Chitinolytic paenibacillus for promoting growth and improving tea quality and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a chitinolytic Paenibacillus which can improve tea yield and tea quality and an application thereof. Paenibacillus chitinolyticus The chitinolytic Paenibacillus has a preservation number of CCTCC NO: M 20241893, has high-efficiency potassium-dissolving activity, strong organic phosphorus-dissolving activity and IAA-producing capacity, can inhibit fungal pathogenic bacteria by secreting chitinase to degrade chitin, can increase the contents of available phosphorus and available potassium in tea garden soil, can improve the total potassium content in tea, can increase the density of tea shoots and the yield of tea, can increase the content of free amino acids in tea, and can reduce the content of tea polyphenols and the phenol-ammonia ratio in tea, thereby improving the quality of tea.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a chitin-solubilizing bacillus with potassium-solubilizing, phosphorus-solubilizing, and growth-promoting properties. Paenibacillus chitinolyticu s) and its application in improving tea yield and quality. Background Technology

[0002] Potassium is one of the three essential nutrients for plant growth and development. The potassium content and supply in tea garden soil have a significant impact on tea yield and quality. However, most tea garden soils in Hubei Province are potassium deficient, with the proportion of available potassium below the critical value reaching 63.4-73.7%. This lack of available nutrients limits the yield and quality of tea trees. Furthermore, long-term excessive application of chemical fertilizers leads to a series of problems, including increased harmful substance residues in tea gardens, imbalance of soil microecology, soil compaction and acidification, and continuous cropping obstacles, thus threatening soil health and the green and sustainable development of agriculture. Rhizosphere growth-promoting bacteria (PGPR) participate in various soil biochemical processes, including nitrogen transformation, phosphorus solubilization, potassium solubilization, iron carrier production, and plant hormone secretion. They can improve nutrient conditions, promote plant growth, and control pests and diseases, playing a vital role in promoting plant growth and maintaining soil ecological health. The development of microbial fertilizers utilizing the phosphorus solubilization, potassium solubilization, and nitrogen fixation capabilities of PGPR has been widely applied.

[0003] Available and potentially available potassium in soil accounts for only 2%–10% of total potassium, while the remaining 90% or more is stably contained in silicate minerals. It is only gradually released through a long process of weathering and decomposition under certain physicochemical factors and the influence of microorganisms. Potassium-solubilizing bacteria are a type of bacteria that can decompose silicate and aluminosilicate minerals, releasing elements such as potassium, phosphorus, and silicon, thereby increasing crop yield and improving soil fertility. Potassium-solubilizing bacteria attach to mineral surfaces by secreting extracellular polysaccharides and weathering minerals with organic acids and enzymes, thus promoting the slow release of exchangeable potassium and increasing the content of available potassium in the environment. They also release silicon, phosphorus, and other substances. Currently, the potassium-solubilizing bacteria identified by previous studies mainly include Bacillus mucilaginosus, Bacillus polymyxa, Bacillus circulans, and Pseudomonas. Therefore, in severely potassium-deficient tea gardens, potassium-solubilizing bacteria, as a microbial fertilizer, can effectively reduce the use of chemical fertilizers, improve the soil ecology of tea gardens, increase crop yield and quality, and promote the healthy development of tea gardens.

[0004] 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 chitin-releasing Bacillus strain that has potassium-releasing, phosphorus-releasing, growth-promoting, and tea-increasing effects, as well as increasing the content of free amino acids in tea and lowering the phenol-amino acid ratio. Paenibacillus chitinolyticu ). Summary of the Invention

[0005] The purpose of this invention is to provide a chitinous spore-forming bacterium with potassium-solubilizing, phosphorus-solubilizing, and growth-promoting properties. Paenibacillus chitinolyticu DFK-17 and its application in improving tea quality.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Chitinous Bacillus ( Paenibacillus chitinolyticu DFK-17, with accession number CCTCC NO: M 20241893, has the following characteristics:

[0008] (1) It can efficiently dissolve potassium minerals in the soil, with a potassium solubilizing activity of 13.65 mg / L;

[0009] (2) Dissolve organophosphates;

[0010] (3) Produces chitinase;

[0011] (4) The IAA production capacity reaches 10.34 mg / L, which can promote tea tree budding and increase the yield of bud tea;

[0012] (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;

[0013] (6) Increase the content of available phosphorus and available potassium in tea garden soil and increase the total potassium content in tea.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] While existing technologies have been reported for the use of Bacillus polymyxa in tea cultivation, such as increasing the polyphenol content of tea leaves (article "..."), Paenibacillus chitinolyticu "Biofertilizer application in a tea plantation reduces soil N2O by changing denitrifier communities", but the chitinous Bacillus strain screened in this invention ( Paenibacillus chitinolyticu DFK-17 has different effects on tea quality. Specifically, it reduces the content of tea polyphenols and effectively reduces the phenol-to-amino acid ratio. It has a significant effect on improving the quality of green tea. At the same time, Bacillus chitinosa DFK-17 can also significantly increase the density of tea buds. Attached Figure Description

[0016] Figure 1 Scanning electron microscope image of chitinous spore-forming bacillus DFK-17.

[0017] Figure 2 Figure 1 is a colony morphology chart of Paenibacillus chitinolyticus DFK-17.

[0018] Figure 3 Figure 2 is a phylogenetic tree of Paenibacillus chitinolyticus DFK-17 based on 16S rDNA.

[0019] Figure 4 Figure 3 is a result of detecting the IAA production ability of Paenibacillus chitinolyticus DFK-17.

[0020] Figure 5 Figure 4 is a result of detecting the chitin degradation ability of Paenibacillus chitinolyticus DFK-17.

[0021] Figure 6 Figure 5 is a result of detecting the organic phosphorus degradation ability of Paenibacillus chitinolyticus DFK-17. DETAILED DESCRIPTION

[0022] Example 1 Screening and purification identification of strains

[0023] (1) Sampling: The sample is the rhizosphere soil of a tea garden in Enshi, Hubei Province. The collected soil is put into a sealed bag and quickly taken back to the laboratory for low-temperature preservation.

[0024] (2) Isolation and culture: After the soil sample is taken 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 flask is placed in a 28°C, 150 rpm shaking incubator for 30 min. After sufficient shaking, it is left to stand for 5 min. The supernatant is used as a 10 -1 dilution soil diluent; the 10 -1 dilution soil diluent is gradient diluted with sterile water to obtain 10 -4 , 10 -5 , and 10 -6 dilutions.

[0025] (3) Colony purification: 0.1 mL of the diluted bacterial solution is taken and added dropwise to the potassium-lysing solid culture medium plate. Immediately, a sterile triangular frame is used to evenly spread it on the surface of the medium, so that there is no water flow. Three plates are prepared for each dilution. After uniform spreading, the plates are inverted and incubated at 28°C. After 48 h, the growth of the colonies is observed and recorded. According to the number, morphology, color, and surface condition of the colonies, representative colonies are selected and picked out with a sterile inoculation loop. At the same time, the colonies are streaked on LB solid culture medium according to aseptic operation requirements until they are purified.

[0026] Among them, the LB solid culture medium is as follows: 10 g / L proteose peptone, 5 g / L yeast powder, 10 g / L sodium chloride, 18 g / L agar, 1000 mL distilled water, and pH 7.0-7.4.

[0027] Potassium-solubilizing solid culture medium: sucrose 10 g / L, magnesium sulfate 0.5 g / L, calcium carbonate 1.0 g / L, ammonium sulfate 1.0 g / L, sodium chloride 0.1 g / L, yeast extract 0.5 g / L, disodium hydrogen phosphate 2.0 g / L, potassium feldspar powder 2 g / L, pH 7.0-7.4, distilled water 1000 mL, agar 15 g / L.

[0028] (4) Appearance and physiological and biochemical characteristics

[0029] The purified strain was named DFK-17, and the electron microscopy results are as follows: Figure 1 As shown, the individual organisms are rod-shaped with pili, and the colony morphology is as follows. Figure 2 As shown, the colonies on LB solid medium 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 DFK-17 are shown in Table 1. DFK-17 showed positive results for methyl red and casein hydrolysis tests, and a weakly positive result for nitrate reduction test.

[0030] Table 1 Physiological and Biochemical Assays

[0031]

[0032] Note: "+" indicates a positive reaction; "W" indicates a weak positive reaction; "-" indicates a negative reaction.

[0033] (5) Molecular biological identification

[0034] The 16S rDNA nucleotide sequence of strain DFK-17 was determined to be 1464 bp. Maximum homology comparison with sequences in databases such as GeneBank revealed that the 16S rDNA sequence of this strain is similar to that of *Bacillus chitinosa* (…). Paenibacillus chitinolyticu The sequence homology reached 99.25%, and the phylogenetic tree constructed based on 16S rDNA is as follows: Figure 3 As shown, strain DFK-17 and Bacillus chitinosa ( Paenibacillus chitinolyticu NBRC-15660 clustered in the same branch, therefore strain DFK-17 was identified as a chitinous spore-forming bacterium (NBRC-15660). Paenibacillus chitinolyticu Paenibacillus chitinolyticu ).

[0035] Chitinous Bacillus ( Figure 4 DFK-17 was deposited at the China Center for Type Culture Collection (CCTCC) on September 2, 2024, with accession number CCTCC NO: M 20241893, and the deposit address is Wuhan University, Wuhan, China.

[0036] Example 2: Determination of potassium solubilizing ability of strain DFK-17

[0037] Potassium-lysing activity determination method: the activated strain was inoculated into potassium-lysing bacterial liquid medium at 5% volume ratio, and the liquid medium without inoculation of bacteria was used as control. After 7 days of culture at 28°C and 150 r / min, the bacterial liquid was centrifuged at 8000 r / min for 10 min. The supernatant was diluted 10 times, and the potassium ion content was determined by flame atomic absorption spectrophotometry. The results are shown in Table 2, and the potassium-lysing activity of strain DFK-17 was 13.65 mg / L.

[0038] Table 2 Potassium-lysing activity of strain DFK17

[0039]

[0040] Example 3 Determination of the IAA production ability of strain DFK-17

[0041] Preparation of IAA standard curve: IAA standard solution was prepared by mixing IAA standard and distilled water at 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L and 50 mg / L, and mixing with Salkowski color reagent at a volume ratio of 1:1. After standing for 30 min in the dark at room temperature, the absorbance of each concentration at 530 nm was determined using distilled water and Salkowski color reagent mixture as blank control. The IAA standard curve was plotted with IAA concentration as abscissa and absorbance as ordinate.

[0042] Strain DFK-17 was inoculated into LB liquid medium with the addition of 100 mg / L L-tryptophan at a volume ratio of 2%, and shaken for 48 h. The fermentation liquid was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. An equal volume of Salkowski color reagent was added, and the operation was repeated 3 times. After standing for 30 min, the absorbance at 530 nm was measured, and the LB liquid medium without inoculation of bacteria was used as blank control. The IAA production was calculated by standard curve. As shown in Table 3, strain DFK-17 has the ability to produce IAA, and the calculated IAA production activity of DFK-17 is 10.34 mg / L, which can promote plant growth. Figure 5

[0043] Example 4 Determination of the chitin degradation characteristics of strain DFK-17

[0044] (1) Preparation of colloidal chitin

[0045] ​Take 10 g chitin powder into 1000 mL glass beaker, add 300 mL concentrated hydrochloric acid, cover the beaker mouth with plastic wrap, put into 35 °C water bath for 1 h, stir several times during the period. When most of the solution is dissolved and a small amount of flocculent remains, add 600 mL distilled water to the beaker. The solution gradually becomes turbid and a white flocculent precipitate is separated out. The white precipitate is colloidal chitin. After static stratification, pour out the supernatant, centrifuge at 8000 r / min for 5 min, then pour out the supernatant. Repeat the process of adding distilled water to form a suspension and centrifuging. Remove the hydrochloric acid by adding distilled water 3 times. Then add 500 mL distilled water to dilute the suspension to colloidal chitin suspension, and adjust to neutral with 4 mol / L NaOH solution.

[0046] (2) Qualitative determination of chitin degradation activity

[0047] Prepare solid chitin medium: 3 g / L proteose peptone, 10% colloidal chitin, 1 g / L NaCl, 0.3 g / L potassium phosphate dibasic, 0.7 g / L potassium phosphate monobasic, 1 g / L magnesium sulfate heptahydrate, 18 g / L agar.

[0048] Inoculate strain DFK-17 on solid chitin medium, and observe whether transparent circles appear after 3 days of incubation at 28 °C to determine whether the strain has chitin degradation effect. As shown in Figure 6 , transparent circles appear around strain DFK-17 after 3 days of incubation, indicating that it has strong chitin degradation activity.

[0049] Example 5: Determination of the phosphorus solubilizing ability of strain DFK-17

[0050] Prepare Mengjina organic and inorganic phosphorus solid medium, and the medium formula is as follows: Mengjina (organic phosphorus) medium: calcium phytate 3.75 g / L, glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate heptahydrate 0.3 g / L, ferrous sulfate heptahydrate 0.03 g / L, manganese sulfate 0.03 g / L, yeast powder 0.5 g / L, adjust pH to 7.0-7.5, add 18 g agar per liter of solid medium according to the proportion.

[0052] Mengjina (inorganic phosphorus) medium: calcium phosphate 5 g / L, glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate heptahydrate 0.3 g / L, ferrous sulfate heptahydrate 0.03 g / L, manganese sulfate 0.03 g / L, yeast powder 0.5 g / L, adjust pH to 7.0-7.5, add 18 g agar per liter of solid medium according to the proportion.

[0053] The clear zone method was used to inoculate the purified strain DFK-17 onto inorganic and organic phosphorus solid plates, with three replicates per group. The plates were incubated at 28°C for 7 days, and the presence or absence of a phosphate-solubilizing zone was used to preliminarily determine the strain's phosphate-solubilizing effect. ​ As shown, the strain formed a clear zone on the Monkina (organophosphate) plate, indicating its organophosphate solubilizing activity. The organophosphate solubilizing activity of strain DFK-17 was quantitatively determined: DFK-17 chitinogenic Bacillus DFK-17 bacterial suspension was inoculated at a 1% ratio into the sterilized organophosphate liquid medium, with no inoculation as a control. Each treatment was repeated three times. After culturing on a shaker (28°C, 150 r / min) for 3 days, the soluble phosphorus content was measured. The cultured bacterial suspension was centrifuged at 8000 r / min 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 strain's phosphorus solubilizing ability. After inoculation with strain DFK-17, the soluble phosphorus content in the Monkina (organophosphate) liquid medium was higher, reaching 139.19 mg / L. The total phosphorus addition in the organic and inorganic phosphorus culture media of Monkina is 1 g / L. The phosphorus dissolution rate is calculated according to the following formula:

[0054] Phosphorus solubility rate = (soluble phosphorus content of inoculated bacteria - soluble phosphorus content of control) / amount of added organic or inorganic phosphorus source × 100%.

[0055] As shown in Table 3, the phosphorus solubility rate of strain DFK-17 for organic phosphorus is 13.92%.

[0056] Table 3. Organophosphate solubilization activity of strain DFK-17

[0057]

[0058] Example 6: Effects of Bacillus chitinosa DFK-17 on tea yield and quality

[0059] (1) Fermentation of microbial agents

[0060] OD was measured after LB fermentation of Bacillus DFK-17 for 48 hours. 600 .

[0061] (2) Experimental setup

[0062] ① Blank control CK (equal volume of water + sterile culture medium)

[0063] ② DFK-17

[0064] There are two treatments, with three replicates for each treatment. Each row of tea bushes is 1.5 m wide and 10 m long, meaning each treatment is 15 m².

[0065] (3) The amount: since the concentration of Lysobacter DFK-17 is low (OD 600 As shown in Table 4), the amount of bacterial agent is selected as 20 mL / m 2 , and when used, it is diluted and irrigated into the roots. On March 6, it is used once, and after 10 days, it is used again. Each plot is set to 15 m 2 , and each treatment has 3 plots.

[0066] After application, the tea bud density, 100-bud weight, and tea yield are investigated for 1, 2, and 4 consecutive months, the average bud density, 100-bud weight, and tea yield are calculated, and the free amino acid content of tea is continuously monitored for 1 month, 2 months, and 4 months. After 4 months, tea leaves with one bud and two leaves are collected and the quality (tea polyphenol, caffeine, free amino acid, and phenol-ammonia ratio) is determined. The tea polyphenol content is determined by the Folin phenol method, according to GB / T 8313-2018; the total amount of free amino acid is determined by the indane trinitro colorimetric method, according to GB / T 8314-2013; caffeine is determined by ultraviolet spectrophotometry, according to GB / T 8312-2013; and the phenol-ammonia ratio = tea polyphenol / free amino acid.

[0067] Table 4 Concentrations of different batches of Lysobacter DFK-17 bacterial agents

[0068]

[0069] As shown in Table 5, in terms of tea bud density, the application of Lysobacter DFK-17 can significantly increase the tea bud density, with an increase of up to 38.25%, promote tea bud emergence, and increase tea yield, compared with the treatment without bacterial agent.

[0070] Table 5 Tea bud density, 100-bud weight, and yield after treatment with Lysobacter DFK-17

[0071]

[0072] As shown in Table 6, compared with the control, after treatment with Lysobacter DFK-17, the tea polyphenol content is reduced by 14.32%, the caffeine content is increased by 9.81%, the amino acid content is increased by 4.47%, and the phenol-ammonia ratio is reduced by 18.81%, which significantly affects the quality of tea.

[0073] Table 6 Tea quality after treatment with Lysobacter DFK-17

[0074]

[0075] As shown in Table 7, the content of free amino acids increased by 3.72%, 11.69% and 4.47% after 1 month, 2 months and 4 months of application of Lysobacter DFK-17, respectively, indicating that Lysobacter DFK-17 has a long-acting effect of increasing the content of free amino acids in tea leaves.

[0076] Table 7 Effect of Lysobacter DFK-17 with different treatment times on free amino acids

[0077]

[0078] The tea leaf sample was digested with nitric acid and perchloric acid, and the total potassium content in the tea leaf was determined by flame photometry. As shown in Table 8, the total potassium content in the tea leaf treated with Lysobacter DFK-17 increased by 5.23%.

[0079] Table 8 Total potassium content in tea leaves treated with Lysobacter DFK-17

[0080]

[0081] The soil samples from the 5-20 cm soil layer of the tea tree rhizosphere were mixed and air-dried, and then passed through a 20-mesh sieve to determine the soil available nutrients. The available phosphorus was determined by sodium bicarbonate extraction-molybdenum antimony anti-spectrophotometry, and the available potassium was determined by ammonium acetate extraction-flame photometry. As shown in Table 9, the available phosphorus content in the soil treated with Lysobacter DFK-17 increased by 67.82%, and the available potassium content in the soil increased by 10.22%.

[0082] Table 9 Available phosphorus and potassium content in the soil treated with Lysobacter DFK-17

[0083]

Claims

1. Chitin-degrading Bacillus ( Paenibacillus chitinolyticus DFK-17, characterized in that, and its preservation number is CCTCC NO: M 20241893.

2. A product containing the chitinolytic Paenibacillus DFK-17 according to claim 1.

3. The product of claim 2, wherein, The product is a microbial fertilizer.

4. The product of claim 3, wherein, The microbial fertilizer includes microbial inoculants, soil conditioners.

5. The use of the Paenibacillus chitinolyticus DFK-17 of claim 1 or the product of claim 2 in tea tree cultivation, characterized in that, Any of the following applications is included: I. Increasing the density of tea buds; II. Increasing the yield of tea; III. Reducing the content of tea polyphenols; IV. Reducing the phenol-ammonia ratio of tea.

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