Burkholderia forest capable of improving tea quality and application of Burkholderia forest

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses burkholderia forest capable of improving tea quality and application of the burkholderia forest. The preservation number of the Burkholderia forest is CCTCC NO: M 20241144, and the Burkholderia forest has the activity of efficiently dissolving organic phosphorus and inorganic phosphorus, has strong IAA and siderophore producing capacity, has certain inhibition capacity on fungal pathogenic bacteria, and can improve the tea bud density, increase the free amino acid content of tea leaves, reduce the tea polyphenol content and the phenol-ammonia ratio of the tea leaves and improve the quality of the tea leaves. Therefore, tea quality is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Burkholderia arboris with the functions of dissolving phosphorus, promoting growth and preventing diseases, and application thereof in improving the quality of tea. Background Art

[0002] The development of microbial fertilizers using the phosphorus, potassium and nitrogen-fixing abilities of growth-promoting rhizobacteria (PGPR) has been widely used. Rhizobacteria have the functions of resisting soil-borne diseases, improving soil environment and improving crop products and quality. Phosphorus is one of the macronutrients required for plant growth. 20% to 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. Inorganic phosphorus in the soil will combine 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 rhizobacteria 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 organic phosphorus is dissolved by producing enzymes to undergo mineralization reactions with them. Common phosphate-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 soup. Tea amino acids are highly correlated with tea quality. The phenol-amino ratio is the ratio of tea polyphenols to free amino acids, which reflects the taste quality of tea. For green tea, the lower the phenol-amino ratio, the better the quality of tea. The present invention aims to provide a Burkholderia strain that has the functions of solubilizing phosphate, promoting growth, increasing the free amino acid content of tea leaves, and reducing the phenol-amino ratio. Summary of the invention

[0004] The invention aims to provide a Burkholderia arboris DFP-24 with the functions of solubilizing phosphate and promoting growth and application thereof in improving the quality of tea leaves.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0007] (1) It can dissolve both organic and inorganic phosphorus, with the dissolution rate of organic phosphorus being 14.67% and that of inorganic phosphorus being 9.01%;

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

[0009] (3) Efficient siderophore production capacity;

[0010] (4) Inhibit the root rot pathogen Fusarium solani;

[0011] (5) Increase the free amino acid content of tea, reduce the content of tea polyphenols, lower the phenol-ammonia ratio of tea, and improve the quality of tea.

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

[0013] Although there are reports in the prior art that Burkholderia is used for tea tree cultivation, such as increasing the tea polyphenol content of tea leaves (CN113151076A) and increasing theanine content (CN110892836A), the forest Burkholderia DFP-24 screened by the present invention has different effects on the quality of tea leaves, specifically increasing the free amino acid content of tea leaves, reducing the tea polyphenol content, effectively reducing the phenol-ammonia ratio, and increasing the caffeine content, especially having a significant effect on improving the quality of green tea. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 This is the colony morphology of Burkholderia forestia DFP-24.

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

[0017] Figure 4 These are the results of the siderophore production ability test of Burkholderia forestii DFP-24.

[0018] Figure 5 The results of the disease resistance test of Burkholderia solani DFP-24 are shown in the left figure. The left figure is the blank control, and the right figure is the confrontation culture result of Burkholderia solani DFP-24 and Fusarium solani. DETAILED DESCRIPTION

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

[0020] (1) Sampling: The samples were rhizosphere soil from tea gardens in Hefeng County, Enshi City, 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 is brought back to the laboratory, 10 g of soil is weighed and poured into a triangular flask containing 90 mL of sterile water and glass beads on a sterile workbench. The soil is shaken on a shaker at 28 °C and 150 rpm for 30 min. After sufficient shaking, the soil is allowed to stand for 5 min. The supernatant is used as the 10 -1 Diluted soil solution; 10 -1 The soil dilution was serially diluted with sterile water to obtain 10 -4 , 10 -5 , 10 -6 Three serial dilutions were made.

[0022] (3) Colony purification: Take 0.1 mL of the diluted bacterial solution and drop it onto a Montana solid culture medium plate. Immediately spread it evenly with a sterile glass spreading rod to ensure that there is no water flow on the surface of the culture medium. Spread three plates for each dilution. After spreading evenly, turn the plate upside down and culture it at a constant temperature of 28°C. After 48 hours, observe and record the growth of the colonies. Select representative colonies with phosphate circles, pick out single colonies with a sterile inoculation loop, and streak them on LB solid culture medium according to sterile operation requirements until purification.

[0023] Montana (organic phosphorus) medium: 3.75g / L calcium phytate, 10g / L glucose, 0.5g / L ammonium sulfate, 0.3g / L sodium chloride, 0.3g / L potassium chloride, 0.3g / L magnesium sulfate heptahydrate, 0.03g / L ferrous sulfate heptahydrate, 0.03g / L manganese sulfate, 0.5g / L yeast powder, adjust pH to 7.0-7.5, add 18g agar per liter of solid culture medium in proportion.

[0024] Montana (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 powder 0.5g / L, adjust pH to 7.0-7.5, add 18g agar per liter of solid culture medium in proportion.

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

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

[0027] Table 1 Physiological and biochemical assays

[0028]

[0029] Note: “+” means positive reaction; “-” means 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. The 16S rDNA sequence of the strain DFP-24 was compared with sequences in databases such as GeneBank for maximum homology. It was found that the 16S rDNA sequence of the strain had a homology of 98.96% with that of Burkholderia arboris. The phylogenetic tree constructed based on 16S rDNA was as follows: Figure 3 As shown, the strain DFP-24 and the Burkholderia arboris strain R-24201 clustered in the same branch, so the strain DFP-24 was identified as Burkholderia arboris.

[0032] Burkholderia arboris DFP-24 was deposited in the China Center for Type Culture Collection on June 4, 2024, with the deposit number CCTCC NO: M 20241144, and the deposit address is Wuhan University, Wuhan, China.

[0033] Example 2 Determination of phosphate solubilization ability of Burkholderia forestii DFP-24

[0034] Phosphate solubilization activity determination method: Prepare the organic phosphorus and inorganic phosphorus liquid culture medium of Montana respectively, sterilize them under high pressure at 121℃ for 20min, and set aside. Inoculate the forest Burkholderia DFP-24 bacterial liquid in the above sterilized liquid culture medium at a ratio of 1%, and take the non-inoculated bacteria as the control. Each treatment is repeated 3 times, and the determination is carried out after shaking culture (28℃, 150r / min) for 3d. Take the cultured bacterial liquid and centrifuge it at 8000r / min for 10min, take the supernatant in a 50mL colorimetric tube, and use the ammonium molybdate colorimetric method to determine the soluble phosphorus content. The phosphorus solubilization rate is used to represent the size of the strain's phosphorus solubilization ability. As shown in Table 2, the soluble phosphorus content in the organic phosphorus liquid culture medium after the strain DFP-24 is treated is 146.6mg / L, and the soluble phosphorus content in the inorganic phosphorus liquid culture medium is 90.13mg / L.

[0035] The total phosphorus addition amount in the organic phosphorus and inorganic phosphorus culture medium of Mengjina is 1g / L, and the phosphorus solubility rate is calculated according to the following formula:

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

[0037] As shown in Table 2, the solubilization rates of organic phosphorus and inorganic phosphorus by strain DFP24 were 14.67% and 9.01%, respectively.

[0038] Table 2 Organic phosphorus decomposition activity and inorganic phosphorus decomposition activity of strain DFP-24

[0039]

[0040] Example 3 Determination of IAA production capacity of Burkholderia forestii DFP-24

[0041] Preparation of IAA standard curve: Prepare 10mg / L, 20mg / L, 30mg / L, 40mg / L, and 50mg / L IAA standard solutions with distilled water, mix with Salkowski colorimetric reagent at a volume ratio of 1:1, and place at room temperature in the dark for 30 minutes. Use a mixture of distilled water and Salkowski colorimetric reagent as a blank control, and use an ultraviolet spectrophotometer to measure the absorbance of each concentration at 530nm. Draw the IAA standard curve with IAA concentration as the horizontal axis and absorbance as the vertical axis.

[0042] Burkholderia forestis DFP-24 was inoculated at a ratio of 2% in LB liquid medium supplemented with 100 mg / L L-tryptophan and shaken for 48 hours. The fermentation broth of the strain was centrifuged at 4000 r / min for 10 minutes, 1 mL of the supernatant was drawn, and an equal volume of Salkowski colorimetric solution was added. This was repeated 3 times. After standing for 30 minutes, the absorbance at 530 nm was measured. The LB liquid medium without bacteria was used as a blank control, and the IAA yield was calculated by drawing a standard curve. The amount of IAA produced by strain DFP-24 reached 5.52 mg / L, which can promote plant growth.

[0043] Example 4 Determination of the ability of Burkholderia forestii DFP-24 to produce siderophore

[0044] CAS detection medium: chrome azuro blue 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, sodium dihydrogen 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] The preliminary screening of siderophore-producing rhizobacteria was performed by CAS plate analysis. The forest Burkholderia DFP-24 was streaked on a CAS test plate and cultured at 28°C. The color change on the plate (from blue to orange) was used to determine whether it was a siderophore-producing strain. Figure 4 As shown, Burkholderia forestii DFP-24 has the ability to produce siderophores. The ability of microorganisms to produce siderophores can improve the biological effectiveness of iron in the environment and promote plant growth. It can also achieve the purpose of controlling plant diseases by competing with pathogenic microorganisms for limited iron ions in the environment.

[0046] Example 5 Inhibitory Effect of Burkholderia silvoli DFP-24 on Fungal Diseases

[0047] The counter-culture method was used to determine the inhibitory effect of forest Burkholderia DFP-24 on fungal diseases. A 5mm bacterial cake was placed around the activated Fusarium solani colony and placed in the center of the PDA plate. Sterile filter paper was placed at two opposite points 2.15cm away from the center of the PDA plate, and 7 microliters of DFP-24 bacterial solution were inoculated. The filter paper plus an equal amount of sterile water was used as a blank control. The plate was cultured at 28°C to observe the antibacterial effect. The results showed that forest Burkholderia DFP-24 had a certain inhibitory effect on the root rot pathogen Fusarium solani ( Figure 5 ).

[0048] Example 6 Effect of Burkholderia forestii DFP-24 on tea quality

[0049] 1. Fermentation with bacterial agents

[0050] The OD of Burkholderia forestii DFP-24 was measured after LB fermentation for 48 hours. 600 .

[0051] 2. Experimental Setup

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

[0053] ②DFP-24

[0054] There are 2 treatments in total, with 3 replicates in each treatment. Each row of the tea field is 1.5m wide and 10m long, i.e. 15m 2 For one treatment.

[0055] 3. Dosage: The concentration of bacterial solution is OD 600 =1.7, dosage is 10mL / m 2 , dilute and irrigate the roots when using, use once on March 6, and apply again after 10 days, set each plot to 15m 2 , 3 plots per treatment.

[0056] The density of tea buds was investigated after one month of application. After four months, 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 free amino acid content of tea leaves was continuously monitored in January, February and April. The tea polyphenol content was determined by the Folin phenol method, with reference to GB / T8313-2018; the total free amino acid content was determined by the ninhydrin colorimetric method, with reference to GB / T8314-2013; caffeine was determined by ultraviolet spectrophotometry, with reference to GB / T8312-2013; phenol-amino acid ratio = tea polyphenols / amino acids.

[0057] As shown in Table 3, in terms of tea bud density, application of Burkholderia forestii DFP-24 was able to increase tea bud density relative to the no-fertilization treatment.

[0058] Table 3 Effect of Burkholderia forestii DFP-24 on tea bud density

[0059]

[0060] As shown in Table 4, compared with the control, 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% after applying Burkholderia forestii DFP24 for 4 months, which improved the quality of tea.

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

[0062]

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

[0064] Table 5 Effects of different treatment times of Burkholderia forestii DFP-24 on free amino acids

[0065]

[0066]

Claims

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

2. A product containing the Burkholderia forestia DFP-24 according to claim 1.

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

4. Use of the Burkholderia forestii DFP-24 described in claim 1 or the product described in claim 2 in tea tree planting.

5. The use according to claim 3, characterized in that: Includes any of the following applications: Ⅰ. Dissolve organic and inorganic phosphorus in soil; Ⅱ. Inhibit the root rot pathogen Fusarium solani; III. Increase tea bud density; IV. Increase the free amino acid content of tea leaves; Ⅴ. Reduce the content of tea polyphenols; Ⅵ. Reduce the phenol-ammonia ratio of tea.

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