IAA-producing strain XY11 and application thereof in inhibition of activity of heavy metal cadmium

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an IAA-producing strain XY11 and application of the IAA-producing strain XY11 in inhibition of activity of heavy metal cadmium. The invention discloses a strain XY11, the taxonomic name of which is Lysinibacillus sp. The strain XY11 is preserved in Guangdong Microbial Culture Collection Center on July 1, 2025, and the preservation number of the strain XY11 is GDMCC No: 66620. According to the present invention, the morphological observation and the molecular biological identification are performed on the strain XY11, such that the strain XY11 is determined to be Lysinibacillus sp. In addition, tests find that the strain XY11 has the effects of selenium resistance, cadmium resistance, IAA production, selenium activation and cadmium reduction, can improve the content of effective selenium in soil and reduce the content of effective cadmium in soil, and is beneficial to selenium enrichment and cadmium reduction of crops; meanwhile, the growth of crops can be promoted, the selenium and cadmium stress resistance of the crops is improved, and the method has important significance for producing selenium-rich green products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an IAA-producing strain XY11 and its use in inhibiting the activity of the heavy metal cadmium. Background Technology

[0002] Selenium (Se) is one of the essential trace elements for human health, possessing antioxidant, anti-aging, and anti-cancer properties. 72% of China's regions are selenium-deficient, with 30% being severely deficient, making it a globally recognized selenium-deficient country. Insufficient selenium intake leads to selenium deficiency, weakened immunity, and can trigger various diseases such as diabetes, Keshan disease, arthritis, cardiovascular disease, and even cancer. Rice ( Oryza sativa Selenium (L.) is a staple food crop for Chinese residents. Consuming selenium-enriched rice to supplement selenium has advantages such as safety and economy, and is considered the best way to increase the selenium intake of the population.

[0003] The selenium content in surface soil is controlled by geological background and parent rock. my country's selenium-rich areas are generally located in regions rich in mineral resources. The parent rocks in these areas typically have high levels of heavy metals, easily leading to selenium-cadmium coexistence or high-selenium-high-cadmium phenomena. At the same time, human activities such as mining, metal smelting, electroplating, and the application of cadmium-containing fertilizers and pesticides release cadmium into the environment, resulting in excessive cadmium levels in the soil of selenium-rich areas.

[0004] Cadmium is highly toxic and can cause various human diseases such as Itai-itai disease, headaches, high blood pressure, kidney disease, lung cancer, and prostate cancer. Rice is a staple food crop consumed daily by urban and rural residents. While rice is highly tolerant of cadmium, it is also a food crop that readily absorbs and accumulates it. Cadmium-contaminated rice enters the human body through daily consumption, posing a threat to human health. Cadmium contamination in rice is primarily caused by cadmium pollution in local paddy field soil. Higher levels of cadmium pollution exist in selenium-rich areas, and soil environmental quality has become a crucial factor restricting the usability of selenium-rich land resources in these selenium-cadmium coexisting areas. Therefore, how to reduce the cadmium content in rice from high-selenium and high-cadmium areas and safely produce selenium-enriched rice is a pressing issue that needs to be addressed.

[0005] Chemical remediation methods (such as adding chemical passivating agents to the soil) can rapidly reduce the content of available cadmium in the soil, but this also reduces the activity of selenium in the soil, produces inorganic precipitates, leads to soil compaction, damages soil structure, degrades the original functions of the soil, and causes secondary pollution. Therefore, more and more researchers are turning their attention to microbial bioremediation.

[0006] The inventors screened several indigenous microorganisms, including strain XY11, from soils used for normal crop cultivation in the Guangxi Zhuang Autonomous Region. These microorganisms possess the properties of activating phosphorus, activating selenium, inhibiting cadmium activity, and producing auxin A (IAA). These microorganisms help improve soil phosphorus utilization, increase available selenium content, and inhibit cadmium activity, thus contributing to selenium enrichment and cadmium reduction in crops. Furthermore, IAA plays a crucial role in plant organ development and various physiological processes, especially in plant growth and development; IAA-producing strains help promote crop growth.

[0007] There are currently no reports on strain XY11 inhibiting soil cadmium activity or producing IAA.

[0008] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide an IAA-producing strain XY11 and its use in inhibiting the activity of the heavy metal cadmium.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A strain XY11, the taxonomic name of which is... Lysinibacillus sp . It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 1, 2025, with accession number GDMCC No: 66620.

[0011] A second object of the present invention is to provide the use of the strain XY11 in inhibiting the activity of the heavy metal cadmium in soil.

[0012] A third object of the present invention is to provide the use of the strain XY11 in the production of IAA.

[0013] A fourth object of the present invention is to provide the use of the strain XY11 in improving the resistance of crops to selenium and cadmium stress.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The strain XY11 of this invention was identified as [the present invention name] through morphological observation and molecular biological identification. Lysinibacillus sp . Furthermore, experiments have revealed that the strain XY11 of this invention exhibits selenium and cadmium tolerance, IAA production, and cadmium reduction effects through active selenium. It can increase the available selenium content in the soil and reduce the available cadmium content, thus contributing to the selenium enrichment and cadmium reduction of crops. Simultaneously, it can promote crop growth and enhance their tolerance to selenium and cadmium stress, which is of great significance for the production of selenium-enriched green products.

[0015] Preservation Information Strain XY04, taxonomically named *Bacillus capsicisae* (… Lysinibacillus capsici It was deposited on July 1, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences, with accession number GDMCC No: 66618.

[0016] Strain XY10, taxonomically named *Bacillus longiformis* (Lysine Bacillus) Lysinibacillus macroides It was deposited on July 1, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences, with accession number GDMCC No: 66619.

[0017] strain XY11, taxonomic name Lysinibacillus sp . It was deposited on July 1, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences, with accession number GDMCC No: 66620. Attached Figure Description

[0018] Figure 1 This is a diagram showing the colony morphology of the bacterial strain. Figure 2 Phylogenetic tree of strain XY04; Figure 3 Phylogenetic tree of strain XY10; Figure 4 Phylogenetic tree of strain XY11; Figure 5 To assess the strain's tolerance to selenium and cadmium; Figure 6 The effect of different pH values ​​on the growth of the strain; Figure 7 The effect of different pH values ​​on the IAA production capacity of the strain; Figure 8 The effect of different temperatures on the IAA production capacity of the strain; Figure 9 The effect of different inoculum amounts on the IAA production capacity of the strain; Figure 10 The effect of different initial selenium concentrations on the IAA production capacity of the strain; Figure 11 The effect of different initial cadmium concentrations on the IAA production capacity of the strain; Figure 12 The effect of the strain on the available phosphorus content in the soil; Figure 13 The effect of the strain on the available cadmium content in the soil; Figure 14 The effect of bacterial strains on the available selenium content in soil. Detailed Implementation

[0019] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0020] 1. Materials and Methods 1.1 Screening soil for bacteria Soil samples were collected from Nanning, Jingxi, Liuzhou and Guiping cities in Guangxi Zhuang Autonomous Region.

[0021] 1.2 Culture medium formulation LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, add 1000mL deionized water, adjust pH to 7.0, boil to dissolve, and then autoclave at 121℃ for 20min.

[0022] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, add 1000mL deionized water, adjust pH to 7.0, boil to dissolve, and then autoclave at 121℃ for 20min.

[0023] Isolation medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, add 1000mL deionized water, adjust pH to 7.0, boil to dissolve, and then autoclave at 121℃ for 20min. After sterilization, while still hot, add cadmium chloride (CdCl2) and sodium selenite (Na2SeO3) solution to neutralize the Cd in the medium. 2+ and Se 4+ The concentration of each medium was 1 mg / L (relative to the unsolidified medium). After mixing, the medium was poured into sterile petri dishes to make plates.

[0024] PVK medium: 15g glucose, 5.0g Ca3(PO4)2, 0.2g NaCl, 0.5g (NH4)2SO4, 0.1g MgSO4, pH 6.8-7.0, dissolved in 1000mL deionized water, autoclaved at 121℃ for 20min.

[0025] NBRIP medium (phosphate-solubilizing medium): (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O 0.3g, glucose 10.0g, MnSO4·2H2O 0.03g, Ca3(PO4)2 5.0g, FeSO4·7H2O 0.03g, KCl 0.3g, pH 7.2-7.4, dissolved in 1000mL deionized water, autoclaved at 121℃ for 20min.

[0026] Nitrogenous medium (YN): 10.0g sucrose, 1.0g ammonium sulfate, 2.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.1g sodium chloride, 0.5g yeast extract, and 0.5g calcium carbonate were dissolved in distilled water in sequence, and the volume was adjusted to 1000mL. The pH was adjusted to 7.2, and the medium was autoclaved at 121℃ for 20min.

[0027] 1.3 Strain screening Weigh 10g of soil sample and add it to an Erlenmeyer flask containing 90mL of sterile water. Shake the flask at room temperature for 1 hour to disperse the microorganisms evenly. After standing for 30 minutes, take 5mL of the supernatant and add it to 150mL of bacterial liquid culture medium. Incubate at 30℃ and 120r / min for 5 days and observe the growth of the microorganisms.

[0028] 1.4 Strain purification Dilute the bacterial suspension cultured for 5 days (1.3): First, take 1 mL of the bacterial suspension and add it to 9 mL of sterile water; this makes a 10⁻⁶ solution. -1 After mixing the bacterial suspension, take 1 mL of 10 -1 Add the bacterial culture to 9 mL of sterile water; this is 10. -2 Bacterial solution, and so on, yielding 10 -3 10 -4 10 -5 Diluted bacterial suspensions; take 10... -3 10 -4 10 -5 Spread 0.5 mL of diluted bacterial solution evenly on the isolation medium and incubate at 30°C inverted position for 48 h.

[0029] Visually inspect and select colonies of different shapes and colors, then continue dilution using the above method. Further isolate and purify using the spread plate method until no abnormal colonies remain on the plate. Inoculate the purified strain onto LB agar slants and store at 4°C.

[0030] 1.5 Strain identification and growth characteristics 1.5.1 Colony morphology characteristics After sterilizing LB solid medium, pour it into sterile petri dishes to prepare plates. Use an inoculation loop to pick up one loopful of the preserved strain and place it in 9 mL of sterile water. Dilute to 10⁻⁶ using the steps in 1.4. -3 10 -4 10 -5 Each of the three dilutions of bacterial culture was taken and spread onto LB solid medium. After incubation at 30°C for 48 hours, the size, shape and color of the colonies were observed.

[0031] 1.5.2 Identification of 16S rRNA in the strain DNA was extracted from the bacterial strain using the Ezup column-based bacterial genomic DNA extraction kit (B518255). Universal primers 27F and 1492R were selected for PCR amplification. The primer sequences of the PCR amplification products are shown in Table 1, and the PCR reaction system is shown in Table 2.

[0032] Table 1 16S rRNA primers

[0033] Table 2 PCR reaction system

[0034] The PCR program was as follows: 95℃, 5 min; 94℃, 30 s; 57℃, 30 s; 72℃, 1 min, 30 cycles; 72℃, 10 min.

[0035] The purified PCR products were sequenced, and the sequencing results are shown in SEQ ID No. 1-SEQ ID No. 3 of the sequence listing. The obtained sequences were compared with nucleic acid data in GenBank using the Blast program for homology analysis, and a phylogenetic tree was constructed.

[0036] 1.5.3 Colony physiological and biochemical characteristics Referring to "Microbiology Experiments", Gram staining, starch hydrolysis test, indole reaction, methyl red test, glucose fermentation test, VP reaction, salt tolerance test and ammonia production test were performed on the target colony.

[0037] 1.6 Experiment on the strain's tolerance to selenium-cadmium One loopful of the isolated and purified bacterial strain, preserved at 4℃, was inoculated onto an LB agar slant and activated at 35℃ for 48 hours. The activated strain was then inoculated onto LB agar plates using the streak plate method and incubated upside down at 35℃ for 48 hours. Colony growth was observed and photographed.

[0038] To investigate the tolerance of each strain to cadmium selenide, LB solid medium was sterilized, and cadmium chloride (CdCl2) and sodium selenite (Na2SeO3) solution was added before the medium solidified, so that the Cd concentration in the medium increased.2+ Se 4+ Cd 2+ +Se 4+ Concentrations of 1 mg / L, 5 mg / L, and 1 mg / L + 5 mg / L were prepared, mixed thoroughly, and poured onto plates. Using the streak plating method, the strains grown on the aforementioned LB solid medium plates (without added selenium and cadmium) were inoculated onto these LB solid medium plates (first round with added selenium and cadmium). The plates were incubated upside down at 35°C for 48 hours, and colony growth was observed and photographed. This process was repeated for a second round of LB solid medium plates with added selenium and cadmium (Cd). 2+ Se 4+ Cd 2+ +Se 4+ Concentrations of 2 mg / L, 10 mg / L, and 2 mg / L + 10 mg / L were added, and the third round of LB solid culture medium plates with added selenium and cadmium (Cd) were used. 2+ Se 4+ Cd 2+ +Se 4+ Concentrations of 5 mg / L, 20 mg / L, and 5 mg / L + 20 mg / L were added in the fourth round of LB solid culture medium plates with added selenium and cadmium (Cd). 2+ Se 4+ Cd 2+ +Se 4+ Concentrations of 10 mg / L, 50 mg / L, and 10 mg / L + 50 mg / L were added in the fifth round of LB solid culture medium plates with added selenium and cadmium (Cd). 2+ Se 4+ Cd 2+ +Se 4+ Concentrations of 20 mg / L, 100 mg / L, and 20 mg / L + 100 mg / L were added, until the ninth round of adding cadmium selenide (Cd) LB solid medium plates. 2+ Se 4+ Cd 2+ +Se 4+ The concentrations were 300 mg / L, 2000 mg / L, and 300 mg / L + 2000 mg / L, respectively.

[0039] 1.7 Effects of different environmental conditions on the growth of the strain and its IAA production capacity 1.7.1 Effect of different pH values ​​on the growth of the strain The bacterial strain was inoculated onto LB solid medium and activated at 30°C for 24 hours. Then, under aseptic conditions, the activated strain was inoculated into LB liquid medium and cultured at 30°C and 120 rpm for 24 hours. The cultured bacterial solution was then diluted with sterile water to prepare OD. 600 Prepare a bacterial suspension with a value of 1.0.

[0040] bacterial suspension (OD) 600 The culture medium was inoculated with a pH of 1.0 at a rate of 5% (v / v) into LB liquid medium and incubated at 33°C with constant temperature shaking at 160 rpm. Uninoculated culture was used as a blank control, and each treatment was repeated three times. The pH of the LB liquid medium was adjusted with 1 mol / L HCl and 1 mol / L NaOH solutions, and six pH gradients (5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) were established. Samples were taken after 36 hours of incubation, and the absorbance (OD) of the bacterial culture at 600 nm was measured using a spectrophotometer. 600 value.

[0041] 1.7.2 The impact of different environmental conditions on IAA production capacity 1.7.2.1 Qualitative determination of IAA production by the strain The strain was activated and cultured at 30°C for 24 hours on LB solid medium.

[0042] Add 5% L-tryptophan solution filtered through a 0.22 μm filter to LB liquid medium to achieve an L-tryptophan concentration of 0.05%. This medium is called LB liquid medium (containing 0.05% L-tryptophan). Inoculate a loopful of single colonies from strains XY04, XY10, and XY11 preserved on slant culture into 100 mL of LB liquid medium (containing 0.05% L-tryptophan) and incubate at 30°C with shaking at 150 rpm for 24 h. Transfer 2 mL of the culture to a test tube and add 2 mL of Alkowski's colorimetric reagent (a mixture of 1 mL of 0.5 mol / L FeCl3 and 50 mL of 35% HClO4). Incubate at room temperature in the dark for 30 min. A red color indicates that the strain has the ability to produce IAA, and the deeper the red, the higher the IAA production.

[0043] 1.7.2.2 Quantitative determination of IAA production by the strain The quantitative determination of IAA production by the strain was performed using the Salkowski colorimetric method.

[0044] A 5% L-tryptophan solution filtered through a 0.22 μm filter was added to LB liquid medium to bring the L-tryptophan content in the liquid medium to 0.05%. This medium was called LB liquid medium (containing 0.05% L-tryptophan). One loopful of strains XY04, XY10, and XY11 preserved from slant culture was inoculated into 50 mL of LB liquid medium using an inoculation loop. The culture was incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. The cultured bacterial suspension was then diluted with sterile water to prepare OD (diethyltoluene) concentration. 600 A bacterial suspension with an OD value of 0.8. Take OD... 600=5 mL of a 0.8% bacterial suspension was inoculated into 95 mL of LB liquid medium (containing 0.05% L-tryptophan, and the pH was adjusted to 7 with 1 mol / L HCl and 1 mol / L NaOH solution), with an inoculation amount of 5% (v / v). The medium was incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. 5 mL of the incubated bacterial suspension was then transferred to a 10 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. 2 mL of the supernatant was mixed with 2 mL of Salkowski reagent and reacted at room temperature in the dark for 30 min. The absorbance was measured at 530 nm using a spectrophotometer, with distilled water used as a blank reference for zeroing. Meanwhile, IAA solutions with concentrations of 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, and 35 mg / L were prepared using distilled water. 2 mL of each IAA standard solution was mixed with 2 mL of Salkowski colorimetric reagent, and the absorbance was measured using the same method. A standard curve was plotted, and the concentration of IAA produced by each strain in the cultured bacterial suspension was calculated by comparing with the standard curve.

[0045] 1.7.2.3 Effect of different pH values ​​on the IAA production capacity of the strain A 5% L-tryptophan solution filtered through a 0.22 μm filter was added to LB liquid medium to bring the L-tryptophan content in the liquid medium to 0.05%. This medium was called LB liquid medium (containing 0.05% L-tryptophan). One loopful of strains XY04, XY10, and XY11 preserved from slant culture was inoculated into 50 mL of LB liquid medium using an inoculation loop. The culture was incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. The cultured bacterial suspension was then diluted with sterile water to prepare OD (diethyltoluene) concentration. 600 A bacterial suspension with an OD value of 0.8. Take OD... 600= Five mL of a 0.8% bacterial suspension was inoculated into 95 mL of LB liquid medium (containing 0.05% L-tryptophan) at different pH values, with an inoculation volume of 5% (v / v). A culture medium without bacterial suspension served as a blank control. Each treatment was repeated three times. The cultures were incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. Absorbance was measured using the same quantitative method, and the corresponding IAA yield was calculated based on the standard curve to determine the optimal pH for each strain. The pH of the LB liquid medium (containing 0.05% L-tryptophan) at different pH values ​​was adjusted using 1 mol / L HCl and 1 mol / L NaOH solutions, setting six pH gradients: 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0.

[0046] 1.7.2.4 Effect of culture temperature on the IAA production capacity of the strain Add a 5% L-tryptophan solution filtered through a 0.22 μm filter to LB liquid medium to achieve an L-tryptophan concentration of 0.05%. This medium is called LB liquid medium (containing 0.05% L-tryptophan). One loopful of strains XY04, XY10, and XY11 preserved from slant culture was inoculated into 50 mL of LB liquid medium and cultured at 30°C with constant shaking at 150 rpm for 24 h. The cultured bacterial suspension was then prepared with sterile water to achieve an OD600 value of 0.8. The OD600 value was then measured. 600 Five mL of a bacterial suspension with a pH of 0.8 was inoculated into 95 mL of LB liquid medium (containing 0.05% L-tryptophan, and adjusted to pH 7 with 1 mol / L HCl and 1 mol / L NaOH solutions), with an inoculation amount of 5% (v / v). Uninoculated culture medium served as a blank control. Each treatment was repeated three times. Five different incubation temperatures were set up: 28℃, 31℃, 34℃, 37℃, and 40℃. The culture was incubated at a constant temperature with shaking at 150 rpm for 24 h. The absorbance was measured using the same quantitative method. The corresponding IAA yield was calculated based on the standard curve to determine the optimal incubation temperature for each strain.

[0047] 1.7.2.5 Effect of inoculum size on the IAA production capacity of the strain A 5% L-tryptophan solution filtered through a 0.22 μm filter was added to LB liquid medium to bring the L-tryptophan content in the liquid medium to 0.05%. This medium was called LB liquid medium (containing 0.05% L-tryptophan). One loopful of strains XY04, XY10, and XY11 preserved from slant culture was inoculated into 50 mL of LB liquid medium using an inoculation loop. The medium was incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. The cultured bacterial suspension was then prepared with sterile water to achieve an OD600 value of 0.8. Different volumes of OD600 were collected... 600 The bacterial suspension with a concentration of 0.8 was inoculated into the corresponding LB liquid medium (containing 0.05% L-tryptophan, and the pH was adjusted to 7 with 1 mol / L HCl and 1 mol / L NaOH solutions). Five gradients of inoculum (v / v) were set up, namely 0.5%, 1%, 2%, 5%, and 10%, with uninoculated culture medium as a blank control. Each treatment was repeated three times. The culture was carried out at 34℃ and 150 r / min for 24 h with constant temperature shaking. The absorbance was measured by the same quantitative method. The corresponding IAA yield was calculated according to the standard curve to determine the optimal inoculum for each strain.

[0048] 1.7.2.6 Effect of different initial selenium-cadmium concentrations on the IAA production capacity of the strain Add 5% L-tryptophan solution filtered through a 0.22 μm filter to LB liquid medium to make the L-tryptophan content in the liquid medium 0.05%. Then add different volumes of 100 mg / L Cd filtered through a 0.22 μm filter. 2+ Solution and Se 4 + A solution (containing cadmium chloride and sodium selenite solution) was added to form LB liquid culture media (containing 0.05% L-tryptophan) with different initial cadmium selenide concentrations. The LB liquid culture medium (containing 0.05% L-tryptophan) contained Cd... 2+ and Se 4+ The initial concentrations were set at 1 mg / L, 3 mg / L, 5 mg / L, 7 mg / L, and 9 mg / L. One loopful of the XY04, XY10, and XY11 strains preserved on slant culture were inoculated into 50 mL of LB liquid medium and cultured at 30°C with constant shaking at 150 rpm for 24 h. The cultured bacterial suspension was then prepared with sterile water to a bacterial suspension with an OD600 value of 0.8. The OD600 value was then measured. 600 Five mL of bacterial suspension with a concentration of 0.8 g / L was inoculated into 95 mL of LB liquid medium (containing 0.05% L-tryptophan, and adjusted to pH 7 with 1 mol / L HCl and 1 mol / L NaOH solutions) with different initial concentrations of cadmium selenide. The medium was incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. The inoculation amount was 5% (v / v). Uninoculated culture medium was used as a blank control. Each treatment was repeated three times. The culture was incubated at 30°C with constant temperature shaking at 150 rpm for 24 h. The absorbance was measured using the same quantitative method, and the corresponding IAA content was calculated based on the standard curve.

[0049] 1.8 Soil Sample Pot Experiment To evaluate the regulatory effect of the strain on the available phosphorus, cadmium and selenium content in rice soil.

[0050] Select a round plastic basin 5cm high and 5cm in diameter, and fill each basin with 300g of soil sample (soil sample collected from paddy fields in Shilong Town, Guiping City) that has been air-dried through a 2mm nylon sieve. Completely saturate the soil and place it in a dry environment for 2 weeks to equilibrate, replenishing water periodically until the moisture content is maintained at 60% of the field's maximum water holding capacity.

[0051] Original bacterial culture of the strain (OD) 600The bacterial solution was applied at two concentration gradients (5% and 10% v / m, i.e., 5 mL or 10 mL of bacterial solution added to 100 g of soil sample) to plastic pots containing 100 g of paddy soil. Six treatments were set up: XY10-5%, XY10-10%, XY11-5%, XY11-10%, XY04-5%, and XY04-10% (the "-" indicates the strain number, and the "-" indicates the inoculum size). The original soil without bacterial solution served as a blank control. Each treatment was replicated in triplicate. All plastic pots were placed in an artificial climate chamber with a temperature of 31℃ and a relative humidity of 60%, simulating the daylight hours of paddy soil (14 h light / 10 h dark) to ensure uniform and sufficient light exposure for each pot.

[0052] On days 7, 14, and 21, 50g of mixed soil samples were collected from each plastic basin using a diagonal sampling method. After natural air drying, grinding, and passing through a 2mm nylon sieve, the available phosphorus, available selenium, and available cadmium contents in the soil were determined.

[0053] The determination of available phosphorus in soil was performed using the sodium bicarbonate extraction-molybdenum antimony spectrophotometric method (reference standard: Determination of Available Phosphorus in Soil - Sodium Bicarbonate Extraction-Molybdenum Antimony Spectrophotometric Method HJ704-2014): 2.5000 g of air-dried soil sample was taken, and 50 mL of 0.5 mol / L NaHCO3 extraction solution (pH 8.5) was added. The mixture was shaken at 25℃ and 180 r / min for 30 min. The sample was filtered through phosphorus-free filter paper, and 10 mL of the filtrate was transferred to a 50 mL colorimetric tube. Water was added to a final volume of 15-20 mL, followed by one drop of 2,4-dinitrophenol indicator. Sulfuric acid solution was then added dropwise until the solution was nearly colorless. 0.75 mL of ascorbic acid solution was added, and the mixture was stirred until dissolved. After 30 seconds, 5 mL of molybdate solution was added, and the volume was adjusted to 50 mL with water. The mixture was stirred until dissolved. The volumetric flask was then placed at room temperature for 30 min. Colorimetric measurements were performed using a 10mm cuvette at a wavelength of 880nm under ambient conditions above 20°C, with deionized water as a reference. The absorbance was measured, and the available phosphorus content in the soil was calculated based on a pre-established standard curve.

[0054] The available selenium and available cadmium contents in the soil were determined using DTPA extraction-atomic fluorescence spectrometry and DTPA extraction-inductively coupled plasma atomic emission spectrometry. 10.00 g of air-dried soil sample (passed through a 2.00 mm sieve) was weighed and placed in a 100 mL plastic bottle. 20.0 mL of diethylenetriaminepentaacetic acid-calcium chloride-triethanolamine (DTPA-CaCl2-TEA) buffer solution was accurately added. The bottle was capped, shaken well, and oscillated at 180 r / min for 2 h at a constant temperature of 20 ± 2 °C. After settling and standing for 30 min, the extract was slowly filtered through medium-speed filter paper and then gravity filtered. The extract was measured within 48 h. Available cadmium content was determined using inductively coupled plasma atomic emission spectrometry, and available selenium content was determined using atomic fluorescence spectrometry.

[0055] 1.9 Hydroponic Experiment To investigate the promoting effect of bacterial solution on rice seed germination and its alleviating effect on selenium and cadmium stress, a hydroponic experiment was conducted.

[0056] S1. Soak rice seeds in 1% CuSO4 solution for 30 min, then soak them in 0.5% ZnSO4 solution for 15 min. After disinfection, rinse them repeatedly with sterile water 3-5 times. The volume ratio of rice seeds to sterile water during rinsing is 1:20. S2. In a sterile operating room, the bacterial strain was inoculated at a 5% inoculum into LB medium (pH adjusted to 8.0 with 1 mol / L HCl and 1 mol / L NaOH solution). After incubation at 34°C for 2 days, the cultured bacterial solution was removed, centrifuged at 4000 rpm for 10 min, and the bacterial cells were collected and adjusted to OD500 with sterile water. 600 The bacterial suspension was obtained with a value of 0.8 and was ready for use. S3. Place sterile filter paper in a sterilized petri dish (9cm in diameter). Divide the rice seeds treated in S1 into two groups: one group is soaked in the bacterial suspension for 2 hours, and the other group is not soaked in the bacterial suspension. Then, spread 50 seeds per dish evenly on the filter paper and set the following treatments for the rice seeds: Treatment 1: Do not soak the bacterial suspension, add 5 mL of sterile water to moisten; Treatment 2: Soak in bacterial suspension with a selenium-cadmium concentration of 0, then add 5 mL of sterile water to moisten; Treatment 3: Without soaking the bacterial suspension, add 5 mL of Se at concentrations of 5 mg / L, 10 mg / L, and 15 mg / L respectively. 4+ Solution and Cd 2+ Solution (prepared with cadmium chloride and sodium selenite solution, concentration expressed as Se) 4+ and Cd 2+ express); Treatment 4: Soak the bacterial suspension and add 5 mL of Se at concentrations of 5 mg / L, 10 mg / L, and 15 mg / L, respectively. 4+ Solution and Cd 2 + Solution (prepared with cadmium chloride and sodium selenite solution, concentration expressed as Se) 4+ and Cd 2+ express); Each of the above treatments was repeated 3 times. The seeds were cultured for 7 days under the conditions of 26±1℃, 70% relative humidity, and 12h photoperiod / 12h dark period. Water was added as needed during the period depending on the dryness and humidity. Seed germination was observed every 24 hours, and seedling length and root length were measured on the 7th day.

[0057] The methods for measuring each indicator are as follows: (1) Germination potential (GE) and germination rate (GR) refer to the percentage of all normally germinated seeds out of the total number of tested seeds at the beginning and end of the germination stages. Germination potential was measured on day 3 after sowing, and germination rate was measured on day 7. The formulas for calculating germination potential and germination rate at this time are: Germination potential (%) = (Number of seeds germinated within 3 days / Total number of seeds tested) × 100; Germination rate (%) = (Number of seeds germinated within 7 days / Total number of seeds tested) × 100; (2) Germination index is a seed vigor indicator. A high germination index means high vigor.

[0058] Germination Index (GI) = ∑Gt / Dt, where Gt represents the number of seeds that germinate on day t, and Dt represents the corresponding number of germination days; (3) Vitality Index (VI) = GI × S, where S represents the average root length; (4) Seedling length and root length: Take 10 seedlings with uniform growth in each dish and measure their seedling length and root length. Take the average value of the results.

[0059] 2. Results and Analysis 2.1 Isolation and purification of strains During the process of strain isolation and purification, strain XY04 was screened from soil samples collected in Nanning City. Strains XY10 and XY11 were screened from soil samples collected from Guiping City. 2.2 Colony morphology characteristics The colony morphology of strains XY04, XY10, and XY11 after incubation on inverted plates at 30°C for 48 hours is shown in the figure. Figure 1 .

[0060] Depend on Figure 1 It can be seen that the single colonies formed by strain XY04 on LB medium plates are regular round, white, with the surface and bottom of the colony being the same color. The surface of the colony is moist and smooth, with flat edges without serrations. The colony is slightly raised, relatively thin, and has a soft texture, making it easy to pick up.

[0061] The single colonies formed by strain XY10 on LB agar plates are regular round, white, with the surface and bottom of the colony being the same color. The surface of the colony is moist and smooth, with flat, smooth and thick edges. The center of the colony is slightly concave, and the thickness is very thin. The colony texture is relatively soft and easy to pick up.

[0062] The single colonies formed by strain XY11 on LB agar plates are irregularly round, white, with the surface and bottom of the colony being the same color. The surface of the colony is moist and smooth, with smooth and even edges without serrations. The center of the colony is slightly convex, and the colony is relatively thin. The texture of the colony is soft and sticky, similar to jelly. It is easy to pick up but not easy to stick to the inoculation loop.

[0063] 2.3 Colony Biochemical Characteristics The results are shown in Table 3.

[0064] Table 3. Colony biochemical characteristics

[0065] Note: In Gram staining, "+" represents positive bacteria and "-" represents negative bacteria; the salt tolerance test is set with sodium chloride solution concentrations of 5%, 9%, and 13%. In the table, "5%" and "9%" represent that the strain grows well at these concentrations, and "-" indicates that it does not grow at the lowest concentration of 5%; in other test items, "+" represents positive, "++" represents a stronger positive reaction, and "-" represents negative.

[0066] 2.4 Phylogenetic Tree See results Figure 2-4 .

[0067] Combining colony morphological characteristics, colony biochemical characteristics and Figure 2-4 The strain XY04 was identified as *Bacillus capsicisae*. Lysinibacillus capsici Strain XY10 is a long-shaped lysine-containing Bacillus. Lysinibacillus macroides Strain XY11 is Lysinibacillus sp . ; 2.5 Strain tolerance to cadmium selenide See results Figure 5 .

[0068] Depend on Figure 5 It can be seen that in the ninth round of adding LB solid medium plates (Cd) to the strain to improve its tolerance to cadmium selenide, the strain's tolerance to cadmium selenide was significantly improved. 2+ Se 4+ Cd 2+ +Se 4+ All three strains were able to grow at concentrations of 300 mg / L, 2000 mg / L, and 300 mg / L + 2000 mg / L.

[0069] strain XY04 in Cd 2+ It grew normally and well on LB solid medium plates with a concentration of 300 mg / L. In Se... 4+ The strain XY04 grew normally and vigorously on LB solid medium plates at a concentration of 2000 mg / L, showing a tendency to expand beyond the streaked area. It also reacted with selenium in the medium, changing the valence state of selenium and thus exhibiting a deep orange-red color, indicating that strain XY04 has high tolerance to selenium. When treated with a combination of selenium and cadmium, strain XY04 grew well, better than with cadmium alone, but worse than with selenium alone, indicating that 300 mg / kg Cd... 2+Concentration had a certain inhibitory effect on its growth, while selenium may have played a role in alleviating the inhibition of cadmium.

[0070] XY10 strain in Cd 2+ The sample could grow on LB solid medium plates with a concentration of 300 mg / L, but the growth was not very good, indicating that this concentration of Cd... 2+ It had a strong inhibitory effect on its growth. In Se 4+ The XY10 strain grew normally and vigorously on LB solid medium plates at a concentration of 2000 mg / L, exhibiting a tendency to spread outwards from the streaked area. It also reacted with selenium in the medium, altering its valence state and producing a deep orange-red color, indicating that the XY10 strain also has high tolerance to selenium. When treated with a combination of selenium and cadmium, the XY10 strain grew well, better than with cadmium alone, but worse than with selenium alone, indicating that 300 mg / kg Cd... 2+ Concentration had a certain inhibitory effect on its growth, while selenium may have played a role in alleviating the inhibition of cadmium.

[0071] XY11 strain in Cd 2+ The sample could barely grow on LB solid medium plates with a concentration of 300 mg / L, and the growth was extremely poor, indicating that this concentration of Cd... 2+ It had a strong inhibitory effect on its growth. In Se 4+ The XY11 strain grew normally and vigorously on LB solid medium plates at a concentration of 2000 mg / L, exhibiting divergent growth. It also reacted with selenium in the medium, changing its valence state and producing a deep orange-red color, indicating that the XY11 strain also has high selenium tolerance. When treated with a combination of selenium and cadmium, the XY11 strain grew well and vigorously, significantly better than with cadmium alone, but worse than with selenium alone. This suggests that 300 mg / kg Cd... 2+ Concentration had a strong inhibitory effect on its growth, while selenium also played a role in alleviating the inhibition of cadmium.

[0072] Overall, the three strains were effective against Cd. 2+ The growth of the three strains on LB solid medium plates with a concentration of 300 mg / L (or mg / kg) was in the following order: XY04 > XY10 > XY11. However, the concentration of selenium was far from reaching the tolerance limit of the three strains. The addition of selenium also greatly alleviated the inhibitory effect of cadmium on the growth of the three strains.

[0073] 2.6 Effects of different pH values ​​on the growth of the strain The effects of inoculum size of 5%, incubation temperature of 33℃, and incubation time of 36 h on the growth of the strain are shown in the figure. Figure 6 .

[0074] Depend on Figure 6 It can be seen that the OD of the bacterial culture of strain XY04 is...600 The OD value decreases slightly at pH 5.0-6.0, rises slowly at pH 6.0-9.0, reaches its maximum at pH 9.0, and then decreases again. At pH 9.0, the OD value... 600 Maximum value (OD) 600 =1.558), which is only slightly better than OD. 600 The lowest pH value is 10.0 (OD). 600 =1.509) when the value was 3.24%, showing a narrow fluctuation trend overall, indicating that strain XY04 is not sensitive to changes in pH value and can grow stably in all pH ranges examined.

[0075] OD of bacterial culture of strains XY10 and XY11 600 The values ​​were highest at pH 5.0, then decreased rapidly. XY10 approached equilibrium at pH 6.0; XY11 only began to decline at pH 9.0. At pH 5.0, the OD value of the XY10 strain was... 600 The value was 1.582 for XY10 and 1.662 for XY11, indicating that these two strains are more suitable for growth in acidic environments, with XY11 showing slightly better growth performance than XY10.

[0076] 2.7 Qualitative and quantitative analysis of IAA production by the strain In the experiment on IAA-producing bacteria to produce IAA activity, three isolated strains were subjected to qualitative and quantitative analysis experiments based on colorimetric reactions. The results are shown in Table 4. Table 4. Qualitative and quantitative results of IAA-induced growth in three strains.

[0077] Note: "+" represents red, "++" represents dark red, and all data in the table are mean ± standard deviation.

[0078] Table 4 shows that, based on the colorimetric results of the qualitative tests, XY11 and XY04 have a deeper color, appearing as a deep red, while XY10 has a slightly lighter color, remaining red. Therefore, it can be preliminarily determined that all three strains have the ability to produce IAA. Quantitative results show that the IAA production capacity of the three different strains varies. Strains XY11 produce the highest IAA content, reaching a maximum yield of 22.56 ± 0.79 mg / L, while strain XY10 produces the lowest, with a yield of 18.69 ± 1.23 mg / L.

[0079] 2.8 Effect of different pH values ​​on the IAA production capacity of the strain See results Figure 7 .

[0080] Depend on Figure 7It can be seen that at pH 5.0, the IAA production of all three strains was the lowest, with strain XY10 producing the lowest IAA at 15.08 mg / L, and strain XY11 producing the highest IAA at 20.9 mg / L. This indicates that under acidic conditions, the IAA synthesis ability of these four strains was inhibited to some extent. At pH 8.0, all three strains exhibited the highest IAA production, with strain XY11 showing the highest yield at 25.42 mg / L. Furthermore, the IAA production at pH 9.0 was higher than at pH 5.0, 6.0, and 7.0, indicating that the IAA synthesis capacity of these three strains was enhanced under alkaline conditions.

[0081] Therefore, it can be concluded that the three strains have a weaker ability to produce IAA under acidic conditions than under alkaline conditions, and the optimal pH value is 8.0.

[0082] 2.9 Effect of different temperatures on the IAA production capacity of the strain See results Figure 8 .

[0083] Depend on Figure 8 It can be seen that with the increase of temperature, the IAA production of all three strains showed a trend of gradually increasing and then decreasing. At a temperature of 28℃, the IAA production of all three strains was the lowest; at a temperature of 34℃, the IAA production of all three strains reached the highest value, with strain XY11 having the highest IAA production at 24.14 mg / L, while XY10 had the lowest IAA production at 20.66 mg / L.

[0084] In addition, as can be seen from the figure, the IAA production of strains XY11 and XY12 varies greatly. For example, at 40℃, the IAA production of strains XY04, XY10, and XY11 decreased by 5.03%, 9.7%, and 13.27% respectively compared with that at 37℃.

[0085] 2.10 Effect of different inoculum amounts on the IAA production capacity of the strain See results Figure 9 .

[0086] Depend on Figure 9 It can be seen that the IAA production of strains XY11 and XY04 was highest at an inoculum size of 5%, with IAA contents of 28.01 mg / L and 24.81 mg / L, respectively; while strain XY10 achieved the highest IAA production at an inoculum size of 10%, at 20.06 mg / L.

[0087] When the inoculum concentration was 0.5-5%, the IAA production of strains XY11 and XY04 increased with increasing inoculum concentration. When the inoculum concentration was 5-10%, the IAA production gradually decreased. This reflects that within the limited space of the conical flask, the strains need to reach a certain concentration to produce IAA at the fastest rate. Strain XY10 showed the highest IAA production at an inoculum concentration of 10%, indicating that strain XY10 may have a longer growth cycle compared to the other three strains. However, its IAA production was only 0.07 mg / L different from that at 5% inoculum concentration, suggesting that inoculum concentrations of 5% and 10% have little effect on XY10.

[0088] 2.11 Effect of different initial selenium concentrations on the IAA production capacity of the strain See results Figure 10 .

[0089] Depend on Figure 10 It was found that selenium inhibited the IAA production capacity of all three strains to varying degrees. The IAA production of strains XY10 and XY04 decreased with increasing selenium content. 4+ The initial concentration gradually decreased with increasing concentration, and the highest yield was consistently found in Se. 4+ The highest values ​​were observed at an initial concentration of 1 mg / L, reaching 16.85 mg / L and 18.19 mg / L respectively. 1 The maximum IAA yield of strain XY11 was at Se... 4+ The initial concentration was 3 mg / L, which was the highest, but it was different from Se. 4+ Compared to the initial concentration of 1 mg / L, the increase was only 0.09 mg∙L. -1 This indicates that strain XY11 is less sensitive to selenium concentration within a lower range, and its IAA production subsequently decreases with increasing selenium content. 4+ The initial concentration gradually decreases with increasing concentration.

[0090] In addition, strains XY4, XY10, and XY11 were found in Se 4+ When the initial concentration is 9 mg / L, it reacts with Se. 4+ Compared with the initial concentration of 1 mg / L, the IAA production decreased by 17.81%, 14.48%, and 16.29%, respectively. Under the same concentration of selenium stress, strain XY11 had the highest IAA production among the three strains, indicating that strain XY11 is more tolerant to selenium than the other two strains.

[0091] 2.12 Effect of different initial cadmium concentrations on the IAA production capacity of the strain See results Figure 11 .

[0092] Depend on Figure 11It was found that cadmium inhibited the IAA production capacity of the three strains to varying degrees, but the IAA production performance of the three strains differed compared to that of selenium. The IAA production of strain XY10 increased with increasing Cd content. 2+ The concentration gradually decreased with increasing concentration; strain XY11 in Cd 2+ The IAA production of strain XY04 gradually increased at concentrations of 1-7 mg / L, reaching a maximum of 23.1 mg / L at 7 mg / L, and decreased at 9 mg / L. 2+ The production of IAA gradually increased at concentrations of 1-5 mg / L, reaching a maximum of 21.27 mg / L at a concentration of 5 mg / L, and then gradually decreased at concentrations of 5-9 mg / L; this suggests that lower concentrations of cadmium may not be sufficient to activate the stress response of these three strains.

[0093] 2.13 Effects of bacterial strains on soil available phosphorus content See results Figure 12 .

[0094] Depend on Figure 12 It was found that after 7 days of treatment, the XY04 treatment group showed the most significant phosphorus solubility effect. The available phosphorus content in the soil of the XY04-10% treatment group reached 29.68 mg / kg, which was 94.13% higher than that of the control group. The XY04-5% treatment group was the second most effective, with an increase of 48.42% in available phosphorus content. The XY10-10% and XY11-5% treatment groups also showed significant phosphorus solubility effects, which were 27.45% and 24.76% higher than that of the control group, respectively. However, the available phosphorus content in the soil of the XY10-5% and XY11-10% treatment groups was actually lower than that of the control group.

[0095] After 14 days of treatment, the available phosphorus content in the soil of all treatment groups was higher than that of the control group, indicating that all strains played a phosphorus-solubilizing role. Among them, the XY04-5% treatment group showed the most outstanding performance, with the available phosphorus content in the soil reaching 38.07 mg / kg, an increase of 123.68% compared with the control group; the XY04-10% treatment group was the second most effective, with an increase of 96.14%. The phosphorus-solubilizing effect of all treatment groups at 14 days was generally better than that at 7 days, indicating that the strains need a certain amount of time to adapt to the soil environment and fully exert their phosphorus-solubilizing function.

[0096] After 21 days of treatment, the phosphorus-solubilizing effect in half of the treatment groups decreased compared to 14 days, but still maintained a positive effect. The XY10-5% treatment group showed the most significant phosphorus-solubilizing effect, with the soil available phosphorus content increasing by 112.86%, far exceeding other treatment groups; the XY10-10% treatment group was second, with an increase of 62.31%. The available phosphorus content in the XY11-10% and XY04-5% treatment groups increased by more than 50% compared to the control group. The outstanding phosphorus-solubilizing effect shown by the XY10-5% treatment group at 21 days indicates that this strain may have long-lasting phosphorus-solubilizing characteristics, making it suitable for agricultural production environments requiring continuous phosphorus supply.

[0097] 2.14 Effects of bacterial strains on available cadmium content in soil See results Figure 13 .

[0098] Depend on Figure 13 It was found that all three strains effectively reduced the content of available cadmium in the soil, demonstrating a significant inhibitory effect on cadmium activity. After 7 days of treatment, the XY11-5% treatment group showed the most significant cadmium inhibition effect, with an available cadmium content reduced by 63.53% compared to the control group, while the XY11-10% treatment group reduced it by 62.34%. Even though the cadmium inhibition effect of strain XY10 was relatively weaker, it still reduced the content by 38.23% (5% inoculum) and 44.72% (10% inoculum), respectively. Among the three strains, the available cadmium content was lowest in the XY11 treatment group, followed by XY04 and XY10.

[0099] After 14 days of treatment, the cadmium inhibition effect of each treatment group was weaker than that after 7 days, but it still remained at a high level overall. Among them, the XY11-10% treatment group performed the best, with the effective cadmium content reduced by 39.05% compared with the control group. Among the three strains, strain XY11 showed the strongest cadmium inhibition ability in the short to medium term, followed by XY04 and XY10.

[0100] After 21 days of treatment, all treatment groups continued to maintain good cadmium inhibition effects. The XY11-10% and XY10-10% treatment groups performed the best, with effective cadmium content decreasing by 30.20% and 28.90% respectively compared with the control group, indicating that these two strains have good long-term cadmium inhibition ability under high inoculum conditions.

[0101] The differences in cadmium inhibition effects among different strains are related to their metabolic characteristics and mechanisms of action. In the long term, the cadmium inhibition effect of strain XY11 is more persistent, which may be related to their ability to continuously secrete cadmium-inhibiting substances or alter the soil microenvironment.

[0102] 2.15 Effects of bacterial strains on soil available selenium content See results Figure 14 .

[0103] Depend on Figure 14It was found that after 7 days of treatment, except for the XY10-5% treatment group, all other treatment groups reduced the content of available selenium in the soil. The available selenium content of the XY10-5% treatment group was 0.149 mg / kg, an increase of 42.96% compared to the control group. The available selenium content of the XY04-10% treatment group decreased by 42.08%, and the XY11-10% treatment group decreased by 35.87%. The available selenium content of the other treatment groups was basically the same as that of the control group. Overall, the 5% inoculum amount reduced the amount of available selenium less than that of the 10% inoculum. This phenomenon may be due to the strain consuming soluble selenium in the environment during the initial growth process, or the secretion of certain substances temporarily reducing the solubility of selenium. Further investigation is needed. Among the three strains, the XY10 treatment group showed the smallest decrease in available selenium content, and even showed an increase.

[0104] After 14 days of treatment, the available selenium content in the XY10-5% treatment group was 0.137 mg / kg, an increase of 1.31% compared to the control group. The available selenium content in all other treatment groups was lower than that in the control group to varying degrees. However, the decrease under high inoculum conditions was generally less than that under 7 days, indicating that the inhibitory effect of high inoculum strains on selenium weakened with prolonged treatment. Among them, the XY04-5% and XY04-10% treatment groups showed the largest decreases in available selenium content compared to the control group, decreasing by 19.91% and 19.40%, respectively. The decreases in other treatment groups were smaller. Among the three strains, strain XY10 showed the lowest decrease in available selenium, even showing a slight increase. The available selenium content was not significantly different under the two inoculum conditions.

[0105] After 21 days of treatment, most treatment groups continued to show selenium inhibition compared to the control group. The XY04-10% treatment group showed selenium activation, with the effective selenium content increasing by 0.23% compared to the control group. Over three time periods, the effective selenium content in the XY04-10% treatment group increased from 0.060 mg / kg (7 days), 0.109 mg / kg (14 days), and 0.147 mg / kg (21 days) to -42.08% (7 days), 19.40% (14 days), and 0.23% (21 days) respectively compared to the control group. This result suggests that strain XY04 may have long-term selenium activation potential. Furthermore, the effect of the XY10-5% treatment group on soil available selenium content was the opposite of that of the XY04-10% treatment group. Over three time periods, the available selenium content in the XY10-5% treatment group increased from 0.149 mg / kg (7d), 0.137 mg / kg (14d), and 0.120 mg / kg (21d) to 42.96% (7d), 1.31% (14d), and 18.46% (21d) in the control group, respectively. This indicates that strain XY10 has the ability to activate soil selenium in the short term and its activation performance is relatively stable.

[0106] Therefore, among these three strains, there is strain XY10, which has the short-term activation potential for soil selenium, and strain XY04, which has the long-term activation potential for soil selenium. In actual agricultural production activities, the appropriate strain can be applied according to actual needs.

[0107] 2.16 Effects of bacterial strains under selenium and cadmium stress on rice seed germination indices The results are shown in Tables 5 and 6.

[0108] Table 5. Effects of strains on rice seed germination potential under selenium-cadmium stress (%)

[0109] Note: All data in the table are mean ± standard deviation. Different lowercase letters in each column indicate significant differences between treatments of different strains (p<0.05).

[0110] Table 6. Effects of strains on rice seed germination rate under selenium-cadmium stress (%)

[0111] Note: All data in the table are mean ± standard deviation. Different lowercase letters in each column indicate significant differences between treatments of different strains (p<0.05).

[0112] As shown in Tables 5 and 6, under selenium- and cadmium-free stress conditions, the germination potential and germination rate of rice seeds soaked by the four bacterial strains were higher than those of the control group (CK, unsoaked bacterial suspension). The germination potential of the groups soaked in bacterial suspensions of strains XY10, XY11, and XY04 increased by 21.82%, 55.76%, and 50.91%, respectively, and the germination rate increased by 5.91%, 11.41%, and 9.84%, respectively, compared to the control group. This indicates that all three strains have a certain growth-promoting ability and exert a positive promoting effect on rice seed germination by secreting IAA. Strains XY11 and XY04A showed the most significant improvement in germination potential, suggesting that strains with higher IAA yields have a more significant promoting effect on seed germination potential.

[0113] Under selenium-cadmium stress, the germination potential and germination rate of the CK group and the group soaked with the three bacterial strains were lower than those under no selenium-cadmium stress, indicating that the selenium-cadmium solution exerted a stress effect on rice seed germination and inhibited it. Compared with the control group (CK) that was not soaked in bacterial solution, the seeds soaked in the bacterial suspension of the three bacterial strains had greater germination potential and germination rate at the same concentration of selenium-cadmium, indicating that the three bacterial strains could alleviate the inhibitory effect of selenium-cadmium stress on seed germination to some extent. Among them, the seeds soaked in the bacterial suspension of strain XY11 showed the highest germination potential and germination rate under all treatments, demonstrating its strong tolerance to selenium and cadmium stress and its ability to promote growth.

[0114] 2.17 Effects of strains under selenium and cadmium stress on germination index and vigor index of rice seeds The results are shown in Tables 7 and 8.

[0115] Table 7. Effects of bacterial strains on rice seed germination index under selenium-cadmium stress.

[0116] Note: All data in the table are mean ± standard deviation. Different lowercase letters in each column indicate significant differences between treatments of different strains (p<0.05).

[0117] Table 8. Effects of strains on rice seed vigor index under selenium-cadmium stress.

[0118] Note: All data in the table are mean ± standard deviation. Different lowercase letters in each column indicate significant differences between treatments of different strains (p<0.05).

[0119] As shown in Tables 7 and 8, under selenium- and cadmium-free stress, the germination indices of strains XY10, XY11, and XY04 increased by 23.67%, 45.40%, and 47.54% respectively compared with the control group, and the vigor indices increased by 62.54%, 298.27%, and 203.19% respectively. This further confirms that the three strains have a positive promoting effect on rice seed germination, with strains XY11 and XY04 showing particularly significant growth-promoting effects, which is consistent with their higher IAA yield.

[0120] Under selenium and cadmium stress, the germination index and vigor index of rice seeds soaked in the four bacterial strains were lower than those in the selenium and cadmium-free environment. However, compared with the control group without bacterial suspension, the germination index and vigor index of seeds soaked in bacterial suspension were still higher than those of the control group (CK). This indicates that even under selenium and cadmium stress, the three bacterial strains can still alleviate the inhibitory effect of selenium and cadmium stress on seed germination to a certain extent, maintaining and improving seed germination ability and vigor. Among them, the seeds soaked in the suspension of strain XY11 showed the highest germination index and vigor index under all selenium and cadmium stress treatments, which again proves that strain XY11 has strong tolerance and growth-promoting ability to selenium and cadmium stress. XY04 was the second highest, while the seeds soaked in XY10 also showed a certain growth-promoting effect under selenium and cadmium stress, but the increase in germination index and vigor index was relatively small, which is also related to the relatively low IAA production capacity of strain XY10.

[0121] 2.18 Effects of strains under selenium and cadmium stress on root length and seedling growth in rice The results are shown in Tables 9 and 10.

[0122] Table 9. Effects of strains on rice root length (cm) under selenium and cadmium stress.

[0123] Table 10. Effects of strains on rice seedling growth (cm) under selenium and cadmium stress.

[0124] As shown in Tables 9 and 10, under selenium- and cadmium-free stress, the root length and seedling length of rice treated with strains XY10, XY11, and XY04A4 were significantly higher than those of the control group (CK). The root length increased by 32.69%, 174.04%, and 98.08%, respectively, and the seedling length increased by 12.11%, 65.79%, and 43.16%, respectively. This indicates that these three strains have a greater effect on promoting root growth than on promoting seedling growth. Among them, the XY11 strain treatment group showed the best performance in rice root length and seedling length, which increased by 174.04% and 65.79% respectively compared with the control group. The XY04A strain treatment group followed, which increased by 98.08% and 43.16% respectively. Although the XY10 strain treatment group also showed a certain growth-promoting effect, its improvement was smaller compared with the XY11 and XY04A strain treatment groups. This further confirms the positive promoting effect of these three strains on rice growth, and the growth-promoting effect is positively correlated with the IAA yield of the strains.

[0125] Under selenium-cadmium stress, except for Cd 2+ Except for a concentration of 5 mg / L, the root length and seedling length of rice in the three strain treatment groups were lower than those in the selenium- and cadmium-free stress environment. However, compared with the control group that was not soaked in bacterial solution, the root length and seedling length of rice soaked in bacterial suspension were still higher than those of the control group. 2+ At a concentration of 5 mg / L, the root length of all treatment groups increased to some extent. Except for the XY04 treatment group, the seedling length of other treatment groups also increased to some extent, but less than the increase in root length.

[0126] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain XY11, characterized in that, The taxonomic name of the strain XY11 is... Lysinibacillus sp . It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 1, 2025, with accession number GDMCC No: 66620.

2. Use of strain XY11 of claim 1 in inhibiting the activity of heavy metal cadmium in soil.

3. The use of strain XY11 of claim 1 in IAA production.

4. Use of strain XY11 of claim 1 in improving crop resistance to selenium and cadmium stress.

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