A Burkholderia gladiolus HM-72 and its application

Abstract

The present invention discloses a gladiolus Burkholderia (Burkholderia gladioli) HM-72 and its application, which has been deposited in China Center for Type Culture Collection on July 13, 2023, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCCNO: M20231291. The strain has strong resistance to Pb stress, has partial phosphate dissolution, and the ability to produce siderophores. After applying the strain in Pb-contaminated soil, the mobility of Pb can be increased, the removal of Pb in the soil is accelerated and accumulated in the above-ground parts of enriched plants, to achieve the problem of repairing soil lead pollution, while the bacteria can inhibit the growth of pathogenic fungi, and its growth-promoting antibacterial properties can also provide favorable conditions for the growth of enriched plants in high-lead environments, promote its biomass accumulation, and have application prospects in environmental remediation of heavy metal pollution.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Burkholderia gladiolus HM-72 isolated from tartary buckwheat and its application in heavy metal pollution treatment and plant growth promotion. Background Art

[0002] 1. Current Status and Hazards of Heavy Metal Lead (Pb) Pollution

[0003] Lead (Pb) is a highly toxic heavy metal pollutant found in nature. Although lead is present in relatively low concentrations in the Earth's crust, industrialization and human activities have significantly altered its distribution and concentration in soils. Lead is widely released through smelting, the burning of leaded gasoline, and the land application of wastes such as sewage sludge and fertilizers, leading to a continuous increase in lead levels in soils and water bodies. Due to the persistence and diverse sources of lead pollution, this pollution has caused serious environmental damage worldwide.

[0004] Lead's prominent characteristics in the environment are its high chemical stability and strong biological toxicity, making it difficult to remove through natural degradation. Lead contamination not only degrades soil quality but also accumulates through the food chain, causing widespread and profound toxic effects on organisms, including their morphology, metabolism, physiology, and biochemical processes. It poses a significant threat to plant, microbial, and human health. The U.S. Environmental Protection Agency (EPA) has designated lead as the second most hazardous environmental substance after arsenic, making it a priority pollutant requiring urgent global control.

[0005] 2. Current Status and Challenges of Remediation Methods for Heavy Metal Pollution

[0006] Traditional methods for remediating heavy metal pollution primarily include physical and chemical methods, such as soil excavation, solidification and stabilization, and chemical precipitation. While these methods can reduce pollutant concentrations in the short term, they are often costly, energy-intensive, and prone to soil deterioration and secondary pollution, limiting their widespread practical application. Consequently, bioremediation, as a low-cost, eco-friendly, and sustainable alternative, has attracted widespread attention in recent years in both scientific research and industry.

[0007] Bioremediation leverages the metabolic activity of organisms, such as microorganisms or plants, to fix or convert heavy metals into less toxic and less mobile forms through various mechanisms, including biosorption, complexation, bioaccumulation, bioprecipitation, and biotransformation, thereby reducing their bioavailability in the soil. Compared to physical and chemical methods, bioremediation offers advantages such as protecting soil structure, reducing secondary pollution, and being relatively low-cost. It is a key area of ​​future development in the field of heavy metal pollution remediation.

[0008] 3. Application of plant endophytes in heavy metal pollution remediation

[0009] Plant endophytes are microorganisms that reside within plant tissues, including bacteria, actinomycetes, and fungi. Through their symbiotic relationship with their host plants, endophytes develop a variety of beneficial physiological functions, such as promoting plant growth, enhancing heavy metal tolerance, inhibiting pathogens, and improving nutrient absorption efficiency. In recent years, the application value of endophyte-based biological agents in plant heavy metal tolerance and remediation has gradually attracted attention. Plant endophytes can bind to heavy metal ions through their metabolites, forming complexes or bioprecipitates, reducing the bioavailability of heavy metals and thus alleviating heavy metal toxicity in plants. Furthermore, the growth-promoting effects of endophytes can increase plant biomass and accelerate the remediation of contaminated soils.

[0010] 4. Potential of Tartary Buckwheat and Its Endophytes in the Remediation of Heavy Metal Pollution

[0011] Tartary buckwheat (Fagopyrum tataricum L.Gaertn.), an annual herb, exhibits remarkable heavy metal tolerance. Its short growth cycle, low nutrient requirements, high biomass, and strong adaptability have been shown to exhibit good tolerance in a variety of heavy metal-contaminated environments. Tartary buckwheat is not only rich in protein, fatty acids, and trace elements, but also considered an important candidate plant for bioremediation of heavy metal pollution due to its significant antioxidant and bioaccumulation abilities. However, in highly polluted environments, the growth of tartary buckwheat is subject to multiple limitations, including insufficient soil nutrients, heavy metal toxicity, and pathogenic infection, resulting in its inability to fully realize its remediation effects. Therefore, in order to improve the remediation efficiency of tartary buckwheat, there is an urgent need to identify auxiliary microbial resources that can assist plant growth and decontamination in high-heavy metal environments. Summary of the Invention

[0012] The invention aims to provide a lead-tolerant Burkholderia gladiolus strain, which has an inhibitory effect on plant pathogens and can be applied to environmental remediation of heavy metal pollution.

[0013] In order to achieve the above-mentioned purpose, the present invention provides a Burkholderia gladioli HM-72, which was deposited in the China Center for Type Culture Collection on July 13, 2023, with the deposit address being Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC NO: M20231291.

[0014] The present invention also provides a heavy metal soil conditioner comprising the aforementioned Burkholderia gladiolus HM-72.

[0015] Preferably, the soil conditioner is a fermentation broth of HM-72, and the preparation method of the fermentation broth is as follows: HM-72 is activated and inoculated into a beef extract peptone liquid culture medium, and the fermentation broth is obtained when OD600=1.0.

[0016] The gladiolus Burkholderia HM-72 or soil conditioner provided by the present invention can be used in any of the following, including:

[0017] (1) Improve the heavy metal tolerance of plants; (2) Increase the mobility of heavy metals in soil; (3) Repair heavy metal pollution in soil; (4) Inhibit the growth of pathogens; (5) Promote the growth of plants in heavy metal contaminated soil.

[0018] Preferably, the heavy metal in the above application is lead (Pb), and the pathogens include tomato early blight (Afternaria solani (Ell. et Mart). Jones et Grout), cucumber late blight (Phytophthorainfestans), and watermelon wilt (Fusarium oxysporum f.sp. Hiveum (EFSmith) Wollen.).

[0019] The present invention has the following advantages:

[0020] The strain HM-72, which was applied for in this application, was derived from the rhizosphere soil of lead-contaminated tartary buckwheat. It possesses multiple biological functional properties, including high lead tolerance, antibacterial activity, phosphate solubilization, and siderophore production. Studies have shown that this strain can enhance the mobility of lead in lead-contaminated soil, promote its absorption and accumulation in accumulating plants, and ultimately achieve effective lead removal from the contaminated soil.

[0021] After applying this strain, lead contamination in soil can be remediated through the following mechanisms:

[0022] Improve lead mobility: This strain can change the chemical form of lead in the soil through its metabolites, thereby enhancing the lead's mobility and making it easier for plants to absorb and utilize it.

[0023] Synergistic repair: This strain forms a synergistic relationship with enrichment plants such as buckwheat, which can not only enhance the plant's ability to accumulate lead, but also reduce the invasion of pathogens on plants through its antibacterial properties, ensuring the healthy growth of plants in high-lead environments.

[0024] Growth-promoting effect: The growth-promoting properties of the strain can provide additional growth support for enriched plants, promote the accumulation of plant biomass, and accelerate the purification of contaminated soil.

[0025] The gladiolus Burkholderia HM-72 provided by the present invention can improve the stress resistance of tartary buckwheat in high-lead contaminated soil, increase the biomass of tartary buckwheat, and has application potential in the field of tartary buckwheat cultivation.

[0026] The HM-72 provided by the present invention has antagonistic effects on tomato early blight pathogen (Afternaria solani (Ell. et Mart). Joneset Grout), cucumber late blight pathogen (Phytophthora infestans), and watermelon wilt pathogen (Fusarium oxysporum f. sp. Hiveum (EF Smith) Wollen.), and also has application prospects in plant disease prevention and control.

[0027] The strain provided by the invention can be used as a biological preparation material for herbaceous Solanaceae plants, and has a good market prospect whether for developing new biological control agents or growth regulators.

[0028] In summary, the application of this strain has significant innovation and practical value, providing an eco-friendly and sustainable solution for the bioremediation of heavy metal-contaminated soils. Using this strain not only effectively improves the remediation efficiency of lead-contaminated soils, but also supports the healthy growth of enrichment plants in the harsh environment of heavy metal pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the results of the antibacterial experiment of the strain HM-72 in the present invention.

[0030] Figure 2 The plate shows the results of the phosphate solubilization experiment of the strain HM-72 in the present invention.

[0031] Figure 3 These are the siderophore production experimental results of the strain HM-72 of the present invention.

[0032] Figure 4 This is the phylogenetic tree constructed based on the 16s rRNA gene of strain HM-72 in the present invention.

[0033] Figure 5 The strain HM-72 of the present invention is in the presence of Pb 2+ and Pb-free 2+ SEM results under treatment.

[0034] Figure 6 This is the EDS energy spectrum scanning result of the strain HM-72 in the present invention.

[0035] Figure 7 The strain HM-72 of the present invention is in the presence of Pb 2+ and Pb-free 2+TEM results under treatment.

[0036] Figure 8 These are the metabolite detection and analysis results of the strain HM-72 in the present invention.

[0037] Figure 9 The strain HM-72 in the present invention is 2+ PCA analysis results of metabolites under treatment.

[0038] Figure 10 This is the result of metabolic pathway enrichment analysis of strain HM-72 in the present invention.

[0039] Figure 11 The strain HM-72 of the present invention is effective against Pb 2+ The effect of potted buckwheat on the fruiting process. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0041] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0042] Experimental Example 1 Isolation and identification of strains

[0043] 1.1 Strain Source

[0044] Buckwheat seedlings were randomly collected from potted buckwheat in high-lead contaminated soil as samples, placed in sterilized sealed plastic bags, labeled, and taken back to the laboratory for storage.

[0045] 1.2 Isolation of endophytic bacteria

[0046] Buckwheat seedlings grown in lead-contaminated soil were used as the material. The soil was removed from the seedlings, rinsed with clean water, and then immersed in 75% ethanol for 30 seconds. The surface was disinfected with 5% NaClO solution for 5 minutes and then rinsed three times with sterile water. The cut surface that came into contact with the disinfectant was cut off, and a 10 mm stem segment of the explant was cut and crushed in a pre-sterilized mortar. Endophytic buckwheat bacteria were isolated on the surface of beef extract peptone medium by smearing plant tissue blocks with tweezers and applying tissue fluid. The plates were cultured in a 30°C incubator. After obvious bacterial colonies grew, single colonies were picked and purified based on their apparent characteristics such as colony size, morphology, and color. A total of 81 single strains were obtained.

[0047] Pb of experimental example 2 strain 2+ Tolerance test

[0048] The 81 isolated strains were inoculated into 7 Pb 2+ In beef extract peptone agar medium with increasing concentration gradient, the strains were 2+ The maximum tolerance is used to screen the strains for subsequent tests. 2+ Prepared from lead nitrate reagent and adjusted to pH 6.5, 7 kinds of Pb 2+ The concentrations were 1.0 mM, 2.0 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, and 5.0 mM, respectively. The maximum Pb tolerance of each strain is shown in Table 1.

[0049] Table 1 Determination of the maximum tolerance of endophytic bacteria of buckwheat to Pb

[0050]

[0051]

[0052] As shown in Table 1, among the 81 strains, 23 strains were sensitive to Pb 2+ The tolerance capacity exceeded 4.0mM, accounting for 28.4%, and there were 12 bacterial strains that were resistant to Pb 2+ The tolerance was higher than 4.5 mM, accounting for 14.8% of the total, but all strains were 2+ When the concentration was 5mM, the growth stopped, indicating that 5mM Pb 2+ This is the lethal concentration of all endophytes isolated from buckwheat.

[0053] Determination of antibacterial activity of experimental example 3 strains

[0054] To investigate the inhibitory activity of the isolated strains against pathogens, the filter paper plate standoff assay was used to determine the antagonistic activity of 81 isolates against the indicator pathogens Afternaria solani (Ell. et Mart). Joneset Grout, Phytophthora infestans, and Fusarium oxysporum f.sp. Hiveum (EF Smith) Wollen. The inhibition zone diameter (HD) and colony diameter (CD) were measured, and the HD / CD ratio was calculated to identify strains with inhibitory activity. Table 2 shows strain 1 against Afternaria solani (Ell. et Mart). Joneset Grout, strain 2 against Phytophthora infestans, and strain 3 against Fusarium oxysporum f.sp. Hiveum (EF Smith) Wollen. '++' indicates that the HD / CD value is between 2-3; '+' indicates that the HD / CD value is between 1-2; and '-' indicates that the HD / CD value is less than 1 or there is no antagonistic ability.

[0055] Table 2 Antibacterial ability of endophytic bacteria in buckwheat

[0056]

[0057]

[0058] As shown in Table 2, 30 of the 81 strains had antagonistic effects against tomato early blight, of which 8 strains showed high resistance; 39 strains had antagonistic effects against cucumber late blight, of which 12 strains showed high resistance; 37 strains had antagonistic effects against watermelon wilt, of which 10 strains showed high resistance; 39.51% of the strains could simultaneously inhibit two pathogens, and 14.81% of the strains could simultaneously inhibit three pathogens. The results of the antibacterial experiment of HM-72 are shown in Figure 1 As shown, plates 1, 2, and 3 from left to right in the figure are pathogens of tomato early blight, cucumber late blight, and watermelon wilt, respectively.

[0059] Experimental Example 4 Determination of Inorganic Phosphorus Degradation Ability of Strain

[0060] To test the phosphate-solubilizing ability of the selected strains, 81 strains were inoculated into Montkina inorganic phosphate medium (PVK) (specifically, the following composition: glucose 10.0 g, (NH₄)₂SO₄ 0.5 g, NaCl 0.3 g, MgSO₄·7H₂O 0.3 g, MnSO₄·H₂O 0.03 g, KCl 0.3 g, FeSO₄·7H₂O 0.03 g, Ca₃(PO₄)₄ 25 g, agar 15.0 g, pH adjusted to 7.0 ± 0.2). The medium was incubated at 28°C for 7 days, and the size of the inorganic phosphate dissolution zone was measured. The dissolution zone sizes of the different strains are shown in Table 3.

[0061] Table 3 Inorganic phosphorus degradation capacity of endophytic bacteria in buckwheat

[0062]

[0063] Note: nd stands for not detected, indicating that the activity was not detected.

[0064] As shown in Table 3, 34 of the 81 strains had the ability to degrade inorganic phosphorus, accounting for 41.98% of the total strains tested. Among them, strain HM-45 had the strongest phosphorus solubilization ability, with a dissolution zone diameter of 2.02 cm. Strain HM-72 had a phosphorus solubilization ability second only to HM-45, and the plate results of its phosphorus solubilization experiment are shown in Figure 3. Figure 2 shown.

[0065] Experimental Example 5 Determination of the ability of strains to produce siderophores

[0066] The screened strains were inoculated onto a siderophore-producing qualitative culture medium (Chrome azurol S, CAS) (CAS culture medium formula: mg / L, 60.5 mg of chrome azurol S (CAS), 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA), 2.645 mg of ferric chloride hexahydrate, 295.25 mg of sodium dihydrogen phosphate dihydrate, 1213.5 mg of disodium hydrogen phosphate dodecahydrate, 125 mg of ammonium chloride, 37.5 mg of potassium dihydrogen phosphate, 62.5 mg of sodium chloride, 9000 mg of agar, pH 6.8±0.1). The ability of the strain to produce siderophores and the strength of the ability were determined based on whether the color of the plate changed from cyan to yellow and the size of the hydrolysis zone. The siderophore production ability was shown in Table 4.

[0067] Table 4 Qualitative ability determination of siderophore production

[0068]

[0069] Note: nd stands for not detected, indicating that the capability was not detected.

[0070] As can be seen from Table 4, in the siderophore production qualitative experiment, there are 18 strains that can produce siderophores, accounting for 22.22% of the total experimental strains. Among them, the strain with the largest siderophore hydrolysis zone is HM-72, with a hydrolysis zone diameter of 1.97 cm. The siderophore production experimental results of HM-72 are shown in Figure 3 .

[0071] Based on the qualitative siderophore production results of the 81 strains described above, quantitative siderophore concentration determination was performed on 18 positive strains. Equal volumes of bacterial culture supernatant were mixed with CAS assay solution and incubated in the dark for 1 h. The absorbance (As) was measured at 680 nm. Simultaneously, the absorbance (Ar) of a blank culture medium reacting with an equal volume of CAS assay solution (CAS assay solution mg / L, CAS 60, FeCl₃·6H₂O₂.7, HDTMA 73) was measured. The siderophore production capacity of the strains was analyzed using the As / Ar ratio. As / Ar ranges from 0 to 1, with a "+" added for every 0.2 decrease. Bacteria with higher siderophore production have an As / Ar ratio below 0.5. Siderophore production is expressed in siderophore activity units (SU), calculated using the formula SU = [(Ar - As) / Ar] × 100%. The results of the capacity determination are shown in Table 5.

[0072] Table 5 Determination of the quantitative ability of siderophore production

[0073]

[0074] Based on the above results, it can be seen that among the 81 isolated strains, the strain that meets the characteristics of Pb stress resistance, strong antibacterial, phosphate solubility and siderophore production is HM-72. Therefore, the strain HM-72 was biologically identified and selected as the target strain for potted verification test.

[0075] Experimental Example 6: Bioinformatics Analysis of HM-72

[0076] Bacterial DNA was extracted using a bacterial genomic DNA extraction kit, and the quality and concentration of the DNA were tested. After passing the test, it was diluted 10 times and used as a template for PCR reaction to amplify 16s rDNA. Universal primers 27F and 1492R were used as amplification primers for amplification. The amplification program was set as follows: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 1.5 minutes, and 72°C final extension for 10 minutes. The three intermediate steps were set for 30 cycles. The amplified product was recovered and sent to a biological company for sequencing. The obtained sequence is detailed in the sequence table. The NCBI gene accession number of its 16s rRNA is OR083585. Based on the 16srRNA gene sequence obtained by sequencing, the phylogenetic tree of the strain was constructed using MEGA11 software and the neighbor-joining method. The results are shown in Figure 4 shown.

[0077] Based on phylogenetic analysis of the 16S rRNA gene, it was found that strain HM-72 had a sequence similarity of 93% with the type strain NBRC 13700 of Burkholderia gladioli, and clustered on the same branch, supporting its classification as Burkholderia gladioli. Therefore, combined with the clustering results of the phylogenetic tree and sequence similarity analysis, strain HM-72 can be preliminarily identified as a member of Burkholderia gladioli. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on July 13, 2023, at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M20231291.

[0078] Experimental Example 7HM-72 Characterization Imaging

[0079] According to Pb 2+ The maximum lethal concentration for HM-72 (4.5mM) and Pb-free 2+ Scanning electron microscope (SEM) samples were prepared for each treatment. The SEM results are shown in Figure 5 As shown, Figure 5 a in the equation is Pb-free 2+ The processed electron microscopy results, Figure 5 b in the equation is 4.5 mM Pb 2+ It can be seen that HM-72 has a high 2+ During treatment, the strain had a complete morphology, showing a rod or rod shape, with an obvious dent in the middle, which may be the process of binary fission of the cells during proliferation. In addition, the cells were aggregated or distributed individually, while at 4.5 mMPb 2+ After treatment, the bacteria are incomplete or completely broken, having basically lost their morphological characteristics and are unable to grow normally and perform biological activities.

[0080] When taking the SEM image, we also used the backscattering + secondary electron mode to randomly select four points in the imaging and perform EDS energy spectrum scanning. Some of the results are shown in Figure 6 As shown, Figure 6 a is the all-element map obtained by SEM-EDS scanning of the sample under lead-free treatment, b is the relative content table of each element obtained by SEM-EDS scanning of the sample under lead-free treatment, c is the all-element map obtained by SEM-EDS scanning of the sample under 4.5mM lead treatment, d is the relative content table of each element obtained by SEM-EDS scanning of the sample under 4.5mM lead treatment. It can be seen that Pb-free 2+ The scan results of the treatment were between 0% and 2.53% Pb, and at 4.5 mM Pb2+ After treatment, the energy spectrum results showed that the Pb content was as high as 54.84%-66.68%, which shows that HM-72 has a high tolerance and absorption capacity for Pb.

[0081] At the same time, the 4.5mM and Pb-free 2+ The treated HM-72 were imaged by transmission electron microscope (TEM), and the results are shown in Figure 7 As shown, Figure 7 a in the figure is the TEM electron microscope result of lead-free treatment. Figure 7 The b in the figure is the TEM result of 4.5mM lead treatment. It can be seen that HM-72 2+ Under the treatment condition, the cell structure is intact, the appearance is plump, the contents are full, and the whole cell imaging shows dark color with no transparent area. 2+ After treatment, the cell contents were almost completely lost and the entire cell image was completely transparent, indicating that 4.5 mM lead posed a serious threat to the growth of the strain, causing cell osmotic pressure imbalance, cell rupture and cell inactivation.

[0082] Experimental Example 8 Identification of metabolites of HM-72

[0083] After activation, HM-72 was inoculated into beef extract peptone liquid medium and cultured for 24 h as seed solution. OD600 was adjusted to 0.8, and 1 mL of the adjusted bacterial solution was inoculated into new 150 mL beef extract peptone liquid medium (CK group) and 4.5 mM Pb was added. 2+ The culture medium was shaken in 150 mL beef extract peptone liquid medium (Pb group) for 24 h, and then the fermentation broth was collected in a sterile centrifuge tube and handed over to the company for metabolomics determination.

[0084] The results of HM-72 metabolite detection and analysis are shown in Figure 8 As shown, it can be seen that the most abundant substances in the metabolites are carboxylic acids and their derivatives, accounting for 22.0%, followed by fatty acids 19.7%, steroids and steroid derivatives, organic oxygen compounds and benzene and substituted derivatives each accounting for 6.3%.

[0085] Among them, PCA analysis was performed on the metabolites of CK group and Pb group, and the results are shown in Figure 9 The results showed that there were certain differences between the samples of CK group and Pb group, indicating that 4.5mM Pb 2+ The metabolism of strain HM-72 was significantly affected, which may change the normal metabolism of HM-72. At the same time, the possible metabolic pathways were enriched and analyzed. The results are shown in Figure 10As shown in the figure, the results show that most metabolites are concentrated in the Metabolism category, with a small number concentrated in the Environmental Information Processing category. The top five KEGG pathways are toluene degradation, arachidonic acid metabolism, ABC transport, caprolactam degradation, and pentose phosphate pathway.

[0086] Experimental Example 9 Potted Plant Experiment Verification

[0087] After activation, HM-72 was inoculated into beef extract peptone liquid culture medium (culture medium formula: g / L, peptone 10, NaCl 5, beef extract 3, pH = 7.0 ± 0.2), and cultured in a constant temperature shaker at 30°C and 180 r / min. After culture for 24 hours, it was used as a seed liquid, and the OD600 was adjusted to 0.8. 1 mL of the adjusted bacterial liquid was aspirated and inoculated into new 150 mL of beef extract peptone liquid culture medium, and then shaken and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 hours. When the bacterial liquid was cultured to OD600 = 1.0, the fermentation bacterial liquid was obtained for use.

[0088] Select buckwheat seeds with full grains, disinfect their surfaces with 2% sodium hypochlorite solution, and place them in a constant temperature and humidity incubator for germination. When the buckwheat embryos grow to 6-8 cm, select seedlings with consistent growth and plant them in flower pots, three plants per pot, and the soil weight in each flower pot is 1.2 kg. The soil was collected from the purple soil in Jiangzhou Town, Huili City, Liangshan Prefecture, Sichuan Province. The following experimental treatments were set up: CK: no treatment was added; T1: only HM-72 bacterial solution was added; T2: only heavy metal Pb was added 2+ 1.2g / kg; T3: heavy metal Pb is added at the same time 2+ 1.2g / kg and HM-72 bacterial solution, including heavy metal Pb 2+ Prepared with lead nitrate (2g / kg lead nitrate), and control Pb according to soil weight 2+ The addition amount was , and each treatment was repeated three times. The bacterial solution of strain HM-72 was inoculated into the buckwheat pot by root irrigation, once every 7 days, 50 mL each time. In addition, in order to maintain the nutrient supply, all treatments added an equal amount of 50% Hoagland nutrient solution (nutrient solution formula: mg / L, calcium nitrate tetrahydrate 945, potassium nitrate 607, ammonium dihydrogen phosphate 115, magnesium sulfate heptahydrate 493, disodium ferrous ethylenediaminetetraacetic acid 20-40, boric acid 2.86, manganese sulfate tetrahydrate 2.13, zinc sulfate heptahydrate 0.22, copper sulfate pentahydrate 0.08, ammonium molybdate tetrahydrate 0.02) once every 7 days, 50 mL each time, for a total of 3 irrigations. After 60 days, the plant growth (such as plant height, root length, aboveground fresh and dry weight, root fresh and dry weight, and leaf area, etc.) was measured. The potted results are shown in Tables 6 and Figure 11 .

[0089] Table 6 Effects of strain HM-72 on the growth of tartary buckwheat

[0090]

[0091] Note: Different lowercase letters indicate significant differences among treatments

[0092] From Table 6 and Figure 11 It can be seen from the results of potted plant experiments that the phenotype and biomass of buckwheat under T1 treatment (HM-72) were not significantly different from those under CK, while those under T2 treatment (Pb 2+ All the parameters of tartary buckwheat under the treatment of Pb 1.2g / kg were significantly lower than those of CK and T1. Among them, plant height and root length decreased by 38.1% and 36.8% respectively, aboveground fresh and dry weight decreased by 59.0% and 55.6% respectively, and leaf area decreased by 20.2%. 2+ Plant traits under the 1.2g / kg (1.2g / kg) HM-72 treatment were significantly improved compared to T2. Plant height and root length increased by 27.2% and 29.4%, respectively, compared to CK. Aboveground fresh and dry weights increased by 45.3% and 42.0%, respectively, and leaf area increased by 7.5%. This suggests that the application of HM-72 significantly alleviated the symptoms of tartary buckwheat poisoning.

[0093] At the same time, the activity of strain HM-72 on Pb in buckwheat was also determined. 2+ The content and distribution of Pb in soil were analyzed by the following procedures: different parts of the dried buckwheat plant (roots and leaves) were ground and passed through a 100-mesh sieve. 0.1000 g of dry powder was weighed into a 50 mL conical flask. 10 mL of HNO3-HClO4 mixed acid (7:3, v / v) was added overnight. The next day, it was heated on a high-temperature graphite hot plate. After covering the bottle mouth with a small funnel, it was first digested at 200 ° C and then gradually increased to 300 ° C. During this period, the color of the digestion liquid was observed to change from dark brown to light green and then to colorless and transparent. If the digestion result is not ideal, H2O2 can be added to promote the reaction. Finally, the liquid was concentrated to about 2 mL. The conical flask was removed from the heating plate to cool. After that, all the digestion liquid was transferred to a 50 mL colorimetric tube, rinsed with ultrapure water and transferred together. Soil Pb 2+ The test digestion method is similar to that used for plant digestion. However, the soil digestion uses a ternary acid mixture of nitric acid, perchloric acid, and hydrofluoric acid. Furthermore, a Teflon crucible must be used instead of a glass Erlenmeyer flask, and the heating temperature must not exceed 300°C. The digestion solution is then analyzed using an inductively coupled plasma mass spectrometer (ICP-MS).

[0094] And the calculation formula of Pb mobility is:

[0095] Pb mobility rate (%) = lead content in the aboveground part of the plant / lead content in the root system of the plant × 100%

[0096] The results of Pb content in soil and buckwheat and Pb mobility are shown in Table 7.

[0097] As can be seen from Table 7, except for the significant difference in migration rate between CK and T1 treatments (HM-72), other indicators did not change. 2+ 1.2g / kg), in T3(Pb 2+ 1.2g / kg+HM-72) treatment, soil Pb 2+ Although the content did not decrease significantly, it still decreased significantly, by 5.30%. 2+ The contents of Pb in leaves and roots increased significantly. 2+ The contents increased by 393.69% and 77.81% respectively, which shows that buckwheat has a significant enrichment and absorption effect on heavy metals under the treatment of strain HM-72. Strain HM-72 can improve the bioavailability of heavy metals in soil and promote the transfer of heavy metals from soil to plants. This shows that the strain can be used for the preparation of heavy metal soil conditioners, for the absorption and reduction of heavy metals in soil, and for biological green restoration of soil ecology.

[0098] Table 7 Effect of strain HM-72 on Pb in tartary buckwheat 2+ Influence of content and distribution

[0099]

[0100] Note: Different lowercase letters indicate significant differences among treatments.

[0101] In summary, the present invention screens buckwheat endophytes and comprehensively evaluates their anti-plant pathogenic fungi properties, phosphate solubilization, and siderophore production abilities, selecting strain HM-72, which can inhibit plant pathogenic fungi and has phosphate solubilization and siderophore production abilities. Application of this strain in Pb-contaminated soil can increase the mobility of Pb, accelerate the removal of Pb from the soil, and accumulate it in the aboveground parts of plants that accumulate it, thereby remediating the problem of soil lead contamination. At the same time, the strain can inhibit the growth of pathogenic fungi, increase the plant height and biomass of buckwheat, improve the plant's tolerance to heavy metal environments, and promote plant growth in heavy metal environments. This growth-promoting and antibacterial property can also provide favorable conditions for the growth of plants that accumulate in high-lead environments, and has application prospects in environmental remediation of heavy metal pollution.

[0102] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A strain of Burkholderia gladiolus ( Burkholderia gladioli ) HM-72, characterized by, The strain was deposited in the China Center for Type Culture Collection on July 13, 2023, with the collection address at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the collection number is CCTCC NO: M20231291.

2. A heavy metal soil conditioner comprising the Burkholderia gladiolus HM-72 according to claim 1, wherein the soil conditioner is a fermentation broth of HM-72.

3. The soil conditioner according to claim 2, characterized in that The preparation method of the fermentation broth is as follows: the HM-72 according to claim 1 is activated and inoculated into a beef extract peptone liquid culture medium, and cultured until OD600=1.0 to obtain the fermentation broth.

4. Use of the Burkholderia gladiolus HM-72 according to claim 1 or the soil conditioner according to claim 2 in any of the following: (1) Improve the heavy metal tolerance of plants; (2) Improve the mobility of heavy metals in soil; (3) Remediation of soil heavy metal pollution; (4) Inhibit the growth of pathogens; (5) Promote the growth of plants in heavy metal contaminated soil; in, The heavy metal is lead Pb; the pathogen is tomato early blight ( Afternaria solani )、Cucumber late blight ( Phytophthora infestans )、Watermelon wilt pathogen ( Fusarium oxysporum f. sp.); The plant is buckwheat.

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