Burkholderia gladioli HM-72 and application thereof

Abstract

The invention discloses Burkholderia gladioli HM-72 and application thereof, the Burkholderia gladioli HM-72 is preserved in China Center for Type Culture Collection on July 13, 2023, the preservation address is Wuhan University, Wuhan, No. 299, eight road, Wuchang District, Wuhan, Hubei, and the preservation number is CCTCC NO: M20231291. The strain is high in Pb stress resistance, and has the capability of partially dissolving phosphorus and producing siderophores. After the strain is applied to the Pb-polluted soil, the migration rate of Pb can be improved, the removal of Pb in the soil is accelerated, the Pb is accumulated in the overground part of the enrichment plant, the problem of repairing the lead pollution of the soil is solved, meanwhile, the strain can inhibit the growth of pathogenic fungi, the growth promoting and bacteriostasis characteristics of the strain can provide favorable conditions for the growth of the enrichment plant in a high-lead environment, and the application prospect is wide. The biomass accumulation is promoted, and the method has an application prospect in heavy metal polluted environment restoration.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Burkholderia gladiolus HM-72 isolated from tartary buckwheat and application thereof 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 one of the most toxic heavy metal pollution sources in nature. Although the content of lead in the earth's crust is relatively low, industrialization and human activities have significantly changed its distribution and concentration in the soil. Lead is widely released in smelting, burning leaded gasoline, and land application of waste such as sewage sludge and fertilizers, resulting in a continuous increase in lead content in soil and water. Due to the persistence and diversity of lead pollution, this pollution has caused serious environmental damage worldwide.

[0004] Lead's prominent features in the environment are its high chemical stability and strong biological toxicity, making it difficult to remove through natural degradation. Lead pollution not only leads to a decline in soil quality, but also accumulates through the food chain, causing extensive and far-reaching toxic effects on organisms, including their morphology, metabolism, physiology and biochemical processes, and especially poses a major threat to plants, microorganisms and human health. Lead has been listed by the U.S. Environmental Protection Agency (EPA) as the second most hazardous environmental substance after arsenic, and has now become one of the priority pollutants that urgently need to be controlled worldwide.

[0005] 2. Current status and challenges of remediation of heavy metal pollution

[0006] Traditional methods for remediating heavy metal pollution mainly include physical and chemical means, such as soil excavation, solidification and stabilization, and chemical precipitation. Although these methods can reduce pollutant concentrations in the short term, they are usually costly, energy-intensive, and prone to soil quality deterioration and secondary pollution, which limits their practical application on a large scale. Therefore, in recent years, bioremediation has gradually attracted widespread attention in the scientific research and industrial fields as a low-cost, eco-friendly and sustainable alternative strategy.

[0007] Bioremediation uses the metabolic activity of organisms such as microorganisms or plants to fix or convert heavy metals into low-toxic and low-mobility forms through various mechanisms such as biosorption, complexation, bioaccumulation, bioprecipitation and biotransformation, thereby reducing their biological effectiveness in the soil. Compared with physical and chemical methods, bioremediation has the advantages of protecting soil structure, reducing secondary pollution and lower costs, and is an important development direction in the field of heavy metal pollution remediation in the future.

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

[0009] Plant endophytes refer to microorganisms that exist inside plant tissues, including bacteria, actinomycetes and fungi. Through a symbiotic relationship with host plants, plant endophytes have formed 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 biological agents based on plant endophytes in plant heavy metal tolerance and remediation has gradually attracted attention. Plant endophytes can combine with heavy metal ions through metabolites to form complexes or biological precipitation, reduce the bioavailability of heavy metals, and thus alleviate heavy metal toxicity in plants. In addition, the growth-promoting effect of endophytes can also increase plant biomass and accelerate the remediation of contaminated soil.

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

[0011] As an annual herb, buckwheat (Fagopyrum tataricum L.Gaertn.) has significant heavy metal tolerance. It has a short growth cycle, low nutrient requirements, large biomass, and strong adaptability. Studies have shown that it exhibits good tolerance in a variety of heavy metal pollution environments. Buckwheat is not only rich in protein, fatty acids, and trace elements, but also considered to be an important candidate plant for heavy metal pollution bioremediation materials due to its significant antioxidant and bioaccumulation abilities. However, in a high heavy metal pollution environment, the growth of buckwheat is subject to multiple restrictions such as insufficient soil nutrients, heavy metal toxicity, and pathogen infection, resulting in its restoration effect being unable to be fully exerted. Therefore, in order to improve the restoration efficiency of buckwheat, it is urgent to find auxiliary microbial resources that can assist plant growth and purify pollution in a high heavy metal environment. Summary of the invention

[0012] The invention aims to provide a gladiolus Burkholderia with lead tolerance, 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 strain of Burkholderia gladioli HM-72, which has been deposited in the China Center for Type Culture Collection on July 13, 2023, and the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M20231291.

[0014] The present invention also provides a heavy metal soil conditioner comprising the gladiolus Burkholderia 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 applied to 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 soil heavy metal pollution; (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 applied for this time originated from the rhizosphere soil of bitter buckwheat contaminated with lead. It has multiple biological functional characteristics such as high lead tolerance, antibacterial activity, phosphate solubilization and iron carrier production. Studies have shown that this strain can increase the mobility of lead in lead-contaminated soil, promote its absorption and accumulation in enriched plants, and ultimately achieve effective removal of lead in contaminated soil.

[0021] After applying this strain, lead pollution 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 metabolites, enhance the mobility of lead, and make it easier for plants to absorb and utilize it.

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

[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 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 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 (EFSmith) 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 fungi or growth regulators.

[0028] In summary, the application of this strain has significant innovation and practical value, and can provide an eco-friendly and sustainable solution for the bioremediation of heavy metal contaminated soil. The use of this strain can not only effectively improve the remediation efficiency of lead-contaminated soil, but also support the healthy growth of enriched plants in the harsh environment of heavy metal pollution. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 strain HM-72 in the present invention.

[0032] Figure 4 This is a 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 spectrum scanning result of 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 This is the metabolite detection and analysis result of strain HM-72 in the present invention.

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

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

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

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0041] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0042] Experimental Example 1 Isolation and identification of strains

[0043] 1.1 Strain source

[0044] Buckwheat seedlings were randomly collected from potted buckwheat grown 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] Using potted buckwheat in lead-contaminated soil as the material, the buckwheat seedlings were freed from soil, rinsed with clean water and immersed in 75% ethanol for 30 seconds, disinfected with 5% NaClO solution for 5 minutes, and then rinsed three times with sterile water. The cut surface in contact with the disinfectant was cut off, and the 10 mm stem segment of the explant was cut and crushed with a pre-sterilized mortar. The endophytic bacteria of buckwheat were isolated on the surface of beef extract peptone culture medium by smearing plant tissue blocks with tweezers and applying tissue fluid. The plates were cultured in an incubator at 30°C. After obvious bacterial colonies grew, single colonies were picked and purified according to the apparent characteristics such as colony size, morphology and color, and a total of 81 single strains were obtained.

[0047] Pb of strains in Experimental Example 2 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 capacity is used to screen strains for subsequent tests. 2+ Prepared from lead nitrate reagent and adjusted to pH 6.5, 7 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 tolerance of Pb 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 strains of bacteria that were resistant to Pb 2+ The tolerance capacity exceeded 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+ It is the lethal concentration of all endophytes isolated from buckwheat this time.

[0053] Experimental Example 3 Determination of antibacterial activity of strains

[0054] In order to investigate the inhibitory ability of isolated strains against pathogens, the antagonistic effect of 81 isolated strains on indicator bacteria of tomato early blight (Afternaria solani (Ell. et Mart). Joneset Grout), cucumber late blight (Phytophthora infestans), and watermelon wilt (Fusarium oxysporum f.sp. Hiveum (EFSmith) Wollen.) was determined by the filter paper plate confrontation method. The diameter of the inhibition zone (HD) and the colony diameter (CD) were determined, and the ratio of HD / CD was calculated to screen out strains with antibacterial effect, as shown in Table 2, where strain 1 is tomato early blight (Afternaria solani (Ell. et Mart). Joneset Grout), strain 2 is cucumber late blight (Phytophthora infestans), and strain 3 is watermelon wilt (Fusarium oxysporum f.sp. Hiveum (EFSmith) Wollen.). The '++' indicates that the HD / CD value is between 2-3; the '+' indicates that the HD / CD value is between 1-2; and the '-' indicates that the HD / CD value is less than 1 or has 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 on tomato early blight, of which 8 showed high resistance; 39 strains had antagonistic effects on cucumber late blight, of which 12 showed high resistance; 37 strains had antagonistic effects on watermelon wilt, of which 10 showed high resistance; strains that could simultaneously inhibit two pathogens accounted for 39.51% of the total, and strains that could simultaneously inhibit three pathogens accounted for 14.81% of the total. The antibacterial test results 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 the Inorganic Phosphorus Degradation Ability of the Bacteria

[0060] In order to test whether the selected strains have the ability to dissolve phosphate, the 81 selected strains were inoculated into the Montkina inorganic phosphate medium (PVK), the specific formula of which was: glucose 10.0 g, (NH 4 ) 2 SO 40.5g, NaCl 0.3g, MgSO 4 7H 2 00.3g, MnSO 4 ·H 2 O 0.03 g, KCl 0.3 g, FeSO 4 7H 2 O 0.03g, Ca 3 (PO 4 ) 2 5g, agar 15.0g, pH adjusted to 7.0±0.2), cultured at 28°C for 7 days, and the size of the inorganic phosphorus dissolution zone was measured. The sizes of the dissolution zones of different strains are shown in Table 3.

[0061] Table 3 Inorganic phosphorus degradation capacity of endophytic bacteria of 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 have the ability to degrade inorganic phosphorus, accounting for 41.98% of the total strains tested. Among them, strain HM-45 has the strongest phosphorus dissolving ability, with a dissolution circle diameter of 2.02 cm. Strain HM-72 has the strongest phosphorus dissolving ability after HM-45, and the plate results of its phosphorus dissolution 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 into a siderophore-producing qualitative culture medium (Chrome azurol S, CAS) (CAS culture medium formula: mg / L, chrome azurol S (CAS) 60.5, hexadecyltrimethylammonium bromide (HDTMA) 72.9, ferric chloride hexahydrate 2.645, sodium dihydrogen phosphate dihydrate 295.25, disodium hydrogen phosphate dodecahydrate 1213.5, ammonium chloride 125, potassium dihydrogen phosphate 37.5, sodium chloride 62.5, agar 9000, pH 6.8±0.1) plate. Whether the strain has the ability to produce siderophores and the strength of the ability is determined based on whether the color of the plate changes from cyan to yellow and the size of the hydrolysis circle. The siderophore-producing ability is shown in Table 4.

[0067] Table 4 Determination of the qualitative ability of producing siderophores

[0068]

[0069] Note: nd stands for not detected, indicating that the capability is 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 experimental results of the siderophore production of the above 81 strains, the quantitative concentration of siderophores was determined for 18 positive strains: an equal volume of bacterial culture supernatant was mixed with CAS detection solution, and the reaction was protected from light for 1 hour. The absorbance As was detected at a wavelength of 680 nm. At the same time, the absorbance Ar of the blank culture medium and the equal volume of CAS detection solution (CAS detection solution mg / L, CAS 60, FeCl3·6H2O2.7, HDTMA 73) was measured. The siderophore production capacity of the strain was analyzed by As / Ar. As / Ar was between 0-1, and a "+" was added for every 0.2 decrease. The As / Ar of bacteria with higher siderophore production capacity was less than 0.5. The siderophore activity unit (SU) was used to represent the siderophore production of the strain. The calculation formula SU = [(Ar-As) / Ar] × 100%, and the results of its capacity determination were shown in Table 5.

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

[0073]

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

[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 for 16s rDNA amplification. Universal primers 27F and 1492R were used as amplification primers for amplification. The amplification program was set as follows: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1.5min, 72℃ final extension for 10min, and 30 cycles for the three intermediate steps. 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 the phylogenetic analysis of the 16S rRNA gene, it can be seen that the sequence similarity between strain HM-72 and the model strain NBRC 13700 of Burkholderia gladioli reached 93%, and they were clustered on the same branch, supporting its attribution to Burkholderia gladioli. Therefore, combined with the clustering results of the phylogenetic tree and the 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. The storage address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the storage number is CCTCC NO: M20231291.

[0078] Experimental Example 7 HM-72 Characterization Imaging

[0079] According to Pb 2+ The maximum lethal concentration for HM-72 (4.5mM) and Pb-free 2+ The SEM samples were prepared for each treatment. The SEM results are shown in Figure 5 As shown, Figure 5 a in the formula is Pb-free 2+ The processed electron microscopy results, Figure 5 b in is 4.5 mM Pb 2+ It can be seen that HM-72 has a good 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 in proliferation. In addition, the cells were aggregated or distributed individually, while at 4.5mMPb 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, four points were randomly selected in the imaging using the backscattering + secondary electron mode for EDS 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 sample under lead-free treatment, b is the relative content table of each element obtained by SEM-EDS scanning sample under lead-free treatment, c is the all-element map obtained by SEM-EDS scanning sample under 4.5mM lead treatment, and d is the relative content table of each element obtained by SEM-EDS scanning sample under 4.5mM lead treatment. 2+ The scan results of the treatments 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%. This result 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 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+ In the case of treatment, the cell structure is intact, the appearance is full, the content is full, and the whole cell image appears dark 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 an imbalance in cell osmotic pressure, cell rupture and cell inactivation.

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

[0083] After activation, HM-72 was inoculated in beef extract peptone liquid medium and cultured for 24 h as seed solution. The OD600 was adjusted to 0.8, and 1 mL of the adjusted bacterial solution was inoculated in 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 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 Fig. 9 The results showed that there were some differences between the samples in the CK group and the Pb group, indicating that 4.5 mM Pb 2+ The metabolism of strain HM-72 was greatly affected, which may change the normal metabolism of HM-72. At the same time, the possible metabolic pathways were enriched and analyzed. Fig.10As shown in the figure, the results show that most of the metabolites are in the Metabolism category, and a small number are 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 culturing for 24 hours, it was used as 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 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 standby use.

[0088] Select buckwheat seeds with full grains, disinfect the surface with 2% sodium hypochlorite solution, and place them in a constant temperature and humidity incubator for germination. When the buckwheat embryo grows to 6-8cm, select seedlings with consistent growth and plant them in pots, three plants per pot, and the soil weight in each pot is 1.2kg. The soil was collected from the purple soil in Jiangzhou Town, Huili City, Liangshan Prefecture, Sichuan Province. Set up the following experimental treatments: CK: no treatment is added; T1: only HM-72 bacterial solution is added; T2: only heavy metal Pb is 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 amount of addition was repeated three times for each treatment. The bacterial solution of strain HM-72 was inoculated into the bitter buckwheat pot by root irrigation, once every 7 days, 50 mL each time. In addition, to maintain nutrient supply, all treatments added an equal amount of 50% Hogland nutrient solution (nutrient solution formula: mg / L, calcium nitrate tetrahydrate 945, potassium nitrate 607, ammonium dihydrogen phosphate 115, magnesium sulfate heptahydrate 493, ethylenediaminetetraacetic acid disodium iron 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 growth of the plants (such as plant height, root length, fresh and dry weight of the aboveground part, fresh and dry weight of the root, and leaf area, etc.) were measured. The potted results are shown in Tables 6 and Fig.11 .

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

[0090]

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

[0092] From Table 6 and Fig.11 It can be seen from the pot experiment verification results that compared with CK, there was no significant difference in the phenotype and biomass of bitter buckwheat under T1 treatment (HM-72), and there was no significant difference in the phenotype and biomass of bitter buckwheat under T2 treatment (Pb 2+ All parameters of bitter buckwheat under Pb 1.2 g / kg were significantly lower than those of CK and T1, among which plant height and root length were reduced by 38.1% and 36.8% respectively, aboveground fresh and dry weight were reduced by 59.0% and 55.6% respectively, and leaf area was reduced by 20.2%. 2+ The plant traits under the treatment of 1.2g / kg+HM-72) were significantly improved compared with T2, with plant height and root length increased by 27.2% and 29.4% respectively, aboveground fresh and dry weight increased by 45.3% and 42.0% respectively, and leaf area increased by 7.5%. This shows that the application of HM-72 significantly improved the poisoning phenomenon of bitter buckwheat.

[0093] At the same time, the activity of strain HM-72 on Pb 2+ The specific operation is as follows: different parts of the dried buckwheat plant (roots and leaves) are ground and passed through a 100-mesh sieve. 0.1000 g of dry powder is weighed into a 50 mL conical flask, and 10 mL of HNO is added. 3 -HClO 4 Mixed acid (7:3, v / v) overnight, and heat on a high-temperature graphite hot plate the next day. Cover the bottle mouth with a small funnel, first digest at 200℃, then gradually increase the temperature to 300℃. During this period, observe the color of the digestion solution change from dark brown to light green and then to colorless and transparent. If the digestion result is not ideal, add H 2 O 2 To promote the reaction, the liquid was concentrated to about 2 mL, the flask was removed from the heating plate to cool, and then all the digestion liquid was transferred to a 50 mL colorimetric tube, rinsed with ultrapure water and transferred together. 2+ The test digestion method is similar to plant digestion. It should be noted that the mixed acid used for soil digestion is a ternary mixed acid of nitric acid, perchloric acid and hydrofluoric acid, and a glass Erlenmeyer flask cannot be used for digestion. A polytetrafluoroethylene crucible must be used instead, and the heating temperature cannot exceed 300°C. The digestion solution is finally measured using an inductively coupled plasma mass spectrometer (ICP-MS).

[0094] And the Pb mobility calculation formula is:

[0095] Pb mobility % = Pb content in the aboveground part of the plant / Pb 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 buckwheat 2+ Effect of content and distribution

[0099]

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

[0101] In summary, the present invention screens tartary buckwheat endophytes, comprehensively evaluates the anti-plant pathogenic fungi characteristics, phosphate solubilization and iron carrier production capabilities of tartary buckwheat endophytes, and selects strain HM-72, which can inhibit plant pathogenic fungi and has phosphate solubilization and iron carrier production capabilities. 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, so as to repair the problem of soil lead pollution. At the same time, the bacteria can inhibit the growth of pathogenic fungi, and it can also increase the plant height and biomass of tartary buckwheat, improve the tolerance of plants in heavy metal environments, and promote the growth of plants in heavy metal environments. This growth-promoting and antibacterial property can also provide favorable conditions for the growth of enriched plants in high-lead environments, and has application prospects in environmental remediation of heavy metal pollution.

[0102] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A strain of Burkholderia gladioli HM-72, characterized in that: 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 as claimed in claim 1.

3. The soil conditioner according to claim 2, characterized in that The soil conditioner is the fermentation liquid of HM-72.

4. The soil conditioner according to claim 3, characterized in that The method for preparing the fermentation liquid is as follows: the HM-72 as claimed in claim 1 is activated and inoculated into a beef extract peptone liquid culture medium, and the fermentation liquid is obtained when OD600=1.

0.

5. Use of the gladiolus Burkholderia 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.

6. The use according to claim 5, characterized in that: The heavy metal is lead (Pb).

7. The use according to claim 5, characterized in that: The pathogens include tomato early blight (Afternaria solani (Ell. et Mart). Jones et Grout), cucumber late blight (Phytophthorainfestans), watermelon wilt (Fusarium oxysporum f.sp. Hiveum (E.F. Smith) Wollen.).

Images