fold sequentially, following the same procedure as above, to obtain 10... -4 and 10-5 Soil dilution solution. Apply 100 μL of the dilution solution to a soil containing Cd. 2+ LB agar plates containing 25 mg / L (10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 20.0 g agar, 1000 mL distilled water, pH 7.0–7.2) were placed on plates and incubated at 28–30 °C for 48 h. After 48 h, vigorously growing colonies were picked and streaked onto fresh plates containing Cd. 2+ Incubate on 25 mg / L LB solid medium plates at 28°C for 48 h. Repeat the operation 3 times. Pick a single colony and add it to LB liquid medium. Place the plates on a shaker and incubate at 28°C and 180 r / min for 28–36 h. Preserve the bacterial culture with 40% glycerol suspension at -80°C for later use.

[0034] Example 2

[0035] Identification of strain YHY20-d

[0036] The strain YHY20-d was tested according to the "Handbook for Systematic Identification of Common Bacteria".

[0037] See Figure 1 When this strain YHY20-d grows on LB solid medium, the colonies are yellow, smooth, with regular edges, opaque, with a raised center, and no halo. The physiological and biochemical characteristics of this bacterium are: Gram-negative, amylase-negative, VP-negative, MR-negative, catalase-positive, and hydrogen sulfide-negative. It tolerates 7% sodium chloride, grows well at 30°C, and can use glucose, lactose, sucrose, starch, maltose, and mannose as carbon sources; and can use ammonium nitrogen and nitrate nitrogen as nitrogen sources.

[0038] DNA from bacterial strain YHY20-d was amplified by PCR using universal primers 27F and 1492R for the bacterial 16S rRNA gene. The obtained PCR products were sequenced. The 16S rDNA sequence was analyzed using the NCBI nucleic acid database BLAST. (See also...) Figure 2 The results showed that strain YHY20-d belongs to the genus *Sphingopyxis* sp. and has the highest similarity (99.38%) to the type strain *Sphingopyxis chilensis* strain S37. A phylogenetic tree based on 16S rRNA showed that strain YHY20-d clustered with *Sphingopyxis chilensis* strain S37 on the same branch.

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

[0040] To further investigate the taxonomic position of strain YHY20-d, whole-genome sequencing analysis was performed. 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] (GGDC; http: / / ggdc.dsmz.de / ggdc.php) The DDH value was calculated. ANI analysis and dDDH results are shown in Table 1. The thresholds for ANI analysis and dDDH in determining a new species were ANI Value < 95%-96% and dDDH < 70%, respectively. The results showed that, based on the whole genome sequence, the ANI value of strain YHY20-d was less than 95% and the dDDH value was less than 70% between it and the highly similar type strain, both below the thresholds for species identification. Therefore, strain YHY20-d can be identified as a new species of the genus Sphingopyxis, distinct from previously reported Sphingopyxis species.

[0042] Table 1. ANI and dDDH analysis results of closely related 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 with different concentration gradients 2+ (5, 25, 50, 75, 100mg / L), Pb 2+ (200, 400, 600, 800, 900, 1000 mg / L) and Cu 2+ LB solid medium with concentrations of 25, 50, 75, 100, 120, 150, and 200 mg / L was used. The strains were streaked onto solid medium containing and without heavy metals, and incubated at 28°C for 72 hours before observing their growth.

[0047] The strain YHY20-d was inoculated into liquid LB medium and cultured at 28℃ with a constant temperature shaker at 160 rpm for 24 h; the bacterial culture was then inoculated at 1% in Cd. 2+ The concentrations were 5, 25, 50, 75, and 100 mg / L; Pb 2+ The concentrations were 200, 400, 600, 800, 900, and 1000, respectively; Cu 2+Liquid LB medium with concentrations of 25, 50, 75, 100, 120, 150, and 200 mg / L was used, with each concentration treatment repeated three times. The medium was incubated in a shaker at 160 r / min and 28 ℃ for 48 h, and the OD value at 600 nm was measured using a spectrophotometer.

[0048] like Figure 3 As shown in Figure A, strain YHY20-d can grow well on LB solid medium free of heavy metals. In Cd... 2+ Colonies or bacterial growth appeared on culture media with concentrations of 5, 25, 50, and 75 mg / L, but with the increase of Cd... 2+ As the concentration increased, the growth of strain YHY20-d gradually weakened. 2+ Growth status on 50 mg / L plates as follows Figure 3 As shown in B. In Cd 2+ When the concentration reached 100 mg / L, no colonies appeared on the culture medium, indicating that Cd... 2+ A concentration of 100 mg / L was lethal to strain YHY20-d. This strain was effective against Pb. 2+ Colonies or bacterial growth appeared on media containing 200, 400, 600, and 800 mg / L, with the growth gradually decreasing as the concentration increased. This strain YHY20-d exhibited good growth on Pb... 2+ Growth status on 600 mg / L plates as follows Figure 3 As shown in Figure C. When the concentration reaches 900 mg / L, no colonies appear on the culture medium, indicating that Pb... 2+ A concentration of 900 mg / L was lethal to strain YHY20-d. This strain was found in Cu... 2+ Colonies or bacterial growth appeared on culture media at concentrations of 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 could still grow. Figure 3 The presence of D indicates that this strain has good copper tolerance.

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

[0050] In summary, the sphingosine sphingolipids YHY20-d exhibit strong tolerance to cadmium, lead, and copper.

[0051] Example 4

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

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

[0054] See Figure 5 The results showed that this strain could thrive in the absence of heavy metals and Cd. 2+ Concentration of 5 mg / L, Pb 2+ Concentration of 100 mg / L, Cu 2 + All samples grew normally at a concentration of 10 mg / L, entering the logarithmic growth phase after approximately 9 hours of incubation and reaching the stationary phase after 30 hours. The growth pattern was as follows: no heavy metals > Cu. 2+ 10 mg / L group > Cd 2+ 5 mg / L group > Pb 2+ 100mg / L group.

[0055] Example 5

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

[0057] Single colonies of strain YHY20-d were picked and inoculated into LB liquid medium and cultured at 28°C with a shaker at 180 rpm for 24 h. The bacterial culture was then inoculated into 5 mL of Cd-containing medium at a 3% inoculation rate. 2+ Concentration of 5 mg / L, Pb 2+ Concentration 100mg / L, Cu 2+The medium was cultured in LB liquid medium at a concentration of 10 mg / L at 28°C and a shaker at 180 rpm for 72 h. After the culture was completed, the supernatant was collected at 6000 rpm, and the heavy metal content was determined by atomic absorption spectrometry. The results are shown in Table 2.

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

[0059]

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

[0061] Example 6

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

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

[0064] The strain was inoculated into 5 mL test tubes and cultured on a shaker at 28℃ and 160 rpm for 30 h. Then, a 5% inoculum was transferred to 10 mL of phosphorus-solubilizing medium (10.0 g glucose, 0.1 g ammonium sulfate, 0.2 g potassium chloride, 0.25 g magnesium sulfate heptahydrate, 5.0 g magnesium chloride hexahydrate, 5.0 g calcium phosphate, 1000 mL distilled water, pH 7.0, 115℃, 30 min). The medium was cultured on a shaker at 28℃ and 160 rpm for 72 h. The culture was centrifuged at 8000 rpm, and 20 μL of the supernatant was collected. 1 mL of molybdenum-antimony antichromic reagent was added, and the volume was adjusted to 10 mL with distilled water. Color development was performed for 15–20 min, and the OD value at 700 nm was measured. A standard curve was prepared by serially diluting a 5 mg / L phosphorus standard solution to phosphorus concentrations of 0.00, 0.25, 0.50, 1.00, 1.25, and 1.50 mg / L. Molybdenum-antimony anti-chromic reagent was added at room temperature, and the mixture was allowed to develop color for 20 minutes. The absorbance was measured, and a standard curve was prepared. The standard curve was calculated as y = 0.3717x - 0.0055, with a correlation coefficient R0. 2 =0.9997( Figure 6 According to the standard curve, the phosphorus content in the culture medium of strain YHY20-d was calculated to be 29.93 mg / L. This indicates that the strain has a certain phosphorus solubilizing ability and can convert insoluble calcium phosphate into soluble phosphorus.

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

[0066] The standard curve was prepared using analytical grade 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, and 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 R0. 2 =0.9992.

[0067] Nitrogenous medium (10.0 g sucrose, 1.0 g ammonium sulfate, 2.0 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.1 g sodium chloride, 0.5 g yeast extract, 0.5 g calcium carbonate, pH 7.2, 1000 mL distilled water, 115℃ for 30 min) was dispensed into test tubes, 4 mL per tube. After sterilization, 1 mL of filtered and sterilized tryptophan was added to bring the tryptophan concentration in the medium to 0.5 mg / mL. The target strain was inoculated into this medium and cultured at 28℃ and 160 rpm for 48 h. After the culture was completed, the culture was centrifuged at 6000 rpm for 10 min. 1 mL of the supernatant was taken and 50 μL of 10 mmol / L orthophosphate was added, followed by 2 mL of Sackowski's colorimetric reagent. The mixture was thoroughly mixed and incubated in the dark at 25℃ 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 containing strain YHY20-d was 5.72 mg / L.

[0068] In summary, strain YHY20-d possesses certain phosphorus solubilization and IAA production capabilities, indicating that the strain has the potential to promote plant growth.

[0069] Example 7

[0070] The inhibitory effect of strain YHY20-d on the absorption of heavy metals in wheat

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

[0072] Using an inoculation loop, pick a loopful of YHY20-d glycerol tube culture and aseptically streak it onto LB solid medium, incubating at 28°C for 72 h. Then, select vigorous YHY20-d strains and inoculate single colonies into LB liquid medium, incubating at 28°C with shaking at 150–180 rpm for 20–30 h to achieve an effective viable count of 2 × 10⁻⁶. 8- 9×10 9 Liquid bacterial agent of strain YHY20-d can be obtained within the range of CFU / mL.

[0073] 2. This embodiment uses a field experiment conducted in a farmland contaminated with heavy metals in Anhui Province. The soil physicochemical characteristics of this farmland are 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 are Zhenmai 15 and Yangmai 28, and wheat seeds with plump grains and uniform size are selected.

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

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

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

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

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

[0079] Cells of 1 square meter size were set up in the field, with each treatment group located in one cell. A randomized complete block design was adopted, and protective rows with a width of 20-40 cm were set between cells. 200-300 mL of liquid inoculant of strain YHY20-d was applied to each cell. When the wheat reached the jointing stage, 100-240 mL of liquid inoculant of strain YHY20-d was added by root irrigation. After the wheat matured, the wheat grains, straw, and roots were collected, dried, and then the cadmium, lead, and copper contents were determined. The determination method was as follows: the wheat grains, straw, and roots were ground into powder, and the contents were determined by 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 different parts of wheat.

[0081]

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

[0083]

[0084]

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

[0086]

[0087] Note: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates a significant difference between treatments at the p < 0.001 level.

[0088] As shown in Tables 3, 4, and 5, compared with control group 1, the cadmium content in the grains of experimental group 1 decreased significantly by 26.88%, the lead content by 43.47%, and the copper content by 48.57% and 48.57%, respectively; the cadmium content in the straw decreased significantly by 17.41%, the lead content by 19.15%, and the copper content by 7.94%; and the cadmium content in the roots decreased significantly by 10.17%, the lead content by 7.05%, and the copper content by 14.15%.

[0089] Compared with control group 2, experimental group 2 showed a significant decrease in cadmium content (15.98%), lead content (23.72%), and copper content (21.16%) in grains; a significant decrease in cadmium content (18.42%), lead content (10.65%), and copper content (10.86%) in straw; and a significant decrease in cadmium content (11.24%), lead content (9.52%), and copper content (15.77%) in roots.

[0090] Accordingly, inoculation with YHY20-d significantly reduced the cadmium and copper content in the grains, straw, and roots of both wheat species, as well as significantly reduced 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 method was used. First, the roots of the plant were gently shaken 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 self-sealing bag or centrifuge tube for later use.

[0094] The method for determining soil organic matter is as follows: Air-dry the soil sample and sieve it. Weigh 0.2 g of rhizosphere soil and add 10 mL of 0.136 mol / L K₂Cr₂O₇-H₂SO₄ standard solution. Gently shake to mix. Heat at 170–190℃, and start timing 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 titration endpoint is reached when the solution color changes from yellow to green and then abruptly 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, and *** indicates a significant difference between treatments at the p < 0.001 level.

[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 were significantly increased, by 59.52% and 25.30%, respectively. Compared with control group 2, the organic matter and available phosphorus contents in experimental group 2 were significantly increased, by 42.00% and 32.35%, respectively. This indicates that inoculation with the strain increased the content of organic matter and available phosphorus in the soil, thereby improving soil fertility.

[0100] Example 9

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

[0102] The rhizosphere soil of the test plants was obtained from Example 7: After the experimental wheat matured, the shaking method was used. First, the roots of the plant were gently shaken 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 self-sealing bag or centrifuge tube for later use.

[0103] The method for determining the nitrate nitrogen content in soil is as follows: Take 5.0g of soil sample, add 50mL of 2mol / L KCl solution, shake for 1h, let the suspension stand for 3-5min and then filter, measure the absorbance of the extract at 220nm and 275nm, 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 10.0g of soil sample, place it in a 250mL Erlenmeyer flask, add 50mL of KCl solution, stopper tightly, shake for 30min, filter, take 2.5mL of the extract into a 25mL colorimetric tube, add 2.5mL of KCl, then add 2.5mL of phenol solution and 2.5mL of NaClO alkaline solution, shake well, and let stand at room temperature (20℃) for 1h. Then add 0.5mL of masking agent to dissolve any possible precipitates, and then dilute to 25mL with water. Measure the absorbance at 625nm.

[0105] The main source of soil ammonium nitrogen is the decomposition products 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 thus crop growth. The changes in soil nitrate nitrogen and ammonium nitrogen content are shown in Table 7.

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

[0107]

[0108] Note: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates a significant difference between treatments at the p < 0.001 level.

[0109] As can be seen, compared with control group 1, the content of nitrate nitrogen in experimental group 1 increased significantly by 41.95%, while the content of ammonium nitrogen decreased significantly by 49.97%. Compared with control group 2, the content of nitrate nitrogen in experimental group 2 increased significantly by 56.70%, while the content of ammonium nitrogen decreased by 34.37%. Therefore, inoculation with the strain converts the less mobile ammonium nitrogen into highly mobile nitrate nitrogen, which is beneficial for supplementing the nitrogen deficit in the root zone, facilitating the absorption of nitrogen nutrients by plants, and improving soil fertility.

[0110] Example 10

[0111] Effects of strain YHY20-d on the content 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 method was used. First, the roots of the plant were gently shaken 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 self-sealing bag or centrifuge tube for later use.

[0113] The method for determining the available heavy metal content in soil is as follows: extract according to a solid-liquid ratio of 1:5, that is, weigh 2g of soil sample, add 10mL of DTPA extractant and mix well, shake thoroughly for 2h at 20℃ and 180r / min, let stand for 30min, filter and make up to volume, and then determine by atomic absorption spectrometry.

[0114] The results are shown in Table 8.

[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, and *** indicates a significant difference between treatments at the p < 0.001 level.

[0118] As shown in Table 8, compared with control group 1, experimental group 1 had significantly lower levels of available cadmium (15.16%), available lead (15.67%), and available copper (10.25%). Compared with control group 1, experimental group 2 had significantly lower levels of available cadmium (16.52%), available lead (14.81%), and available copper (9.94%).

[0119] Therefore, inoculation can reduce the content of available heavy metals in the soil, alleviate soil heavy metal pollution, and thus improve the soil ecological environment.

[0120] In summary, the strain YHY20-d screened in this invention can grow well under heavy metal stress (cadmium, lead, and copper) and can adsorb and remove soluble cadmium, lead, and copper from the culture medium. Simultaneously, strain YHY20-d can produce indoleacetic acid, which has a certain dissolving effect on insoluble tricalcium phosphate, thus improving soil phosphorus nutrition. Inoculating heavy metal-contaminated soil with the strain of this invention can effectively control the absorption of heavy metals cadmium, lead, and copper by wheat, reducing the accumulation of cadmium, lead, and copper in wheat grains, straw, and roots; increasing the organic matter and available phosphorus content in the rhizosphere soil; promoting the conversion of ammonium nitrogen to nitrate nitrogen in the soil; increasing soil fertility; and simultaneously reducing the content of available heavy metals in the soil, thus 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sphingopyxis sp., named YHY20-d, was deposited on December 30, 2024, at the China General Microbiological Culture Collection Center with accession number CGMCC NO.33226.

2. A microbial agent, characterized in that, The bacterial agent includes the sphingosine box bacteria YHY20-d as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The bacterial agent is a liquid bacterial agent, which is produced by fermentation of *Sphingosine Box Bacterium* YHY20-d.

4. The microbial agent according to claim 3, characterized in that, The liquid bacterial agent contains 2 × 10⁶ live bacteria. 8 -9×10 9 CFU·mL -1 Within the range.

5. A method for preparing the microbial agent as described in claim 3 or 4, characterized in that, The preparation method of the bacterial agent is as follows: Sphingosine monocytogenes YHY20-d is inoculated into LB liquid medium and cultured at 28℃ with shaking at 150-180 r / min for 20-30 h to achieve an effective viable count of 2×10⁻⁶. 8 ~9×10 9 The desired bacterial agent can be obtained within the CFU / mL range.

6. The application of the sphingosine box bacterium YHY20-d as described in claim 1 or the bacterial agent as described in any one of claims 2-4 in controlling the absorption of heavy metals by plants.

7. The application as described in claim 6, characterized in that, The heavy metals are cadmium and / or lead and / or copper.

8. The application of the sphingosine box bacterium YHY20-d as described in claim 1 or the bacterial agent as described in any one of claims 2-4 in increasing the content of organic matter and / or available phosphorus in soil.

9. A method for controlling the absorption of heavy metals by plants, characterized in that, Apply an effective amount of the fungicide as described in any one of claims 2-4 to the plant or seeds.

10. The method as described in claim 9, characterized in that, The inoculant used as described in any one of claims 2-4 can be used to water the roots of the plants, or to soak or coat the seeds.

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