A strain of Staphylococcus epidermidis and its applications, as well as the microbial fertilizer for kidney beans prepared therefrom, the preparation method thereof, and the cultivation method for kidney beans

Abstract

The present invention discloses a strain of Staphylococcus epidermidis, its application, the microbial fertilizer for kidney beans prepared therefrom, the preparation method thereof, and the cultivation method for kidney beans, belonging to the field of microbial technology. The present invention solves the problem that the current traditional chemical fertilizers and pesticides lead to soil degradation and affect the yield and quality of kidney beans. The Latin name of the Staphylococcus epidermidis is Staphylococcus epidermidis, and the Staphylococcus epidermidis is preserved in the China Center for Type Culture Collection of Wuhan University, with the preservation number of CCTCC NO: M 2024036 and the preservation time of January 8, 2024. The Staphylococcus epidermidis can be used to prepare the microbial fertilizer for kidney beans. The preparation method is to inoculate the Staphylococcus epidermidis into a beef extract peptone medium and culture it in a constant temperature shaker for 24 h to obtain an activated bacterial solution, and the activated bacterial solution is the microbial fertilizer for kidney beans, and the method for cultivating kidney beans using the microbial fertilizer for kidney beans. The Staphylococcus epidermidis of the present invention is applicable to the field of preparation of microbial fertilizers for kidney beans.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a strain of Staphylococcus epidermidis and its application. Background Art

[0002] Phaseolus vulgaris var. sesquipedalis is a high-quality climbing pod bean variety unique to Northeast China and is one of the "local advantageous characteristic agricultural products" in Jilin and Heilongjiang provinces. In recent years, with the reform of the agricultural product supply system and the development of the green vegetable industry in China, the production of Phaseolus vulgaris var. sesquipedalis has developed rapidly. With the spread of the national food culture, Phaseolus vulgaris var. sesquipedalis is also well-known to consumers. Because of its unique bean fragrance that cannot be compared with other vegetables, it is favored by consumers at home and abroad, and the market demand is gradually increasing.

[0003] Due to the influence of the planting of Phaseolus vulgaris var. sesquipedalis on the cultivated land area and cultivation environment, the industrial development of Phaseolus vulgaris var. sesquipedalis in our province is severely restricted. Traditional practices such as applying chemical fertilizers and pesticides to increase the yield of Northeast Phaseolus vulgaris var. sesquipedalis will cause a series of soil degradation problems such as soil compaction, continuous cropping obstacles, salinization, and heavy metal pollution, seriously affecting crop yields and food security.

[0004] Research results show that soil microorganisms play an important role in the processes of organic matter decomposition, nutrient cycling, and plant nutrient utilization. Plant growth-promoting rhizobacteria are of great significance for promoting plant growth and maintaining soil ecological health through beneficial functions such as regulating plant hormone balance, nutrient absorption, adapting to abiotic stress environments, and suppressing diseases. Therefore, how to use microorganisms to improve the soil environment, alleviate the harm of chemical fertilizers and pesticides to the land, and effectively promote the growth and development of Northeast Phaseolus vulgaris var. sesquipedalis, and further improve the yield and quality of Phaseolus vulgaris var. sesquipedalis on the premise of stable production is an urgent problem to be solved at present. Summary of the Invention

[0005] The present invention discloses a strain of Staphylococcus epidermidis and its application, which improves the problem of land degradation caused by traditional chemical fertilizers and pesticides at present and effectively promotes the growth and development of Phaseolus vulgaris var. sesquipedalis.

[0006] The technical solution of the present invention is as follows:

[0007] A strain of Staphylococcus epidermidis, the taxonomic name of the Staphylococcus epidermidis is Staphylococcus epidermidis XGZN20, the Staphylococcus epidermidis is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, the preservation number is CCTCC NO: M 2024036, and the preservation time is January 8, 2024.

[0008] The application of the Staphylococcus epidermidis in the preparation of a microbial fertilizer for Phaseolus vulgaris var. sesquipedalis.

[0009] A preparation method of a kidney bean microbial fertilizer. The above-mentioned Staphylococcus epidermidis is inoculated into a beef extract peptone medium and cultured in a constant temperature shaker for 24 h to obtain an activated bacterial solution, and the activated bacterial solution is the kidney bean microbial fertilizer.

[0010] Further, the temperature of the constant temperature shaker culture is 25 °C.

[0011] Further, the rotation speed of the constant temperature shaker culture is 140 r·min -1 .

[0012] Further, the OD600 value of the activated bacterial solution is 1.0.

[0013] A kidney bean microbial fertilizer, which is obtained by the above-mentioned preparation method.

[0014] A kidney bean cultivation method, in which the above-mentioned kidney bean microbial fertilizer is used. In the cultivation method, after the kidney bean seeds germinate to 1-2 cm, they are planted. After planting, each kidney bean seedling with two true leaves is watered with 10 mL of the kidney bean microbial fertilizer, and it is re-watered every 10 days for a total of 3 times.

[0015] Beneficial effects

[0016] The preparation method of the kidney bean microbial fertilizer of the present invention is simple and fast. It only needs to activate and culture Staphylococcus epidermidis and then dilute it, and it can be watered on the roots of kidney beans during use. The present invention compares the kidney bean microbial fertilizer with two chemical fertilizers and a water group, and finds that the group watered with Staphylococcus epidermidis has the most obvious growth-promoting effect on kidney beans, which can increase the plant height, dry and fresh weights of upper and lower parts, stem diameter, main root length, root volume, connection number, node number, root tip number, bifurcation number, and the synthesis of chlorophyll, SOD, POD and CAT of kidney beans. Watering Staphylococcus epidermidis can provide supplementary nutrition for kidney beans in the seedling stage and replace the use of chemical fertilizers, which has good social and economic value. Description of the drawings

[0017] Figure 1 It is a phylogenetic tree strain sequence alignment result diagram of XGZN20 based on the 16S rRNA sequence and other strains;

[0018] Figure 2 It is the root system scanning morphology diagram of 15 d and 30 d of treatment; A is the root system morphology of the water treatment, B is the root system morphology of the chemical fertilizer (potassium dihydrogen phosphate fertilizer) treatment, and C is the root system morphology of the Staphylococcus epidermidis XGZN20 treatment;

[0019] Figure 3Diagram showing the effects of different treatments on the root growth of Phaseolus vulgaris var. humilis. Among them, A shows the root growth after 15 days of different treatments, and B shows the root growth after 30 days of different treatments; CK is the control group treated with water, F is the treatment group treated with chemical fertilizer (potassium dihydrogen phosphate), and Staphylococcus epidermidis XGZN20 is the treatment group treated with Staphylococcus epidermidis.

[0020] Figure 4 Diagram showing the changes in chlorophyll content, SOD, POD, and CAT activities in the leaves of Phaseolus vulgaris var. humilis under different treatments. Among them, A shows the effects of three treatments on the chlorophyll of Phaseolus vulgaris var. humilis at different days; B shows the effects of three treatments on the SOD activity of Phaseolus vulgaris var. humilis at different days; C shows the effects of three treatments on the POD activity of Phaseolus vulgaris var. humilis at different days; D shows the effects of three treatments on the CAT enzyme activity of Phaseolus vulgaris var. humilis at different days; different letters in the diagram represent significant differences.

[0021] Figure 5 Diagram showing the growth promotion effects of three different treatments on Phaseolus vulgaris var. humilis plants; CK is the control group treated with water, F is the treatment group treated with chemical fertilizer (potassium dihydrogen phosphate), XGZN20 is the treatment group treated with Staphylococcus epidermidis XGZN20, A and D are Xiaguan, B and E are Qingguan, C and F are Jinguan, and the growth effects 15 days after inoculating the bacteria are shown in A, B, and C, and the growth effects 30 days after inoculating the bacteria are shown in D, E, and F.

[0022] Figure 6 Diagram showing the scanned root morphology at 15 days and 30 days of treatment. Among them, A, B, and C are the scanned root morphology diagrams of the Xiaguan variety treated with CK, F, and XGZN20 at 15 days and 30 days respectively; D, E, and F are the scanned root morphology diagrams of the Qingguan variety treated with CK, F, and XGZN20 at 15 days and 30 days respectively.

[0023] Figure 7 Diagram showing the effects of different treatments on the root growth of different varieties of Phaseolus vulgaris var. humilis. Among them, A and B are the root growth conditions of the Xiaguan variety after 15 days and 30 days of different treatments respectively; C and D are the root growth conditions of the Qingguan variety after 15 days and 30 days of different treatments respectively; CK is the control group treated with water, F is the treatment group treated with chemical fertilizer (potassium dihydrogen phosphate), and XGZN20 is Staphylococcus epidermidis XGZN20.

[0024] Figure 8It is a graph showing the changes in chlorophyll content, SOD, POD, and CAT activities in the leaves of Phaseolus vulgaris var. humilis under different varieties and treatments. Among them, A, B, C, and D respectively represent the effects of three treatments on chlorophyll, SOD activity, POD activity, and CAT enzyme activity in Phaseolus vulgaris var. humilis of Xiaguan variety at different days; E, F, G, and H respectively represent the effects of three treatments on chlorophyll, SOD activity, POD activity, and CAT enzyme activity in Phaseolus vulgaris var. humilis of Qingguan variety at different days; different letters in the graph represent significant differences.

[0025]

Preservation Information

[0026] Next, the technical solutions in the embodiments of the present invention will be described in conjunction with the accompanying drawings in the embodiments of the present invention, but not limited thereto.

[0027] In the following embodiments, the Staphylococcus epidermidis XGZN20 will be named XGZN20.

[0028] The required culture media and reagents are as follows:

[0029] Beef extract peptone liquid medium: 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of NaCl, and 1 L of water.

[0030] LB liquid medium: 5.0 g of yeast extract powder, 10.0 g of peptone, 10.0 g of NaCl, and 1 L of water.

[0031] Nitrogen-free liquid medium: KH 2 PO 4 0.2 g, MgSO 4 0.2 g, NaCl 0.2 g, CaCO 3 5.0 g, CaSO 4 0.1 g, 10.0 g of mannitol, and 1 L of water.

[0032] NBRIP medium: Ca 3 (PO4) 2 5.0 g, MgCl 2 ·6H 2 O 0.2 g, (NH4) 2 SO 4 0.1 g, 10.0 g of glucose, and 1 L of water.

[0033] Alexandrov medium: MgSO 4 ·7H 2 O 0.5 g, Na 2 HPO 4 2.0, CaCO 3 0.1 g, FeCl 3 ·6H 2 O 0.005 g, potassium feldspar powder 1.0 g, sucrose 5.0 g, water 1 L.

[0034] MKB liquid medium: K 2 HPO 4 2.5 g, MgSO 4 ·7H 2 O 2.5 g, casein amino acids 5 g, glycerol 15 mL, water 1 L.

[0035] Salkowski colorimetric solution: 50 mL of 35% perchloric acid, 1 mL of 0.5 mol / L FeC1 3 solution.

[0036] CAS detection solution: Dissolve 0.07 g of chrome azurol S in 50 mL of water, then add 10 mL of 1 mmol / L FeCl3 solution to make solution A; dissolve 0.06 g of cetyltrimethylammonium bromide in 40 mL of water, this is solution B; slowly pour solution A along the wall of the cup into solution B and mix well to obtain the CAS detection solution.

[0037] Example 1 Isolation and identification of plant growth-promoting rhizobacteria XGZN20 from Phaseolus vulgaris var. hortensis.

[0038] Take the rhizosphere soil from the Phaseolus vulgaris var. hortensis planting area of Qiqihar University, place the collected rhizosphere soil in an ice box and transport it back to the laboratory and immediately store it in an 80°C refrigerator.

[0039] Weigh 1.0 g of Phaseolus vulgaris var. hortensis rhizosphere soil, prepare a soil suspension in a triangular flask under sterile conditions, after gradient dilution, place the soil suspensions at different concentrations (10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 ) on a vortex shaker to mix well, then take 50 μL of the suspensions at the above gradients and spread them on solid Mongina medium, solid Alexandrov medium, solid Ashby medium, and solid associative nitrogen-fixing culture medium plates respectively. The spread plates are inverted and cultured in an incubator at 30°C for 1 - 2 days, and finally this strain of free-living nitrogen-fixing bacteria is isolated and purified from the solid Ashby medium and named XGZN20.

[0040] The sequencing results of strain XGZN20 were subjected to gene BLAST alignment in the NCBI (National Center of Biotechnology Information) database, and a phylogenetic tree was constructed.

[0041] The results are as Figure 1 shown. The isolated strain had the closest genetic evolutionary distance to Staphylococcus epidermidis FL27. Combining its cell morphology and colony characteristics, XGZN20 was identified as Staphylococcus epidermidis, and classified and named as Staphylococcus epidermidis XGZN20. It was deposited in the China Center for Type Culture Collection (CCTCC), Wuhan University, with the deposit number CCTCC NO: M 2024036, and the deposit date was January 8, 2024.

[0042] Example 2 Growth-promoting characteristics of XGZN20.

[0043] 1) Determination of nitrogen fixation ability.

[0044] XGZN20 was inoculated into a nutrient broth liquid medium and cultured in a constant temperature shaker at 120 r·min -1 , 30 °C for 24 h to obtain a seed solution. It was inoculated into 100 mL of nitrogen-free liquid medium at an inoculation amount of 1%, and cultured in a constant temperature shaker at 120 r·min -1 , 30 °C for 7 d. Centrifuge, retain the supernatant, and measure the nitrogen content in the supernatant using a carbon and nitrogen analyzer.

[0045] 2) Determination of phosphorus solubilization ability.

[0046] The soluble phosphorus content in the fermentation broth was determined by the molybdenum-antimony anti-colorimetric method. The XGZN20 seed solution was inoculated into 100 mL of NBRIP liquid medium at an inoculation amount of 1%, and cultured in a constant temperature shaker at 120 r·min -1 , 30 °C for 7 d. Measure the absorbance values of standard solutions with phosphorus contents of 0, 0.2, 0.4, 0.8, 1.0 mg·L -1 at a wavelength of 700 nm, draw a standard curve, and calculate the phosphorus content of the corresponding strain.

[0047] 3) Determination of potassium solubilization ability.

[0048] The XGZN20 seed solution was inoculated into 100 mL of potassium-solubilizing liquid fermentation medium at an inoculation amount of 1%, and cultured in a constant temperature shaker at 120 r·min -1, Incubate it on a constant temperature shaker at 30 °C for 7 days with shaking. Take 10 mL of XGZN20 culture solution, add 2 mL of 6% hydrogen peroxide, and then nitrify it in a boiling water bath for 1 h. Take the nitrified solution and centrifuge it at 10000 r·min -1 for 10 min, and take the supernatant to measure the soluble potassium content with a flame spectrophotometer.

[0049] 4) Determination of IAA secretion ability.

[0050] Use the Salkowski colorimetric solution colorimetry method. Prepare a 200 mg·L -1 standard solution with analytical pure IAA and dilute it to obtain concentrations of 0, 25, 50, 75, 100, 125, 150, 175 μg·mL -1 . Take 5 mL of each, use distilled water as a control, add an equal volume of Salkowski colorimetric solution, let it stand in the dark for 30 min, measure the absorbance value (OD530), and draw a standard curve. Inoculate the XGZN20 seed solution at an inoculation amount of 1% into LB liquid medium containing 200 mg / L tryptophan and culture it for 2 d (at 30 °C, 140 r·min -1 ), then centrifuge it at 4000 r·min -1 for 10 min. Take 5 mL of the supernatant and add an equal volume of Salkowski colorimetric solution. Repeat each group 3 times, and measure the absorbance value (OD530) after standing in the dark for 30 min.

[0051] 5) Determination of siderophore synthesis ability.

[0052] Use the CAS detection solution detection method. Inoculate the XGZN20 seed solution at an inoculation amount of 1% into MKB liquid medium, and culture it at 30 °C, 140 r·min -1 for 2 d, then centrifuge it at 4000 r·min -1 for 10 min. Take 3 mL of the supernatant, add an equal volume of CAS detection solution, mix well and let it stand for 1 h, then measure the absorbance value (OD630) (As). The control group is deionized water and CAS detection solution (Ar), and repeat each group 3 times. The determination of siderophore content is calculated according to the formula: siderophore activity unit (%) = (Ar - As) / Ar × 100 to calculate the siderophore yield.

[0053] 6) Determination of gibberellin secretion ability.

[0054] Dissolve analytical pure gibberellin in absolute ethanol with a volume fraction of 70% to prepare a gibberellin standard solution of 100 μg / mL and gradient dilute it to 0, 10, 30, 40, 50, 60 μg / mL. Take 0.5 mL of each concentration of gibberellin standard solution, mix it with 4.5 mL of concentrated sulfuric acid, and then place it in an ice bath for 10 min. After water bath at 28 °C for 1 h, let it stand at room temperature for 15 min, measure the absorbance value (OD412) and draw a standard curve. Transfer the XGZN20 seed solution to LB liquid medium according to an inoculation amount of 1%, and culture it at 30 °C and 140 r·min -1 After culturing for 2 days at 10000 r·min -1 Centrifuge for 10 min, measure the gibberellin content in its supernatant, and set 3 parallels for each group.

[0055] 7) The measurement results are shown in Table 1:

[0056] Table 1

[0057]

[0058] Example 3 Growth promotion effect of XGZN20 on kidney bean.

[0059] 1) Materials: Heidajinguan

[0060] Select seeds with plump grains, complete embryos, and no mildew. Soak them in 75% alcohol for 1 min, then soak them in 5% NaClO solution for 3 - 5 min, and then rinse the seeds with sterile water until the smell of NaClO is no longer detectable. Place the seeds in a sterilized glass plate lined with filter paper, spray sterile water to moisten the filter paper, keep it moist, and place it in a light incubator at 24 °C for dark culture for 3 days. When the seeds germinate to 1 - 2 cm, plant them in flower pots with vermiculite: nutrient soil = 2:1. After the plants grow two true leaves, irrigate 10 mL of bacterial liquid per plant, and re-irrigate once every 10 days for a total of 3 times. Observe and measure the morphological indexes such as the plant height, upper and lower dry and fresh weights, stem diameter, and main root length of kidney bean at 15 days and 30 days, and physiological indexes such as chlorophyll, SOD, POD, and CAT. Analyze the root volume, connection number, node number, root tip number, and bifurcation number through a root scanner and observe the root morphology.

[0061] 2) Influence of XGZN20 on the morphological indexes of kidney bean

[0062] Collect the Jinguanyou kidney bean plants 15 days and 30 days after applying the bacterial agent, measure the plant height, upper fresh weight, lower fresh weight, stem diameter, upper dry weight, and lower dry weight of the seedlings, and analyze their root volume, surface area, total root length, root tip number, and bifurcation number with a root scanner.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] Its growth-promoting effect is shown in Table 1 and Table 2. 15 days and 30 days after treatment, the plant height, fresh weight of upper part, fresh weight of lower part, stem diameter, dry weight of upper part, and dry weight of lower part of the kidney beans treated with XGZN20 were significantly higher than those of the control group. Compared with the control group, the plant height of XGZN20 increased by 28.7% - 63.0%, the fresh weights of the upper and lower parts increased by 43.8% - 94.2% and 37.4% - 123.0% respectively, the dry weights of the upper and lower parts increased by 77.1% - 117.9% and 52.6% - 123.1% respectively, and the stem diameter increased by 17.2% - 38.9%. There was no significant difference in the fresh weight of the upper part, fresh weight of the lower part, and dry weight of the upper part between the chemical fertilizer group F (potassium dihydrogen phosphate fertilizer) and the water group CK. There was no significant difference in the stem diameter between F and CK at 15 days.

[0068] Figure 2 They are the root scanning morphology diagrams at 15d and 30d of treatment, and it can be seen that there are significant differences in their root morphology.

[0069] Figure 3 The results showed that the main root length, root volume, connection number, node number, root tip number, and bifurcation number of the kidney beans treated with XGZN20 were significantly higher than those of the control group, and the chemical fertilizer group was higher than the water group.

[0070] In summary, XGZN20 has the best growth-promoting effect on kidney beans.

[0071] 3) The effect of XGZN20 on the physiological indexes of kidney beans.

[0072] Collect the fresh leaves of kidney beans at 105d and 30d after applying XGZN20 bacterial fertilizer, and measure the chlorophyll, SOD, POD, and CAT indexes of the kidney bean leaves.

[0073] Determination of chlorophyll content:

[0074] Weigh 5g of leaves, cut them into small pieces, place them in a mortar, and add 5ml of 95% ethanol solution to grind them into a homogeneous paste. Make a cotton ball with absorbent cotton and place it at the neck of the funnel. The size and looseness should be such that water can flow through, and water can drip down without washing away the dough. Rinse the filter residue and mortar with 95% ethanol solution and collect them into the filtrate. Dilute the filtrate to 25ml with 95% ethanol solution, mix well, and set aside. Take an appropriate amount of the diluted solution and measure the absorbance at wavelengths of 663nm and 645nm. Use 95% ethanol solution as a control to calculate the chlorophyll content (mg·g -1 ).

[0075] Chl a = 12.7A663 - 2.69A645

[0076] Chl b = 22.9A645 - 4.86A663

[0077] CV = Chl a + Chl b

[0078] Determination of superoxide dismutase (SOD) activity by nitroblue tetrazolium photoreduction method:

[0079] (1) Solution preparation

[0080] 14.5 mM methionine solution: Take 2.1637 g of Met and make up the volume to 1000 ml with phosphate buffer (pH 7.8).

[0081] 30 μM EDTA-Na 2 solution: Take 0.001 g of EDTA-Na 2 and make up the volume to 100 ml with phosphate buffer.

[0082] 60 μM riboflavin solution: Take 0.0023 g of riboflavin and make up the volume to 100 ml with phosphate buffer, store in the dark.

[0083] 2.25 mM nitroblue tetrazolium (NBT) solution: Take 0.1840 g of NBT and make up the volume to 100 ml with PBS, store in the dark.

[0084] (2) Enzyme solution preparation

[0085] Take 0.2 g of kidney bean leaves, place them in a pre-cooled mortar, add 2 ml of 0.05 mol·L -1 phosphate buffer (pH = 7.8), grind them into a homogenate in an ice bath, make up the volume to 10 ml, transfer them to a centrifuge tube, and centrifuge at 4°C and 12000 g for 20 min. The supernatant is the enzyme solution.

[0086] (3) Preparation of reaction mixture (for 10 samples)

[0087] Take 2.7 ml of Met solution, 0.1 ml of EDTA-Na 2 solution, 0.9 ml of phosphate buffer, 1 ml of NBT solution, and 1 ml of riboflavin solution respectively, mix well and set aside for use.

[0088] (4) Enzyme activity determination

[0089] Take 3 ml of reaction mixture and 30 μl of enzyme solution in test tubes respectively, repeat three times. Place the test tubes in a light incubator and react for 20 min under 4000 lux light. At the same time, make two control tubes. One test tube takes 3 ml of reaction mixture and adds 30 μl of PBS (without enzyme solution), and measure it after light irradiation as the maximum light reduction tube. The other test tube only adds buffer and is used for zero adjustment when measured in the dark. Measure the absorbance value at 560 nm wavelength in the dark.

[0090] (5) Calculation of enzyme activity

[0091]

[0092] The total activity of SOD is expressed as enzyme units per gram of fresh weight (U·g -1 ·FW).

[0093] Where: ACK: Absorbance value of the control cup (illuminated); AE: Absorbance value of the sample; V: Total volume of the sample solution (ml); Vt: Volume of the sample during determination (ml); W: Sample weight (g)

[0094] Determination of peroxidase (POD) activity by guaiacol method:

[0095] (1) Preparation of enzyme solution

[0096] Weigh 0.2 g of Phaseolus vulgaris leaves, add 2 mL of 0.2 mol·L -1 phosphate buffer (pH = 6.0), put it into a pre-cooled mortar and grind it into a homogenate, then make up the volume to 10 ml, transfer it to a centrifuge tube, centrifuge at 4 °C and 12000 g for 20 min, and the supernatant is the enzyme solution.

[0097] (2) Preparation of reaction mixture (for 10 samples)

[0098] Take 33.33 ml of PBS, add 0.0127 ml of guaiacol stock solution, heat and stir to dissolve, after cooling, add 0.019 ml of 30% H2O2, mix well and store in the refrigerator for later use.

[0099] (3) Sample determination

[0100] Use PBS as a control to zero the instrument, measure the absorbance at a wavelength of 470 nm (measure for 40 s). Take 3 ml of the reaction solution and add 30 μl of the enzyme solution (add the sample while measuring, wait for 5 s before measurement, and be quick. If it is slow, ensure that the time difference from adding the sample to starting the timer for each sample is not large), and read the value every 30 s.

[0101] (4) Calculation of enzyme activity

[0102]

[0103] The unit is (U·g-1·FW).

[0104] Where: △A470: Change in absorbance during the reaction time; t: Reaction time (min); VT: Total volume of the extracted enzyme solution (mL); VS: Volume of the enzyme solution used during determination (mL); m: Fresh weight of the sample

[0105] Determination of catalase (CAT) activity:

[0106] (1) Preparation of enzyme solution

[0107] Take 0.2 g of Phaseolus vulgaris var. hultianum leaves and place them in a pre-cooled mortar. Add 2 mL of 0.15 mol·L -1 phosphate buffer solution (pH = 7.0), grind them into a homogenate in an ice bath, make up the volume to 10 mL, transfer it to a centrifuge tube, and centrifuge at 4 °C and 12,000 g for 20 min. The supernatant is the enzyme solution.

[0108] (2) Preparation of reaction solution

[0109] Take 200 mL of PBS, add 0.3092 mL of 30% H 2 O 2 (stock solution), and shake well.

[0110] (3) Sample determination

[0111] Zero the absorbance with PBS as a control, and measure the absorbance at a wavelength of 240 nm (measure for 40 s). Take 3 mL of the reaction solution and add 0.1 mL (adjustable according to the situation) of the enzyme solution. (Add the sample while measuring, wait for 5 s before measurement,) and read the value every 30 s.

[0112] (4) Calculation of enzyme activity

[0113]

[0114] The unit is (U·g -1 ·FW).

[0115] In the formula: △A470: the change in absorbance during the reaction time; T: the reaction time (min); VT: the total volume of the extracted enzyme solution (mL); VS: the volume of the enzyme solution used during measurement (mL); m: the fresh weight of the sample

[0116] The growth-promoting effect of XGZN20 on the physiological indexes of Phaseolus vulgaris var. hultianum:

[0117] The results are as Figure 4 shown. The chlorophyll content, SOD, POD, and CAT activities of Phaseolus vulgaris var. hultianum after treatment with XGZN20 are significantly higher than those of the control group. Except for the non-significant difference in CAT activity between the fertilizer group F ((potassium dihydrogen phosphate fertilizer)) and the CK group at 30 days of treatment, there are significant differences in the other groups. In summary, XGZN20 has the best growth-promoting effect on Phaseolus vulgaris var. hultianum.

[0118] The strain described in the present invention has growth-promoting characteristics such as nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA production, and siderophore production, can promote the growth of Phaseolus vulgaris var. hultianum, and is manifested in the increase in plant height, upper and lower dry and fresh weights, stem diameter, main root length, root volume, connection number, node number, root tip number, and bifurcation number, as well as the increase in chlorophyll content, AOD, POD, and CAT activities.

[0119] Example 4

[0120] Growth promotion effect of XGZN20 on different varieties of kidney beans

[0121] Place the seeds of Xiaguan and Qingguan into a sterilized glass plate lined with filter paper, spray sterile water to moisten the filter paper, keep it moist, and place it in a light incubator at 24°C for dark culture for 3 days. When the seeds germinate to 1-2 cm, soak them in the XGZN20 fermentation broth for 2 hours, and then plant them in flower pots with vermiculite: nutrient soil = 2:1. After two true leaves grow, pour 10 mL of the bacterial liquid per plant, and re-pour it every 10 days for a total of 3 times. The effect diagrams are as shown in Figure 5 , which respectively show the effect diagrams of kidney bean growth at 15 and 30 days. Among them, ABC are the effect diagrams at 15 days, and DEF are the effect diagrams at 30 days. Measure the growth indexes (plant height, upper fresh weight, lower fresh weight, stem diameter, upper dry weight, lower dry weight), root morphology (root volume, surface area, total root length, root tip number, and bifurcation number), and physiological indexes (chlorophyll content, SOD, POD, CAT) of Xiaguan and Qingguan at 15d and 30d

[0122] As shown in Figure 5 , in addition to promoting the growth of Jinguan, XGZN20 also has an obvious growth promotion effect on Xiaguan and Qingguan

[0123] Collect the Xiaguan and Qingguan kidney bean plants at 15d and 30d after applying the bacterial agent, and measure the plant height, upper fresh weight, lower fresh weight, stem diameter, upper dry weight, and lower dry weight of the seedlings; use a root scanner to analyze their root volume, surface area, total root length, root tip number, and bifurcation number

[0124] Table 4

[0125]

[0126] Table 5

[0127]

[0128] Table 6

[0129]

[0130] Table 7

[0131]

[0132] Its growth promotion effects are shown in Tables 4 to 7, which are the growth indexes of seedlings of Xiaguan and Qingguan 15 days and 30 days after treatment. The plant height, fresh weight above ground, fresh weight below ground, stem diameter, dry weight above ground, and dry weight below ground of the kidney beans treated with XGZN20 are significantly higher than those of the control group and the chemical fertilizer group. Tables 4 and 5 show the growth of kidney beans of the Xiaguan variety 15 days and 30 days after treatment. XGZN20 is significant compared with the control group and the chemical fertilizer group. The plant height increased by 29.1% - 60.1%, the fresh weight above and below ground increased by 17.7% - 110.3% and 23.0% - 93.9% respectively, the dry weight above and below ground increased by 24.0% - 125.0% and 34.8% - 191.7% respectively, and the stem diameter increased by 13.3% - 45.4%. Tables 5 and 6 are for the Qingguan variety. The plant height increased by 38.0% - 54.2%, the fresh weight above and below ground increased by 25.6% - 80.9% and 34.9% - 81.0% respectively, the dry weight above and below ground increased by 21.9% - 92.5% and 25.0% - 118.8% respectively, and the stem diameter increased by 23.0% - 93.9%.

[0133] Figure 6 Figures are the root scanning morphology diagrams for treatments at 15d and 30d, and it can be seen that there are significant differences in their root morphology. Among them, A, B, and C are the root scanning morphology diagrams of the Xiaguan variety CK, F, and XGZN20 at 15d and 30d respectively; D, E, and F are the root scanning morphology diagrams of the Qingguan variety CK, F, and XGZN20 at 15d and 30d respectively. It can be seen from the figures that there are significant differences in their root morphology, and XGZN20 has the best effect on promoting root growth.

[0134] Figure 7 Figure shows the effects of different treatments on the root growth of kidney beans of different varieties. Among them, A and B are the root growth conditions of the Xiaguan variety after different treatments for 15 days and 30 days respectively; C and D are the root growth conditions of the Qingguan variety after different treatments for 15 days and 30 days respectively; CK is the water treatment group, F is the chemical fertilizer (potassium dihydrogen phosphate) treatment group, and XGZN20 is the Staphylococcus epidermidis treatment group. The results show that the main root length, root volume, connection number, node number, root tip number, and bifurcation number of the kidney beans treated with XGZN20 are significantly higher than those of the control group, and the chemical fertilizer group is higher than the water group, indicating that XGZN20 has the best effect.

[0135] Figure 8Figure showing the changes in chlorophyll content, SOD, POD, and CAT activities in the leaves of different varieties of kidney beans under different treatments. Among them, A, B, C, and D respectively represent the effects of three treatments on chlorophyll, SOD activity, POD activity, and CAT enzyme activity in the Xiaguan variety of kidney beans at different days; E, F, G, and H respectively represent the effects of three treatments on chlorophyll, SOD activity, POD activity, and CAT enzyme activity in the Qingguan variety of kidney beans at different days; different letters in the figure represent significant differences. The results show that after the treatment of the XGZN20 group, the chlorophyll content, SOD, POD, and CAT activities of the Xiaguan and Qingguan kidney beans are significantly higher than those of the control group; compared with the chemical fertilizer group F, except for the non-significant differences in chlorophyll and CAT activities in the Xiaguan variety at 30 days of treatment and non-significant differences in chlorophyll in the Qingguan variety at 30 days of treatment, there are significant differences in the other groups. In summary, XGZN20 has the best growth-promoting effect on kidney beans.

Claims

1. A strain of Staphylococcus epidermidis, characterized in that: The classification name of the Staphylococcus epidermidis is Staphylococcus epidermidis XGZN20, and the Staphylococcus epidermidis is deposited in the China Center for Type Culture Collection, the preservation address is Wuhan University, the preservation number is CCTCC NO: M 2024036, and the preservation time is January 8, 2024.

2. Use of the Staphylococcus epidermidis according to claim 1 in preparing carob microbial fertilizer.

3. A method for preparing carob microbial fertilizer, characterized in that: The Staphylococcus epidermidis described in claim 1 is inoculated into a beef extract peptone culture medium and cultured in a constant temperature shaker for 24 hours to obtain an activated bacterial solution, which is a carob microbial fertilizer.

4. The preparation method according to claim 3, characterized in that: The temperature of the constant temperature shaker culture is 25°C.

5. The preparation method according to claim 3, characterized in that The rotation speed of the constant temperature shaker culture is 140 r / min.

6. The preparation method according to claim 3, characterized in that: The OD600 value of the activated bacterial solution is 1.

0.

7. A carob microbial fertilizer, characterized in that: The carob microbial fertilizer is obtained by the preparation method described in any one of claims 3 to 6.

8. A method for cultivating snap beans, characterized in that: The cultivation method uses the carob microbial fertilizer according to claim 7. In the cultivation method, the carob seeds are planted after they germinate to 1-2 cm. After planting, each carob seedling that has grown two true leaves is watered with 10 mL of the carob microbial fertilizer, and watered again every 10 days for a total of 3 times.

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