Phosphorus-dissolving and potassium-dissolving low-temperature-resistant burkholderia sp. And application thereof

Abstract

The invention discloses low-temperature-resistant burkholderia capable of dissolving phosphorus and potassium and application of the low-temperature-resistant burkholderia capable of increasing nutrition of green vegetables, the low-temperature-resistant burkholderia capable of increasing nutrition of the green vegetables is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.27769. The low-temperature-resistant burkholderia sp. Capable of dissolving phosphorus and potassium is obtained by carrying out low-temperature stress screening and ultraviolet mutagenesis on phosphorus-dissolving and potassium-dissolving bacteria, the ultraviolet mutagenesis power is 15W, the irradiation distance is 30cm, and the irradiation time is 30s. The low-temperature-resistant burkholderia capable of dissolving phosphorus and potassium has the effects of enriching soil nutrition and increasing nutrition of green vegetables.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to low-temperature-resistant Burkholderia bacteria capable of solubilizing phosphorus and potassium and an application thereof. Background Art

[0002] Phosphorus-solubilizing and potassium-solubilizing bacteria are microorganisms that can decompose available phosphorus and potassium in the soil, which can promote plant growth and development and the absorption and utilization of these nutrients by plants, and play an important role in improving vegetable yield and quality. Studies have shown that there are three ways for bacteria to dissolve phosphorus: the first is to secrete H + , phosphate and H + exchange; the second is to secrete organic acids, or to convert Ca 2+ and Fe 3+ etc. to complex with organic acids; the third is to produce phosphatase. All three methods can reduce the pH value in the system and increase the acidity. The first and second methods promote the dissolution reaction of originally insoluble phosphates, degrading insoluble phosphates into effective phosphorus, and the third method degrades organic phosphorus into effective phosphorus through enzymatic reactions. Potassium-dissolving bacteria can decompose aluminum silicates, potassium feldspar and apatite minerals in the soil, promote the conversion of insoluble nutrient elements such as potassium, phosphorus, and magnesium into soluble nutrients, and increase the content of fast-acting nutrients in the soil. Therefore, bacteria with the function of dissolving phosphorus and potassium play an important role in microbial fertilizers.

[0003] Studies have shown that the application of phosphate-solubilizing and potassium-solubilizing bacteria can significantly improve the yield and quality of leguminous vegetables. However, there are not many varieties of phosphate-solubilizing and potassium-solubilizing bacteria that are resistant to low temperatures. According to Morito's definition, low-temperature bacteria usually refer to microorganisms whose optimal growth temperature does not exceed 20°C. They can survive under extreme low temperature conditions and can adapt to and respond to environmental changes. Especially in northern my country, the low temperature period is relatively long. The lack of low-temperature resistant Burkholderia fertilizers has affected the yield and quality of crops. Therefore, the study of low-temperature microbial fertilizers is not only of great theoretical significance, but also has important application value in production practice and environmental protection. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a low-temperature resistant Burkholderia that can solubilize phosphorus and potassium and its application in view of the deficiencies of the above-mentioned prior art. The low-temperature resistant Burkholderia that can solubilize phosphorus and potassium can significantly increase the content of available phosphorus and available potassium in the soil, significantly promote the activities of urease, protease and catalase in the soil, reduce the content of nitrite in green vegetables, increase the content of vitamin C in green vegetables and increase the content of soluble protein in green vegetables, and has the effect of enriching soil nutrition and increasing the nutrition of green vegetables.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperature, characterized in that it is classified and named as Burkholderia, Burkholderia, and is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 27769 and a deposit date of July 3, 2023.

[0006] The above-mentioned Burkholderia that can solubilize phosphate and potassium and can withstand low temperatures is characterized in that the Burkholderia that can solubilize phosphate and potassium and can withstand low temperatures is a low-temperature resistant Burkholderia obtained after low-temperature stress screening and ultraviolet mutagenesis of phosphate and potassium solubilizing bacteria; the ultraviolet mutagenesis power is 15W, the irradiation distance is 30cm, and the irradiation time is 30s.

[0007] The application of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperature in promoting plant growth.

[0008] The use of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth is characterized in that it is used to increase the enzyme content in soil.

[0009] The use of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth is characterized in that the enzyme includes urease, protease and / or catalase.

[0010] The use of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth is characterized by increasing the content of available nitrogen, available phosphorus and / or available potassium in the soil.

[0011] The use of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth is characterized by increasing the nutrition of green vegetables.

[0012] The use of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth is characterized by reducing the nitrite content in green vegetables, increasing the vitamin C content in green vegetables and / or increasing the soluble protein content in green vegetables.

[0013] The use of the above-mentioned Burkholderia that can solubilize phosphorus and potassium and can withstand low temperatures in promoting plant growth is characterized in that it includes irrigating the roots of green vegetable seedlings with a bacterial liquid containing Burkholderia that can solubilize phosphorus and potassium and can withstand low temperatures to improve the nutrition of the green vegetables.

[0014] The above-mentioned application of a Burkholderia phosphate-solubilizing and potassium-solubilizing low-temperature-resistant bacterium in promoting plant growth is characterized in that the concentration of the Burkholderia phosphate-solubilizing and potassium-solubilizing low-temperature-resistant bacterium in the bacterial solution containing the Burkholderia phosphate-solubilizing and potassium-solubilizing low-temperature-resistant bacterium is 10 6 CFU / mL~10 3CFU / mL.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. The low-temperature resistant Burkholderia spp. of the present invention can significantly increase the content of available phosphorus and available potassium in the soil, significantly promote the activities of urease, protease and catalase in the soil, reduce the content of nitrite in green vegetables, increase the content of vitamin C in green vegetables and / or increase the content of soluble protein in green vegetables, and has the effect of enriching soil nutrition and increasing the nutrition of green vegetables.

[0017] 2. The low-temperature-resistant Burkholderia of the present invention can adapt to extreme low-temperature environments after being subjected to low-temperature stress, and has wide application and promotion value.

[0018] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0019] Instruction Manual

[0020] Figure 1.1 For the morphological identification of potassium-solubilizing bacteria; Figure 1.2 is the potassium standard curve; Figure 1.3 is the phosphorus standard curve; Figure 1.4 Comparison of the survival rate of BZY1-2 bacteria induced by ultraviolet light; Figure 1.5 This is the PCR picture of BZY1-B strain; Figure 1.6 The OD value was measured for the available phosphorus content of BZY1-B bacteria after subculture; Figure 1.7 The OD value was measured for the effective potassium content of BZY1-B bacteria after subculture; Figure 1.8 is the survival rate of BZY1-B bacteria; Figure 2.1 The nitrogen regression equation in the effect of low temperature resistant mutant strain BZY1-B on soil urease activity; Figure 2.2 The nitrogen regression equation for the effect of low temperature resistant mutant BZY1-B strain on soil protease activity; Figure 3.1 is the standard curve of available phosphorus; Figure 3.2 is the standard curve of effective potassium; Figure 3.3 is the standard curve of ammonium nitrogen; Figure 3.4 is the standard curve of nitrate nitrogen; Figure 4.1 is the standard curve of nitrite content; Figure 5 The standard curve of soluble protein content was obtained. DETAILED DESCRIPTION

[0021] 1. Selection of strains

[0022] 1.1 Isolation, purification and screening of strains

[0023] Step 101: Place 1 g of soil in 9 mL of sterile water, let stand for 30 minutes, and then shake at room temperature until fully mixed to obtain 10 -1 Dilute the suspension; the soil used is 4 portions, all from the wheat field of Fengjia Village, Lantian County, Xi'an City, Shaanxi Province. The soil is yellow sandy, and the soil is taken from 10 cm below the surface and dried for later use;

[0024] Step 102, take 1.0 mL of 10 -1 Dilute the suspension, add 9 mL of sterile water, and shake at room temperature until fully mixed to obtain 10 -2 Dilute the suspension, and so on, to prepare 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 and 10 -10 dilute the suspension;

[0025] Step 103, using the spread plate method, inoculating 0.1 mL of the diluted suspension at each dilution multiple onto a potassium-dissolving bacteria culture medium, culturing at 30° C. until a potassium-dissolving zone is formed around the plate, separating and screening, and obtaining 9 strains of potassium-dissolving bacteria; the potassium-dissolving bacteria culture medium comprises: 10 g of glucose, 5 g of CaCO3, 0.2 g of Na2HPO4, 0.2 g of MnSO4 7H2O, 0.2 g of CaSO42H2O, 0.2 g of NaCL, 18 g of agar, 10 g of potassium feldspar, adding distilled water to 1000 mL, and the pH of the potassium-dissolving bacteria culture medium is 7.2;

[0026] Step 104, according to the dilution multiple and the plate number, they are named BZY1-2, BZY1-3, BZY3-2, BZY5-1, BZY6-2, BZY7-1, BZY7-2, BZY7-3 and BZY7-4 respectively; for example, BZY1-2 is the dilution multiple of 10 -1 , strain isolated from plate No. 2;

[0027] The soil is from the wheat field of Fengjia Village, with an ammonium nitrogen content of 2.08 ppm, a nitrate nitrogen content of 0.15 ppm, an available phosphorus content of 33.59 ppm, and an available potassium content of 57.72 ppm.

[0028] The following experiment also involves the soil of Fengjia Village Cherry Orchard. The ammonium nitrogen content in the soil of Fengjia Village Cherry Orchard is 0.02ppm, the available phosphorus content is 34.29ppm, and the available potassium content is 34.29ppm;

[0029] According to the second national soil survey and relevant standards, the above soil is grade five soil (there are six levels in the soil main nutrient classification standard, with grade six being the lowest and grade one being the highest).

[0030] 1.2 Morphological identification and physiological and biochemical detection of potassium-solubilizing bacteria

[0031] According to observation, Figure 1.1 As shown, the colonies of the screened potassium-solubilizing bacteria are round, with neat edges and moistness. The colonies are bright light yellow, sticky, and elastic. Gram staining shows that they are Gram-negative bacteria.

[0032] 1.3 Rescreening of potassium-solubilizing bacteria using the transparent circle method

[0033] Step 201, using the transparent circle method to measure the potassium-dissolving circle diameter (D / mm) and the colony diameter (d / mm) of potassium-dissolving bacteria, and calculating the ratio of potassium-dissolving circle to colony diameter (D / d), as shown in Table 1.1. According to Table 1.1, the (D / d) of the BZY1-2 strain is 1.32, and that of the BZY7-1 strain is 1.30, which is larger than that of other strains, indicating that the BZY1-2 and BZY7-1 strains have strong potassium-dissolving abilities.

[0034] Step 202, inoculating the two strains screened out on a nutrient agar medium to obtain a rescreened bacterial solution, and storing the rescreened bacterial solution at -4°C for subsequent experiments; the inoculation rate is 2%; the nutrient agar medium comprises: 10g of peptone, 3g of beef extract, 5g of NaCl, 15-20g of agar, and 1000mL of distilled water; the preparation method comprises: dissolving the remaining components except agar with distilled water, adding a sodium hydroxide solution with a mass percentage of 15% to correct the pH to 7.2-7.4, adding agar, heating and boiling to dissolve the agar, dispensing in conical flasks, and autoclaving at 121°C for 15 minutes to obtain a nutrient agar medium.

[0035] Table 1.1 Determination of potassium solubilization capacity of strains by transparent zone method

[0036]

[0037] 1.4 Determination of potassium dissolving capacity by sodium tetraphenylborate method

[0038] Step 301, put the improved potassium liquid fermentation medium into a triangular flask and sterilize it under high temperature and high pressure; the improved potassium liquid fermentation medium includes: 0.75g of carbon source, 10g of potassium feldspar, 0.15g of (NH4)2SO4, 0.30g of Na2HPO4, and distilled water is added to 1000mL; the preparation method includes sterilizing the distilled water system containing the carbon source, potassium feldspar, (NH4)2SO4 and Na2HPO4 under high pressure at 121°C for 15min to obtain a potassium bacterial liquid culture medium; the carbon source is starch, glucose or sucrose; in this determination process, the carbon source is sucrose;

[0039] Step 302, inoculating the stored rescreened bacterial solution into the sterilized improved potassium liquid fermentation medium according to a preset inoculation amount, and culturing the culture in a shaking flask at 30° C. and 180 rpm for 7 days to obtain a seed solution; the preset inoculation amount is 2%;

[0040] Step 303, centrifuge the seed solution at 2000 r / min for 20 min to remove the bacteria and residues, add 1 mL of masking agent formaldehyde-EDTA, shake well, accurately and quickly add 1 mL of sodium tetraphenylborate with a pipette, shake well immediately, let it stand for 15 min and shake well again, and perform colorimetry at a wavelength of 420 nm.

[0041] The standard curve drawing process includes:

[0042] Pipette 0, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mL of 3 wt% KCl standard solution into a 25 mL volumetric flask, add 1 mL of formaldehyde-EDTA masking agent and shake well, use a pipette to quickly and accurately add 1 mL of 0.01 mol / L sodium tetraphenylborate solution, shake well immediately, let stand for 15 min and shake well again, perform colorimetry at a wavelength of 420 nm, and use transmittance (OD 420 ) as the ordinate and potassium content (mg / L) as the abscissa to draw a standard curve, such as Figure 1.2 shown; according to Figure 1.2 It can be seen that the formula of potassium standard curve is y=0.3469x+0.0293(R 2 =0.9962).

[0043] The absorbance value at 420nm was compared with the standard curve, and the results are shown in Table 1.2. The results showed that the potassium contents of BZY1-2 and BZY7-1 strains were 12.249mg / L and 11.198mg / L, respectively, indicating that the two strains had a high potassium solubility ability (P<0.01).

[0044] CK is the blank control group, and CK is the bacterial solution after rescreening replaced with an equal amount of distilled water and inoculated into the sterilized modified potassium liquid fermentation medium.

[0045] Table 1.2 Determination of dissolved potassium content by sodium tetraphenylborate method

[0046]

[0047] 1.5 Determination of soluble phosphorus content by molybdenum antimony colorimetric method

[0048] The methods for determining the soluble phosphorus content of strains include:

[0049] Step 501, 150mL of improved phosphorus culture medium is placed in a conical flask for sterilization, and the rescreened bacterial solution is inoculated into the conical flask according to an inoculum amount of 2%, and cultured at 30°C and 180r / min for 7d, 5mL of the cultured system is taken, 50mL of a 10% NaHCO3 solution is added, and the mixture is shaken and placed in a constant temperature oscillating box at a temperature of 25°C and a frequency of about 180r / min for 30min, and the oscillated system is filtered with a phosphorus-free filter paper to obtain a filtrate; the improved phosphorus culture medium comprises: 10g of glucose, 20g of agar, 0.3g of sodium chloride, 0.3g of magnesium sulfate, 0.3g of potassium chloride, 0.03g of manganese sulfate tetrahydrate, 12g of tricalcium phosphate, 0.03g of ferrous sulfate heptahydrate, 0.5g of ammonium sulfate, and distilled water is added to 1000mL, and the pH value is 7.2;

[0050] Step 502, take 10 mL of the filtrate into a 50 mL colorimetric tube, add 5 mL of molybdenum antimony anti-color developing agent, shake, dilute to 25 mL with distilled water, place for color development for 30 minutes, and measure its absorbance at 700 nm with an ultraviolet spectrophotometer;

[0051] The drawing of phosphorus standard curve includes: measuring 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6mL of phosphorus standard solution respectively into a 50mL volumetric flask, adding 10mL of 10% NaHCO3 extract and 5mL of molybdenum antimony anti-color developing agent in turn, shaking, constant volume, leaving at room temperature for 30min, measuring its absorbance with an ultraviolet spectrophotometer with a wavelength of 700nm, and drawing a standard curve of absorbance and phosphorus content. The phosphorus standard curve is shown in Figure 1.3 , the formula y=0.6132x-0.0115(R 2 =0.9965).

[0052] The available phosphorus content was calculated according to the phosphorus standard curve formula, and the results are shown in Table 1.3. The results showed that the phosphorus solubilization capacity of the BZY1-2 strain reached 7.539 mg / L, and the phosphorus solubilization capacity of the BZY7-1 strain reached 6.119 mg / L. The BZY1-2 strain was 2.67 times that of the CK, indicating that the two strains had strong ability to dissolve available phosphorus.

[0053] CK is the blank control group, and CK is the bacterial solution after rescreening replaced with an equal amount of distilled water and placed in a 250 mL conical flask.

[0054] Table 1.3 Soluble phosphorus content of three strains

[0055]

[0056] 1.6 Screening of low-temperature resistant strains

[0057] The screening process of low-temperature resistant strains specifically includes:

[0058] Step 601, placing 1 μL of the preserved rescreened bacterial liquid in a culture bottle containing 10 mL of nutrient agar medium, placing it in a constant temperature oscillator at three temperatures of 4°C, 15°C, and 20°C, respectively, and culturing it at a speed of 30 r / min, with culturing at a temperature of 23°C as a control, observing the growth status after culturing for 48h and 72h, and recording the growth of the low-temperature stress strain, as shown in Table 1.4; the nutrient agar medium includes: 10g of peptone, 3g of beef extract, 5g of NaCL, 15-20g of agar, and 1000mL of distilled water; the preparation method includes: dissolving the remaining ingredients except agar with distilled water, adding a sodium hydroxide solution with a mass percentage of 15% to correct the pH to 7.2-7.4, adding agar, heating and boiling to dissolve the agar, dispensing it in conical flasks, and autoclaving it at 121°C for 15min to obtain a nutrient agar medium.

[0059] According to Table 1.4, after low temperature stress, the germination time of each group of strains was prolonged, up to 15 days at most. The growth state of BZY1-2 under low temperature stress of 4℃ and 15℃ was better than that of CK. The growth of strain BZY7-1 was severely inhibited under low temperature stress. BZY1-2 was initially screened as a low temperature resistant strain.

[0060] CK is the blank control group, and CK is the culture medium containing beef extract peptone in which the bacterial solution after rescreening is replaced with an equal amount of distilled water.

[0061] Table 1.4 Survival rate of three strains under low temperature stress

[0062]

[0063] 1.7 Results of UV mutagenesis of strains

[0064] The specific UV mutagenesis of strains includes:

[0065] Step 701: dilute the bacterial solution to 10 -6 , spread on the plate, add 0.1 mL of diluted bacterial solution to each plate; the bacterial solution is the bacterial solution corresponding to BZY1-2 bacteria after low temperature stress;

[0066] Step 702: Place the plate containing the bacterial solution in a clean bench, preheat it with a 15W ultraviolet lamp for 20 minutes, and irradiate it with the ultraviolet lamp for 0 seconds, 15 seconds, 30 seconds, 45 seconds, 60 seconds, 90 seconds, or 120 seconds at a preset distance; the preset distance is the distance from the plate to the ultraviolet lamp, and the preset distance is 20 cm or 30 cm;

[0067] Step 703: Wrap the irradiated plate with black cloth and place it in a 27°C incubator for 48 hours. Count the bacteria and calculate the survival rate under each condition. Perform a preliminary screening of the induced strains based on the survival rate. The results are as follows: Figure 1.4 As shown in Table 1.5;

[0068] Survival rate = (number of surviving colonies after treatment / number of surviving colonies in the control group) × 100%

[0069] According to Table 1.5, the ultraviolet mutagenesis BZY1 bacteria had an irradiation distance of 30 cm and an irradiation time of 30 s, and the highest survival rate reached 0.953, which was significantly different from the control group (P<0.05). The bacteria obtained after ultraviolet mutagenesis were named BZY1-B.

[0070] CK was the blank control group, and the bacterial solution after rescreening was replaced with an equal amount of distilled water and coated on the plate.

[0071] Table 1.5 Survival rate of BZY1-2 bacteria induced by ultraviolet radiation at an irradiation distance of 30 cm

[0072]

[0073] 1.8 Identification of 16s rDNA strain of cold-resistant mutant strain BZY1-B

[0074] BZY1-B obtained by UV mutagenesis was subjected to 16s rDNA identification. The results are as follows Figure 1.5 The identification process includes strain DNA extraction, PCR amplification, gel electrophoresis, purification and recovery, ligation, preparation of competent cells, transformation of ligation products, plasmid extraction and sequencing, and comparison of the purified 16SrDNA sequence with the ribosomal database. The identification work was completed by Sangon Biotechnology Co., Ltd. (Shanghai). The PCR reaction system is as follows: bacterial 16srDNA universal primers 27 (AGTTTGATCMTGGCTCAG), 1492

[0075] (GGTTACCTTGTTACGACTT) PCR amplification system was established for amplification. PCR reaction system Template (genomic DNA 20-50ng / nL) was 0.5μL, 10x buffer 2.5μL, dNTP (2.5mM each) 1μL, enzyme 0.2μL, F each (10μM) 1μL, R (10μM) 1μL plus double distilled water to 25μL, amplification program, 94℃ pre-denaturation for 4min, 94℃ denaturation for 45s, 55℃ annealing for 45s, 72℃ extension for 1min, a total of 30 cycles, 72℃ extension after 10min, and the reaction product was terminated at 4℃.

[0076] The sequencing results showed that the strain BZY1-B was 1392bp in size. The classification information of the strain was identified by the 16srDNA gene sequence analysis method based on molecular biological detection methods. The results of comparison in the ribosome database showed that the sequence similarity with Burkholderia reached 100%, indicating that the BZY1-B strain belongs to the Burkholderia genus.

[0077]

[0078] 1.9 Genetic stability of cold-resistant mutagenic strain BZY1-B

[0079] The BZY1-B bacteria were cultured continuously, and the effective phosphorus and potassium contents were determined by the molybdenum antimony colorimetric method and the sodium tetraphenylborate method. The genetic stability was also observed under various low temperature gradients, including:

[0080] Step 901, using the plate streak method to perform continuous subculture, specifically comprising: in a sterile environment, cooling the flame-sterilized inoculation loop, dipping the loop in the bacterial solution and streaking the surface of the plate culture medium for multiple times, culturing, obtaining a single colony, which is the first generation, picking up the first generation single colony, transferring it to a new plate culture medium for culturing, and obtaining a colony, which is the second generation, and so on;

[0081] Step 902, measuring 2.5 mL of the bacterial solution of the 5th, 10th, 15th, 20th and 25th generations, respectively, and determining the effective phosphorus content according to the above-mentioned molybdenum antimony colorimetric method; the inoculation amount is 2%;

[0082] Step 903, measuring 10 mL of the bacterial solution of the 5th, 10th, 15th, 20th and 25th generations, respectively, and determining the effective potassium content according to the above-mentioned sodium tetraphenylborate method; the inoculation amount is 2%;

[0083] The results showed that the OD value of the effective phosphorus content of the BZY1-B strain after UV mutagenesis was 0.890±0.033 ( Figure 1.6 ), the average OD value of the effective potassium content was 0.512±0.05( Figure 1.7 ). This indicates that after UV-induced mutagenesis, the BZY1-B strain still has the ability to solubilize phosphorus and potassium in the 5th, 10th, 15th, 20th and 25th generations.

[0084] Depend on Figure 1.8 It can be seen that after ultraviolet mutagenesis, the survival rate of BZY1-B strain at 4°C was 0.61±0.034, the survival rate at 10°C was 0.738±0.047, the survival rate at 15°C was 0.82±0.036, and the survival rate at 20°C was 0.961±0.059. This shows that the BZY1-B strain still has the ability to withstand low temperatures at the 5th, 10th, 15th, 20th, and 25th generations of passage, and is a low-temperature resistant strain.

[0085] 2 Effects of low temperature resistant mutant strain BZY1-B on soil enzyme content

[0086] After the BZY1-B bacteria were activated and cultured, they were diluted with a nutrient liquid culture medium to obtain BZY1-B bacterial suspensions with different dilution multiples; the dilution multiples were 100 times, 200 times, 300 times and 400 times. In the BZY1-B bacterial suspension with a dilution multiple of 100 times, the live bacterial count was 5.54×103 CFU / mL, in the 200-fold diluted BZY1-B bacterial suspension, the viable bacterial count was 2.77×10 3 CFU / mL, in the 300-fold diluted BZY1-B bacterial suspension, the viable bacterial count was 1.85×10 3 CFU / mL, the viable bacterial count in the 400-fold diluted BZY1-B bacterial suspension was 1.39×10 3 CFU / mL; nutrient liquid medium includes peptone 10g, beef extract 3g, NaCL 5g, distilled water 1000mL;

[0087] The enzyme activity index of the soil sample in the above-mentioned BZY1-B bacterial solution is determined; the soil sample includes experimental cultivation soil and soil from Fengjia Village wheat field, and the experimental cultivation soil and the soil from Fengjia Village wheat field are coarsely ground in ceramic mortars respectively to refine soil particles and remove large stones and plant branches and stems; the experimental cultivation soil is a mixture of nutrient soil and sandy soil in a ratio of 1:1, and the nutrient soil is composed of 50% peat, 20% coconut bran, 10% carbonized rice husks and 20% perlite.

[0088] 2.1 Effect of cold-tolerant mutant strain BZY1-B on soil urease activity

[0089] 2.1.1 Nitrogen standard curve

[0090] With the nitrogen standard solution concentration gradient X as the horizontal axis and the OD value Y as the vertical axis, draw the nitrogen standard curve and obtain the regression equation, as shown in Table 2.1 and Figure 2.1 shown.

[0091] The methods for drawing the standard curve include:

[0092] Dissolve 0.4717g of ammonium sulfate in distilled water and dilute to 1000mL to obtain a nitrogen standard solution with a concentration of 0.1mg / mL. Take 0, 1, 3, 5, 7, 9, and 11mL of the nitrogen standard solution and put them into conical flasks respectively. Add 4ml of sodium phenol solution and 3mL of 0.9% sodium hypochlorite solution by mass, shake well, add distilled water to the 20mL mark of the conical flask, wait for 20min, and obtain a nitrogen standard solution with a concentration gradient of 0, 0.2, 0.6, 1.0, 1.4, 1.8, and 2.2μg / mL. Use a spectrophotometer to compare colors at 578nm, and draw a standard curve based on the density value and solution concentration. According to Figure 2.1 From Table 2.1, we can see that the regression equation is Y = 0.6732X + 0.0024 (R 2 =0.9999).

[0093] Table 2.1 OD values ​​of nitrogen standard solution

[0094]

[0095] 2.1.2 Sample determination

[0096] The cultivated soil and wheat field soil after refinement and impurity removal were divided into two groups, each of which was divided into 6 subgroups; the soil weight in each subgroup was 5g;

[0097] Add 1 mL of toluene to each group, let it stand for 15 minutes, add 10 mL of urea solution with a mass percentage of 10% and 20 mL of citrate buffer with a mass percentage concentration of 20% at pH = 6.7, stir evenly, add 1 mL of BZY1-B bacterial solution with different dilution multiples, and culture in a 37°C constant temperature box. After culturing for a preset time, take a sample, distill and filter at 38°C, and take the filtrate; the preset time is 3h, 6h, 9h or 12h;

[0098] The urease content of the soil sample was measured every three hours, and the results are shown in Table 2.2 and Table 2.3; the method for determining the enzyme activity index includes: placing 3 mL of the filtrate in a 50 mL volumetric flask, adding 17 mL of distilled water, 4 mL of a 1.35 mol / L sodium phenol solution and 3 mL of a 0.9% mass percent sodium hypochlorite solution, leaving it for 20 minutes, diluting it to the 50 mL mark with distilled water, and sampling for colorimetry at 578 nm;

[0099] Urease activity calculation formula: U = A × B

[0100] Where: U is urease activity mg; A is the concentration of nitrogen standard curve mg / mL; B is the coefficient converted to 1g soil;

[0101] CK was the blank group, i.e., an equal amount of distilled water was added to the soil, and the no-matrix group was sandy soil;

[0102] Table 2.2 Effect of BZY1-B bacterial agent on urease activity in experimental cultivation soil

[0103]

[0104] (Table * Indicates significant difference P < 0.05; ** Indicates extremely significant difference (P<0.01)

[0105] Table 2.2 shows that different dilution multiples have significant differences in the analysis results (P < 0.05). Compared with CK and no matrix, after adding the dilution of BZY1-B bacterial agent, the urease activity in the cultivation soil generally increased, and the promotion effect increased with the increase of concentration. When the 100-fold diluted BZY1-B bacterial agent was added to the cultivation soil, the urease activity was the highest at 12h of cultivation, with an activity of 0.782IU, which was 20% higher than the 100-fold dilution at 3h of cultivation, and the results were significantly affected by CK (P < 0.01).

[0106] Table 2.3 Effect of BZY1-B on soil urease activity in Fengjia Village wheat fields

[0107]

[0108] (Table * Indicates significant difference P < 0.05; ** Indicates extremely significant difference (P<0.01)

[0109] Table 2.3 shows that different dilution multiples have significant differences in the analysis results (P < 0.05). Compared with CK and no matrix, after adding the diluted solution of BZY1-B bacterial agent, the urease activity in the soil of Fengjia Village wheat field generally increased, and the promotion effect increased with the increase of concentration. When the 100-fold diluted BZY1-B bacterial agent was added to the soil solution of Fengjia Village wheat field, the urease activity was the highest at 12h of culture, with an activity of 0.577IU, which was 20% higher than the 100-fold diluted solution at 3h of culture, and the result had a very significant effect (P < 0.01) compared with CK.

[0110] 2.2 Effect of low-temperature-resistant mutant BZY1-B on soil protease activity

[0111] 2.2.1 Nitrogen standard curve

[0112] The drawing of the nitrogen standard curve includes:

[0113] Dissolve 10 g of casein in 1000 mL of phosphate buffer with a pH of 7.4 to obtain a glycine solution, take 0, 1, 3, 5, 7, 9, and 11 mL of the glycine solution and place them in 50 mL volumetric flasks, add 1 mL of a 2% ninhydrin solution by mass to each flask, heat in a boiling water bath for 10 min, add water to the scale value, and obtain gradient standard solutions with glycine concentrations of 0, 0.2, 0.6, 1.0, 1.4, 1.8, and 2.2 μg / mL, respectively. The gradient standard solutions are colorimetrically compared at 560 nm.

[0114] Table 2.4 OD values ​​of nitrogen standard solution

[0115]

[0116] According to Table 2.4 and Figure 2.2 It can be seen that the regression equation Y = 0.7198X + 0.0005 (R 2 =1). This indicates that the standard curve of nitrogen has a good linear relationship in the range of 0 to 2.2 ug / mL.

[0117] 2.2.2 Sample determination

[0118] Preparation of sample solution, including:

[0119] 5g of ground and air-dried soil for experimental cultivation and soil from Fengjia Village were placed in 50mL conical flasks, and 20mL of 1% casein solution and 1mL of toluene were added;

[0120] Add 1 mL of BZY1-B bacterial solution with different dilution multiples, culture in a 30°C incubator for 24 h, and filter;

[0121] To 5 mL of filtrate, add 0.5 mL of 0.1 N dilute sulfuric acid, 3 mL of 20% sodium sulfate solution and 1 mL of 2% ninhydrin solution. Boil in boiling water bath for 10 min. After cooling, dilute to volume in a 50 mL volumetric flask. Compare color at 560 nm, using test tube No. 1 as blank control. The results are shown in Tables 2.5 and 2.6.

[0122] Soil protease activity was expressed as micrograms of glycine in 1g of soil after 24 hours, and the calculation formula was:

[0123] X=(AB)×C

[0124] In the formula: X is the protease activity in the soil, mg; A is the amino nitrogen concentration in mg obtained from the standard curve; B is the amino nitrogen concentration in mg of the sample control group obtained from the standard curve; C is the coefficient converted to 1g of soil.

[0125] Table 2.5 Effect of BZY1-B on the activity of soil protease used in experimental cultivation

[0126]

[0127] (Table * Indicates significant difference P < 0.05; ** Indicates extremely significant difference (P<0.01)

[0128] Table 2.5 shows that different dilution multiples have significant differences in the analysis results (P < 0.05). Compared with CK and no matrix, after adding the dilution of BZY1-B bacterial agent, the protease activity in the experimental cultivation soil generally increased, and the promotion effect increased with the increase in concentration. The protease activity of BZY1-B bacterial agent was the highest when it was diluted 100 times in the cultivation soil solution and cultured for 12 hours, with an activity of 0.772IU, which was 26% higher than the 100-fold dilution after 3 hours of culture, and the results were significantly affected by CK (P < 0.01).

[0129] Table 2.6 Effect of BZY1-B on soil protease activity in Fengjia Village wheat fields

[0130]

[0131] (Table * Indicates significant difference P < 0.05; **Indicates extremely significant difference (P<0.01)

[0132] Table 2.6 shows that different dilution multiples have significant differences in the analysis results (P < 0.05). Compared with CK and no matrix, after adding the dilution of BZY1-B bacterial agent, the protease activity in the soil of Fengjia Village wheat field generally increased, and the promotion effect increased with the increase of concentration. When BZY1-B bacterial agent was diluted 100 times and cultured in the soil solution of Fengjia Village wheat field for 12 hours, the protease activity was the highest, with an activity of 0.701IU, which was 24% higher than that of 100 times diluted for 3 hours, and the result was significantly affected by CK (P < 0.01).

[0133] 2.3 Effect of low temperature resistant mutagenesis BZY1-B on soil catalase activity

[0134] 2.3.1 Potassium permanganate calibration

[0135] Add 0.2g sodium oxalate to 250mL distilled water, add 10mL 98% sulfuric acid, heat to 75℃~85℃, and calibrate with 0.02mol / L potassium permanganate solution while hot until the solution turns pink and does not change color within half a minute, which means the titration endpoint is reached.

[0136] The potassium permanganate titration results are shown in Table 2.7.

[0137] Table 2.7 Potassium permanganate three titration results

[0138]

[0139] From Table 2.7, we can see that T = 0.144, which means that 1 mL of dye is equivalent to 0.144 g of sodium oxalate.

[0140] 2.3.2 Sample determination

[0141] The above-mentioned cultivated soil and wheat field soil after refinement and impurity removal were divided into two groups, each of which was divided into 6 subgroups; the soil mass in each subgroup was 2g;

[0142] To each group, 40 mL of distilled water and 5 mL of 0.3% hydrogen peroxide solution were added, mixed and then 1 mL of BZY1-B bacterial solution with different dilution multiples was added, and the mixture was placed on a shaker and slowly shaken for 20 minutes. The mixture was cultured in a 30°C incubator. After the preset time, 5 mL of 1.5 mol / L sulfuric acid was added, filtered, and the filtrate was titrated with potassium permanganate standard solution. The consumption was recorded and the catalase activity was calculated. The results are shown in Tables 2.8 and 2.9; the preset time was 3 h, 6 h, 9 h or 12 h.

[0143] Catalase activity calculation formula: W = (AB) × T

[0144] Where: W is catalase activity mL; T is potassium permanganate titration correction value mL; A is the volume of potassium permanganate consumed mL; B is the volume of potassium permanganate consumed by titration without soil mL

[0145] Table 2.8 Effect of BZY1-B on catalase activity in cultivation soil

[0146]

[0147] (Table * Indicates significant difference P < 0.05; ** Indicates extremely significant difference (P<0.01)

[0148] As shown in Table 2.8, different dilution multiples had significant effects on the analysis results (P < 0.05). Compared with CK and no matrix, after adding the dilution of BZY1-B bacterial agent, the catalase activity in the experimental cultivation soil generally increased, and the promotion effect increased with the increase of concentration. When the 100-fold diluted BZY1-B bacterial agent was added to the cultivation soil, the catalase activity was the highest at 8.85 IU after 12 hours of cultivation, which was 32% higher than the 100-fold dilution after 3 hours of cultivation, and the results were significantly affected by CK (P < 0.01).

[0149] Table 2. Effect of 9BZY1-B bacterial agent on catalase activity in wheat field soil of Fengjia Village

[0150]

[0151] (Table * Indicates significant difference P < 0.05; ** Indicates extremely significant difference (P<0.01)

[0152] Table 2.9 shows that different dilution multiples have significant differences in the analysis results (P < 0.05). Compared with CK and no matrix, after adding the dilution of BZY1-B bacterial agent, the catalase activity in the soil of Fengjia Village wheat field generally increased, and the promotion effect increased with the increase of concentration. When the 100-fold diluted BZY1-B bacterial agent was added to the soil solution of Fengjia Village wheat field, the catalase activity was the highest at 8.12 IU after 12 hours of cultivation, which was 36% higher than the 100-fold dilution after 3 hours of cultivation. Compared with CK, the results had a very significant effect (P < 0.01).

[0153] 3 Effects of low temperature resistant mutant strain BZY1-B on available N, P and K contents in soil

[0154] Experimental Materials

[0155] The soil is from the cherry orchard in Fengjia Village, Lantian County, Xi'an, and the wheat field in Fengjia Village, Lantian County, Xi'an;

[0156] BZY1-2 and BZY1-B were activated and cultured and then diluted to obtain bacterial solutions diluted 100 to 400 times. The corresponding bacterial solution concentrations after dilution were 5.25×10 3 CFU / mL, 2.62×10 3 CFU / mL, 1.75×10 3 CFU / mL, 1.31×10 3 CFU / mL;

[0157] The cherry orchard soil and wheat field soil were divided into two groups, each of which was divided into three subgroups; the soil sample in each subgroup was 2.5g;

[0158] Each group of soil samples was placed in a dry conical bottle, 50 mL of distilled water was added to make a suspension, and the suspension was oscillated at 25°C at a rate of 180 times / min for 30 minutes. 0.3 mL of diluted bacterial solution was added and the suspension was placed at 25°C for 72 hours to obtain soil samples.

[0159] 3.1 Effects of low-temperature-resistant mutant BZY1-B on soil P

[0160] Phosphorus standard curve

[0161] The method for determining the standard curve of phosphorus element includes: drying potassium dihydrogen phosphate in an oven at 105° C. for 2 hours, dissolving 0.4394 g of the dried potassium dihydrogen phosphate in water, adding 5 mL of sulfuric acid and then filling up to 1 L with water to obtain a phosphorus standard stock solution with a concentration of 100 mg / L;

[0162] Using the phosphorus standard stock solution and 5 mL of antimony molybdate colorimetric solution as main raw materials and dinitrophenol as an indicator, a series of phosphorus standard colorimetric solutions with concentrations of 0.00 mg / L, 0.10 mg / L, 0.20 mg / L, 0.30 mg / L, 0.40 mg / L, 0.50 mg / L and 0.60 mg / L were prepared;

[0163] After the phosphorus standard colorimetric solution was allowed to stand for half an hour in an environment above 20°C, an absorption spectrum was measured at a wavelength of 700 nm using a spectrophotometer to draw a standard curve of phosphorus absorbance, as shown in Table 3.1 and Figure 3.1 shown.

[0164] Table 3.1 Absorbance of available phosphorus standard solution at 700nm wavelength

[0165]

[0166] Determination of phosphorus content by molybdenum-antimony colorimetry

[0167] The soil sample was air-dried and sieved with a 2 mm pore size sieve, 5 g of the sieved soil sample was placed in 25.0 mL of the double acid extraction solution, and filtered at a speed of 160 revolutions per minute for 5 minutes to obtain a filtrate for detection;

[0168] The detection filtrate was measured by molybdenum antimony colorimetry at a wavelength of 700 nm to determine the effective phosphorus content, and the results are shown in Tables 3.2 and 3.3; in the double acid extraction solution, the concentration of HCl was 0.05 mol / L, and the concentration of H2SO4 was 0.025 mol / L;

[0169] Among them, the control group (CK) was prepared by adding an equal amount of distilled water to the suspension;

[0170] The calculation formula for soil available phosphorus content is:

[0171] W P =(C-C0)×V / (m×k)(4.1)

[0172] W p represents the available phosphorus content in the soil, and its unit is mg / kg; c represents the phosphorus content in the test sample obtained from the standard spectrum, and its unit is mg / L; c0 refers to the phosphorus content in the control solution obtained from the standard spectrum, and its unit is mg / L; V is the total volume of the color developer, and the application amount is set at 50mL; m refers to the weight of the soil sample before drying, and its unit is g; and k is the moisture correction factor required to convert the dry soil sample to the air-dried state.

[0173] Table 3.2 Effect of BZY1-B on available phosphorus content in soil of Fengjia Village (unit: mg / kg)

[0174]

[0175] (* indicates significant difference P < 0.05; ** indicates extremely significant difference P < 0.01)

[0176] Table 3.3 Effect of BZY1-2 on available phosphorus content in soil of Fengjia Village (unit: mg / kg)

[0177]

[0178] (* indicates significant difference P < 0.05; ** indicates extremely significant difference P < 0.01)

[0179] Using SPSS27 statistical analysis software for descriptive analysis, it can be determined that different irrigation treatments have significant effects on the analysis results (*). The soil added with the diluted solution of BZY1-B microbial agent was significantly different from the CK group (P < 0.05), and the microbial agent with a higher concentration had a more significant effect. The 100-fold diluted BZY1-B microbial agent increased the available phosphorus in the cherry orchard soil by 78% and the available phosphorus in the wheat field soil by 75%. Compared with the 100-fold diluted BZY1-2 microbial agent irrigation treatment, the available phosphorus content in the cherry orchard soil increased by 37% and the available phosphorus content in the wheat field soil increased by 39%. This shows that the BZY1-B microbial agent can effectively increase the content of available phosphorus, thereby improving soil fertility.

[0180] 3.2 Effect of cold-tolerant mutant BZY1-B on soil K

[0181] Potassium standard curve

[0182] The method for drawing the potassium standard curve includes: adding 10 mL of 10 g / L sodium chloride liquid and 20 mL of 2 mol / L nitric acid to 1.00, 2.00, 3.00, 4.00 and 5.00 mL of potassium ion standard solution, respectively, and adding water to make up the volume to obtain a series of potassium standard solutions with potassium ion concentrations of 0, 1.00, 2.00, 3.00, 4.00 and 5.00 mg / L, respectively. The absorbance of the potassium standard solution is measured and the standard curve is drawn. The results are shown in Table 3.4 and Figure 3.2 shown.

[0183] Table 3.4 Absorbance of effective potassium standard solution

[0184]

[0185] Determination of potassium content

[0186] The soil sample was air-dried and sieved with a 100-mesh sieve. 2.5 g of the sieved soil sample was placed in a 250 mL conical flask, 50 mL of a 2 mol / L nitric acid solution was added, and the flask was shaken at a rate of 180 times / min for 30 min at 25° C. The flask was allowed to stand for 30 min, and filtered. 2 mL of the filtrate was added with 1 mL of a 10 g / L sodium chloride solution and 7 mL of distilled water, and the mixture was shaken thoroughly to obtain a sample solution.

[0187] The sample solution was tested for effective potassium content by flame spectrometry, and the results are shown in Table 3.5 and Table 3.6.

[0188] The formula for calculating available potassium is:

[0189] W k =(C-C0)×V / (m×k)(4.2)

[0190] In formula (4.2), W k It refers to the mass fraction of soluble potassium element, expressed in mg / kg; c represents the density of soluble potassium in the test sample, which is determined according to the standard curve and the unit is mg / L; c0 is the concentration of soluble potassium in the blank control solution, which is also determined according to the standard curve and the unit is also mg / L; V represents the volume of the extract, which is set to 50mL; m refers to the weight of the soil sample after air-drying, in g; k is the moisture conversion coefficient required to convert the air-dried soil sample to a dry state.

[0191] Table 3.5 Effect of BZY1-B on the available potassium content in the soil of Fengjia Village (mg / kg)

[0192]

[0193] (* indicates significant difference P < 0.05; ** indicates extremely significant difference P < 0.01)

[0194] Table 3.6 Effect of BZY1-2 on the effective potassium content in the soil of Fengjia Village (mg / kg)

[0195]

[0196] (* indicates significant difference P < 0.05; ** indicates extremely significant difference P < 0.01)

[0197] Using SPSS27 statistical analysis software for descriptive analysis, it can be determined that different irrigation treatments have significant effects on the analysis results (*). The soil added with BZY1-B bacterial agent dilution was significantly different from the CK group (P < 0.05), and the higher concentration of the bacterial agent had a more significant effect. The 100-fold diluted mutagenic B bacterial agent increased the effective potassium in the cherry orchard soil by 297% and the effective potassium in the wheat field soil by 300%. Compared with the 100-fold diluted BZY1-2 bacterial agent irrigation treatment, the effective potassium content in the cherry orchard soil increased by 98%, and the effective potassium content in the wheat field soil increased by 120%. It shows that BZY1-B bacterial agent can effectively increase the content of effective potassium, thereby improving soil fertility.

[0198] 3.3 Effect of cold-resistant mutant BZY1-B on ammonium nitrogen in soil

[0199] Ammonium nitrogen standard curve

[0200] The method for drawing the ammonium nitrogen standard curve includes: adding 10 mL of potassium chloride solution to 0 mL, 2 mL, 4 mL, 6 mL, 8 mL and 10 mL of ammonium nitrogen standard solution, respectively, and then adding 5 mL of phenol reagent, 5 mL of sodium hypochlorite and 1 mL of masking agent, and making up the volume with water to obtain a series of standard solutions with concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / L, respectively, measuring the absorbance of the standard solution at a wavelength of 625 nm and drawing a standard curve. The results are shown in Tables 3.7 and Figure 3.3 shown.

[0201] Table 3.7 Absorbance of ammonium nitrogen standard working solution at 625nm

[0202]

[0203] Determination of ammonium nitrogen content

[0204] The soil sample was air-dried, sieved with a 2 mm pore size sieve, 20 g of the sieved soil sample was placed in 100 mL of a 3 g / L potassium chloride solution, shaken and immersed for 1 h, and filtered to obtain a soil filtrate;

[0205] Place 5 mL of the soil filtrate in a 50 mL measuring cylinder, add 5 mL of KCl solution, 5 mL of phenol and 5 mL of alkaline NaClO solution, stir until well mixed, let stand at room temperature for 60 minutes, then add 1 mL of sunscreen to dissolve potential precipitation, add distilled water to the 50 mL measuring cylinder mark, measure the absorbance at a wavelength of 625 nm, and the results are shown in Tables 3.8 and 3.9.

[0206] The calculation formula for ammonium nitrogen is:

[0207] W N =(C-C0)×V / (m×k)(4.3)

[0208] In equation (4.3), W N Represents ammonium nitrogen (NH3 - -N) concentration, and measured in mg / kg;

[0209] c refers to the ammonium nitrogen content in the test solution calculated according to the standard curve, and its unit of measurement is mg / L;

[0210] c0 refers to the concentration of ammonium nitrogen in the blank control solution determined by the standard curve, and the unit of measurement is also mg / L;

[0211] V represents the total volume of the solution involved in the color development reaction, measured in mL;

[0212] m represents the mass of the fresh soil sample collected, in g; k is the moisture content adjustment coefficient required for the soil sample to change from a wet state to a dry state.

[0213] Table 3.8 Effect of BZY1-B on the ammonium nitrogen content in the soil of Fengjia Village (mg / kg)

[0214]

[0215] (* indicates significant difference P < 0.05; ** indicates extremely significant difference P < 0.01)

[0216] Table 3. Effect of 9BZY1-2 bacterial agent on the ammonium nitrogen content in the soil of Fengjia Village (mg / kg)

[0217]

[0218] (* indicates significant difference P < 0.05; ** indicates extremely significant difference P < 0.01)

[0219] SPSS27 statistical analysis software was used for descriptive analysis. The soil added with BZY1-B bacterial agent dilution had significant differences from the CK group (P < 0.05), and the higher concentration of the bacterial agent had a more significant effect. The 100-fold diluted mutagenic B bacterial agent reduced the ammonium nitrogen in the cherry orchard soil by 23% and the ammonium nitrogen in the wheat field soil by 31%. Compared with the 100-fold diluted BZY1-2 bacterial agent irrigation treatment, the ammonium nitrogen in the cherry orchard soil decreased by 130%, and the ammonium nitrogen content in the wheat field soil decreased by 8%. This shows that BZY1-B bacterial agent can effectively increase the conversion rate of ammonium nitrogen, thereby improving soil fertility.

[0220] 3.4 Determination results and analysis of nitrate nitrogen

[0221] Nitrate nitrogen standard curve

[0222] The method for drawing a nitrate nitrogen standard curve comprises: adding an equal amount of potassium chloride to 0.00, 0.50, 1.00, 2.00, 3.00 and 4.00 mL of nitrate nitrogen calibration solution, stirring to dissolve, and then adding water to make up the volume to obtain nitrate nitrogen standard solutions with concentrations of 0.00, 0.50, 1.00, 2.00, 3.00 and 4.00 mg / L, respectively, measuring the wavelengths of the nitrate nitrogen standard solutions at 220 nm and 275 nm and drawing a standard curve. The results are shown in Tables 3.10 and Figure 3.4 shown.

[0223] Table 3.10 Absorbance of nitrate nitrogen standard working solution at 220nm and 275nm

[0224]

[0225] Determination of nitrate nitrogen content

[0226] 40.00 g of the soil sample was placed in a 500 mL Erlenmeyer flask, and 200 mL of a 0.3% KCl solution was poured into the flask. The flask was sealed tightly, and the flask was vibrated at a frequency of 220 times per minute for 1 h at room temperature to obtain a suspension.

[0227] Take 60 mL of the suspension and place it in a 100 mL centrifuge tube. Centrifuge at 3000 rpm for 10 min to obtain the supernatant.

[0228] Using an ultraviolet and visible light spectrophotometer, the absorbance of the supernatant at wavelengths of 220nm and 275nm was measured, and the content of nitrate nitrogen in the soil extract was calculated. The results are shown in Tables 3.11 and 3.12.

[0229] W N =(ρ N -ρ0)×R(4.4)

[0230] According to equation (4.4), the value ρ obtained on the calibration curve is N represents the nitrate nitrogen concentration in the soil sample solution, measured in mg / L; ρ0 refers to the nitrate nitrogen concentration in the blank solution determined on the same calibration curve, and its unit is also mg / L; and R represents the sample volume ratio, that is, the volume ratio of the leachate volume plus the soil inherent water volume to the dry soil mass, and its unit is mL / g. This value is calculated based on equations (4.5) to (4.7).

[0231] R=R1+R2 (4.5)

[0232] R1=v / m×(1+W W / 100) (4.6)

[0233] R2=W W / (d W ×100) (4.7)

[0234] In formulas (4.5) to (4.7), the coefficient R1 represents the volume ratio of the extract to the dry soil sample, measured in mL / g; the coefficient R2 refers to the volume ratio of the water content in the soil to the dry soil sample, also expressed in mL / g; V refers to the volume of the extract used, in mL; m refers to the mass of the wet soil sampled, in g; W W It indicates the proportion of soil moisture content calculated on the basis of dry soil, expressed as a percentage; d W is the mass per unit volume of water at the experimental ambient temperature, expressed in g / cm 3 Assume that the value is 1.00 g / cm3 If the nitrate nitrogen content in the soil is less than 1 mg / kg, the result should be retained to two decimal places; if the content is greater than or equal to 1 mg / kg, the result should be ensured to have three significant figures.

[0235] Table 3. Effect of 11BZY1-B bacterial agent on nitrate nitrogen content in soil of Fengjia Village (mg / kg)

[0236]

[0237] (* in the table indicates significant difference P < 0.05, Note: some data in 4.4.2 are 0.00)

[0238] Table 3. Effect of 12BZY1-2 bacterial agent on nitrate nitrogen content in soil of Fengjia Village (mg / kg)

[0239]

[0240] (* in the table indicates significant difference P < 0.05, Note: some data in 4.4.2 are 0.00)

[0241] Using SPSS27 statistical analysis software for descriptive analysis, it can be determined that different irrigation treatments have significant effects on the analysis results (*). The soil added with BZY1-B bacterial agent dilution was significantly different from the CK group (P < 0.05), and the higher concentration of bacterial agent had a more significant effect. The 100-fold diluted BZY1-B bacterial agent increased the nitrate nitrogen in the wheat field soil by 540%, and the nitrate nitrogen content in the wheat field soil increased by 42% compared with the 100-fold diluted BZY1-2 bacterial solution irrigation treatment. This shows that BZY1-B bacterial agent can effectively increase the content of nitrate nitrogen, thereby improving soil fertility.

[0242] Effects of 4BZY1-B bacteria on nutritional components of pakchoi

[0243] 4.1 Planting and management of pakchoi

[0244] The pakchoi seeds were rinsed with sterile distilled water and placed in a culture dish containing sterile wet filter paper discs, and cultured at 37°C in the dark to germinate; the pakchoi seeds were Huamei Siji Qinggengcai, which were provided by greenhouse cultivation;

[0245] Transplant the germinated seeds into a plastic tray filled with peat, cover with 0.5-1 cm of soil and culture until the four-leaf stage;

[0246] Transplanting the four-leaf seedlings into a plastic pot filled with soil for cultivation; the mass percentage of each component in the soil is: sand 51%, clay 19%, silt 30%, organic matter 1%, and total organic carbon 0.39%;

[0247] After the seedlings grew steadily, they were divided into groups of 40 plants each. The roots were irrigated with bacterial solution at an average temperature of 27°C, an average relative humidity of 67.5%, and a light intensity of 408 μm. -2 s -1 The culture is carried out under the following conditions, and the nutrient components of the pakchoi are tested after the culture is completed for a preset time; the strains in the bacterial solution include phosphate- and potassium-solubilizing bacteria BZY1-2 or ultraviolet-induced phosphate- and potassium-solubilizing bacteria BZY1-B.

[0248] The root irrigation scheme of bacterial solution is shown in Table 4.1.

[0249] Table 4.1 Experimental grouping scheme

[0250]

[0251] 4.2 Determination of nitrite content by UV spectrophotometry

[0252] Nitrite standard curve drawing

[0253] To draw the nitrite standard curve, the reagents to be added are shown in Table 4.2:

[0254] Table 4.2 Configuration of nitrite standard solution

[0255]

[0256] Nitrite content and OD 538 The data are shown in Table 4.3, and the nitrite standard curve is shown in Figure 4.1 shown.

[0257] Table 4.3 Nitrite content and OD 538

[0258]

[0259] From the data table and graph, the regression equation of nitrite concentration and OD value is Y = 0.0405X + 0.6133 (R 2 =0.9728), indicating that the nitrite content is related to OD 538 The linear relationship was good in the range of 0-5μg / g.

[0260] Determination of nitrite content in samples

[0261] According to the root irrigation culture method in the table above, accurately weigh 0.500 g of fresh pakchoi leaves after 7, 14, and 21 days of culture as samples, mash and stir evenly to a homogenous state, and use them as test samples;

[0262] Place the sample in a 50mL stoppered conical flask, add 1.25mL saturated borax solution and 15mL 70℃ distilled water, mix well and heat in a boiling water bath for 15min, take out and cool in a cold water bath to room temperature;

[0263] The cooled system was placed in a 40 mL volumetric flask, 0.50 mL of 100 mg / L potassium ferrocyanide solution was added, and the mixture was shaken well. 0.50 mL of 0.01 mol / L zinc acetate solution was added to precipitate protein. Water was added to make up the volume, and the mixture was shaken well and allowed to stand for 30 min. The mixture was filtered with filter paper, and 3 mL of the initial filtrate was discarded to obtain the sample solution to be tested.

[0264] The sample solution to be tested was prepared into a detection solution, and the absorbance at a wavelength of 538 nm was measured. The nitrite content was obtained by comparing it with the standard curve. The results are shown in Tables 4.4 and 4.5.

[0265] Table 4.4 Nitrite content of green vegetables in treatment 1 (μg / g)

[0266]

[0267]

[0268] Table 4.5 Nitrite content of green vegetables in treatment 2 (μg / g)

[0269]

[0270] Note: Different capital letters indicate significant differences between different concentrations of the same strain (P<0.01); Different lowercase letters indicate significant differences between different strains of the same concentration (P<0.01).

[0271] It can be seen that compared with the control group without the mutagenic agent, the nitrite content of the pakchoi treated with the BZY1-B agent after ultraviolet mutagenesis was reduced. 5 CFU / mL irrigated roots for 7, 14, and 21 days, and the nitrite content decreased by 48.25%, 88.73%, and 78.21%, respectively (P<0.01). 5 After root irrigation with 500 μg / mL CFU / mL for 7, 14, and 21 days, the nitrite content in the Brassica pekinensis seedlings decreased by 67.78%, 79.96%, and 86.86%, respectively (P<0.01).

[0272] 4.3 Determination of Vitamin C Content by 2,6-Dichloroindophenol Titration

[0273] Standardization of 2,6-dichloroindophenol standard solution

[0274] In a 25mL conical flask, add 1mL of 1mg / mL ascorbic acid solution (vitamin C solution) and 9mL of 1% oxalic acid, and titrate with 0.1% 2,6-dichlorophenol indophenol until the color turns light red and does not disappear within 15s. The titration endpoint is determined to be reached, and the amount of ascorbic acid equivalent to 1mL of dye is calculated. The results are shown in Table 4.6.

[0275] Table 4.6 Consumption of Vitamin C Volume

[0276]

[0277] The volume consumed for vitamin C is 0.125 mL. It is calculated that 1 mL of dye is equivalent to 0.8 mg of ascorbic acid.

[0278] Determination of vitamin C content in samples

[0279] 0.500 g of fresh pakchoi leaves after 7, 14 and 21 days of culture were respectively placed in a mortar, 10 mL of 1% oxalic acid was added in portions, and the slurry was ground into a homogenate, the homogenate was filtered through gauze to obtain a homogenate solution, and the homogenate solution was placed in a centrifuge and centrifuged at 3000 rpm for 10 min to obtain a supernatant;

[0280] Take 5 mL of the supernatant and place it in a 50 mL conical flask. Titrate with 0.1% by mass 2,6-dichlorophenol indophenol until it turns light red and the color does not disappear within 15 seconds. Record the titration volume and calculate the vitamin C content using the following formula:

[0281] Vitamin C = (volume of dye consumed by sample - volume of dye consumed by blank) × volume after extraction and volume fixation × number of milligrams of ascorbic acid that can be oxidized by 1 mL of dye / volume of sample used for titration × sample mass;

[0282] The results are shown in Tables 4.7 and 4.8.

[0283] Table 4.7 Vitamin C content of green vegetables in treatment 1 (mg / g)

[0284]

[0285] Table 4.8 Vitamin C content of green vegetables in treatment 2 (mg / g)

[0286]

[0287] Note: Different capital letters indicate significant differences between different concentrations of the same strain (P<0.01); Different lowercase letters indicate significant differences between different strains of the same concentration (P<0.01).

[0288] It can be seen that compared with the control group without mutation, the vitamin C content of the pakchoi treated with BZY1-B after ultraviolet mutation increased. 5 After irrigating roots with CFU / mL for 7, 14, and 21 days, the vitamin C content of Brassica chinensis increased by 25.00%, 5.27%, and 11.11%, respectively (P<0.01). 5 After root irrigation with CFU / mL for 7, 14, and 21 days, the vitamin C content of Brassica chinensis increased by 4.85%, 12.50%, and 50.00% (P<0.01).

[0289] 4.4 Determination of soluble protein content by Coomassie Brilliant Blue method

[0290] Drawing of standard curve

[0291] The reagents required to draw the soluble protein standard curve are shown in Table 4.9:

[0292] Table 4.9 Configuration of protein solution

[0293]

[0294] The OD value at 595 nm was measured. 595 The soluble protein standard curve was drawn with OD as the ordinate and protein content as the abscissa. 595 The data are shown in Table 4.10 and the standard curve is shown in Figure 5 shown.

[0295] Table 4.10 Soluble protein content and OD595

[0296]

[0297] From the data table and graph, the regression equation of the soluble protein standard curve is Y=0.0051X+0.8576(R 2 =0.9650), indicating that the soluble protein content is closely related to OD 595 The linear relationship was good in the range of 0-100 μg / g.

[0298] Determination of soluble protein content in samples

[0299] 0.500 g of fresh pakchoi leaves after 7, 14, and 21 days of culture were taken in a mortar and ground, then 2 mL of 0.02% phosphate buffer with a pH of 7.0 was added, transferred to a centrifuge tube, allowed to stand, and centrifuged at 4000 rpm for 15 min to collect the supernatant;

[0300] Take 0.10 mL of the supernatant, add 3.00 mL of Coomassie Brilliant Blue G-250 reagent, shake well and let stand for 5 minutes, measure the OD value at 595 nm, and calculate the soluble protein content of the sample. The results are shown in Tables 4.11 and 4.12.

[0301] Table 4.11 Soluble protein content of green vegetables in treatment 1 (μg / g)

[0302]

[0303] Table 4.12 Soluble protein content of green vegetables in treatment 2 (μg / g)

[0304]

[0305] Note: Different capital letters indicate significant differences between different concentrations of the same strain (P<0.01); Different lowercase letters indicate significant differences between different strains of the same concentration (P<0.01).

[0306] The results showed that compared with the control group without mutagenic agents, the soluble protein content of pakchoi treated with BZY1-B agent after ultraviolet mutagenesis increased. 5 After irrigating roots with 10% CFU / mL for 7, 14, and 21 days, the soluble protein content of Brassica chinensis increased by 5.70%, 6.32%, and 5.21% (P < 0.01). 5 After root irrigation with CFU / mL for 7, 14, and 21 days, the soluble protein content of Brassica chinensis increased by 8.02%, 5.22%, and 2.23% (P<0.01).

[0307] In summary, it can be seen that the BZY1-B bacterial agent of the present invention has a more obvious effect on the nutritional components of plants after ultraviolet mutation, indicating that the BZY1-B of the present invention has good performance in promoting the nutritional components of plants.

[0308] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A low-temperature resistant Burkholderia species capable of solubilizing phosphorus and potassium, characterized in that: It is classified and named as Burkholderia Burkholderia, and is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 27769 and the deposit date of July 3, 2023.

2. The low-temperature resistant Burkholderia species capable of solubilizing phosphorus and potassium according to claim 1, characterized in that: The low-temperature-resistant Burkholderia phosphate-and-potassium-solubilizing bacteria is obtained by screening phosphate-and-potassium-solubilizing bacteria under low-temperature stress and subjecting them to ultraviolet mutagenesis; the ultraviolet mutagenesis power is 15W, the irradiation distance is 30cm, and the irradiation time is 30s.

3. Use of the low-temperature resistant Burkholderia bacteria that can solubilize phosphorus and potassium as described in claim 1 in promoting plant growth.

4. The use of a low-temperature resistant Burkholderia species capable of solubilizing phosphorus and potassium according to claim 3 in promoting plant growth, characterized in that: Includes use to increase soil enzyme content.

5. The use of a Burkholderia species that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth according to claim 4, characterized in that: The enzymes include urease, protease and / or catalase.

6. The use of a low-temperature resistant Burkholderia species capable of solubilizing phosphorus and potassium according to claim 3 in promoting plant growth, characterized in that: This includes increasing the available nitrogen, available phosphorus and / or available potassium content in the soil.

7. The use of a Burkholderia species that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth according to claim 3, characterized in that: Including increasing the nutrition of green vegetables.

8. The use of a Burkholderia species that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth according to claim 7, characterized in that: The method comprises reducing the nitrite content in the green vegetables, increasing the vitamin C content in the green vegetables and / or increasing the soluble protein content in the green vegetables.

9. The use of a Burkholderia species that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth according to claim 7, characterized in that: The method includes irrigating the roots of green vegetable seedlings with a bacterial solution containing Burkholderia bacteria that can dissolve phosphorus and potassium and is resistant to low temperatures, so as to improve the nutrition of the green vegetables.

10. The use of a Burkholderia species that can solubilize phosphorus and potassium and is resistant to low temperatures in promoting plant growth according to claim 9, characterized in that: In the bacterial solution containing Burkholderia spp. that can dissolve phosphorus and potassium and can tolerate low temperatures, the concentration of Burkholderia spp. that can dissolve phosphorus and potassium and can tolerate low temperatures is 10 6 CFU / mL~10 3 CFU / mL.

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