CN115868506A - Application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion and soil enzyme system metabolism regulation - Google Patents
Application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion and soil enzyme system metabolism regulation Download PDFInfo
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- 239000011574 phosphorus Substances 0.000 title claims abstract description 31
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Abstract
The invention provides application of bacillus thuringiensis in dissolving phosphorus in soil, promoting plant growth and regulating soil enzyme system metabolism, and belongs to the technical field of plant growth-promoting bacteria. The invention firstly provides that the bacillus thuringiensis has strong inorganic phosphorus dissolving effect, and the phosphorus dissolving effect is more than 3 times of that of the common commercial bacillus thuringiensis, bacillus licheniformis and bacillus subtilis and more than 10.7 times of that of the bacillus amyloliquefaciens. The fermentation liquor of the bacillus thuringiensis can obviously improve the biomass and the chlorophyll content of plants, and has the effect of regulating the metabolism of a soil enzyme system.
Description
Technical Field
The invention belongs to the technical field of plant growth-promoting bacteria, and particularly relates to application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion and soil enzyme system metabolism regulation.
Background
Phosphorus is one of essential nutrient elements for plant growth and development, more than 95% of phosphorus in soil exists in a form which is not easily absorbed and utilized by plants, and a large amount of phosphorus required by plants can be provided only by applying phosphate fertilizer in production. However, the applied phosphate fertilizer has the seasonal utilization efficiency of only 5-25%, high cost, low effective conversion rate and serious waste, and the excessive use of the phosphate fertilizer can cause serious pollution to soil and water environment.
The rhizosphere is the area of the soil near the roots of the plants, which is the interface between the soil and the roots of the plants. Some soil microorganisms can convert phosphorus that is difficult to be absorbed and utilized by plants into a form that can be absorbed and utilized, and such microorganisms are called phosphate solubilizing microorganisms or phosphate solubilizing bacteria, and these beneficial microorganisms are collectively called PGPR (Plant growth-promoting rhizobacteria). The PGPR can promote the release and the effectiveness of soil accumulated phosphorus by utilizing a microbial way, explore the potential of crops, reduce the fixation of the soil to the phosphorus, exert a mechanism of activating the soil phosphorus by root exudates, and have great practical significance. More and more researchers carry out deep research on phosphate solubilizing microorganisms, and at present, the screening and application of phosphate solubilizing bacteria mainly focus on the soil with higher fertility such as corn and wheat, and the research of screening the phosphate solubilizing bacteria in the poor soil is still fresh.
Disclosure of Invention
In view of the above, the invention aims to provide application of bacillus thuringiensis, which is a high-efficiency phosphate solubilizing bacterium screened from drought-barren soil, in soil phosphorus solubilization, plant growth promotion and soil enzyme system metabolism regulation.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion or soil enzyme system metabolism regulation.
Preferably, the bacillus thuringiensis comprises bacillus thuringiensis TG-5.
Preferably, the promotion of plant growth comprises increasing plant root length, dry fresh weight and chlorophyll content.
Preferably, the fermentation broth of bacillus thuringiensis is used for dissolving phosphorus in soil, promoting plant growth or regulating metabolism of soil enzymes.
Preferably, the fermentation liquor is prepared by the following steps: inoculating Bacillus thuringiensis into a fermentation culture medium, and performing shaking culture at 36-39 deg.C and 180-220 r/min for more than 8 h.
Preferably, the fermentation medium comprises a source of C, a source of N and inorganic salts, the source of C comprising yeast extract, soluble starch, maltose, glucose, sucrose or corn meal.
Preferably, the N source comprises tryptone, urea, soy flour, ammonium sulfate or beef extract.
Preferably, the inorganic salt comprises sodium chloride, calcium chloride, magnesium nitrate, potassium nitrate, ferrous sulfate heptahydrate or aluminum phosphate.
Preferably, the concentration ratio of the C source to the N source to the inorganic salt in the fermentation medium is 5-10.
The invention has the beneficial effects that:
the bacillus thuringiensis has strong inorganic phosphorus dissolving effect, which is more than 3 times that of common bacillus thuringiensis, bacillus licheniformis and bacillus subtilis and more than 10.7 times that of bacillus amyloliquefaciens. The fermentation liquor of the bacillus thuringiensis can obviously improve the biomass and the chlorophyll content of plants, and has the effect of regulating the metabolism of a soil enzyme system. Taking spinach as an example, the fermentation broth of the bacillus thuringiensis can obviously improve the biomass of the spinach, wherein the plant height, the fresh weight and the dry weight are respectively improved by 30.6 percent, 46.8 percent and 96.0 percent; the total amount of chlorophyll a, chlorophyll b and chlorophyll of the spinach is remarkably increased, and the increasing rates are respectively 7.0%, 23.4% and 1.3%; the activity of soil sucrase and urease is obviously improved, and the growth rates are respectively 30.3 percent and 688.6 percent; the activity of spinach root dehydrogenase and catalase is obviously improved, and the growth rates are respectively 165.5 percent and 15 percent.
Drawings
FIG. 1 is a photograph showing the culture of TG-5 strain;
FIG. 2 shows a bacterial developmental tree constructed based on the 16S rRNA gene sequence;
FIG. 3 shows the variation of the absorbance of phosphorus decomposition in different strains;
FIG. 4 shows the absorbance changes of different carbon sources and nitrogen sources;
FIG. 5 shows the absorbance change for different inorganic salts and incubation times;
FIG. 6 shows the effect of different inoculum sizes and culture temperatures on absorbance;
FIG. 7 is a graph showing the effect of different pH values and rotation speeds on absorbance;
FIG. 8 shows the results of orthogonal experiments with optimized ratios of the components of the culture medium;
FIG. 9 the influence of Bacillus thuringiensis fermentation broth treatment on spinach plant height, root length, dry fresh weight;
FIG. 10 shows the effect of Bacillus thuringiensis fermentation broth treatment on spinach chlorophyll;
FIG. 11 shows the effect of Bacillus thuringiensis fermentation broth on spinach rhizosphere soil enzyme metabolism;
FIG. 12 is the effect of Bacillus thuringiensis broth treatment on the physicochemical properties of spinach soil.
Detailed Description
The invention provides application of bacillus thuringiensis in soil phosphorus dissolution, plant growth promotion or soil enzyme system metabolism regulation.
The bacillus thuringiensis is separated from the perennial growing plant rhizosphere in Tianxian, taian, shandong, has the capabilities of dissolving phosphorus, decomposing lignocellulose and generating auxin, and is beneficial to nutrient activation and promoting plant growth. In the invention, the bacillus thuringiensis is preferably bacillus thuringiensis TG-5. In the invention, the whole genome of Bacillus thuringiensis TG-5 is disclosed, wherein the registration number is GWHBOWN00000000, and the whole genome nucleotide sequence of the Bacillus thuringiensis TG-5 is shown in SEQ ID NO. 1. (the 16S rRNA gene sequence of TG-5 of the present invention was stored in NCBI database under accession number RID-8MG9P79F013 QUERY-39355, and the bacterium was preliminarily identified as Bacillus thuringiensis). The applicant has promised free of charge to the public for the public release of the Bacillus thuringiensis TG-5 biological material. In the present invention, the promotion of plant growth preferably includes increasing plant root length, dry fresh weight and chlorophyll content, and the soil enzyme system preferably includes sucrase, urease, dehydrogenase and catalase.
When the bacillus thuringiensis is used for soil phosphorus dissolving, plant growth promotion or soil enzyme system metabolism regulation, the fermentation liquor of the bacillus thuringiensis is preferably used for playing a role. In the present invention, the fermentation broth of bacillus thuringiensis is preferably prepared by the following steps: inoculating Bacillus thuringiensis into a fermentation culture medium, and performing shaking culture at 36-39 deg.C and 180-220 r/min for more than 8 h.
In the present invention, the fermentation medium preferably comprises a C source, an N source and inorganic salts, and the concentration ratio of the C source, the N source and the inorganic salts is preferably 5-10. The C source preferably comprises yeast extract, soluble starch, maltose, glucose, sucrose or corn flour; the N source preferably comprises tryptone, urea, soybean meal, ammonium sulfate or beef extract; the inorganic salt preferably includes sodium chloride, calcium chloride, magnesium nitrate, potassium nitrate, ferrous sulfate heptahydrate or aluminum phosphate. In the invention, the temperature of the fermentation is preferably 37-38 ℃, and the rotation speed of the fermentation is preferably 220r/min.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Taking 5g of perennial plant rhizosphere soil of Tianwamura city, taian, shandong province under aseptic condition, dissolving in 45mL of sterile distilled water, performing shake culture for 30min at a speed of 220r/min. Pipette 100. Mu.L of soil dilutionThe center of each separation culture medium plate is evenly coated by a coater and cultured for 1-3 days at the constant temperature of 37 ℃. The bacteria separation adopts LB solid culture medium, the actinomycetes separation adopts Gao's I culture medium, the selected dilution is 10 -4 、10 -5 And 10 -6 (ii) a The fungus is separated by adopting a Martin culture medium, and the selected dilution degree is 10 -3 、10 -4 And 10 -5 。
92 strains of bacteria are separated from the LB solid culture medium and the PDA solid culture medium. The method takes a cellulose Congo red culture medium, a cellulase-producing fermentation culture medium, an Ashby nitrogen-free culture medium, an inorganic phosphorus bacteria separating culture medium and a silicate bacteria separating culture medium as screening conditions, and the one meeting the five conditions is a high-efficiency growth promoting target strain Bacillus thuringiensis TG-5.
The screened growth-promoting strains are inoculated on an LB solid culture medium, cultured for 48 hours at 37 ℃, and observed according to bacterial colony of Bergey's Manual of bacteriological appraisal of systems, and gram staining and spore staining are carried out, the result is shown in figure 1, and morphological appraisal shows that: TG-5 gram-positive stain, and after culturing for 24h at 37 ℃ on an LB culture medium, a single bacterium is milky white, opaque, irregular, raised and wavy in edge. The colony is observed by a microscope to be round or oval, is light yellow, has irregular edges, and is opaque and slightly bulged to be in the shape of a drop wax.
Then, physiological and biochemical identification is carried out according to the microbiological experiment course and the soil agricultural chemical analysis method. The results are shown in Table 1.
TABLE 1 TG-5 physiological and biochemical identification results
Note: in the table, "+" indicates positive and "-" indicates negative.
As can be seen from Table 1, after the partial growth promoting function of the strain TG-5 is identified, transparent circles appear in a starch culture medium in a starch hydrolysis experiment, and the transparent circles are positive; the solution appeared yellow in the methyl red experiment and was negative. The phosphorus dissolving capacity is determined as that a transparent circle appears in the organophosphorus culture medium, and the result is positive. The strain TG-5 has the capability of dissolving phosphorus, decomposing lignocellulose and generating auxin, and is beneficial to nutrient activation and plant growth promotion.
The bacterial genomic DNA extraction kit of Escire company is adopted to extract the bacterial genomic DNA. The 16SrRNA gene of the strain is subjected to PCR amplification by using the extracted DNA as a template and a universal primer 27F/1492R of the bacteria. And sending the PCR product to Beijing Optimalaceae Biotechnology Limited for sequencing, comparing sequencing results in NCBI blast, performing sequence similarity analysis through software MEGA-X, and constructing a phylogenetic tree. The results are shown in FIG. 2. The bacillus TG-5 obtained by preliminary identification and screening is bacillus thuringiensis.
Example 2
After activating the Bacillus thuringiensis TG-5 isolated in example 1, it was inoculated at a rate of 1% to a liquid inorganic phosphorus medium (g/L): glucose 10.0, yeast extract 0.5, calcium phosphate 5.0, ammonium sulfate 0.5, potassium chloride 0.2, magnesium sulfate 0.1, manganese sulfate 0.0001, ferrous sulfate 0.0001), shaking at 37 ℃ and 180r/min for 5 days, sampling, centrifuging at 4000r/min for 10min, and taking the supernatant to measure the concentration of soluble phosphorus by Mo-Sb colorimetry. Commercially available conventional Bacillus thuringiensis (abbreviated BT), bacillus licheniformis (abbreviated BL), bacillus amyloliquefaciens (abbreviated BA) and Bacillus subtilis (abbreviated BS) were set as control groups. The results are shown in FIG. 3.
As can be seen from FIG. 3, the soluble phosphorus content in the Bacillus thuringiensis TG-5 medium of the invention is 248.2mg/L, which is more than 3 times of that of the commercially available conventional Bacillus thuringiensis (79.1 mg/L), bacillus licheniformis (51.1 mg/L) and Bacillus subtilis (34.5 mg/L), and more than 10.7 times of that of Bacillus amyloliquefaciens (23.2 mg/L).
Example 3
In liquid medium (in 950ml ddH) 2 Adding 10g of tryptone, 5g of Yeast extract and 10g of NaCl into O, adjusting the pH value to 7.0 by using 1M NaOH, and fixing the volume to 1L) to serve as a control, and respectively replacing the Yeast extract (Yeast extract) in a control liquid culture medium by an equal amount (5.0 g/L) of Soluble starch (Soluble stage), maltose (Malt), glucose (Glucose), sucrose (Sucrose) and Corn flour (Corn flour); and (3) filling 100mL of the mixture into a triangular bottle of every 250mL, inoculating bacillus thuringiensis according to the inoculation amount of 2% seed solution, oscillating the mixture on a shaker at 30 ℃ and 180r/min for 12 hours, measuring the OD 600 value of the fermentation reaction solution, repeating the steps for 3 times, and screening the optimal C source. The results are shown on the left of FIG. 4.
And (3) respectively replacing Tryptone (Tryptone) in a control liquid culture medium with equal amount (10 g/L) of Urea (Urea), soybean meal (Soybeanpowder), ammonium sulfate (Ammonium sulfate) and beef extract (Beefpaste), and screening the fermentation reaction conditions with the same C source to screen out the optimal nitrogen source. The results are shown on the right of FIG. 4.
Sodium chloride (Sodium chloride) in a control liquid culture medium is respectively replaced by equal amounts (5.0 g/L) of Calcium chloride (Calcium chloride), magnesium nitrate (magnesium nitrate), potassium nitrate (Potassium nitrate), ferrous sulfate heptahydrate (iron sulfate) and Aluminum phosphate (Aluminum phosphate), and the fermentation reaction conditions are the same as those of C source screening, so that the optimal inorganic salt is screened. The results are shown on the left of FIG. 5.
As can be seen from FIGS. 4 and 5, the optimal carbon source of the fermentation medium of Bacillus thuringiensis of the present invention is corn flour, the optimal nitrogen source is soybean flour, and the optimal inorganic salt is calcium chloride.
Example 4
The influence of fermentation culture speed, pH, culture temperature, culture time and inoculation amount on the growth amount of the strain is researched by taking the growth amount of the bacillus thuringiensis strain as an index.
The optimal medium (ddH at 950 ml) obtained in example 3 was used 2 Adding 10g of soybean meal, 5g of corn meal and 10g of calcium chloride into O, adjusting the pH value to 7.0 by using 1M NaOH, fixing the volume to 1L) to serve as a fermentation culture medium, filling 100mL of triangular bottles into each 250mL of bottles, inoculating bacillus thuringiensis according to the inoculation amount of 2% seed solution, oscillating the bottles on a shaking table at 30 ℃ and 180r/min for 12h, and determining the fermentationOD 600 of the reaction solution was repeated 3 times. The results are shown in FIG. 5, right-hand FIG. 7, in which different rotation speeds, pH values, incubation temperatures, incubation times, and inoculum sizes were set. The best culture condition for the fermentation culture of the bacillus thuringiensis is that the inoculation amount is 5 percent, the temperature is 37 ℃, the pH value is 7.0, the rotating speed is 220r/min, and the strain culture enters the logarithmic phase for 2-8 hours.
Example 5
A three-level three-factor orthogonal test was conducted using the optimum C source, N source and inorganic salts obtained in example 3 as components of the fermentation medium, and the OD 600 value of the fermentation reaction solution was measured for 3 repetitions using the optimum fermentation conditions obtained in example 4 as an index of growth. Wherein the best C source: corn flour and N source: soybean flour and inorganic salts: the amounts of calcium chloride used are shown in table 2. The results are shown in FIG. 8. The best combination is A3B3C3, namely corn flour (10.0 g/L), soybean flour (15.0 g/L) and calcium chloride (10.0 g/L).
TABLE 2 orthogonal Experimental factors and levels of Components in fermentation Medium
Example 6
Pot experiment
The growth-promoting strain Bacillus thuringiensis was inoculated into liquid medium (in 950ml ddH) 2 Adding soybean powder 10g, corn powder 5g and calcium chloride 10g into O, adjusting pH to 7.0 with 1M NaOH, diluting to 1L), culturing at 37 deg.C under shaking at 180r/min for 48 hr to obtain viable count of 2 × 10 11 CFU/ML, obtaining Bacillus thuringiensis fermentation liquor, and performing a pot culture test for later use.
Experiment design:
the test is carried out in 2022, 1-5 months, spinach is used as a target plant of the test, the spinach is uniformly sown in a plastic flowerpot with the diameter of 20 cm and the height of 30 cm, the pot is respectively filled with 2.5 kg and 1 kg of sandy soil and loam, and 6 treatments are set in total: regular soil (control, CK); 0.2X 10 8 CFU/ML；0.5×10 8 CFU/ML；1×10 8 CFU/ML；2×10 8 CFU/ML；5×10 8 CFU/MLAnd 3 times of treatment are repeated, no fertilizer is added in the treatment period, drip irrigation watering is adopted, and normal management is performed.
After 30 days of potted seedling growth, soil and plant samples were collected at 3 pots per treatment. Simultaneously removing the treated soil sample, sieving with a 2mm sieve, mixing uniformly in equal amount, and air drying for measuring the activity of the soil enzyme; collecting plant samples in a whole plant, cleaning root soil with clear water, measuring the plant height and the root length of each plant with a ruler, measuring the fresh weight with an electronic balance, deactivating enzymes at 105 ℃ for 30min for the plants with the measured weights, drying the plants in a 70 ℃ oven to constant weight, and measuring the dry weight. The results are shown in FIG. 9. The application of the bacillus thuringiensis fermentation liquor has obvious promotion effect on the spinach plant height and root length, and the spinach plant height and root length are respectively increased by 30.6 percent and 16.7 percent; the fresh weight and the dry weight are respectively increased by 46.8 percent and 96.0 percent.
Determination of chlorophyll content: collecting leaf tips of the homotopic leaves in each treatment group, cutting into pieces, weighing 0.2 g each, extracting chlorophyll, injecting chlorophyll extract into a cuvette, and measuring absorbance at 665 nm and 649nm with 95% ethanol as blank control. As shown in FIG. 10, the effect of the administration of the Bacillus thuringiensis fermentation broth on chlorophyll in spinach increased with the T1 gradient, and chlorophyll tended to decrease with increasing concentration.
And (3) soil enzyme activity determination: the determination of soil enzyme activity refers to a method for determining the Guansong shade. The soil sucrase activity was measured by a colorimetric method, and the mass (mg) of glucose produced in 1g of soil after 1 day was expressed as the enzyme activity of sucrose in mg/g/d. The soil urease activity is measured by a colorimetric method, and NH generated by the soil after 1d 3 The mass of-N (mg) represents the urease activity, expressed in mg/g/d. The activity of the alkaline phosphatase in the soil is measured by adopting a disodium phenyl phosphate colorimetric method, and taking 1g of P in the soil after 1d 2 O 5 The number of milligrams indicates the phosphatase activity in mg/g/d. The activity of soil catalase is determined by potassium permanganate titration method, and H consumed by 1g soil per minute 2 O 2 The number of mg indicates the catalase activity, and mg/g/d indicates the catalase activity.
And (3) measuring the activity of the root system enzyme: the activity of root superoxide dismutase (SOD) is determined by adopting a Nitrogen Blue Tetrazole (NBT) method, and taking the enzyme quantity required for inhibiting NBT photochemical reduction by 50 percent as an enzyme activity unit (U); peroxidase (POD) activity is determined by guaiacol method, and enzyme A470 change of 0.01 per minute is used as a peroxidase change unit (U); catalase (CAT) activity is measured by adopting an ultraviolet absorption method, and the enzyme amount of which A240 is reduced by 0.1 per minute is taken as an enzyme activity unit (U); the content of Malondialdehyde (MDA) is measured by a thiobarbituric acid (TBA) method. The results are shown in FIG. 11. The influence of the application of the bacillus thuringiensis fermentation liquor on spinach rhizosphere soil enzyme metabolism is as follows: urease, sucrase and catalase show a parabolic trend of firstly increasing and then decreasing on the whole; the phosphatase content and the whole bacillus thuringiensis fermentation liquor application form an inverse proportion relation; the dehydrogenase content and the application of the bacillus thuringiensis fermentation liquor present a direct proportional relationship.
Influence of bacillus thuringiensis fermentation broth treatment on spinach soil physicochemical properties: the results are shown in FIG. 12. The influence of the application of the bacillus thuringiensis fermentation liquor on the spinach rhizosphere soil nutrients is as follows: the content of the quick-acting phosphorus in the soil is in a parabolic trend, and the content of the quick-acting phosphorus in the soil is increased and then decreased; the total nitrogen, organic matter and pH value of the soil are increased obviously.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (9)
1. The application of the bacillus thuringiensis in dissolving phosphorus in soil, promoting plant growth or regulating soil enzyme system metabolism.
2. The use of claim 1, wherein the bacillus thuringiensis comprises bacillus thuringiensis TG-5.
3. The use according to claim 1, wherein said promoting plant growth comprises increasing plant root length, dry fresh weight and chlorophyll content.
4. Use according to any one of claims 1 to 3, wherein a fermentation broth of Bacillus thuringiensis is used to effect soil phosphorus solubilization, plant growth promotion or regulation of soil enzyme system metabolism.
5. The use according to claim 4, wherein the fermentation broth is prepared by: inoculating Bacillus thuringiensis into a fermentation culture medium, and performing shaking culture at 36-39 deg.C and 180-220 r/min for more than 8 h.
6. The use according to claim 5, wherein the fermentation medium comprises a C source comprising yeast extract, soluble starch, maltose, glucose, sucrose or corn meal, an N source and inorganic salts.
7. Use according to claim 6, wherein the N source comprises tryptone, urea, soy flour, ammonium sulphate or beef extract.
8. Use according to claim 6, wherein the inorganic salt comprises sodium chloride, calcium chloride, magnesium nitrate, potassium nitrate, ferrous sulphate heptahydrate or aluminium phosphate.
9. The use according to claim 6, wherein the concentration ratio of the C source, the N source and the inorganic salt in the fermentation medium is 5-10.
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| CN102598909A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Method for regulating activity of soil matrix enzyme with Bacillus thuringiensis fermentation liquid |
| CN102599196A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Tall fescue growth adjusting method of employing bacillus thuringiensis fermentation broth |
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| CN102191205A (en) * | 2011-04-17 | 2011-09-21 | 南京农业大学 | Bacterial strain B1 for converting insoluble phosphate into soluble phosphate |
| CN102598909A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Method for regulating activity of soil matrix enzyme with Bacillus thuringiensis fermentation liquid |
| CN102599196A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Tall fescue growth adjusting method of employing bacillus thuringiensis fermentation broth |
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