Application of DL-alanine in promoting colonization of Pseudomonas aeruginosa and their synergistic effect in enriching selenium and reducing cadmium

Abstract

The present invention relates to the field of microbial technology, and in particular to an application of DL-alanine to promote the colonization of Pseudomonas aeruginosa and the synergistic selenium-enriched and cadmium-reduced use of the two. The present invention provides a novel composition for selenium-enriched and cadmium-reduced use, the composition comprising independently packaged DL-alanine and Pseudomonas aeruginosa. The composition is applied to the rhizosphere soil of crops, which can increase the selenium content of the crops, reduce the cadmium content of the crops, and promote the increase in the effectiveness of selenium and the decrease in the effectiveness of cadmium in the rhizosphere soil. The present invention also proposes the application of DL-alanine and Pseudomonas aeruginosa to synergistically enrich selenium and reduce cadmium in crops and their rhizosphere soils in areas with high natural selenium and cadmium backgrounds. Therefore, the present invention will have significant application prospects in the utilization of actual selenium-cadmium soils, the production of selenium-enriched and cadmium-reduced crops, and the preparation of selenium-enriched and cadmium-reducing conditioners.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an application of DL-alanine promoting the colonization of Pseudomonas aeruginosa and the synergistic use of the two to enrich selenium and reduce cadmium. Background Art

[0002] Pseudomonas aeruginosa is widely distributed in soil, water, and various environmental niches. It generally has a flagellum at one end of its body and cilia on the surface. It is active and is a type of Gram-negative bacteria. Pseudomonas aeruginosa isolated from rhizosphere or contaminated soil is an important plant rhizosphere growth-promoting bacterium. The effective colonization of rhizosphere growth-promoting bacteria in the rhizosphere of plants is an important prerequisite for the growth-promoting bacteria to control crop diseases, promote plant growth, increase the utilization rate of nutrients by plants, and enhance the effect of plant restoration.

[0003] Plant root exudates can identify and attract beneficial rhizosphere microorganisms. The flagella assembly, group movement, biofilm formation and colonization of rhizosphere microorganisms are regulated by root exudates. In addition, plant root exudates play an important role in the element cycle of soil-plant system. Root exudates contain organic acids, amino acids, alcohols, amines and other substances, which have a direct impact on the transformation of soil heavy metal forms.

[0004] Selenium (Se) is a trace nutrient essential to the human body. It has the functions of improving human immunity and anti-cancer. Eating selenium-rich crops is one of the main ways for the human body to supplement selenium. There are large areas of selenium-rich soil in Enshi, Hubei, Ankang, Shaanxi, Guangxi and other regions in my country. In recent years, many studies and investigations have found that cadmium (Cd) is associated with the soil in some selenium-rich areas. The accumulation of cadmium in crops puts residents who eat local crops for a long time at risk of cadmium poisoning.

[0005] At present, the development of selenium-rich compound fertilizers for foliar cadmium reduction using rhizosphere growth-promoting bacteria or root secretions has been widely studied. However, these studies mainly focus on the effects and mechanisms of exogenous selenium-rich compound fertilizers on cadmium accumulation in crops in cadmium-contaminated areas. Exogenous selenium-rich compound fertilizers have complex ingredients, high acquisition costs, low utilization rates, and are prone to secondary pollution. In addition, the research objects of exogenous selenium-rich compound fertilizers are crops in cadmium-contaminated areas, not crops in areas with high natural selenium and cadmium backgrounds. Summary of the invention

[0006] In view of this, the present invention provides a novel selenium-enriched cadmium-reducing composition which utilizes DL-alanine and Pseudomonas aeruginosa to synergistically perform selenium-enriched cadmium-reducing.

[0007] DL-alanine is an amino acid containing chiral carbon, with the molecular formula C5H 11 NO2 is easily soluble in water. The present invention finds that DL-alanine can also be found in rapeseed root secretions.

[0008] The present invention finds that DL-alanine in rapeseed root secretions can promote the colonization of Pseudomonas aeruginosa ATCC27853, and applying the composition to crop rhizosphere soil can increase the selenium content of the crop, reduce the cadmium content of the crop, and promote the increase of selenium effectiveness and the decrease of cadmium effectiveness in the rhizosphere soil.

[0009] The technical solution of the present invention is achieved in this way:

[0010] In a first aspect, the present invention provides a selenium-enriched cadmium-reducing composition, characterized in that the composition comprises independently packaged DL-alanine and Pseudomonas aeruginosa.

[0011] In a second aspect, the present invention provides applications of the composition, which are one or more of the following: (1) application in the production of selenium-enriched and / or cadmium-reduced crops; (2) application in the preparation of selenium-enriched and / or cadmium-reduced products; (3) application in crop planting in selenium-cadmium soil; (4) application in improving or repairing selenium-cadmium soil.

[0012] In a third aspect, the present invention provides a method for promoting colonization of Pseudomonas aeruginosa, the method comprising: first adding not less than 10 μmol·L -1 The concentration of DL-alanine was used to treat Pseudomonas aeruginosa, and then the treated Pseudomonas aeruginosa was colonized in the roots of the plants.

[0013] The promoting the colonization of Pseudomonas aeruginosa includes at least one of promoting the growth of the strain, the formation of biofilm, and the colonization in the rhizosphere soil of crops.

[0014] In a fourth aspect, the present invention provides a method for promoting selenium-enrichment and cadmium-reduction in crops, the method comprising applying the selenium-enriched and cadmium-reducing composition to the rhizosphere soil of the crops.

[0015] In a fifth aspect, the present invention also provides a method for enriching at least one of available selenium in soil and reducing available cadmium in soil, the method comprising applying the selenium-enriched and cadmium-reducing composition to the rhizosphere soil of crops.

[0016] In the present invention, the Pseudomonas aeruginosa includes Pseudomonas aeruginosa ATCC27853, which was purchased from Shanghai Jinpan Biotechnology Co., Ltd.

[0017] The present invention has the following beneficial effects compared with the prior art:

[0018] (1) The present invention provides an application of DL-alanine, a root secretion of rapeseed, in promoting the reproduction of Pseudomonas aeruginosa, biofilm formation and colonization in rhizosphere soil. In the present invention, GFP fluorescent labeling technology is first used, and the three-parent hybridization method is used to successfully label Pseudomonas aeruginosa, and it is confirmed that the GFP plasmid of the labeled strain can be stably inherited. The strains before and after labeling were used to carry out rapeseed rhizosphere colonization assays, and the results showed that the labeled strain can colonize in the roots of plants. The present invention selected a series of compounds of different categories of rapeseed root secretions, and conducted interaction tests with Pseudomonas aeruginosa. The screening results showed that DL-alanine significantly promoted the growth, biofilm formation and colonization of Pseudomonas aeruginosa.

[0019] (2) The present invention applies the selenium-enriched cadmium-reducing composition to the natural selenium-enriched cadmium-containing soil in Enshi, and uses potted experiments to plant rapeseed. The selenium-cadmium content of each part of the rapeseed and the form of selenium-cadmium in the rhizosphere soil are measured to illustrate the application effect of Pseudomonas aeruginosa and DL-alanine. The results show that the combination of Pseudomonas aeruginosa and DL-alanine significantly promotes the absorption of selenium and reduces the absorption of cadmium. In addition, in the rhizosphere soil of rapeseed, the effectiveness of selenium increases and the effectiveness of cadmium decreases. Compared with the existing selenium-enriched compound fertilizer and selenium-enriched cadmium-reducing technology, its composition is simple, which can reduce the cost of repair to a certain extent, reduce the low utilization rate of selenium-enriched compound fertilizer, and avoid the problem of secondary pollution that may be caused. According to the characteristics that Pseudomonas aeruginosa and DL-alanine can promote the selenium-enriched cadmium reduction of crops, and increase the effective state of selenium in the soil and reduce the effective state of cadmium, this method has important potential in the actual utilization of selenium-cadmium soil, the production of selenium-enriched cadmium-reducing crops, the planting of crops in selenium-cadmium soil, the improvement or repair of selenium-cadmium soil, and the preparation of selenium-enriched cadmium-reducing conditioners. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :A / A1 are the pictures of the bacterial solution of the unlabeled strain under bright field and fluorescence microscope respectively; B / B1 are the pictures of the bacterial solution of the unlabeled strain under bright field and fluorescence microscope respectively (fluorescence conditions: exposure time 500ms, gain 2.2); C / C1 are the pictures of the colonies of the unlabeled strain under bright field and fluorescence microscope respectively; D / D1 are the pictures of the 17th generation of the labeled strain under bright field and fluorescence microscope (fluorescence conditions: exposure time 100ms, gain 2.2); E is the verification result of the bacterial solution of the labeled strain and the plasmid PCR (CK is a blank control with sterile culture medium, TJ-0, TJ-2, TJ-5, TJ-6 represent the PCR results of the bacterial solution of the 0th, 2nd, 5th and 6th generations, respectively, and TJ-1 plasmid and TJ-4 plasmid represent the plasmid PCR results of the 1st and 4th generations of strains respectively); F is the fragment comparison result of the plasmid sequencing result of the labeled strain and the donor strain;

[0021] Figure 2:A is a picture of the bacterial colonization determination method; B is the amount of bacterial colonization in plants (the number of bacteria is calculated per gram of root dry weight); CF are the colonization conditions of bacteria in plants (where: C is a bright field picture of rapeseed roots without bacterial treatment, D is a fluorescent picture of rapeseed roots without bacterial treatment, E is a bright field picture of rapeseed roots with bacterial treatment, and F is a fluorescent picture of rapeseed roots with bacterial treatment; fluorescence conditions: exposure time 100ms, gain 2.2);

[0022] Figure 3 : Non-targeted metabolic analysis of rapeseed root exudates (where A is the non-targeted metabolic results of exudates, and B is the types and proportions of exudates);

[0023] Figure 4 :Effects of vegetable root secretions on the biomass and growth of Pseudomonas aeruginosa (where: A / B is the amount of biofilm of bacteria under the action of secretions; C is the growth of bacteria under the action of secretions that significantly promote biofilm formation; D / E is the colonization plate and counting results of bacteria under the action of secretions that significantly promote biofilm formation);

[0024] Figure 5 : Biomass and selenium and cadmium content of rapeseed under the action of rapeseed root secretions and Pseudomonas aeruginosa (where: A is the fresh weight of the aboveground part of rapeseed; B is the fresh weight of the underground part of rapeseed; C is the selenium content of the aboveground part of rapeseed; D is the selenium content of the underground part of rapeseed; E is the cadmium content of the aboveground part of rapeseed; F is the cadmium content of the underground part of rapeseed);

[0025] Figure 6 : Changes in selenium forms in the rhizosphere soil of rapeseed under the action of rapeseed root secretions and Pseudomonas aeruginosa (where: A is the proportion of selenium content in different forms in the soil, B is the sum of water-soluble and exchangeable selenium content in the soil, C is the exchangeable selenium content in the soil, D is the iron-manganese oxidized selenium content in the soil, E is the organically bound selenium content in the soil, and F is the residual selenium content in the soil);

[0026] Figure 7 : Changes in cadmium forms in the rhizosphere soil of rapeseed under the action of rapeseed root secretions and Pseudomonas aeruginosa (where: A is the exchangeable cadmium content in the soil, B is the carbonate-bound cadmium content in the soil, C is the iron-manganese oxidation cadmium content in the soil, D is the organic-bound cadmium content in the soil, E is the residual cadmium content in the soil, and F is the proportion of different cadmium forms in the soil). DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] (I) Preparation of GFP-labeled Pseudomonas aeruginosa and detection of its colonization in rapeseed roots

[0029] The present invention firstly tests whether Pseudomonas aeruginosa can successfully colonize rapeseed roots. The test process has the following four steps:

[0030] (1) The three-parent hybridization method was used to fluorescently label Pseudomonas aeruginosa, and the labeled strains were observed using a fluorescence microscope.

[0031] (2) Extract the plasmid of the marker strain, amplify the plasmid by PCR, and compare the sequence determined after amplification with the sequence of the donor strain.

[0032] (3) Subculture the labeled strain to verify the genetic stability of its plasmid.

[0033] (4) The strains before and after labeling were used to conduct rhizosphere colonization assays in rapeseed.

[0034] The specific steps and experimental results are as follows:

[0035] 1 Experimental steps:

[0036] (1) Fluorescent labeling of Pseudomonas aeruginosa using the three-parent hybridization method

[0037] A. Sources of the three strains and their resistance testing:

[0038] The sources of Pseudomonas aeruginosa (recipient bacteria), donor bacteria with GFP labels, and helper bacteria are shown in Table 1. The three strains were tested for antibiotic resistance, and LB solid culture medium was prepared. After sterilization and cooling to about 30 degrees, gentamicin, kanamycin, and ampicillin were added to different culture media to make their final concentrations of 50 μg mL -1 ,25μg·mL -1 ,50μg·mL -1. Donor bacteria, auxiliary bacteria, and recipient bacteria were inoculated on a culture medium containing antibiotics. After being placed in a biochemical incubator for 24 hours, the growth of the strains was observed. The results showed that the donor bacteria could grow on a culture medium containing gentamicin, but could not grow on a culture medium containing kanamycin and ampicillin. The auxiliary bacteria could grow on a culture medium containing kanamycin, but could not grow on a culture medium containing gentamicin and ampicillin. The recipient bacteria could grow on a culture medium containing ampicillin, but could not grow on a culture medium containing gentamicin and kanamycin. The specific results are shown in Table 2.

[0039] Table 1 Relevant characteristics and sources of donor bacteria, helper bacteria and recipient bacteria

[0040]

[0041]

[0042] Table 2 Resistance and concentration of donor bacteria, helper bacteria and recipient bacteria

[0043]

[0044] The components of LB liquid medium are: peptone 10g·L -1 , yeast extract 5g·L -1 and NaCl 10 g·L -1 ; Add 2% agar by volume to LB liquid culture medium to obtain LB solid culture medium.

[0045] B. Principle of three-parent hybridization: According to the resistance results of the strains, Pseudomonas aeruginosa uses the method of three-parent hybridization to introduce exogenous genes into the recipient bacteria. The three-parent hybridization technology is that when these three bacteria are mixed, the auxiliary plasmid pRK2013 swims into Escherichia coli, provides swimming and transfer functions, and transfers the donor's intermediate plasmid into Pseudomonas aeruginosa. In this system, the intermediate plasmid needs to have a specific transfer starting point and activation site so that the swimming and transfer genes of the auxiliary plasmid can act on it and be driven to transfer. The recipient Pseudomonas aeruginosa used in the three-parent hybridization has resistance, and the donor plasmid and the auxiliary plasmid carry resistance markers of gentamicin and kanamycin, respectively, so that gentamicin and ampicillin are used to select the transfer conjugate. The plasmid contained in the conjugate Pseudomonas aeruginosa should be the plasmid carried by the donor bacteria.

[0046] C. Specific operation of three-parent hybridization:

[0047] a) Bacterial activation: Activate the recipient bacteria on the LB plate, pick a single colony into LB liquid culture medium, and culture at 37°C for 15 hours until the middle and late logarithmic phase. Activate the donor bacteria and helper bacteria on the LB plate (containing the corresponding antibiotic concentration), pick a single colony into 5 mL LB liquid culture medium (containing the corresponding antibiotic concentration), and culture at 37°C for about 12 hours until the middle of the logarithmic growth period.

[0048] b) Mixed hybridization: Take 750 μL of donor bacteria and auxiliary bacteria and add them to a centrifuge tube, take 375 μL of recipient bacteria and put them in another centrifuge tube, centrifuge at 10,000 rpm for 3 minutes to collect the bacteria and discard the supernatant. Add 1 mL of LB liquid culture medium to the recipient bacteria, pipette several times, add it to the centrifuge tube containing donor bacteria and auxiliary bacteria, pipette and mix, centrifuge at 10,000 rpm for 3 minutes, discard the supernatant, and let it stand for 5 minutes to make the bacterial mud soft. Gently aspirate 200 μL of bacterial mud and apply it to the 0.22 μm pore filter membrane on the pre-placed LB plate and the surface of which has been dried, first culture it upright for 1.5 hours, and then culture the plate inverted overnight.

[0049] c) Screening of zygotes: Place the filter in LB liquid culture medium, vortex and oscillate, dilute 10 times with LB liquid culture medium and spread on the plate containing Amp and Gen antibiotics. After overnight culture, pick a single colony and culture it in liquid culture medium containing antibiotics overnight. Detect whether the bacterial solution is fluorescent under a fluorescence microscope. The luminescent bacteria are sequenced for 16S rRNA genes to further detect zygotes.

[0050] d) Determination of the stability of the labeled strain: The labeled strain carrying the plasmid was inoculated on a plate containing two antibiotics and streaked for subculture. The fluorescence expression was then observed under a fluorescence microscope (Olympus BX51, Japan), and the ratio of fluorescent expressing colonies to total colonies was calculated to determine the subculture stability of the labeled vector in the strain.

[0051] (2) Perform PCR amplification on the plasmid and compare the sequence determined after amplification with the sequence of the donor strain:

[0052] A single colony of the 0th generation was selected and subcultured, cultured overnight in LB liquid medium containing antibiotics, and PCR amplification was performed directly on the bacterial solution.

[0053] Primers were designed for the EGFP fragment of the PMP2463 plasmid strain. The designed primer sequences are shown in Table 3. The primer fragment size is 232 bp. The bacterial solution and the extracted plasmid were PCR amplified and sent to Qingke Biotech Co., Ltd. for sequencing. PCR amplification was performed using the 2×SuperTaq PCR StarMix (Dye) system. The specific reaction system and reaction procedure are shown in Tables 4 and 5.

[0054] Table 3 PCR amplification primer sequences

[0055]

[0056] Table 4 PCR reaction system

[0057]

[0058] Table 5 PCR reaction program

[0059]

[0060]

[0061] (3) Subculture the labeled strain to verify the genetic stability of its plasmid

[0062] A single fluorescent colony was randomly selected from the subculture, cultured overnight in LB liquid medium containing antibiotics, and the plasmid was extracted from the bacterial liquid using a plasmid extraction kit (HiPure Plasmid MicroKit). After extraction, the plasmid concentration was determined using Nanodrop2000 and stored in a -20 degree refrigerator for future use.

[0063] (4) Qualitative and quantitative determination of strain colonization

[0064] For the determination of the colonization of the strain, 7-day-old sterile rapeseed seedlings were cultured in a culture medium experiment. The rapeseed seedlings were inoculated with bacteria, and the seedlings were placed in a saline solution (0.45% NaCl) as a control. After the inoculated seedlings were cultured for 24 h, the roots of the seedlings infected with bacteria were separated from the aerial parts and soaked in ethanol (75%; volume / volume) for 1 min, then washed four times with sterile water, ground, and diluted with 0.45% NaCl solution to prepare 10 -1 -10 -6 Serial dilutions were plated on LB agar plates containing two antibiotics. The ground stock solution was freeze-dried and weighed to calculate the number of bacteria per mg of root dry weight. Antibiotics were added to LB solid medium to distinguish root endophytes from colonizing bacteria. After the plate was incubated at 37°C for 24 hours, the colonies were counted and expressed as CFU mg -1 Root dry weight. The colonization of the labeled strain was quantitatively determined by plate spreading.

[0065] 2 Experimental results

[0066] (1) GFP labeling results: Pseudomonas aeruginosa was successfully labeled with green fluorescent protein ( Figure 1 B1). PCR amplification results are shown in Figure 1As shown in E, the bacterial solution of Pseudomonas aeruginosa and the amplified bands of the extracted plasmid are brighter and free of mixed bands, indicating that the fluorescent plasmid was successfully transferred. The plasmid of the conjugate was extracted using a plasmid extraction kit and the concentration was determined. The plasmid concentrations of the two extracted samples of Pseudomonas aeruginosa were 32.9 μg·μL -1 and 45.2 μg·μL -1 .

[0067] (2) Comparison of sequencing results: Sequencing was performed using T3 universal primers, and the snapgene software showed that the sequencing sequence was consistent with the EGFP fragment sequence. Figure 1 As shown in F, it means the transfer is successful.

[0068] (3) Subculture results: The 17th generation strain can still emit fluorescence ( Figure 1 D1), indicating that the marker strain can be stably inherited.

[0069] (4) Results of colonization assay: On the medium supplemented with two antibiotics, no single colony grew in the control group, but after the roots colonized with the labeled strain of Pseudomonas aeruginosa were coated, a single colony grew on the medium containing antibiotics, indicating that the labeled strain successfully colonized the roots ( Figure 2 B) Fluorescence microscopy results: Figure 2 As shown in CF, under the microscope bright field observation, it can be found that the bacteria are aggregated on the root surface, and under fluorescence, the strains colonized in the roots are observed to fluoresce, which further shows that Pseudomonas aeruginosa can successfully colonize in the roots of rapeseed.

[0070] (II) Screening of rapeseed root exudates that promote Pseudomonas aeruginosa colonization

[0071] The screening process includes the following:

[0072] (1) Rapeseed root exudates were collected and then subjected to non-targeted metabolic analysis. 67 exudates were identified by comparing with the database. Then, 25 exudates were screened out based on the classification of metabolites in rapeseed exudates.

[0073] (2) Four secretions that significantly promoted the biofilm formation of Pseudomonas aeruginosa were screened out from 25 rapeseed root secretions.

[0074] (3) From these four rapeseed root exudates, the secretion with the greatest effect on promoting the growth and colonization of Pseudomonas aeruginosa was further screened.

[0075] The specific steps and experimental results are as follows:

[0076] 1 Experimental steps

[0077] (1) Collection and identification of rapeseed root exudates

[0078] The root secretions of rapeseed were collected by hydroponic test method. After the rapeseed seeds were disinfected, they were germinated in sterile water for 3 days. After germination, 1 / 2 Hogland nutrient solution was used to continue culture for 7 days, and then full nutrient solution was used for culture. The nutrient solution was replaced every 3 days to ensure that the pH of the nutrient solution was about 5.8. After the rapeseed has grown for 2 months, the roots were washed with deionized water, and then washed with sterile water 3 times. The washed rapeseed roots were placed in a sterile container filled with sterile water for growth. After 24 hours, the secretions in the container were collected, quickly frozen with liquid nitrogen, and stored at -80 degrees. Before the sample was measured, the collected secretions were freeze-dried, and then a liquid chromatography-mass spectrometry analyzer (GC-MS) was used to perform non-targeted metabolic analysis of the rapeseed secretions.

[0079] (2) Detection of the effect of rapeseed root secretions on the biomass of Pseudomonas aeruginosa

[0080] According to the classification of metabolites in rapeseed exudates, a series of compounds of different categories of rapeseed root exudates were configured, as shown in Table 6.

[0081] Table 6 25 secretions and concentrations

[0082]

[0083]

[0084] Inoculate a single colony of the strain into 5 mL of LB liquid medium and culture it overnight. Then inoculate 1% of the colony into a new 200 mL of LB liquid medium and culture it overnight until the OD 600＝ Prepare a sterile 96-well plate, add 198 μL of sterile LB liquid medium, and add overnight cultured bacteria to make the OD 600 = 0.1, and then 2 μl of the prepared rapeseed secretion compound was added to make the final concentration of the metabolite 100 μmol·L -1 , with no compound added as a control. After growing at 37°C for 48 hours, the liquid in each well was removed and the wells were washed with sterile water. Each well was then stained with 0.1% crystal violet for 20 minutes and washed three times with sterile water. The biofilm in the well was removed from the well wall by adding 2 mL of 4:1 (volume:volume) ethanol and acetic acid and gently stirred until completely dissolved. The OD value at 530 nm (OD 530 =The amount of biofilm). Each metabolite treatment was repeated three times. The four metabolites with the most significant effects were selected from the results of biofilm amount. (The composition of LB liquid culture medium is: peptone 10g·L -1 , yeast extract 5g·L -1 and NaCl 10 g·L -1; Add 2% agar by volume to LB liquid medium to obtain LB solid medium)

[0085] (4) Detecting the effects of the four metabolites on the growth and colonization of Pseudomonas aeruginosa

[0086] The method for promoting colonization of Pseudomonas aeruginosa provided by the present invention (first adding not less than 10 μmol·L -1 The specific implementation method is as follows:

[0087] In some preferred embodiments, the concentration of DL-alanine is not less than 100 μmol·L -1 .

[0088] In some preferred embodiments, the viable count of Pseudomonas aeruginosa is not less than 10 7 CFU·mL -1 .

[0089] In some preferred embodiments, the culture medium is LB liquid culture medium or LB solid culture medium, and the components of the LB liquid culture medium include: peptone 10g·L -1 , yeast extract 5g·L -1 and NaCl 10 g·L -1 The components of the LB solid culture medium are 2% agar added to the LB liquid culture medium by volume.

[0090] step:

[0091] 100 μmol·L -1 The four metabolites of Pseudomonas aeruginosa (10 7 CFU·mL -1 ) in LB solid medium (grown at 37°C for 48 hours) to observe the effect of metabolites on bacterial growth.

[0092] 100 μmol·L -1 The four metabolites of Pseudomonas aeruginosa (10 7 CFU·mL -1 ) in LB liquid medium at 37°C. After 48 hours, the OD value at 600 nm was measured.

[0093] At the same time, a culture medium test was used to determine the colonization of bacteria under the action of metabolites. Specifically, the rapeseed seeds were sterilized and germinated on a 1 / 2 MS medium containing agar. After germination for 3 days, the seedlings were transferred to a new 1 / 2 MS medium containing agar for growth. After the seedlings grew for 5 days, the seedlings were taken out and placed in a sterile triangular bottle prepared in advance and inoculated with Pseudomonas aeruginosa. The initial OD in the sterile bottle was adjusted. 600 =0.1, at 28 degrees, 150 rpm min -1 After continuous shaking cultivation for 24 hours, the whole rapeseed seedling was taken out, and the roots were washed with ethanol for 10 seconds, and then washed with sterile water for 3 times. The washed roots were vortexed in 1x PBS solution for 20 seconds, and then the vortexed solution was continuously diluted for smearing and counting.

[0094] 2 Experimental results

[0095] (1) Rapeseed metabolite detection results Figure 3 As shown in the figure, a total of 67 different metabolites were detected based on GC-MS and the metabolite database TMSMW1.0. Quantitative analysis and classification of the detected metabolites revealed that alcohols and amines accounted for 20.6% of the secretions in the rapeseed seedling stage, organic acids and their derivatives accounted for 16.2%, carbohydrates and their metabolites accounted for 13.2%, benzene and its derivatives accounted for 11.8%, amino acids and their metabolites accounted for 8.8%, glycerides, hydrocarbons, heterocyclic compounds, coenzymes and vitamins accounted for 2.9%, 1.5%, 1.5%, 1.5% respectively, and other compounds accounted for 5.9%.

[0096] (2) Biofilm assay results Figure 4 As shown in A and 4B, compared with the control, the addition of metabolites significantly promoted the biofilm formation of Pseudomonas aeruginosa. When sucrose, DL-alanine, L-phenylalanine, and inositol were added, the amount of biofilm was 3.16, 2.73, 3.19, and 2.27 times that of the control, respectively.

[0097] (3) The test results of the effects of these four metabolites on bacterial growth and colonization are as follows Figure 4 As shown in C, compared with the control, the OD of the bacteria under the action of sucrose, DL-alanine, and L-phenylalanine increased significantly. 600 They increased by 53.7%, 65.4% and 23.5% respectively. Figure 4 As shown in D and E, the roots of rapeseed were smeared and counted after colonization, and it was found that DL-alanine had a greater promoting effect on bacterial colonization.

[0098] (III) Application of selenium-enriched cadmium-reducing composition in crops and soil

[0099] The selenium-enriched cadmium-reducing composition provided in the embodiment of the present invention comprises independently packaged DL-alanine and Pseudomonas aeruginosa. The independently packaged DL-alanine is a liquid preparation containing DL-alanine. The independently packaged Pseudomonas aeruginosa is a liquid or solid preparation containing a Pseudomonas aeruginosa bacterial agent.

[0100] In some preferred embodiments, the concentration of DL-alanine in the composition is not less than 10 mmol·L -1 .

[0101] In some preferred embodiments, the viable count of Pseudomonas aeruginosa in the composition is not less than 10 7 CFU·mL -1 .

[0102] In some preferred embodiments, the composition is applied to the rhizosphere soil of crops, and it is found that the selenium content of the crops can be increased, the cadmium content of the crops can be reduced, and the effectiveness of selenium in the rhizosphere soil can be increased and the effectiveness of cadmium can be reduced.

[0103] Based on this, the inventors used selenium-rich soil from Enshi farmland to conduct potted experiments, applied the selenium-rich cadmium-reducing composition provided by the present invention to the rhizosphere soil of crops, and detected and analyzed the fresh weight of crops and the selenium and cadmium content of crops under each treatment. Since the heavy metal form of the soil directly determines the heavy metal content of the plant, the inventors further analyzed the changes in the selenium and cadmium forms of the rhizosphere soil under each treatment.

[0104] The specific steps and experimental results are as follows:

[0105] 1 Experimental steps (1) Application of selenium-enriched cadmium-reducing composition and detection of crop fresh weight and crop selenium and cadmium content

[0106] The experiment was conducted in a potted plantation, and the soil was selenium-rich soil from Enshi farmland. The topsoil was removed, and soil was dug to a depth of 0-20 cm. The soil was placed in a cool place to dry naturally, and stones and plant residues were removed. The soil was ground and passed through a 1 cm sieve for later use. The total selenium content of the soil was 2.8 mg kg -1 .

[0107] The pot experiment was conducted at the experimental base of Huazhong Agricultural University. Each pot was filled with 0.5 kg of soil, and 4 seeds disinfected with 0.5% sodium hypochlorite were sown. After 2 weeks of stable growth, the seedlings were transplanted to 2 plants, and treatment was added after transplanting. Before sowing, 0.2 g N, 0.15 g P2O5, and 0.2 g K2O were applied to each kg of potting soil. Urea, potassium dihydrogen phosphate, and potassium sulfate were used as fertilizer sources for N, P, and K fertilizers, respectively. In addition, 1 mL of Anong nutrient solution was added to each kg of soil. Before sowing, each nutrient element was evenly mixed in the soil in the form of a solution. After sowing, it was placed in a growth chamber for cultivation (22°C, 16h photoperiod), routine management was performed, and the pot placement was randomly changed regularly to ensure consistent growth conditions for each plant.

[0108] The experiment has 4 treatments:

[0109] T1 is CK (control);

[0110] T2 is the Pseudomonas aeruginosa alone treatment group (B represents Pseudomonas aeruginosa);

[0111] T3 is the composition (B+DL-Alanine) treatment group (B+DL-Alanine represents Pseudomonas aeruginosa and DL-alanine);

[0112] T4 is the DL-Alanine treatment group (DL-Alanine means DL-alanine). The specific treatment method is as follows:

[0113] T1: 10 mL of sterile water was applied to each pot soil of T1 as a control;

[0114] T2: 10 mL 10 7 CFU·mL -1 The Pseudomonas aeruginosa bacterial solution was applied to the soil of each pot of T2;

[0115] T3: 10mL 10 7 CFU·mL -1 Pseudomonas aeruginosa bacterial solution and 10mL 10mmol·L -1 A composition of DL-alanine was applied to the soil of each pot of T3;

[0116] T4: 10mL 10mmol·L -1 of DL-alanine was applied to the soil of each pot of T4.

[0117] In each treatment, bacteria and DL-alanine were added once every 3 weeks, for a total of 2 times. After 8 weeks of cultivation, samples were collected. After disinfecting with alcohol, a small shovel was dug down along the root of the plant to remove the whole plant. The rhizosphere soil of the plant was brushed with a sterile small brush for soil selenium and cadmium morphology analysis. The whole plant was washed with tap water, rinsed with deionized water three times and wiped dry. The fresh weight was weighed separately. A part of the root was quickly frozen in liquid nitrogen and then stored in a -80℃ refrigerator for RNA extraction. The remaining roots and aboveground parts were placed in an oven at 105℃ for 30 minutes, and then dried in an oven at 55℃ to constant weight. The dry weight was weighed and the selenium content of each part was determined.

[0118] Determination of selenium and cadmium content in various parts: Determination of selenium content in the aboveground and underground parts of rapeseed was carried out according to GB500993-2010 National Food Safety Standard "Determination of Selenium in Food". Weigh 0.3g (accurate to 0.0001g) of rapeseed sample into a 50mL digestion conical flask, add 10mL of a mixed acid solution of nitric acid-perchloric acid with a volume ratio of 9:1, put a glass funnel in a fume hood and cold digest overnight. The next day, digest at no more than 200℃ on a hot plate until white smoke comes out of the bottle mouth, remove the bottle when there is about 2mL of liquid left, cool to room temperature, and add 10mL of 6mol·L -1 Continue digesting with hydrochloric acid until the solution becomes clear and transparent. When the remaining liquid is about 1 mL, remove the sample, cool it, transfer the digestion solution to a 25 mL volumetric flask, make up the volume, mix well, pour it into a 10 mL centrifuge tube, and measure it with an atomic fluorescence spectrophotometer.

[0119] (2) Determination of selenium and cadmium forms in soil

[0120] Soil selenium forms were extracted using a five-step sequential extraction method:

[0121] Selenium forms can be divided into soluble selenium (SOL-Se), exchangeable selenium (EXC-Se), iron-manganese oxide bound selenium (FMO-Se), organically bound selenium (OM-Se) and residual selenium (RES-Se).

[0122] 2.000 g of soil sample passed through a 0.149 mm sieve was placed in a 100 mL centrifuge tube and extracted with different solutions (solid-liquid ratio of 1:10) in each step. Se(VI) and Se(IV) valence states were determined in soil SOL-Se and EXC-Se. Se(IV) concentration was determined by centrifuging the soil suspension at 4000 r·min -1 The concentration of Se(VI) was determined directly after centrifugation at 100°C for 10 minutes. The concentration of Se(VI) was the sum of Se(VI) and Se(IV) measured after the extract was reduced with 12 mol / L HCl solution (1:1) at 100°C for 30 minutes. The concentration of Se(VI) was obtained by subtracting the above directly measured Se(IV).

[0123] The soil cadmium forms were also extracted using a five-step continuous extraction method

[0124] Weigh 1g of soil sample and add 8mL of 1mol·L -1 MgCl2 solution was used for leaching, and the cadmium content of the leaching solution was exchangeable cadmium. -1 The residue from the previous step was extracted with NaAc solution. The cadmium in the extract was carbonate-bound cadmium. -1NH2OH·HCl 25% HAc solution was used for extraction, and cadmium bound to iron and manganese oxides was extracted in a water bath. -1 HNO3 and 5mL 30% H2O2 were used to extract the organically bound and sulfide-bound cadmium, and aqua regia was used to digest and determine the residual cadmium content.

[0125] 2 Experimental results

[0126] (1) Results of rapeseed fresh weight test: Compared with the control, the fresh weight of rapeseed increased by 38.4% and 25.7% when Pseudomonas aeruginosa was added alone or when DL-alanine was used in combination with Pseudomonas aeruginosa, respectively. There was no significant difference in the fresh weight of the roots among the treatments.

[0127] (2) Rapeseed selenium content test results: Figure 5 As shown in C and 6D, compared with the control, the selenium content in the aerial part of rapeseed increased by 32.3% and 55.8% under the conditions of adding Pseudomonas aeruginosa alone and DL-alanine combined with Pseudomonas aeruginosa, respectively. There was no significant difference in the Se content in the roots among the treatments.

[0128] (3) Results of cadmium content test in rapeseed: Figure 5 As shown in E and 5F, compared with the control, the Cd content in the aerial part of rapeseed was reduced by 47.1% and 66.3% under the conditions of adding Pseudomonas aeruginosa alone and DL-alanine combined with Pseudomonas aeruginosa, respectively. Compared with the control, the Cd content in the root of rapeseed was significantly reduced by 67.9% when DL-alanine and Pseudomonas aeruginosa were combined.

[0129] (4) Analysis results of soil selenium forms:

[0130] The results of selenium speciation are as follows Figure 6 As shown in Figure 2. For soil selenium binding forms, water-soluble selenium and exchangeable selenium are selenium forms that are easily absorbed and utilized by plants and are often considered to be effective selenium. Iron-manganese oxides, organic matter-bound forms, and residual forms are considered to be stable and not easily directly absorbed and utilized by plants.

[0131] Compared with the control, when Pseudomonas aeruginosa was used in combination with DL-alanine, the effective selenium content (the sum of water-soluble selenium and exchangeable selenium) was significantly increased by 12.4%, among which the exchangeable selenium was significantly increased by 13% ( Figure 6 B / C).

[0132] The above results show that under the action of Pseudomonas aeruginosa and DL-alanine, the transformation of soil selenium forms is manifested as an increase in the content of available selenium.

[0133] (5) Results of soil cadmium forms:

[0134] The results are as follows Figure 7As shown. Exchangeable Cd is a form that can be directly absorbed and utilized by plants, and organically bound Cd is a form that cannot be directly utilized by plants. Compared with the control, when Pseudomonas aeruginosa was used in combination with DL-alanine, the content of exchangeable Cd in the rhizosphere soil of rapeseed was significantly reduced by 10%. When Pseudomonas aeruginosa was used in combination with DL-alanine, the organically bound Cd increased significantly by 98.9% compared with the control.

[0135] The above results indicate that the combination of Pseudomonas aeruginosa and DL-alanine reduces the effectiveness of soil Cd, which is consistent with the result that the Cd content in plants decreased when Pseudomonas aeruginosa was used in combination with DL-alanine.

[0136] 3 Experimental conclusions

[0137] When DL-alanine and Pseudomonas aeruginosa were used together, it promoted the enrichment of selenium in crops and reduced cadmium, and increased the available selenium in the soil, while reducing the available cadmium in the soil. This result also echoes the result that when Pseudomonas aeruginosa and DL-alanine were used together, the Se content in plants increased and the Cd content decreased.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for promoting colonization of Pseudomonas aeruginosa, characterized in that: The method comprises: firstly adding no less than 10 μmol·L -1 The concentration of DL-alanine was used to treat Pseudomonas aeruginosa, and then the treated Pseudomonas aeruginosa was colonized in the roots of the plants.

2. The method for promoting colonization of Pseudomonas aeruginosa according to claim 1, characterized in that: The concentration of DL-alanine is not less than 100 μmol·L -1 .

3. The method for promoting colonization of Pseudomonas aeruginosa according to claim 1 or 2, characterized in that: The viable count of Pseudomonas aeruginosa is not less than 10 7 CFU·mL -1 .

4. The method for promoting colonization of Pseudomonas aeruginosa according to claim 1 or 2, characterized in that: The culture medium is LB liquid culture medium or LB solid culture medium, and the components of the LB liquid culture medium include: peptone 10g·L -1 , yeast extract 5g·L -1 and NaCl 10 g·L -1 The components of the LB solid culture medium are 2% agar added to the LB liquid culture medium by volume.

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