Chitin-solubilizing paenibacillus capable of promoting growth and improving tea quality and application of chitin-solubilizing paenibacillus

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a strain of Paenibacillus amyloliquefaciens (Paenibacillus amyloliquefaciens) capable of increasing the yield of tea leaves and improving the quality of the tea leaves and an application of the Paenibacillus amyloliquefaciens (Paenibacillus amyloliquefaciens) capable of increasing the yield of the tea leaves and improving the quality of the tea leaves. The preservation number of the chitin-dissolving paenibacillus is CCTCC NO: M 20241893, and the strain has efficient potassium-dissolving activity, strong organophosphorus-dissolving activity and IAA (Indoleacetic Acid) production capacity, and can degrade chitin by secreting chitinase, so that the strain has certain inhibition capacity on fungal pathogenic bacteria, and can be applied to the field of agricultural production. The bacterial strain can increase the content of rapidly available phosphorus and rapidly available potassium in tea garden soil, increase the total potassium content of tea leaves, increase the density of tea buds and the yield of the tea leaves, increase the content of free amino acids in the tea leaves and reduce the content of tea polyphenols and the phenol-ammonia ratio of the tea leaves, so that the quality of the tea leaves is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a chitin-solubilizing Paenibacillus chitinolyticus with the functions of dissolving potassium, dissolving phosphorus and promoting growth, and application thereof in improving tea yield and quality. Background Art

[0002] Potassium is one of the three basic nutrients for plant growth and development. The potassium content and supply of tea garden soil have a great impact on tea yield and quality. However, most of the soil in tea gardens in Hubei Province is in a potassium-deficient state, and the proportion of available potassium below the critical value is 63.4-73.7%. The lack of effective nutrients limits the yield and quality of tea trees. However, long-term excessive application of chemical fertilizers will lead to a series of problems such as increased residual harmful substances in tea gardens, imbalance of soil microecological balance, soil compaction and acidification, and continuous cropping obstacles, thus threatening soil health and green and sustainable development of agriculture. Rhizosphere growth-promoting bacteria (PGPR) participate in various biochemical processes in the soil, including nitrogen transformation, phosphorus solubilization, potassium solubilization, production of iron carriers, secretion of plant hormones, etc., which can improve nutrient conditions, promote plant growth, prevent and control the occurrence of diseases and insect pests, etc., which is of great significance for promoting plant growth and maintaining soil ecological health. The development of microbial fertilizers using the phosphorus solubilization, potassium solubilization and nitrogen fixation capabilities of rhizosphere growth-promoting bacteria has been widely used.

[0003] The available and potentially available potassium in the soil for plants only accounts for 2% to 10% of the total potassium. The remaining 90% of potassium is stably present in silicate minerals. It can only be gradually released after a long period of weathering and decomposition under the action of certain physical and chemical factors and microorganisms. Potassium-solubilizing bacteria are a type of bacteria that can decompose silicate and aluminosilicate minerals, releasing elements such as potassium, phosphorus and silicon, which can increase crop yields and improve soil fertility. Potassium-solubilizing bacteria attach to the surface of minerals by secreting extracellular polysaccharides, and weathering minerals by secreting organic acids and enzymes, thereby promoting the slow release of exchangeable potassium, increasing the content of available potassium in the environment, and also releasing substances such as silicon and phosphorus. The potassium-solubilizing bacteria discovered by predecessors mainly include Bacillus colloidus, Bacillus polymyxa, Bacillus circulans, Pseudomonas, etc. Therefore, in tea gardens with severe potassium deficiency, potassium-solubilizing bacteria as microbial fertilizers can effectively reduce the use of chemical fertilizers, improve the soil ecology of tea gardens, increase crop yields and quality, and promote the healthy development of tea gardens.

[0004] 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 chitin-solubilizing Paenibacillus chitinolyticus that has the functions of dissolving potassium, dissolving phosphorus, promoting growth, increasing the free amino acid content of tea and reducing the phenol-amino ratio. Summary of the invention

[0005] The purpose of the present invention is to provide a chitin-solubilizing Paenibacillus chitinolyticus DFK-17 with the functions of dissolving potassium, dissolving phosphorus and promoting growth, and application thereof in improving the quality of tea leaves.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Paenibacillus chitinolyticus DFK-17, with a deposit number of CCTCC NO: M 20241893, has the following characteristics:

[0008] (1) Highly efficient in dissolving mineral potassium in soil, with a potassium-dissolving activity of 13.65 mg / L;

[0009] (2) Dissolved organic phosphorus;

[0010] (3) Chitinase production;

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

[0012] (5) Increase the free amino acid content of tea, reduce the content of tea polyphenols, reduce the phenol-ammonia ratio of 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 leaves.

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

[0015] Although there are reports in the prior art that Paenibacillus polymyxa is used in tea cultivation, for example, to increase the polyphenol content of tea leaves (article "Paenibacillus polymyxa biofertilizer application in a tea plantation reduces soil N 2However, the effects of the chitinogenic Bacillus (Paenibacillus chitinolyticus) DFK-17 screened by the present invention on the quality of tea leaves are different, specifically, the content of tea polyphenols is reduced, the phenol-ammonia ratio is effectively reduced, and the quality of green tea is particularly improved. At the same time, the chitinogenic Bacillus DFK-17 can also significantly increase the density of tea buds. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a scanning electron microscope image of chitinogenic Bacillus DFK-17.

[0017] Figure 2 This is the colony morphology of chitinogenic Bacillus DFK-17.

[0018] Figure 3 Phylogenetic tree of Paenibacillus chitinogenicus DFK-17 constructed based on 16S rDNA.

[0019] Figure 4 Results of IAA production capacity test of Paenibacillus chitinosinolates DFK-17.

[0020] Figure 5 These are the results of the chitin degradation ability test of Chitinolytic Bacillus DFK-17.

[0021] Figure 6 These are the results of the test on the ability of strain DFK-17 to degrade organic phosphorus. DETAILED DESCRIPTION

[0022] Example 1 Screening, purification and identification of strains

[0023] (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.

[0024] (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; for 10 -1 The soil dilution was serially diluted with sterile water to obtain 10 -4 , 10 -5 , 10 -6 Three serial dilutions were made.

[0025] (3) Colony purification: Take 0.1 mL of the diluted bacterial solution and drop it onto a potassium-dissolving solid culture medium plate. Immediately spread it evenly with a sterile tripod 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 plates upside down and culture them at a constant temperature of 28°C. After 48 hours, observe and record the growth of the colonies. Select representative colonies based on the number, morphology, color, surface condition and other characteristics of the colonies. Pick out a single colony with a sterile inoculation loop and streak it on LB solid culture medium according to the sterile operation requirements until purification.

[0026] Among them, LB solid culture medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, agar 18g / L, distilled water 1000mL, pH7.0-7.4.

[0027] Potassium-dissolving 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. The electron microscopy results are shown in Figure 1 As shown, the individual morphology is 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 convex and sticky surface, an irregular round shape, and a light yellow color. The physiological and biochemical test results of DFK-17 strain are shown in Table 1. The results of DFK-17 in methyl red and casein hydrolysis tests were positive, and the results of nitrate reduction test were weakly positive.

[0030] Table 1 Physiological and biochemical assays

[0031]

[0032]

[0033] Note: “+” means positive reaction; “W” means weak positive reaction; “-” means negative reaction

[0034] (5) Molecular biological identification

[0035] The 16S rDNA nucleotide sequence of strain DFK-17 was determined to be 1464 bp. The maximum homology comparison between the 16S rDNA sequence of the strain and the sequence of Paenibacillus chitinolyticus was found to be 99.25% homologous. The phylogenetic tree constructed based on 16S rDNA is shown in Figure 1. Figure 3 As shown, strain DFK-17 and Paenibacillus chitinolyticus NBRC-15660 were clustered in the same branch, so strain DFK-17 was identified as Paenibacillus chitinolyticus.

[0036] Paenibacillus chitinolyticus DFK-17 was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC NO: M 20241893, and the deposit address is Wuhan University, Wuhan, China.

[0037] Example 2 Determination of potassium solubilization ability of strain DFK-17

[0038] Potassium-dissolving activity determination method: The activated strain was inoculated into potassium-dissolving bacteria liquid culture medium at a volume ratio of 5%, and no strain was inoculated into the liquid culture medium as a control. After culturing at 28°C and 150r / min for 7 days, the bacterial solution was centrifuged at 8000r / min for 10min, and the supernatant was diluted 10 times, and the potassium ion content was determined by flame atomic absorption spectrophotometer. The results are shown in Table 2. The potassium-dissolving activity of strain DFK-17 was 13.65mg / L (Table 1).

[0039] Table 2 Potassium-solubilizing activity of strain DFK17

[0040]

[0041] Example 3 Determination of IAA production capacity of strain DFK-17

[0042] 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 the mixed solution 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.

[0043] The strain DFK-17 was inoculated into LB liquid medium supplemented with 100 mg / L L-tryptophan at a volume ratio of 2% and shaken for 48 hours. The fermentation broth was centrifuged at 4000 r / min for 10 minutes, the supernatant was aspirated, 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 uninoculated LB liquid medium was used as a blank control, and the IAA yield was calculated using the standard curve. Figure 4 As shown, strain DFK-17 has the ability to produce IAA. After calculation, the IAA production activity of DFK-17 is 10.34 mg / L, which can promote plant growth.

[0044] Example 4 Determination of chitin degradation characteristics of strain DFK-17

[0045] (1) Preparation of colloidal chitin

[0046] Weigh 10g chitin powder into a 1000mL glass beaker, add 300mL concentrated hydrochloric acid, cover the beaker mouth with plastic wrap, put it in a 35℃ water bath to keep warm and dissolve for 1h, stirring several times during the period. When most of it is dissolved and a small amount of flocs remain, add 600mL distilled water to the beaker. The solution gradually becomes turbid and a milky white flocculent precipitate is precipitated. The white precipitate is colloidal chitin. After static stratification, pour out the supernatant, centrifuge at 8000r / min for 5min, pour out the supernatant, add distilled water to mix into a suspension, and centrifuge. Repeat adding distilled water 3 times to remove hydrochloric acid, and then add 500mL distilled water to dilute it to obtain a colloidal chitin suspension, and adjust it to neutral with 4mol / L NaOH solution.

[0047] (2) Qualitative determination of chitin degradation activity

[0048] Prepare solid chitin medium: 3 g / L peptone, 10% colloidal chitin, 1 g / L NaCl, 0.3 g / L dipotassium hydrogen phosphate, 0.7 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, and 18 g / L agar.

[0049] The strain DFK-17 was inoculated on solid chitin medium and cultured at 28℃ for 3 days to observe whether a transparent circle was produced to determine whether the strain had a chitin-degrading effect. Figure 5 As shown, a transparent zone appeared around the strain DFK-17 after three days of cultivation, indicating that it has a strong chitin degradation activity.

[0050] Example 5 Determination of phosphate solubilization ability of strain DFK-17

[0051] Prepare the Montgena organic phosphorus and inorganic phosphorus solid culture medium. The culture medium formula is as follows:

[0052] Montgina (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.

[0053] 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.

[0054] The purified strain DFK-17 was spotted onto inorganic phosphorus and organic phosphorus culture medium solid plates using the transparent circle method, with three replicates per group. The culture was performed at 28°C for 7 days, and the phosphate solubilization effect of the strain was preliminarily determined by observing the presence or absence of phosphate solubilization circles. Figure 6 As shown, the strain forms a transparent circle on the Montana (organic phosphorus) plate, indicating that it has the activity of solubilizing organic phosphorus. The solubilizing organic phosphorus activity of strain DFK-17 was quantitatively determined: the chitin-solubilizing Bacillus DFK-17 bacterial liquid was inoculated at a ratio of 1% in the above-mentioned sterilized organic phosphorus liquid culture medium, and the uninoculated bacteria were used as the control. Each treatment was repeated 3 times, and the soluble phosphorus was determined after shaking culture (28°C, 150r / min) for 3 days. The cultured bacterial liquid was centrifuged at 8000r / min for 10 minutes, and the supernatant was taken in a 50mL colorimetric tube. The soluble phosphorus content was determined by ammonium molybdate colorimetry, and the solubilizing phosphorus rate was used to represent the size of the strain's phosphorus solubilizing ability. After inoculating strain DFK-17, the soluble phosphorus content in the Montana (organic phosphorus) liquid culture medium was higher, and the soluble phosphorus content was determined to be 139.19mg / L. The total phosphorus addition amount in the Montana organic phosphorus and inorganic phosphorus culture medium was 1g / L, and the solubilizing phosphorus rate was calculated according to the following formula:

[0055] Phosphorus solubility rate = (soluble phosphorus content of inoculum - soluble phosphorus content of control) / amount of organic or inorganic phosphorus source added × 100%.

[0056] As shown in Table 3, the solubilization rate of organic phosphorus by strain DFK-17 was 13.92%.

[0057] Table 3 Organic phosphorus decomposition activity of strain DFK-17

[0058]

[0059] Example 6 Effect of Chitinogenic Bacillus DFK-17 on Tea Yield and Quality

[0060] (1) Fermentation with bacterial agents

[0061] The OD of Bacillus chitinosinolates DFK-17 was measured after LB fermentation for 48 hours. 600 .

[0062] (2) Experimental setup

[0063] ①Blank control CK (equal amount of water + sterile culture medium)

[0064] ②DFK-17

[0065] There are 2 treatments in total, and 3 replicates are set for each treatment. Each row of the tea field is 1.5m wide and 10m long, i.e. 15m 2 For one treatment.

[0066] (3) Dosage: Since the concentration of chitin-solubilizing Bacillus DFK-17 is low (OD 600 As shown in Table 4), the dosage of the selected microbial agent is 20 mL / 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.

[0067] After application, tea bud density, 100 bud weight and tea yield were investigated for 1, 2 and 4 months, and the average bud density, 100 bud weight and tea yield were calculated. The free amino acid content of tea leaves was continuously monitored after 1, 2 and 4 months. After 4 months, tea leaves with one bud and two leaves were collected and the quality (tea polyphenols, caffeine, free amino acids and phenol-amino acid ratio) was determined. 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 / free amino acids.

[0068] Table 4 Concentration of different batches of chitin-dissolving Bacillus DFK-17 inoculants

[0069]

[0070] As shown in Table 5, in terms of tea bud density, compared with the treatment without bacterial agent, the application of chitinogenic Bacillus DFK-17 can significantly increase the tea bud density by 38.25%, promote tea tree germination, and increase tea yield.

[0071] Table 5 Density, 100-bud weight and yield of tea buds after treatment with Bacillus chitinosporus DFK-17

[0072]

[0073] As shown in Table 6, compared with the control, after treatment with Paenibacillus chitinosuccinate DFK-17, the content of tea polyphenols decreased by 14.32%, the content of caffeine increased by 9.81%, the content of amino acids increased by 4.47%, and the phenol-ammonia ratio decreased by 18.81%, which significantly affected the quality of tea.

[0074] Table 6 Quality of tea leaves after treatment with Paenibacillus chitinosporus DFK-17

[0075]

[0076] As shown in Table 7, the free amino acid content increased by 3.72%, 11.69% and 4.47% after applying Paenchitinolyticus DFK-17 for 1 month, 2 months and 4 months, respectively, indicating that Paenchitinolyticus DFK-17 has a long-term effect of increasing the free amino acids in tea leaves.

[0077] Table 7 Effect of different treatment time of Paenibacillus chitinosporus DFK-17 on free amino acids

[0078]

[0079] After the tea samples were digested with nitric acid and perchloric acid, the total potassium content of the tea was determined by flame photometry. As shown in Table 8, the total potassium content of the tea increased by 5.23% after treatment with Paenibacillus chitinosuccinate DFK-17.

[0080] Table 8 Total potassium content of tea leaves after treatment with Bacillus chitinosporus DFK-17

[0081]

[0082] The soil samples of 5-20cm soil layer in the rhizosphere of tea trees were collected by 5-point sampling method, mixed and air-dried, and then passed through a 20-mesh sieve to determine the available nutrients in the soil. 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, after treatment with Bacillus chitinosuccinate DFK-17, the available phosphorus content in the soil increased by 67.82%, and the available potassium content in the soil increased by 10.22%.

[0083] Table 9 Available phosphorus and available potassium content in soil after treatment with Bacillus chitinospora DFK-17

[0084]

Claims

1. Paenibacillus chitinosinolates Paenibacillus chitinolyticus )DFK-17, characterized in that, Its deposit number is CCTCC NO: M 20241893.

2. A product containing the chitinogenic bacillus DFK-17 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 chitin-solubilizing Bacillus DFK-17 according to claim 1 or the product according to claim 2 in tea tree planting.

5. The use according to claim 3, characterized in that: Includes any of the following applications: Ⅰ. Dissolve mineral potassium and organic phosphorus in the soil; Ⅱ. Produces chitinase; III. Increase tea bud density; IV. Increase tea production; Ⅴ. Increase the free amino acid content of tea leaves; Ⅵ. Reduce the content of tea polyphenols; VII. Reduce the phenol-ammonia ratio of tea leaves; VIII. Increase the content of available phosphorus and available potassium in tea garden soil and increase the total potassium content in tea leaves.

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