Tea tree endophyte with capabilities of dissolving phosphorus, fixing nitrogen and decomposing cellulose and preparation method of microbial inoculum of tea tree endophyte

Abstract

The invention discloses a tea tree endophyte capable of dissolving phosphorus, fixing nitrogen and decomposing cellulose and a preparation method of a fungicide of the tea tree endophyte, the tea tree endophyte is classified and named as Paenibacillus amyloliquefaciens CPNts501 and is preserved in the China Center for Type Culture Collection, the strain preservation number is CCTCC NO: M20241583, and the preservation date is July 17, 2024. The result shows that compared with CK treatment, the chlorophyll content of tea tree leaves treated by the microbial inoculum prepared from the Paenibacillus amyloliquefaciens CPNts501 strain is remarkably increased by 63.3%, and the nitrate nitrogen content is remarkably increased by 29.1%. Experimental results show that the microbial inoculum prepared from the strain can significantly increase the potassium content of soil, increase the content of organic matters and ammonium nitrogen in the soil, promote absorption and utilization of nitrogen and phosphorus by tea trees, increase the chlorophyll content of the tea trees, accelerate degradation of litters of the tea trees, improve and increase the content of the organic matters and nutrients in the soil and the like; the method can be used for preparing the nitrogen-fixing cellulose-decomposing growth-promoting microbial inoculum special for the mountain tea garden.

Description

Technical Field

[0001] The invention relates to a tea tree endophyte capable of dissolving phosphorus, fixing nitrogen and decomposing cellulose and a preparation method of a bacterial agent thereof, belonging to the technical field of microorganisms and biofertilizers. Background Art

[0002] Tea itself is rich in alkaloids, tea polyphenols and other substances, which are beneficial to the human body and are a popular drink. Endophytes refer to a type of microorganisms that invade the plant body, do no obvious harm to the host plant, form a mutually beneficial relationship with the host plant, and promote plant growth and metabolism. In the process of modern agricultural development, due to the influence of long-term agricultural activities, the abuse of pesticides and chemical fertilizers and other factors, agricultural planting faces problems such as soil fertility loss, soil compaction and continuous cropping obstacles. Soil acidification and fertility loss in tea garden planting are particularly obvious. In the face of problems such as the abuse of chemical fertilizers and pesticides, "green, safe and pollution-free" has become an inherent requirement for the sustainable development of modern agriculture. The use of plant endophytes to make microbial fertilizers has become a research hotspot that is increasingly concerned, and green and efficient new fertilizers are an inevitable trend. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a tea tree endophyte with the ability to dissolve phosphorus, fix nitrogen and decompose cellulose and a preparation method of a bacterial agent thereof, which has a growth-promoting effect on tea trees and can accelerate the degradation of tea garden litter, and can be used as a tea tree-specific growth-promoting bacterial agent or microbial fertilizer. The strain can effectively promote the growth of tea trees and can be used to prepare excellent tea tree-specific bacterial fertilizers. Its cellulose-decomposing ability can accelerate the degradation of tea garden litter and accelerate the return of organic matter to rhizosphere soil, thus overcoming the shortcomings of the prior art.

[0004] The technical solution of the present invention: The endophytic bacteria of tea trees are classified and named Paenibacillus amylolyticus CPNts501, which is preserved in the China Center for Type Culture Collection, with the strain preservation number of CCTCC NO:M20241583 and the preservation date of July 17, 2024.

[0005] The aforementioned Paenibacillus amylolyticus CPNts501 is used as a cellulose decomposing bacterium.

[0006] The aforementioned use of Paenibacillus amylolyticusCPNts501 according to claim 1 as phosphate-solubilizing bacteria.

[0007] The aforementioned use of Paenibacillus amylolyticusCPNts501 according to claim 1 as nitrogen-fixing bacteria.

[0008] The aforementioned Paenibacillus amylolyticus CPNts501 is used as a growth-promoting bacterial agent.

[0009] Paenibacillus amylolyticus CPNts501 was isolated and purified from tea seeds.

[0010] The method for separating and purifying Paenibacillus amylolyticus CPNts501 from tea seeds is as follows:

[0011] a. Sterilize the tea seeds, grind them in a sterile mortar and dilute them with pure water;

[0012] b. using inorganic phosphorus medium, organic phosphorus medium, sodium carboxymethyl cellulose medium, and Winogradsky nitrogen-free medium to separate and purify the functional endophytes obtained in step a;

[0013] c The isolated and purified strains were inoculated into the selective culture medium corresponding to the functions of the strains in turn to identify the functions and preliminarily screen out the functional endophytes with the ability to dissolve phosphorus, fix nitrogen and decompose cellulose. According to the preliminary screening results, the selected functional endophytes were subjected to filter paper enzyme activity (FPA) method, molybdenum antimony resistance colorimetric method and the method of measuring the growth of strains in nitrogen-free culture medium according to their different functions to determine the phosphorus solubilizing ability, nitrogen fixation ability and cellulose degradation ability of the strains, and the dominant strain CPNts501 was re-screened.

[0014] The aforementioned method for separating and purifying Paenibacillus amylolyticus CPNts501 from tea seeds is that the inorganic phosphorus culture medium, organic phosphorus culture medium, sodium carboxymethyl cellulose culture medium and nitrogen-free culture medium are PKO culture medium, Montgina culture medium, CMC-Na culture medium and Winogradsky nitrogen-free culture medium respectively.

[0015] The method for separating and purifying the aforementioned Paenibacillus amylolyticus CPNts501 from tea seeds is as follows: the tea seeds are disinfected by rinsing them with running water and then soaking them in sterile distilled water to soften the shell of the tea seeds, wiping them dry with sterile paper after soaking, and then soaking the dried tea seeds in 75% alcohol in a clean bench for sterilization. After 3 minutes, the dried tea seeds are taken out and rinsed with sterile water for 3-4 times, then soaked in iodine for 3 minutes, and finally rinsed with sterile water for 3-4 times.

[0016] The aforementioned method for separating and purifying Paenibacillus amylolyticus CPNts501 from tea seeds is to first stain the sodium carboxymethyl cellulose culture medium with Congo red dye in an ultra-clean workbench when using sodium carboxymethyl cellulose culture medium for separation and purification, and then select a strain with a transparent zone for separation and purification.

[0017] Compared with the prior art, the present invention uses selective culture media such as CMC-Na culture medium, Montana culture medium, PKO culture medium and Winogradsky nitrogen-free culture medium to gradually separate and purify functional endophytes, and screens out four functional endophytes, namely CPNts501, Cts502, Nts903 and Nts904, in the preliminary screening. The selected functional endophytes are subjected to filter paper enzyme activity (FPA) method, molybdenum antimony colorimetric method and method for measuring the growth of strains in nitrogen-free culture medium according to the preliminary screening results, and the cellulose degradation ability, phosphorus solubilization ability and nitrogen fixation ability of the strains are determined, and the dominant strain CPNts501 is screened out again. The pure culture of the bacteria is identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry using a fully automatic microbial mass spectrometry detection system (Autof Ms1000), and the screened dominant strain CPNts501 with cellulose degradation and phosphorus solubilization and nitrogen fixation ability is Paenibacillus amylolyticus. CPNts501 was inoculated into LB medium and cultured for 10-18 h to prepare a medium containing 1×10 9 ~10 10 CFU / mL of liquid inoculant was used to treat tea trees and set up a control. After 50 days, the ammonia nitrogen, available phosphorus, available potassium, organic matter of tea tree soil treated with different treatments, as well as the nitrate nitrogen, available phosphorus, available potassium, chlorophyll and tea polyphenols of tea trees were measured. The results showed that the chlorophyll content of old leaves of tea trees treated with Paenibacillus amylolyticus CPNts501 bacterial liquid was significantly increased by 63.3% and the nitrate nitrogen content was significantly increased by 29.1% compared with the CK treatment. The experimental results show that Paenibacillus amylolyticus CPNts501 can promote the absorption and utilization of nitrogen by tea trees, increase the chlorophyll content of tea trees, and improve the supply capacity of soil organic matter and potassium. It has a significant growth-promoting effect on tea trees and can be used to prepare growth-promoting bacterial agents specifically for degraded tea gardens. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 CPNts501 in cellulose-degrading medium (left);

[0019] Figure 2Nitrogen-free medium (medium);

[0020] Figure 3 The colonies on the organophosphate-dissolving Montinna medium (right) and the clear cellulose-dissolving / phosphate-dissolving transparent zone. Colony No. 2 in the right picture is a reference strain without the ability to dissolve phosphate;

[0021] Figure 4 Comparison bar graph of tea chlorophyll content;

[0022] Figure 5 Tea polyphenols content comparison bar graph;

[0023] Figure 6 Comparison bar graph of nitrate nitrogen content in tea trees;

[0024] Figure 7 Comparison bar graph of available potassium content in tea tree;

[0025] Figure 8 Comparison bar graph of available phosphorus content in tea trees;

[0026] Note: Figure 4-Figure 8 Different lowercase letters indicate significant differences among treatments (Duncan method, P<0.05)

[0027] The classification name of the deposited strain in this application is Paenibacillus amylolyticusCPNts501, which is deposited in China Center for Type Culture Collection with the strain accession number of CCTCC NO:

[0028] M20241583, the deposit date is July 17, 2024, and the deposit address is Wuhan, China. DETAILED DESCRIPTION

[0029] The following experimental process is used to describe in detail the preparation process of Paenibacillus amylolyticusCPNts501 and the function and application of the bacterium.

[0030] 1 Experimental Materials and Methods

[0031] 1.1 Experimental Materials

[0032] 1.1.1 Overview of plant materials and experimental sites

[0033] The tea seeds were selected from Fuding Dabai tea seeds collected in Fenggang County, Guizhou Province in October 2022, and the well-preserved and undamaged tea seeds were selected for the experiment.

[0034] The field experiment site is located in the tea field of Xinbao Village, Yangchang Town, Guiyang City, Guizhou Province (106°51′5″E, 26°49′0″N). The climate of the site belongs to the subtropical monsoon climate, with humid climate and abundant precipitation.

[0035] 1.1.2 Culture medium

[0036] Inorganic phosphorus (PKO) medium, organic phosphorus (Montgena) medium, sodium carboxymethylcellulose (CMC-Na) medium, Winogradskyi nitrogen-free medium, LB medium.

[0037] 1.2 Experimental methods

[0038] 1.2.1 Sample disinfection

[0039] Rinse the tea seeds with running water and soak them in sterile distilled water to soften the shell of the tea seeds. After soaking, wipe them dry with sterile paper. Soak the dried tea seeds in 75% alcohol in a clean bench for sterilization. After 3 minutes, take them out and rinse them with sterile water for 3-4 times, soak them in iodine for 3 minutes, and finally rinse them with sterile water for 3-4 times.

[0040] 1.2.2 Separation and purification

[0041] After disinfection, tea seeds were ground in a sterile mortar and 10 mL of distilled water was added to make a grinding solution. 1 mL of the grinding solution was added to 9 mL and 99 mL of sterile water to dilute 10 times and 100 times respectively. 0.2 mL of the diluted and mixed solution was spread on sodium carboxymethyl cellulose (CMC-Na) culture medium, inorganic phosphorus (PKO) culture medium, organic phosphorus (Montkina) culture medium and Winogradskyi nitrogen-free culture medium, and the sterile water of the last rinse was used as a control. Each group was paralleled three times. The culture medium after coating was cultured in a constant temperature incubator at 28°C for 3 days. If there was no growth of strains in the blank culture medium, it meant that the disinfection was thorough and there was no contamination by miscellaneous bacteria.

[0042] After a stable number of single colonies grow in the culture medium, select the strains that produce transparent circles in the inorganic phosphorus (PKO) medium and the organic phosphorus (Mongkina) medium, and pick single colonies for separation and purification under a sterile environment; dye the sodium carboxymethyl cellulose culture medium with Congo red dye in an ultra-clean workbench, and select strains with transparent circles for separation and purification; select strains with better growth in Win nitrogen-free culture medium for separation and purification.

[0043] The isolated and purified strains were inoculated into the selection medium corresponding to the strain function using the plate streak method, and cultured at 28°C for 3 days. The size of the phosphate-dissolving zone of the strain in PKO medium and Montkina organic phosphorus medium was observed and its D / d value was recorded. After Congo red staining, the size of the transparent zone of the strain in sodium carboxymethyl cellulose (CMC-Na) medium was observed and its D / d value was recorded. The growth of the strain in Winogradskyi nitrogen-free medium was observed and recorded. The data were analyzed to initially screen out the superior strains with stronger abilities.

[0044] 1.2.3 Enzyme activity assay

[0045] 1.1 Determination of cellulase activity

[0046] The filter paper enzyme activity (FPA) of the strain was determined and a glucose standard curve was drawn.

[0047] The enzyme activity conversion formula is as follows: Enzyme activity (U / mL) = (a×b×5.56)÷(n×t)

[0048] Wherein, a is the content of glucose (mg), b is the total volume of the enzyme solution (mL), n is the amount of enzyme solution added to the reaction solution (mL), t is the time (min), and 5.56 is the number of μmol of 1 mg of glucose (1000 / 180=5.56).

[0049] 1.2 Determination of phosphate dissolving effect (molybdenum antimony colorimetric method)

[0050] Prepare the test solution and blank control and draw a phosphorus standard curve.

[0051] 1.2.4 Strain identification

[0052] The representative strains that have the ability to solubilize phosphorus, fix nitrogen and decompose cellulose were classified and identified using microbial mass spectrometry-fully automatic microbial mass spectrometry detection system (Autofms 1000).

[0053] 1.2.5 Field experiments

[0054] The dominant strain was inoculated into 50 mL LB liquid culture medium, and cultured at 28°C, 150 r·min-1 for 1 day. The seed liquid was inoculated into LB liquid culture medium at a volume ratio of 1:100, and liquid bacterial agent was obtained after constant temperature shaking culture at 28°C for a period of time.

[0055] In the field experiment, three groups of treatments were carried out: CK (sterile water), LB liquid culture medium (sterile), and CPNts501 bacterial solution (OD680nm=0.554). Each plant in the treatment was irrigated with 20mL of the corresponding solution, and the experiment was repeated 5 times. After 50 days of growth, the content of soil organic matter, available potassium, effective phosphorus, and ammonia nitrogen in each treatment was determined. Four plants were selected from each treatment to determine the content of available potassium, effective phosphorus, nitrate nitrogen, chlorophyll, and tea polyphenols.

[0056] 1.2.6 Determination of soil available phosphorus, available potassium, ammonia nitrogen, available potassium and nitrate nitrogen

[0057] The nutrient content was measured according to the instructions of the HM-TYD full-project soil fertilizer tester (Shandong Hengmei).

[0058] 1.2.8 Determination of Available Phosphorus, Tea Polyphenols and Chlorophyll in Plants

[0059] The available phosphorus content of the plants was determined by the molybdenum antimony anti-absorbance method in NY / T 2017-2011 "Determination of nitrogen, phosphorus and potassium in plants", and the tea polyphenol content of the plants was determined by GB / T8313-2018 "Test method for tea polyphenols and catechins in tea". The leaf chlorophyll was determined using a handheld chlorophyll meter (Zhongke Weihe SPAD-502).

[0060] 1.2.9 Data Processing

[0061] Excel 2003 software was used to organize the data, and SPSS 17.0 statistical analysis software was used for data statistical analysis.

[0062] 2 Results and analysis

[0063] 2.1 Isolation of Phosphate-solubilizing, Nitrogen-fixing and Cellulose-degrading Bacteria

[0064] like Figure 1 As shown, it can be seen that Paenibacillus amylolyticus CPNts501 can be used in cellulose decomposition medium ( Figure 1 ), nitrogen-free medium ( Figure 2 ) and soluble organophosphate montgina medium ( Figure 3 ) formed colonies and clear cellulose / phosphate dissolving transparent zones.

[0065] Table 1 Cellulose degradation ability of Bacillus amylolyticus CPNts501 in CMC-Na medium

[0066]

[0067] Note: Different lowercase letters indicate significant differences among treatments (LSD method, P<0.05).

[0068] Table.2 Phosphate solubilization ability of Bacillus amylolyticus CPNts501 in Montana organic medium

[0069]

[0070] Table.3 Cellulase activity, phosphorus solubilization and nitrogen fixation ability of CPNts501

[0071]

[0072] Note: Different lowercase letters indicate significant differences among treatments (LSD method, P<0.05).

[0073] Depend on Figure 1-Figure 3 As shown in Tables 1-3, CPNts501 showed cellulose-degrading / phosphate-dissolving transparent zones in both CMC-Na medium and Montana organic medium, and could form typical colonies on nitrogen-free medium, indicating that the strain has good abilities in cellulose degradation, organic phosphorus dissolution and nitrogen fixation.

[0074] 2.2 Strain identification results

[0075] Table 2.2.1 Strain identification results

[0076]

[0077] After identification, the strain CPNts501 is a Gram-positive bacterium, and its classification name is Paenibacillus amylolyticus CPNts501. It is preserved in the China Center for Type Culture Collection, the strain preservation number is CCTCC NO:M20241583, and the preservation date is July 17, 2024.

[0078] 2.3 Analysis of field experiment results

[0079] Experimental Table 1 Contents of soil ammonia nitrogen, available phosphorus, available potassium and organic matter

[0080]

[0081] Note: Different lowercase letters indicate significant differences among treatments (Duncan method, P<0.05).

[0082] As shown in Table 1, 50 days after inoculation, the soil ammonia nitrogen and organic matter content in the CPNts501 treatment was slightly higher than that in the control treatment, but the difference was not significant. The organic matter residues such as tea tree litter in the rhizosphere were decomposed faster, and the potassium in them was released, making the soil available potassium content significantly higher than the control by 57.19% (P<0.05).

[0083] 2 Analysis of tea tree nutritional components

[0084] Depend on Figure 4 It can be seen that there is no significant difference in the chlorophyll content of new leaves of tea trees treated with the inoculant compared with the control, but after the CPNts501 inoculant treatment, the chlorophyll content of middle-aged and old leaves, which are mainly responsible for photosynthesis, is significantly higher than that of the control (CK) without the inoculant (P<0.05) by 63.3%. This shows that after the application of CPNts501 inoculant, the chlorophyll content of middle-aged and old leaves increases, which means that the photosynthetic capacity and dry matter accumulation efficiency of tea trees increase.

[0085] Depend on Figure 5 It can be seen that the content of tea polyphenols in the new leaves treated with CPNts501 and CK increased, but the difference was not significant; while the content of tea polyphenols in the old leaves treated with CPNts501 was significantly higher than that of CK by 14.3%, indicating that CPNts501 can promote the accumulation of tea polyphenols in tea leaves.

[0086] Depend on Figure 6 It can be seen that CPNts501 can significantly increase the nitrogen accumulation in tea leaves. The nitrate nitrogen content of old leaves treated with bacterial solution was significantly higher than that of the uninoculated control treatment by 29.1% (P<0.05).

[0087] Depend on Figure 7 It can be seen that the content of available potassium in new leaves treated with CPNts501 bacterial solution is slightly higher than that in the control, and slightly lower in old leaves than that in the control. This shows that after the application of bacterial agent, the photosynthesis and nitrogen absorption of plants increased, and because the effective potassium content in the soil is sufficient, the absorption of potassium by new leaves of tea trees increased, but the difference was not significant.

[0088] Depend on Figure 8 It can be seen that after treatment with CPNts501, there was no significant difference in the available phosphorus content between the new leaves and the control (CK); but the old leaves were significantly lower than the control treatment, which may be due to the accelerated growth of the plants and the rate at which the plants consumed available phosphorus faster than the phosphorus solubilization ability of the strain. The lack of phosphorus nutrients in the soil also confirms this inference.

[0089] In summary, the 50-day field inoculation experiment showed that after the inoculation of Paenibacillus amylolyticus CPNts501 in the rhizosphere soil of Fuding white tea trees, the contents of chlorophyll, nitrate nitrogen and tea polyphenols in the old leaves of the tea plants were significantly higher than those in the control (CK) treatment. Among them, the chlorophyll content of the leaves after the treatment with the inoculum increased by 63.3% compared with the control, and the nitrate nitrogen increased significantly by 29.1%. This shows that P. amylolyticus CPNts501 can effectively promote the growth of tea trees and can be used to prepare excellent tea tree-specific bacterial fertilizers. Its cellulose-degrading ability can accelerate the degradation of tea garden litter and accelerate the return of organic matter to the rhizosphere soil, which has the advantages of being green, safe and efficient.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A tea tree endophyte, which is classified and named as Bacillus amyloliquefaciens Paenibacillus amylolyticus CPNts501 is deposited in the China Center for Type Culture Collection, the strain accession number is CCTCC NO: M20241583, and the deposit date is July 17, 2024.

2. The bacillus amyloliquefaciens according to claim 1 Paenibacillus amylolyticus Application of CPNts501 as a cellulose-degrading bacterium.

3. The Bacillus amyloliquefaciens according to claim 1 Paenibacillus amylolyticus CPNts501 Application as phosphate-solubilizing bacteria.

4. The Bacillus amyloliquefaciens according to claim 1 Paenibacillus amylolyticus CPNts501 Application as nitrogen-fixing bacteria.

5. The bacillus amyloliquefaciens according to claim 1 Paenibacillus amylolyticus Application of CPNts501 as a growth-promoting agent and tea tree litter-degrading agent.

6. The Bacillus amyloliquefaciens according to any one of claims 1 to 5 Paenibacillus amylolyticus CPNts501 is isolated and purified from tea plant seeds.

7. The bacillus amyloliquefaciens according to claim 6 Paenibacillus amylolyticus The method for separating and purifying CPNts501 from tea seeds comprises the following steps: a. Sterilize the surface of tea seeds, grind them in a sterile mortar and dilute with pure water; b. using PKO inorganic phosphorus-soluble medium, Montana organic phosphorus-soluble medium, sodium carboxymethyl cellulose medium and nitrogen-free medium to perform functional identification and separation and purification of excellent strains of the endophytic bacteria obtained in step a; c The isolated and purified strains were inoculated into the selective culture medium corresponding to the function of the strains for preliminary screening of functional endophytes. The selected functional endophytes were subjected to filter paper enzyme activity (FPA) method, molybdenum antimony resistance colorimetric method and the method of measuring the growth of strains in nitrogen-free culture medium according to the preliminary screening results, to determine the cellulose degradation ability, phosphate solubilization ability and nitrogen fixation ability of the strains, and the dominant strain CPNts501 was re-screened.

8. The bacillus amyloliquefaciens according to claim 7 Paenibacillus amylolyticus The method for separating and purifying CPNts501 from tea seeds is characterized by: The inorganic phosphorus culture medium, organic phosphorus culture medium, sodium carboxymethyl cellulose culture medium and nitrogen-free culture medium are respectively inorganic phosphorus (PKO) culture medium, organic phosphorus (Montkina) culture medium, sodium carboxymethyl cellulose (CMC-Na) culture medium and Winogradsky nitrogen-free culture medium.

9. The Bacillus amyloliquefaciens according to claim 7 Paenibacillus amylolyticus The method for separating and purifying CPNts501 from tea seeds is characterized by: The tea seed disinfection is to rinse the tea seeds with running water and then soak them in sterile distilled water to soften the shell of the tea seeds, wipe them dry with sterile paper after soaking, soak the dried tea seeds in 75% alcohol in a clean bench for sterilization, take them out after 3 minutes and rinse them with sterile water for 3-4 times, then soak them in iodine for 3 minutes, and finally rinse them with sterile water for 3-4 times.

10. The Bacillus amyloliquefaciens according to claim 7 Paenibacillus amylolyticus The method for separating and purifying CPNts501 from tea seeds is characterized by: When using sodium carboxymethyl cellulose culture medium for separation and purification, the sodium carboxymethyl cellulose culture medium is first stained with Congo red dye in an ultra-clean workbench, and then the strain with a transparent circle is selected for separation and purification.

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