Buchnera forestii with improved tea quality and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a Burkholderia gladioli with improved tea quality and an application thereof. The preservation number of the Burkholderia gladioli is CCTCC NO: M 20241144. The strain has high-efficiency activity of dissolving organic phosphorus and inorganic phosphorus, strong ability of producing IAA and iron carrier, certain inhibiting capacity on fungal pathogenic bacteria, and can improve tea bud density, increase the content of free amino acids in tea, reduce the content of tea polyphenols in tea and the phenol-ammonia ratio, so as to improve the quality of tea.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Burkholderia arboris with phosphorus solubilizing, growth-promoting and disease-preventing properties, and its application in improving tea quality. Background Technology

[0002] The development of microbial fertilizers utilizing the phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing capabilities of rhizosphere growth-promoting bacteria (PGPR) has been widely applied. PGPR possesses functions such as resisting soil-borne diseases, improving the soil environment, and enhancing crop yield and quality. Phosphorus is one of the macroelements required for plant growth. 20%–80% of phosphorus in the soil is organic phosphorus, which needs to be converted into inorganic phosphorus by soil microorganisms or soil enzymes before it can be absorbed by plants. However, inorganic phosphorus in the soil combines with elements such as calcium, magnesium, iron, and aluminum to form insoluble phosphates that are fixed in the soil, affecting plant absorption. Phosphorus-solubilizing microorganisms in the rhizosphere can convert insoluble phosphorus into soluble phosphorus through acidification, chelation, and exchange reactions. Phosphorus-solubilizing microorganisms dissolve inorganic phosphorus mainly by producing organic acids to acidify the surrounding environment, while they dissolve organic phosphorus by producing enzymes that undergo mineralization reactions with it. Common phosphorus-solubilizing microorganisms in soil include Bacillus, Pseudomonas, Enterobacter, Burkholderia, Penicillium, Aspergillus, and Streptomyces.

[0003] Free amino acids are important components of the freshness and aroma of tea infusion. The amino acid content of tea is highly correlated with tea quality. The phenol-amino acid ratio, which is the ratio of tea polyphenols to free amino acids, reflects the flavor quality of tea. For green tea, a lower phenol-amino acid ratio generally indicates better tea quality. This invention aims to provide a Burkholderia strain with phosphate-solubilizing, growth-promoting, and free amino acid-increasing effects, as well as a reduction in the phenol-amino acid ratio of tea. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Burkholderia arcoris DFP-24 with phosphate-solubilizing and growth-promoting properties and its application in improving tea quality.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Burkholderia arboris DFP-24, with accession number CCTCC NO: M20241144, has the following characteristics:

[0007] (1) It can dissolve both organic and inorganic phosphorus at the same time. The phosphorus dissolution rate of organic phosphorus is 14.67%, and the phosphorus dissolution rate of inorganic phosphorus is 9.01%.

[0008] (2) The amount of IAA produced reaches 5.52 mg / L, which can promote the budding of tea trees;

[0009] (3) Highly efficient iron-producing capacity;

[0010] (4) Inhibits the root rot pathogen Fusarium solanum;

[0011] (5) Increase the content of free amino acids in tea, reduce the content of tea polyphenols, reduce the ratio of phenols to amino acids in tea, and improve the quality of tea.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects:

[0013] While there are existing reports of Burkholderia being used in tea cultivation, such as increasing the polyphenol content of tea leaves (CN113151076A) and increasing the theanine content (CN110892836A), the Burkholderia forestica B.DFP-24 screened in this invention has a different effect on tea quality. Specifically, it increases the content of free amino acids in tea leaves, reduces the content of polyphenols, effectively reduces the phenol-to-amino acid ratio, and also increases the caffeine content, especially showing a significant effect on improving the quality of green tea. Attached Figure Description

[0014] Figure 1 This is a scanning electron microscope image of Burkholderia forestosa DFP-24.

[0015] Figure 2 This is a colony morphology diagram of Burkholderia viridissima DFP-24.

[0016] Figure 3 Phylogenetic tree of Burkholderia DFP-24 constructed based on 16S rDNA.

[0017] Figure 4 The results show the siderogenic capacity of Burkholderia viridissima DFP-24.

[0018] Figure 5 The results show the disease resistance of Burkholderia forestii DFP-24. The left image is the blank control, and the right image shows the confrontation culture results of Burkholderia forestii DFP-24 and Fusarium oxysporum. Detailed Implementation

[0019] Example 1: Screening, purification, and identification of strains

[0020] (1) Sampling: The sample was the rhizosphere soil of a tea garden in Hefeng County, Enshi, Hubei Province. The collected soil was placed in a sealed bag and quickly brought back to the laboratory for low-temperature storage.

[0021] (2) Isolation and Culture: After the soil sample was brought back to the laboratory, 10g of soil was weighed and poured into a triangular flask containing 90mL of sterile water and glass beads on a sterile workbench. The flask was placed in a shaker at 28℃ and 150rpm for 30min. After thorough shaking, it was allowed to stand for 5min. The supernatant was used as 10g of soil. -1 Soil dilution solution; for 10 -1 The soil dilution was serially diluted with sterile water to obtain a 10-fold dilution. -4 10 -5 10 -6 Three gradient dilutions.

[0022] (3) Colony purification: Take 0.1 mL of diluted bacterial solution and drop it onto a Monkina solid medium plate. Immediately spread it evenly with a sterile glass spreader to ensure that there is no water flow on the surface of the medium. Spread three plates for each dilution. After spreading evenly, invert the plates and incubate at 28°C. Observe and record the growth of the colonies after 48 hours. Select representative colonies with phosphate-solubilizing zones and pick out individual colonies with a sterile inoculation loop. At the same time, streak the colonies on LB solid medium according to aseptic operation requirements until purification.

[0023] Monkina (organophosphate) culture medium: 3.75 g / L calcium phytate, 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate heptahydrate, 0.03 g / L ferrous sulfate heptahydrate, 0.03 g / L manganese sulfate, 0.5 g / L yeast extract. Adjust the pH to 7.0-7.5. Add 18 g agar per liter of solid culture medium according to the ratio.

[0024] Monkina (inorganic phosphorus) medium: calcium phosphate 5g / L, glucose 10g / L, ammonium sulfate 0.5g / L, sodium chloride 0.3g / L, potassium chloride 0.3g / L, magnesium sulfate heptahydrate 0.3g / L, ferrous sulfate heptahydrate 0.03g / L, manganese sulfate 0.03g / L, yeast extract 0.5g / L. Adjust the pH to 7.0-7.5. Add 18g of agar per liter of solid medium according to the ratio.

[0025] (4) Appearance and physiological and biochemical characteristics

[0026] The purified strain was named DFP-24, and the electron microscopy results are as follows: Figure 1 As shown, the individual cells are rod-shaped, and the colony morphology is as follows. Figure 2As shown, on LB solid medium, the colonies are small, with a smooth, moist, slightly raised, and sticky surface; they are irregularly round in shape and pale yellow in color. The physiological and biochemical results of strain DFP-24 are shown in Table 1. The starch hydrolysis, VP test, and methyl red test results of DFZ24 were negative, while the nitrate test result was positive.

[0027] Table 1 Physiological and Biochemical Assays

[0028]

[0029] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0030] (5) Molecular biological identification

[0031] The 16S rDNA nucleotide sequence of strain DFP-24 was determined as shown in SEQ ID NO.1. Maximum homology comparison with sequences in databases such as GeneBank revealed that the 16S rDNA sequence of this strain shared 98.96% homology with the sequence of Burkholderia arboris. A phylogenetic tree constructed based on the 16S rDNA is shown below. Figure 3 As shown, strain DFP-24 clustered with Burkholderia arboris strain R-24201 in the same branch, so strain DFP-24 was identified as Burkholderia arboris.

[0032] Burkholderia arboris DFP-24 was deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2024, with accession number CCTCC NO: M 20241144, at Wuhan University, Wuhan, China.

[0033] Example 2: Determination of Phosphate-Solubilizing Ability of Burkholderia Floodirizum DFP-24

[0034] Phosphate-solubilizing activity assay: Organic and inorganic phosphorus liquid culture media were prepared separately and autoclaved at 121℃ for 20 min. Burkholderia forestica DFP-24 bacterial suspension was inoculated at a 1% ratio into the sterilized liquid culture media, with no inoculation as a control. Each treatment was repeated three times. After culturing on a shaker (28℃, 150 rpm) for 3 days, the soluble phosphorus content was measured. The cultured bacterial suspension was centrifuged at 8000 rpm for 10 min, and the supernatant was collected in a 50 mL colorimetric tube. The soluble phosphorus content was determined using the ammonium molybdate colorimetric method. The phosphorus solubilization rate was used to represent the phosphorus-solubilizing ability of the strain. As shown in Table 2, the soluble phosphorus content in the organic phosphorus liquid culture medium after treatment with strain DFP-24 was 146.6 mg / L, and the soluble phosphorus content in the inorganic phosphorus liquid culture medium was 90.13 mg / L.

[0035] The total phosphorus addition in the organic and inorganic phosphorus culture medium of Monkina is 1 g / L. The phosphorus dissolution rate is calculated according to the following formula:

[0036] Phosphorus solubility rate = (soluble phosphorus content of inoculated bacteria - soluble phosphorus content of control) / amount of added organic or inorganic phosphorus source × 100%.

[0037] As shown in Table 2, strain DFP24 had phosphorus solubility rates of 14.67% for organic phosphorus and 9.01% for inorganic phosphorus.

[0038] Table 2. Organic and inorganic phosphorus phosphate solubilization activities of strain DFP-24

[0039]

[0040] Example 3: Determination of IAA Production Capacity of Burkholderia forestii DFP-24

[0041] Preparation of IAA standard curve: IAA standard solutions of 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L were prepared with distilled water. These solutions were then mixed with Salkowski colorimetric reagent at a 1:1 volume ratio and allowed to stand at room temperature for 30 min in the dark. A mixture of distilled water and Salkowski colorimetric reagent was used as a blank control. The absorbance at 530 nm for each concentration was measured using a UV spectrophotometer. An IAA standard curve was plotted with IAA concentration on the x-axis and absorbance on the y-axis.

[0042] Burkholderia forestosa DFP-24 was inoculated at a 2% ratio into LB liquid medium supplemented with 100 mg / L L-tryptophan and cultured with shaking for 48 h. The fermentation broth was then centrifuged at 4000 rpm for 10 min, and 1 mL of the supernatant was collected. An equal volume of Salkowski colorimetric solution was added, and this process was repeated three times. After standing for 30 min, the absorbance at 530 nm was measured. Uninoculated LB liquid medium was used as a blank control. IAA yield was calculated using a standard curve. The DFP-24 strain produced 5.52 mg / L of IAA, which promoted plant growth.

[0043] Example 4: Determination of the siderophore-producing capacity of Burkholderia forestii DFP-24

[0044] CAS detection medium: Chromium azurite S (CAS) 60.5 mg / L, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg / L, ferric chloride hexahydrate 2.645 mg / L, sodium dihydrogen phosphate dihydrate 295.25 mg / L, disodium hydrogen phosphate dodecahydrate 1213.5 mg / L, ammonium chloride 125 mg / L, potassium dihydrogen phosphate 37.5 mg / L, sodium chloride 62.5 mg / L, agar 9000 mg / L, pH 6.8 ± 0.1.

[0045] Preliminary screening of siderogenic rhizosphere bacteria was performed using the CAS plate assay. Burkholderia forestii DFP-24 was streaked onto CAS detection plates and incubated at 28°C. The color change (from blue to orange) on the plate was used to determine whether the bacteria were siderogenic. Figure 4 As shown, Burkholderia forestii DFP-24 possesses the ability to produce siderophores. The ability of microorganisms to produce siderophores can increase the bioavailability of iron in the environment, thereby promoting plant growth. It can also control plant diseases by competing with pathogenic microorganisms for limited iron ions in the environment.

[0046] Example 5: Inhibitory effect of Burkholderia forestosa DFP-24 on fungal diseases

[0047] The inhibitory effect of Burkholderia forestii DFP-24 on fungal diseases was determined using a confrontation culture method. A 5mm mycelial cake was collected around a activated Fusarium solani colony and placed in the center of a PDA plate. Sterile filter paper discs were placed at two opposite points 2.15cm from the center of the PDA plate, and 7 μL of DFP-24 bacterial suspension was inoculated on each disc. A filter paper disc with an equal volume of sterile water served as a blank control. The plates were incubated at 28℃, and the inhibitory effect was observed. The results showed that Burkholderia forestii DFP-24 had a certain inhibitory effect on the root rot pathogen Fusarium solani. Figure 5 ).

[0048] Example 6: Effects of Burkholderia forestosa DFP-24 on tea quality

[0049] 1. Microbial fermentation

[0050] OD was measured after Burkholderia forestica DFP-24 was fermented with LB for 48 hours. 600 .

[0051] 2. Experimental Setup

[0052] ① Blank control (equal volume of water + sterile culture medium)

[0053] ②DFP-24

[0054] There are two treatments, each with three replicates. Each row of tea bushes is 1.5m wide and 10m long, totaling 15m. 2 For processing.

[0055] 3. Dosage: The bacterial solution concentration is OD 600 =1.7, dosage is 10mL / m 2 Dilute before use and apply as a root drench. Use once on March 6th, and then apply again 10 days later. Set the dosage for each plot to 15m. 2 Each process handles 3 cells.

[0056] One month after application, the density of tea buds was investigated. Four months later, tea leaves with one bud and two leaves were collected and their quality (tea polyphenols, caffeine, free amino acids, and phenol-amino acid ratio) was determined. The content of free amino acids in the tea leaves was continuously monitored in January, February, and April. The content of tea polyphenols was determined using the Folin-Ciocalteu method, referring to GB / T8313—2018; the total amount of free amino acids was determined using the ninhydrin colorimetric method, referring to GB / T8314—2013; caffeine was determined using ultraviolet spectrophotometry, referring to GB / T8312—2013; the phenol-amino acid ratio was calculated as tea polyphenols / amino acids.

[0057] As shown in Table 3, compared with the no-fertilization treatment, the application of Burkholderia virgaurea DFP-24 can increase the density of tea buds.

[0058] Table 3. Effects of Burkholderia simulans DFP-24 on tea bud density.

[0059]

[0060] As shown in Table 4, compared with the control, after 4 months of application of Burkholderia virgaurea DFP24, the free amino acid content of tea increased by 7.20%, the tea polyphenol content decreased by 12.14%, the tea phenol-amino acid ratio decreased by 19.06%, and the caffeine content increased by 13.08%, which has the effect of improving tea quality.

[0061] Table 4. Effects of Burkholderia filiis DFP-24 on tea quality

[0062]

[0063] As shown in Table 5, the application of Burkholderia virgaurea DFP24 at different times can increase the content of free amino acids in tea. After 1 month, 2 months and 4 months of application, the content of free amino acids increased by 3.15%, 12.26% and 7.20% respectively, indicating that the strain has a long-term effect of increasing the content of free amino acids in tea.

[0064] Table 5. Effects of Burkholderia virgaurea DFP-24 treatment for different durations on free amino acids.

[0065]

[0066]

Claims

1. Burkholderia forestosa ( Burkholderia arboris DFP-24, characterized in that, Its accession number is CCTCC NO: M 20241144.

2. A product containing Burkholderia forestica DFP-24 as described in claim 1.

3. The product according to claim 2, characterized in that, The products include microbial inoculants, soil conditioners, or microbial fertilizers.

4. The application of Burkholderia forestosa DFP-24 as described in claim 1 or the product as described in claim 2 in tea tree cultivation, characterized in that, Includes any of the following applications: I. Reduce the content of tea polyphenols; II. Reduce the phenol-to-amino acid ratio in tea; III. Increase caffeine content.

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