CN110564637B

CN110564637B - Composite microbial inoculum for promoting wheat growth and application thereof

Info

Publication number: CN110564637B
Application number: CN201910620587.9A
Authority: CN
Inventors: 李哲斐; 王娟娟
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2019-07-10
Filing date: 2019-07-10
Publication date: 2022-07-12
Anticipated expiration: 2039-07-10
Also published as: CN110564637A

Abstract

The invention discloses a compound microbial inoculum for promoting wheat growth and application thereof. The composite microbial inoculum is prepared by mixing and fermenting Azotobacter (Azotobacter chlorococcus) N24 bacterial liquid, Rhizobium (Rhizobium radiobacter) J16 bacterial liquid and Klebsiella (Klebsiella quasivariicola) P5 bacterial liquid. The nitrogen-fixing bacteria N24, the potassium-dissolving bacteria J16 and the phosphorus-dissolving bacteria P5 are separated from wheat rhizosphere soil, and the composite microbial inoculum prepared by mixing and fermenting the three bacteria liquids has the functions of fixing nitrogen, dissolving phosphorus and dissolving potassium, can greatly increase the contents of effective nitrogen, effective phosphorus and effective potassium in soil, remarkably improve the plant height, fresh weight, dry weight, nitrogen content, phosphorus content and potassium content of plants, promote the growth of the plants, reduce the use amount of chemical fertilizers and improve the yield of crops, and has important significance in agricultural production.

Description

Composite microbial inoculum for promoting wheat growth and application thereof

Technical Field

The invention relates to the technical field of plant rhizosphere growth-promoting bacteria, and particularly relates to a compound microbial inoculum for promoting wheat growth and application thereof.

Background

Nitrogen, phosphorus and potassium are necessary nutrient elements for plant growth and metabolic activity, and play a key role in high yield of crops. The relevant literature indicates that 95% of phosphorus in soil is combined with ions such as calcium, aluminum, iron and the like to form compounds which are difficult to dissolve; potassium and alkali metals such as sodium and calcium are also present in the form of aluminosilicates, reducing the solubility of potassium. Therefore, the plants in the soil can absorb and utilize less active phosphorus and potassium elements. In addition, although a large amount of fertilizers are put into farmlands every year, the utilization rate of the fertilizers by plants is only about 35%, and redundant nitrogen fertilizers enter a groundwater system to cause certain damage to the environment. Therefore, the method reduces the use of chemical fertilizers, improves the high-efficiency utilization of nitrogen, phosphorus and potassium in the soil, and has very important significance on the aspects of crop high yield, soil ecological maintenance and the like.

Microorganisms can play an important role in soil nutrient element conversion, for example, phosphorus-dissolving bacteria can convert insoluble phosphorus into a form which can be used by plants, silicate bacteria can release potassium ions from potassium feldspar, and nitrogen-fixing bacteria can convert nitrogen in the air into ammonium. The plant probiotics not only enhance the absorption of nitrogen, phosphorus and potassium of plants by activating nutrient elements in soil, but also can synthesize and secrete substances which have a promoting effect on plant growth, such as indole-3-acetic acid (IAA), siderophore, 1-aminocyclopropane-carboxylic Acid (ACC) deaminase and the like, so as to promote the growth and development of the plants.

For example, the Korean yang and the like (2018) collect soil from a tea garden and separate to obtain a bacillus subtilis strain which has strong capacity of transforming and releasing soil potassium. After the separated potassium-decomposing bacteria are inoculated to soil, the content of quick-acting potassium in the tea garden soil is increased by about 28 percent. The bud weight of the tea tree is measured, and the bud weight is obviously increased after the potassium-solubilizing bacterium is used; meanwhile, the tea polyphenol content of the tea is reduced, the concentration of amino acid is obviously improved, and the potassium bacteria can improve the tea yield and the tea quality. Although the technology can promote the growth of plants to a certain extent, as only one potassium-dissolving bacterium is added, although the available potassium content in the soil is increased, phosphorus still becomes a factor for limiting the growth of plants, so that the single strain has limited capability of improving the soil nutrient conversion and the plant growth.

Also, for example, chinese patent application No. CN201610155718.7 discloses a highly efficient phosphate solubilizing bacterium capable of promoting growth of overground tissue of trifolium repens and its application, specifically, trifolium repens rhizosphere soil is collected, a phosphate solubilizing bacterium is separated and purified from a sample by using PKO selective medium, and identified as enterobacter cloacae (enteobacter cloacae), which can form a larger phosphate solubilizing ring on PKO medium. After the bacterium is inoculated on the white clover, the biomass of the host plant is greatly increased. The technology only contains one kind of phosphate solubilizing bacteria, has single function and can only improve the content of phosphorus in soil. The growth of plants needs a large amount of nitrogen, phosphorus and potassium, and a single phosphate solubilizing bacterium cannot meet the growth requirement of the plants, so that the phosphate solubilizing bacterium has certain limitation in popularization and use.

Also, as Reinhold et al (1993), isolate azotobacter sp from the pioneer plant Laocapsa grass (Leptochlo tusca) in Baystein saline-alkali soil, which proves that the biological yield per year of the Kala grass growing in the saline-alkali soil without applying any nitrogen fertilizer all the year round can reach 20-40 t/hm²It is closely related to the action of nitrogen-fixing vibrio. The endogenous nitrogen-fixing bacteria existing in some gramineous plants and host plants form a high-efficiency nitrogen-fixing system-an endogenous nitrogen-fixing system in a long-term co-evolution process. Researches prove that the endogenetic nitrogen fixation bacteria avoid the inhibition of compound nitrogen and the competition of indigenous microorganisms, are more favorable for fully exerting the nitrogen fixation efficiency, secrete the nitrogen fixation product to be directly supplied to plants for absorption, and show higher nitrogen fixation efficiency. Meanwhile, the plant growth promoter also has the functions of secreting auxin, dissolving phosphorus, enhancing disease resistance of plants, resisting stress and the like. The technology only contains one nitrogen fixing bacterium, has single function, can play a role in fixing nitrogen and dissolving phosphorus to a certain extent, but still cannot meet the requirement of large amount of nitrogen, phosphorus and phosphorus for plant growth,Potassium requirement.

Therefore, screening related functional microorganisms and researching the growth promoting effect of the functional microorganisms on plants have important significance in the aspects of reducing the use of potassium fertilizer, improving the yield of crops and the like.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a compound microbial inoculum for promoting the growth of wheat. The azotobacter N24, the potassium bacteria J16 and the phosphorus-solubilizing bacteria P5 are separated from the wheat rhizosphere soil, the three bacteria have good adaptability to wheat, can colonize the wheat rhizosphere and promote the growth of the wheat, and the mixed bacteria liquid prepared by mixing and fermenting the three bacteria liquids can obviously improve the growth performance of plants.

The invention also aims to provide the application of the composite microbial inoculum in improving the growth performance of plants.

In order to achieve the purpose, the invention is realized by the following scheme:

the invention separates a Azotobacter N24 from wheat rhizosphere soil, identifies the Azotobacter N24 in aspects of morphology, physiological and biochemical characteristics and genetics, and verifies that the Azotobacter N24 is Azotobacter in the identification result of 16S rDNA (Azotobacter sp. CCNWSX1904). The azotobacter N24 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: and M2019281. The preservation address is Wuhan university in China. The result of the determination of the azotobacter activity shows that the azotobacter activity of the strain is higher and is 152.56nmol/gh, nitrogen in the atmosphere can be effectively fixed, the content of quick-acting nitrogen in soil can be greatly increased, the utilization rate of nitrogen elements by plants is improved, the growth of the plants is further promoted, the using amount of a chemical fertilizer is reduced, and the yield of crops is improved.

The invention also separates a kalium-solubilizing bacterium J16 from wheat rhizosphere soil, identifies the kalium-solubilizing bacterium J16 in aspects of morphology, physiological and biochemical characteristics and genetics, and verifies that the kalium-solubilizing bacterium J16 is Rhizobium (Rhizobium sp. CCNWSX1901) according to the 16S rDNA identification result. The potassium-solubilizing bacterium J16 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, wherein the preservation number is CCTCC NO: m2019279. The preservation address is Wuhan university. The potassium content is measured, and the result shows that the strain can effectively dissolve the insoluble potassium in the plant rhizosphere soil, greatly increase the content of the quick-acting potassium in the soil, improve the utilization rate of the plant to the potassium in the soil, further promote the plant growth, reduce the use amount of the fertilizer and improve the crop yield.

The invention also separates a phosphorus-solubilizing bacterium P5 from wheat rhizosphere soil, identifies the phosphorus-solubilizing bacterium P5 in aspects of morphology, physiological and biochemical characteristics and genetics, and verifies that the phosphorus-solubilizing bacterium P5 is Klebsiella through 16S rDNA identification results (Klebsiella sp. CCNWSX1902). The phosphorus-solubilizing bacterium P5 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, wherein the preservation number is CCTCC NO: m2019280. The preservation address is Wuhan university in China. The phosphorus content is measured, and the result shows that the strain can effectively dissolve insoluble phosphorus in the plant rhizosphere soil, greatly increase the content of quick-acting phosphorus in the soil, improve the utilization rate of the plants on the phosphorus in the soil, further promote the growth of the plants, reduce the use amount of chemical fertilizers and improve the crop yield.

The bacterial liquid of the three bacteria is inoculated into an LB liquid culture medium according to a certain proportion, and the mixed bacterial liquid obtained after fermentation has the functions of fixing nitrogen, dissolving phosphorus and dissolving potassium, so that the content of effective nitrogen, effective phosphorus and effective potassium in soil can be greatly increased, the plant height, fresh weight, dry weight, nitrogen content, phosphorus content and potassium content of plants are obviously improved, and the function of promoting plant growth is achieved.

Therefore, the invention requests a compound microbial inoculum for promoting the growth of wheat, which is prepared by mixing and fermenting Azotobacter sp.CCNWSX1904N 24 bacterial liquid, Rhizobium sp.CCNWSX1901J 16 bacterial liquid and Klebsiella sp.CCNWSX1902P 5 bacterial liquid.

Preferably, the volume ratio of the Azotobacter (Azotobacter sp.CCNWSX1904) N24 bacterial liquid, Rhizobium sp.CCNWSX1901) J16 bacterial liquid and Klebsiella sp.CCNWSX1902P 5 bacterial liquid is (1-2): (1-2): (1-2).

More preferably, the volume ratio of the Azotobacter (Azotobacter sp. CCNWSX1904) N24 bacterial liquid, rhizobiam (Rhizobium sp. CCNWSX1901) J16 bacterial liquid and Klebsiella sp (Klebsiella sp. CCNWSX1902) P5 bacterial liquid is 2:1: 1.

preferably, the total effective viable count of the three bacteria in the composite microbial inoculum is 2 multiplied by 10⁸～9×10⁸One per mL.

Preferably, the Azotobacter (Azotobacter sp. CCNWSX1904) N24 is preserved in China Center for Type Culture Collection (CCTCC) at 4 and 22 months in 2019, with the preservation number of CCTCC NO: m2019281.

Preferably, the rhizobiaum (Rhizobium sp. ccnwsx1901) J16 is deposited in the chinese type culture collection (CCTCC) at 22/4/2019 with the collection number of CCTCC NO: m2019279.

Preferably, the Klebsiella sp.ccnwsx1902P 5 is deposited in the chinese culture collection center (CCTCC) at 4 months and 22 days in 2019, with the deposit number of CCTCC NO: m2019280.

Preferably, the fermentation medium is LB liquid medium, which consists of the following components: yeast extract with the final concentration of 3-7 g/L, tryptone with the final concentration of 8-12 g/L, NaCl with the final concentration of 5-15 g/L and the balance of water.

More preferably, the LB liquid medium consists of: yeast extract at a final concentration of 5g/L, tryptone at a final concentration of 10g/L, NaCl at a final concentration of 10g/L, and the balance water.

Preferably, the culture condition of the fermentation is 25-30 ℃ and 150-180 rpm for 24-48 h.

More preferably, the culture condition of the fermentation is 18 ℃ and 180rpm for 24-48 h.

The invention also requests to protect the application of the complex microbial inoculum in improving the growth performance of plants.

Preferably, the improvement of the plant growth performance comprises the promotion of plant growth, the improvement of plant height, the improvement of plant dry weight, and the improvement of plant nitrogen content, potassium content and phosphorus content.

More preferably, the plant is wheat.

The invention also provides application of the composite microbial inoculum in dissolving plant rhizosphere soil insoluble potassium and insoluble phosphorus.

Wherein the insoluble potassium is a silicate mineral containing potassium, and the insoluble phosphorus is inorganic phosphorus and/or organic phosphorus.

The invention also provides application of the composite microbial inoculum in improving the contents of quick-acting nitrogen, quick-acting potassium and quick-acting phosphorus in plant rhizosphere soil.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention separates azotobacter N24, potassium bacteria J16 and phosphorus bacteria P5 from wheat rhizosphere soil, and the three strains have good adaptability to wheat and can colonize in wheat rhizosphere and promote wheat growth.

(2) The composite microbial inoculum prepared by mixing and fermenting the three bacterial liquids has the functions of fixing nitrogen, dissolving phosphorus and dissolving potassium, can greatly increase the contents of effective nitrogen, effective phosphorus and effective potassium in soil, remarkably improve the plant height, fresh weight, dry weight, nitrogen content, phosphorus content and potassium content of plants, promote the growth of the plants, reduce the using amount of chemical fertilizers and improve the yield of crops, and has important significance in agricultural production.

Drawings

FIG. 1 shows the results of the enzyme activities of different azotobacteria.

FIG. 2 is a phylogenetic tree of strain N24.

FIG. 3 shows the variation of plant height, dry weight and nitrogen content of wheat inoculated with the strain N24.

FIG. 4 shows the potassium ring formation of strain J16.

FIG. 5 shows the results of colorimetric detection of IAA production by strain J16.

FIG. 6 is a phylogenetic tree of strain J16.

FIG. 7 shows the variation of plant height, dry weight, potassium content and phosphorus content after wheat inoculation with strain J16.

FIG. 8 shows the formation of a phosphate solubilizing loop by strain P5.

FIG. 9 is a phylogenetic tree of strain P5.

FIG. 10 shows the variation of plant height, dry weight, nitrogen content and phosphorus content of wheat inoculated with strain P5.

FIG. 11 shows the growth of wheat after the compound microbial inoculum is applied.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Ashby medium: glucose 10g, K₂HPO₄ 0.2g，NaCl 0.2g，MgSO₄·7H₂O 0.2g，K₂SO₄0.2g，CaCO₃5g, 15g of agar and 1000mL of distilled water.

Potassium-dissolving culture medium: sucrose 5g, Na₂HPO₄ 2g，MgSO₄·7H₂O 0.5g，FeCl₃ 0.005g，CaCO₃0.1g, 1g of potassium feldspar powder (which is sieved by a 100-mesh sieve and then is soaked in sterile water for 3 days to remove water-soluble potassium), 15g of agar, 1000mL of distilled water and pH of 7.0-7.5.

PKO inorganic phosphorus medium: glucose 10g, Ca₃(PO₄)₂ 5g，(NH₄)SO₄ 0.5g，NaCl 0.2g，KCl 0.2g，MgSO₄·7H₂O 0.3g，MnSO₄ 0.03g，FeSO₄·7H₂0.03g of O, 0.5g of yeast extract, 15g of agar and 1000ml of distilled water, wherein the pH value is 6.8-7.0.

LB solid Medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl, 15g of agar and 1000mL of water.

LB liquid medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl and 1000mL of water.

Salkowski developing solution: 0.5M FeCl₃ 1mL，H₂SO₄30mL and 50mL of distilled water.

Monkina organophosphorus culture medium: glucose 10g, (NH)₄)₂SO₄ 0.5g，NaCl 0.3g，KCl 0.3g，FeSO₄·7H₂O 0.03g，MnSO₄·4H₂0.03g of O, 0.2g of lecithin, CaCO₃5g, 0.4g of yeast extract, 15g of agar, l000mL of distilled water and the pH value of 7.0-7.2.

Example 1 screening and identification of Azotobacter

Screening of azotobacteria

1. Azotobacteria prescreening

Wheat plants growing for about 7 months are collected from farmlands in Yanling areas of Shaanxi province in 9 months in 2017, and soil with loosely combined wheat roots is shaken off by force. The plant roots are cut off and put into a 10mL sterilized centrifuge tube, 5mL sterile water is injected, and then ultrasonic treatment is carried out, so that rhizosphere soil tightly combined with the plant roots is dissolved in the water. The collected soil sample is yellow loam, the sample has 24.03mg/kg of quick-acting phosphorus, 126.17mg/kg of quick-acting potassium, 56.28mg/kg of alkaline hydrolysis nitrogen, 21.06g/kg of organic matters and 8.27 of pH.

Diluting the aqueous solution by a series of 10 times, and respectively diluting 5 μ L to 10^-5、10^-6And 10^-7The suspension was spread on an Ashby medium and cultured at 28 ℃ for 5 days. Transferring the grown strain to LB solid culture medium for storage.

2. Azotase activity assay

A10 mL bottle with a rubber stopper was filled with Ashby medium and inclined, and the 11 strains grown on the Ashby medium were inoculated on the inclined surfaces, respectively, and cultured at 28 ℃ for 24 hours. 1mL of air was evacuated from the bottle with a syringe, and 1mL of C was injected with the syringe₂H₂And reacting at 28 ℃ for 12 h. Then, 1mL of the gas was evacuated from the bottle, and C was measured by Shimadzu GC-14C gas chromatograph₂H₄And C₂H₂Simultaneously measuring the peak area of known concentration C₂H₄And C₂H₂And making a standard curve, converting the standard curve to obtain the amount of the generated ethylene, and according to a formula: azotase activity ═ C₂H₄Number of moles (nmol)/[ cell weight (g). The reaction time (h)]The nitrogenase activity was calculated.

The test result shows that the nitrogen-fixing enzyme activity of the strain N24 (namely CCNWSX1904) is high and is 152.56nmol/gh (shown in figure 1).

3. Production of IAA Standard Curve

Accurately weighing 10mg of IAA, dissolving with a small amount of ethanol, and then diluting to 10mL with deionized water to obtain 1mg/mL IAA standard stock solution. Accurately sucking a certain amount of standard stock solution into a 10mL volumetric flask, fixing the volume to scale by using deionized water, so that IAA series standard solutions with standard concentrations of 0, 40, 80, 120, 160 and 200 mu g/mL are obtained, and storing in a dark place after preparation. And (3) taking 1mL of standard solution with each concentration, adding equivalent Salkowski colorimetric solution, carrying out dark reaction for 30min, detecting a light absorption value at 530nm by using an ultraviolet visible spectrophotometer, and drawing a standard curve by taking the light absorption value as a vertical coordinate and the IAA concentration as a horizontal coordinate.

4. N24 IAA-producing ability measurement

The nitrogen-fixing strain N24 is inoculated into a conical flask containing 20mL of TY liquid culture medium and cultured for 48h at the temperature of 28 ℃ under the condition of 150r/min shaking. Centrifuging the culture solution at 10000rpm for 10min, sucking 1mL of supernatant, adding equal volume of Salkowski developing solution, reacting at room temperature in dark place for 30min, and measuring OD with ultraviolet spectrophotometer₅₃₀。

The results showed that strain N24 was able to produce IAA at a concentration of 149.23mg/L in the broth.

II, identification of the strain N24

1. Morphological characteristics

The N24 strain is prepared into a bacterial suspension, diluted and coated on an Ashby culture medium, and the bacterial colony and the bacterial morphology are observed after the culture is cultured for 24 hours at 28 ℃.

The result shows that the bacterium is negative in gram staining, oval in cell shape, capsular and free from spore production; after 24 hours of culture on the Ashby culture medium, the colony is circular, the edge is neat and semitransparent, and the colony appears dark brown when growing to the later stage.

2. Physiological and biochemical characteristics

The physiological and biochemical characteristics of the strain N24, such as VP reaction, carbon source utilization, indole test and the like, are determined by referring to a bacteria identification manual, and the result shows that the strain can grow by using glucose, citric acid and lactose as carbon sources, the indole test, catalase and catalase reaction are positive, and the VP reaction and methyl red test are negative.

3. 16S rDNA sequencing

Extracting genome DNA of the strain N24 by using a bacterial genome extraction kit, amplifying 16S rDNA by using bacterial universal primers 27F (5 '-AGAGTTTGATCCTGGCTCAG) and 1492R (5' -TACCTTGTTACGACTT), carrying out electrophoresis detection, then sending the amplified product to a company for sequencing, and comparing the sequence in an NCBI database. The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

Strain N2416S rDNA sequence (SEQ ID NO: 1):

GTGAGTAATGCCTAGGAATCTGCCCGATAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGCTCTTCGGACCTCACGCTATCGGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCTGTAAGCGAATACCTTGCAGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGGTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGCCTGACTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGCTCCTTGAGAGCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGTCGCGAGGCGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC

and (3) identification result: the strain N24 belongs to Azotobacter in classification, the similarity with Azotobacter chlorococcus is 99.92%, and phylogenetic tree of the strain N24 is shown in figure 2.

Azotobacteria (Azotobacter sp. CCNWSX1904) N24 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: and M2019281. The preservation address is Wuhan university in China.

Wheat growth promotion test

Selecting plump wheat seeds, performing surface sterilization by using 70% ethanol, washing with sterile water for at least 6 times, coating the sterile water washed for the last time on a beef extract peptone solid culture medium, and checking whether the surface sterilization of the wheat is complete. The sterilized wheat seeds were sown in 6 pots of soil, the surface of which was covered with approximately 1cm of soil. Experiment set 2 treatments, 1 treatment of 1 plant inoculated with 1mL sterile medium, 2 treatment of 2 plants inoculated with N24 inoculum (about 10)⁸one/mL) 1mL, 3 replicates per treatment. The flowerpot is placed in a greenhouse, and the culture conditions are set to be 16h/8h under illumination and 25 ℃/18 ℃. After wheat seedlings emerge, water is poured once every 5 days, and the plant height, dry weight and nitrogen content of the plants are measured after 60 days.

As shown in Table 1 and FIG. 3, it can be seen from Table 1 that the growth vigor of wheat inoculated with azotobacter is significantly better than that of the non-inoculated control group, and the height, dry weight and nitrogen content of the inoculated plant are respectively increased by 33.7%, 41.8% and 57.7% compared with those of the blank control group.

TABLE 1 variation of plant height, dry weight and nitrogen content before and after inoculation of wheat with N24 bacterial liquid

	Plant height (cm)	Plant Dry weight (g)	Plant nitrogen content (g/kg)
				Control	20.1	0.966	7.9
Inoculation treatment	26.87	1.37	12.46

Example 2 screening and identification of Potassium-solubilizing bacteria

Screening of potassium-decomposing bacteria

1. Preliminary screening for potassium-decomposing bacteria

Wheat plants growing for about 7 months are collected from farmlands in Yangling areas of Shaanxi province in 9 months in 2017, and soil loosely combined with wheat roots is shaken off by force. The plant roots are cut off and put into a 10mL sterilized centrifuge tube, 5mL sterile water is injected, and then ultrasonic treatment is carried out, so that rhizosphere soil tightly combined with the plant roots is dissolved in the water. The collected soil sample is yellow loam, the sample has 24.03mg/kg of quick-acting phosphorus, 126.17mg/kg of quick-acting potassium, 56.28mg/kg of alkaline hydrolysis nitrogen, 21.06g/kg of organic matters and 8.27 of pH.

Diluting the aqueous solution by a series of 10 times, and respectively diluting 5 μ L to 10^-5、10^-6And 10^-7The soil suspension was spread on a potassium-solubilizing medium and cultured at 28 ℃ for 5 days. Transferring the strain capable of producing the transparent potassium-decomposing ring to an LB solid culture medium for storage and standby.

2. Determination of Potassium-resolving Capacity

And (3) purifying the strain with the potassium-solubilizing ring, then re-inoculating the strain on a potassium-solubilizing culture medium, and measuring the diameter D of the potassium-solubilizing ring and the diameter D of a bacterial colony. Colonies with D/D greater than 2 were picked up in LB liquid medium and cultured at 28 ℃ for 24 h. 1mL of the above bacterial suspension was pipetted into 50mL of potassium feldspar liquid medium, and 3 replicates of each treatment were set using 1mL of LB medium instead of the bacterial suspension as a control. After culturing at 28 deg.C and 200rpm for 3 days, the content of available potassium in the supernatant of the potassium-solubilizing bacteria culture solution was determined by flame photometry.

The test results show that the potassium ring-releasing and soluble effective potassium content of the strain with the number of J16 (namely CCNWSX1901) is larger and is respectively 3.85 and 224.7 mg/L. The potassium ring formed by strain J16 is shown in FIG. 4.

3. Inorganic phosphorus dissolving capacity

The J16 strain with the potassium ring is inoculated into LB liquid medium according to 1 percent and cultured for 24h at 28 ℃. 1mL of the above bacterial suspension was aspirated into 50mL of phosphate solubilizing liquid medium, and 3 replicates of each treatment were set using 1mL of LB medium instead of bacterial suspension as a control. Culturing at 28 deg.C and 200rpm for 5 days, centrifuging 5mL of culture solution at 10000rpm, collecting 1mL of the above supernatant, adding 5mL of molybdenum-antimony anti-reagent, diluting to 50mL with ultrapure water, reacting for 20min, and measuring OD₆₅₀And calculating the phosphorus content.

Test results show that the strain J16 has phosphorus dissolving capacity, and the content of soluble phosphorus in fermentation liquor reaches 156.89 mg/L.

4. Determination of IAA-producing ability of J16

The J16 strain with potassium-dissolving capacity is inoculated into a conical flask containing 20mL of TY liquid culture medium and cultured for 48h at 28 ℃ under the condition of 150r/min shaking. Pipetting 50. mu.L of the culture solution onto a white ceramic plate, adding 50. mu.L of Salkowski color developing solution, using 50. mu.L of 50mg/L IAA standard solution as a positive control, reacting at room temperature in a dark place for 30min, and observing the color.

The results are shown in FIG. 5, which shows that the bacterial suspension of the strain J16 turns red after reacting with the developing solution, and the strain can produce IAA.

II, identification of the Strain J16

1. Morphological characteristics

The J16 strain is prepared into bacterial suspension, diluted and coated on an LB solid culture medium, and after 24 hours of culture at 28 ℃, the colony and the thallus morphology are observed.

The result shows that the gram staining of the bacterium is negative, the cell is in a short rod shape, and no spore is produced; after 24 hours of culture on an LB culture medium, the colony is circular, the edge is neat, and the colony is milky white.

2. Physiological and biochemical characteristics

The physiological and biochemical characteristics of the strain J16, such as VP reaction, carbon source utilization, indole test and the like, are determined by referring to a bacteria identification manual, and the results show that the strain can grow by taking glucose and citric acid as carbon sources, cannot utilize lactose and malonate, is negative in VP reaction, indole test, methyl red test and starch hydrolysis test, and can produce urease.

3. 16S rDNA sequencing

Extracting genome DNA of the strain J16 by using a bacterial genome extraction kit, amplifying 16S rDNA by using bacterial universal primers 27F (5 '-AGAGTTTGATCCTGGCTCAG) and 1492R (5' -TACCTTGTTACGACTT), carrying out electrophoresis detection, then sending to a company for sequencing, and comparing the sequences in an NCBI database. The 16S rDNA sequence of the strain J16 is shown in SEQ ID NO 2.

Strain J1616S rDNA sequence (SEQ ID NO: 2):

GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCATGCTGATCTGCGATTACTAGCGATTCCAACTTCATGCACTCGAGTTGCAGAGTGCAATCCGAACTGAGATGGCTTTTGGAGATTAGCTCGACATCGCTGTCTCGCTGCCCACTGTCACCACCATTGTAGCACGTGTGTAGCCCAGCCCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCGGCTTATCACCGGCAGTCCCCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTTCTGGGGCCAGCCTAACTGAAGGACATCGTCTCCAATGCCCATACCCCGAATGTCAAGAGCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGTTTAATGCGTTAGCTGCGCCACCGAACAGTATACTGCCCGACGGCTAACATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCAGTAATGGACCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCGAATATCTACGAATTTCACCTCTACACTCGGAATTCCACTTACCTCTTCCATACTCAAGATACCCAGTATCAAAGGCAGTTCCAGAGTTGAGCTCTGGGATTTCACCCCTGACTTAAATATCCGCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAACGCTAGCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCTTCTCCGGATACCGTCATTATCTTCTCCGGTGAAAGAGCTTTACAACCCTAAGGCCTTCATCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCTGATCATCCTCTCAGACCAGCTATGGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCAACGCGGGCCAATCCTTCCCCGATAAATCTTTCCCCCGTAGGGCGTATGCGGTATTAATTCCAGTTTCCCGGAGCTATTCCGCAGGAAAGGGTATGTTCCC

and (3) identification result: strain J16 was classified as Rhizobium and was 99.68% similar to Rhizobium radiobacter, and the phylogenetic tree of strain J16 is shown in FIG. 6.

Rhizobium (Rhizobium sp. CCNWSX1901) J16 is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 4 months and 22 days, with the preservation number of CCTCC NO: m2019279. The preservation address is Wuhan university in China.

Wheat growth promotion test

Selecting plump wheat seeds, performing surface sterilization by using 70% ethanol, washing with sterile water for at least 6 times, coating the sterile water washed for the last time on a beef extract peptone solid medium, and checking whether the surface sterilization of the wheat is complete. The sterilized wheat seeds were sown in 6 pots of soil, the surface of which was covered with approximately 1cm of soil. Experiment set 2 treatments, 1 treatment of 1 plant inoculated with 1mL sterile medium, 2 treatment of 2 plants inoculated with J16 inoculum (about 10)⁸one/mL) 1mL, 3 replicates per treatment. The flowerpot is placed in a greenhouse, and the culture conditions are set to be 16h/8h of illumination and 25 ℃/18 ℃. After wheat seedlings emerge, water is poured once every 5 days, and the plant height, fresh weight, dry weight and nitrogen, phosphorus and potassium contents of the plants are measured after 60 days.

The results are shown in table 2 and fig. 7, and it can be seen from table 2 that the phosphorus and potassium absorption of the wheat inoculated with the potassium-solubilizing bacteria is significantly higher than that of the non-inoculated control group, and the growth vigor of the plants is better. The height, dry weight, potassium content and phosphorus content of the inoculated plant are respectively improved by 30.2 percent, 51.1 percent, 24.8 percent and 42.9 percent compared with the blank control group.

TABLE 2 variation of plant height, dry weight, potassium content and phosphorus content before and after inoculation of J16 bacterial liquid into wheat

	Plant height (cm)	Plant Potassium content (g/kg)	Plant Dry weight (g)	Plant phosphorus content (g/kg)
					Control	21.03	24.82	0.95	2.42
Inoculation treatment	27.38	30.98	1.435	3.46

Example 3 screening and identification of phosphate solubilizing bacteria

Screening of phosphate solubilizing bacteria

1. Primary screen for phosphorus-dissolving bacteria

Diluting the aqueous solution by a series of 10-fold dilutions, and respectively taking 5 μ L of dilution with 10^-5、10^-6And 10^-7The soil suspension was spread on PKO inorganic phosphorus medium and cultured at 28 ℃ for 5 days. And (3) transferring the bacterial strain capable of generating the transparent phosphorus-dissolving ring to an LB solid culture medium for storage and standby.

2. Determination of inorganic phosphorus dissolving ability

Purifying the above strain with phosphorus-solubilizing ringAnd (4) inoculating the strain on a PKO inorganic phosphorus culture medium again after the strain is changed, and measuring the diameter D of a phosphorus dissolving ring and the diameter D of a bacterial colony. Colonies with D/D greater than 2 were picked up in LB liquid medium and cultured at 28 ℃ for 24 h. 1mL of the above bacterial suspension was pipetted into 50mL of phosphate solubilizing medium (NBRIP medium), and 3 replicates of each treatment were used as controls in place of the bacterial suspension in 1mL of LB medium. Culturing at 28 deg.C and 200rpm for 5 days, centrifuging 5mL of culture solution at 10000rpm, collecting 1mL of the above supernatant, adding 5mL of molybdenum-antimony anti-reagent, diluting to 50mL with ultrapure water, reacting for 20min, and measuring OD₆₅₀And calculating the phosphorus content.

The test result shows that the phosphorus-dissolving ring of the strain with the number of P5 (namely CCNWSX1902) and the content of soluble phosphorus in the fermentation liquor are larger and are respectively 2.56 mg/L and 231.68 mg/L. The formation of the phosphate solubilizing loop by strain P5 is shown in FIG. 8.

3. Determination of IAA-producing ability of P5

The P5 strain with phosphorus-dissolving capacity is inoculated into an Erlenmeyer flask containing 20mL of LB liquid medium and cultured at 28 ℃ for 48h under the condition of 150r/min shaking. 50 mu L of culture solution is sucked by a pipette onto the white ceramic plate, 50 mu L of Salkowski developing solution is added, 50 mu L of 50mg/L IAA standard solution is used as a positive control, and the color is observed after the reaction is carried out for 30min at room temperature in a dark place.

The result shows that the bacterial suspension of the strain P5 turns red after reacting with the developing solution, which indicates that the strain can produce IAA.

4. Dissolving organic phosphorus

The P5 strain is inoculated on LB liquid culture medium according to 1 percent for shaking culture for 24h, 5 mu L of the suspension is dripped on Monkina organophosphorus culture medium, and the culture is carried out for 5 days at 28 ℃.

The results showed that clear circles of dissolved phosphorus appeared around the colonies, indicating that strain P5 was also able to dissolve the organic phosphorus.

5. Nitrogen fixation capacity detection

The strain P5 is inoculated on LB culture medium for 24h, colonies are picked out and dissolved in sterile water to prepare P5 bacterial suspension, 5 mu L of bacterial suspension is taken by a pipette and dripped on the Ashby culture medium, and the culture is carried out for 3 days at 28 ℃.

The result shows that the strain can normally grow on the Ashby nitrogen-free culture medium, and the strain has certain nitrogen fixation capacity.

II, identification of the strain P5

1. Morphological characteristics

The P5 strain is prepared into bacterial suspension, diluted and coated on an LB solid culture medium, and after 24 hours of culture at 28 ℃, the colony and the thallus morphology are observed.

The result shows that the gram staining of the bacterium is negative, the cell is in a short rod shape, and no spore is produced; after the bacterial colony is cultured on an LB culture medium for 24 hours, the bacterial colony is circular, the edge is neat, the diameter is 2-3 mm, the bacterial colony is milky white, and the center is slightly yellowish.

2. Physiological and biochemical characteristics

The physiological and biochemical characteristics of the strain P5, such as VP reaction, carbon source utilization, indole test and the like, are determined by referring to a bacteria identification manual, and the result shows that the strain can grow by taking glucose, citric acid, malonate and lactose as carbon sources, the indole test and the methyl red test are negative, the VP reaction is positive, and amylase and urease can be produced.

3. 16S rDNA sequencing

Extracting genome DNA of the strain P5 by using a bacterial genome extraction kit, amplifying 16S rDNA by using bacterial universal primers 27F (5 '-AGAGTTTGATCCTGGCTCAG) and 1492R (5' -TACCTTGTTACGACTT), carrying out electrophoresis detection, then sending to a company for sequencing, and comparing the sequences in an NCBI database. The 16S rDNA sequence of the strain P5 is shown in SEQ ID NO. 3.

Strain P516S rDNA sequence (SEQ ID NO: 3):

ACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTGGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTGAGGTTAATAACCTCATCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTG

and (3) identification result: strain P5 was classified as belonging to Klebsiella, with a 99.85% similarity to Klebsiella quaivaricola, and the phylogenetic tree of strain P5 is shown in FIG. 9.

Klebsiella sp.CCNWSX1902P 5 was deposited in the China Center for Type Culture Collection (CCTCC) at 22 months 4 in 2019 with the preservation number of CCTCC NO: m2019280. The preservation address is Wuhan university in China.

Wheat growth promotion test

Selecting plump wheat seeds, performing surface sterilization by using 70% ethanol, washing with sterile water for at least 6 times, coating the sterile water washed for the last time on a beef extract peptone solid culture medium, and checking whether the surface sterilization of the wheat is complete. The sterilized wheat seeds were sown in 6 pots of soil, the surface of which was covered with approximately 1cm of soil. Experiment 2 treatments, 1 treatment with 1mL sterile medium per plant, 2 treatment with P5 inoculum per plant (about 10%⁸one/mL) 1mL, 3 replicates per treatment. The flowerpot is placed in a greenhouse, and the culture conditions are set to be 16h/8h under illumination and 25 ℃/18 ℃. After wheat seedlings emerge, water is poured once every 5 days, and the plant height, fresh weight, dry weight and nitrogen, phosphorus and potassium contents of the plants are measured after 60 days.

The results are shown in table 3 and fig. 10, and it can be seen from table 3 that the absorption of nitrogen and phosphorus by the wheat inoculated with the phosphate solubilizing bacteria is obviously higher than that of the non-inoculated control group, and the growth vigor of the plants is better. The height, dry weight, nitrogen content and phosphorus content of the inoculated plant are respectively improved by 20.2 percent, 55.3 percent, 31.8 percent and 51.2 percent compared with the blank control group.

TABLE 3 variation of plant height, dry weight, nitrogen content and phosphorus content before and after wheat inoculation of P5 bacterial liquid

	Plant height (cm)	Plant nitrogen content (g/kg)	Plant Dry weight (g)	Plant phosphorus content (g/kg)
					Control	21.12	8.57	0.94	2.44
Inoculation treatment	25.39	11.3	1.46	3.69

Example 4 screening of Complex microbial Agents

1. Preparation of composite microbial inoculum and proportioning screening

The three strains of N24 with strong nitrogen fixation capacity, J16 with strong phosphorus dissolving capacity and P5 with strong potassium dissolving capacity are inoculated into an LB liquid culture medium and cultured for 24-48 hours at 28 ℃ and 180 rpm. According to N24: j16: the P5 is combined at a volume ratio of 1:1:1, 2:1:1, 1:2:1 and 1:1:2, and the contents of N24: j16: the best effect was obtained with the combination P5 ═ 2:1:1 (table 4).

TABLE 4 influence of different compounding ratios of composite microbial inoculum on nitrogen, available phosphorus and potassium in soil

N24：J16：P5	Soil alkaline hydrolysis nitrogen (mg/kg)	Soil available potassium (mg/kg)	Soil available phosphorus (mg/kg)
				1:1:1	19.28	98.24	13.09
1:2:1	19.54	100.51	14.19
				2:1:1	21.98	104.42	14.92
1:1:2	18.71	90.38	11.49

Preparation of liquid N24 microbial inoculum:

the strain N24 is inoculated into a beef extract peptone liquid medium according to the proportion of 1 percent, and the strain is cultured for 36 hours under the conditions of 28 ℃ and 180rpm to obtain the N24 microbial inoculum.

Preparation of liquid P5 microbial inoculum:

inoculating the P5 into a beef extract peptone liquid medium according to the proportion of 1%, and culturing at 28 ℃ and 180rpm for 36 hours to obtain the P5 microbial inoculum.

Preparation of liquid J16 microbial inoculum:

the J16 is inoculated into a beef extract peptone liquid medium according to the proportion of 1 percent, and the mixture is cultured for 36 hours under the conditions of 28 ℃ and 180rpm, thus obtaining the J16 microbial inoculum.

Preparing a complex microbial inoculum:

culturing P5, N24 and J16 in beef extract peptone liquid medium for 36 hours, mixing at a ratio of 2:1:1, and making the total effective viable count of three bacteria in the mixed solution be 2X 10⁸～9×10⁸And (2) taking the mixed bacterial liquid per mL as the composite bacterial agent.

2. Wheat potting test

Selecting plump wheat seeds, performing surface sterilization by using 70% ethanol, washing with sterile water for at least 6 times, coating the sterile water washed for the last time on a beef extract peptone solid culture medium, and checking whether the surface sterilization of the wheat is complete. The sterilized wheat seeds were sown in flowerpot soil with a surface covering of about 1cm of soil. The experiment was set to 2 treatments, 1 treatment with 5mL sterile medium per pot, 2 treatments with 5mL complex inoculum per pot, 3 replicates per treatment. The flowerpot is placed in a greenhouse, and the culture conditions are set to be 16h/8h of illumination and 25/18 ℃ of temperature. After wheat seedlings emerge, water is poured once every 5 days, and after 60 days, the effective nitrogen, phosphorus and potassium of the soil, the plant height, fresh weight, dry weight and nitrogen, phosphorus and potassium contents of the plants are measured. The results are shown in tables 5 and 6.

As can be seen from Table 5, the transformation effect of the complex microbial inoculum on nutrient elements in soil is obviously higher than that of a non-inoculated control group. The total nitrogen, alkaline hydrolysis nitrogen, quick-acting phosphorus, quick-acting potassium and organic matters in the soil inoculated with the composite microbial inoculum are respectively improved by 18.4 percent, 81.2 percent, 99.5 percent, 19.4 percent and 55.7 percent compared with the blank control group (table 5).

TABLE 5 Effect of inoculum treatment on soil nutrients

As can be seen from Table 6, the absorption of NPK by wheat inoculated with the compound microbial inoculum is obviously higher than that of the non-inoculated control group, and the growth vigor of plants is better. The plant height, dry weight, nitrogen content, potassium content and phosphorus content of the inoculated composite microbial inoculum are respectively increased by 59.8%, 105%, 65.6%, 50.3% and 75.1% compared with the blank control group (Table 6).

TABLE 6 Effect of inoculation treatment on wheat growth

3. Method for reducing fertilizer usage amount by utilizing compound microbial agent

And (3) applying a local conventional compound fertilizer or compound microbial inoculum to the wheat-planted soil, verifying whether the compound fertilizer can be replaced by the compound microbial inoculum, and reducing the using amount of the fertilizer.

75% compound fertilizer was applied to the soil as a blank control and 100% compound fertilizer was applied as a control. In addition, 75% of compound fertilizer and compound microbial inoculum are applied to the soil to serve as a test group.

As shown in FIG. 11, it can be seen from FIG. 11 that the use of chemical fertilizer was reduced, the growth of wheat was poor, and the color of leaves was yellow. However, after the compound microbial inoculum is added into the soil with 25 percent of fertilizer reduced, the growth condition of wheat is obviously improved, compared with a control group which completely uses the fertilizer, the growth condition of wheat in a test group with reduced fertilizer application and microbial inoculum addition is even better than that in the control group, which shows that the compound microbial inoculum can reduce the use amount of the fertilizer under the condition of not influencing the growth of the wheat.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> composite microbial inoculum for promoting wheat growth and application thereof

<140> 2019106205879

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1299

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<213> Azotobacter chromococcus

<400> 1

gtgagtaatg cctaggaatc tgcccgatag tgggggacaa cgtttcgaaa ggaacgctaa 60

taccgcatac gtcctacggg agaaagtggg ggctcttcgg acctcacgct atcggatgag 120

cctaggtcgg attagctagt tggtggggta aaggctcacc aaggcgacga tccgtaactg 180

gtctgagagg atgatcagtc acactggaac tgagacacgg tccagactcc tacgggaggc 240

agcagtggga atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag 300

aaggtcttcg gattgtaaag cactttaagt tgggaggaag ggctgtaagc gaataccttg 360

cagttttgac gttaccgaca gaataagcac cggctaactt cgtgccagca gccgcggtaa 420

tacgaagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggtttgg 480

taagttggat gtgaaagccc cgggctcaac ctgggaactg catccaaaac tgcctgacta 540

gagtacggta gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag 600

gaacaccagt ggcgaaggcg accacctgga ctgatactga cactgaggtg cgaaagcgtg 660

gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgtc gactagccgt 720

tgggctcctt gagagcttag tggcgcagct aacgcattaa gtcgaccgcc tggggagtac 780

ggccgcaagg ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg 840

gtttaattcg aagcaacgcg aagaacctta cctggccttg acatgctgag aactttccag 900

agatggattg gtgccttcgg gaactcagac acaggtgctg catggctgtc gtcagctcgt 960

gtcgtgagat gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca 1020

cctcgggtgg gcactctaag gagactgccg gtgacaaacc ggaggaaggt ggggatgacg 1080

tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggt cggtacagag 1140

ggttgccaag tcgcgaggcg gagctaatcc cagaaaaccg atcgtagtcc ggatcgcagt 1200

ctgcaactcg actgcgtgaa gtcggaatcg ctagtaatcg cgaatcagaa tgtcgcggtg 1260

aatacgttcc cgggccttgt acacaccgcc cgtcacacc 1299

<210> 2

<211> 1250

<212> DNA

<213> Rhizobium radiobacter

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ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcagcat gctgatctgc 60

gattactagc gattccaact tcatgcactc gagttgcaga gtgcaatccg aactgagatg 120

gcttttggag attagctcga catcgctgtc tcgctgccca ctgtcaccac cattgtagca 180

cgtgtgtagc ccagcccgta agggccatga ggacttgacg tcatccccac cttcctctcg 240

gcttatcacc ggcagtcccc ttagagtgcc caactaaatg ctggcaacta agggcgaggg 300

ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga cagccatgca 360

gcacctgttc tggggccagc ctaactgaag gacatcgtct ccaatgccca taccccgaat 420

gtcaagagct ggtaaggttc tgcgcgttgc ttcgaattaa accacatgct ccaccgcttg 480

tgcgggcccc cgtcaattcc tttgagtttt aatcttgcga ccgtactccc caggcggaat 540

gtttaatgcg ttagctgcgc caccgaacag tatactgccc gacggctaac attcatcgtt 600

tacggcgtgg actaccaggg tatctaatcc tgtttgctcc ccacgctttc gcacctcagc 660

gtcagtaatg gaccagtaag ccgccttcgc cactggtgtt cctccgaata tctacgaatt 720

tcacctctac actcggaatt ccacttacct cttccatact caagataccc agtatcaaag 780

gcagttccag agttgagctc tgggatttca cccctgactt aaatatccgc ctacgtgcgc 840

tttacgccca gtaattccga acaacgctag cccccttcgt attaccgcgg ctgctggcac 900

gaagttagcc ggggcttctt ctccggatac cgtcattatc ttctccggtg aaagagcttt 960

acaaccctaa ggccttcatc actcacgcgg catggctgga tcaggcttgc gcccattgtc 1020

caatattccc cactgctgcc tcccgtagga gtttgggccg tgtctcagtc ccaatgtggc 1080

tgatcatcct ctcagaccag ctatggatcg tcgccttggt aggcctttac cccaccaact 1140

agctaatcca acgcgggcca atccttcccc gataaatctt tcccccgtag ggcgtatgcg 1200

gtattaattc cagtttcccg gagctattcc gcaggaaagg gtatgttccc 1250

<210> 3

<211> 1313

<212> DNA

<213> Klebsiella (Klebsiella quasivariicola)

<400> 3

acgggtgagt aatgtctggg aaactgcctg atggaggggg ataactactg gaaacggtag 60

ctaataccgc ataacgtcgc aagaccaaag tgggggacct tcgggcctca tgccatcaga 120

tgtgcccaga tgggattagc tggtaggtgg ggtaacggct cacctaggcg acgatcccta 180

gctggtctga gaggatgacc agccacactg gaactgagac acggtccaga ctcctacggg 240

aggcagcagt ggggaatatt gcacaatggg cgcaagcctg atgcagccat gccgcgtgtg 300

tgaagaaggc cttcgggttg taaagcactt tcagcgggga ggaaggcggt gaggttaata 360

acctcatcga ttgacgttac ccgcagaaga agcaccggct aactccgtgc cagcagccgc 420

ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagcgc acgcaggcgg 480

tctgtcaagt cggatgtgaa atccccgggc tcaacctggg aactgcattc gaaactggca 540

ggctagagtc ttgtagaggg gggtagaatt ccaggtgtag cggtgaaatg cgtagagatc 600

tggaggaata ccggtggcga aggcggcccc ctggacaaag actgacgctc aggtgcgaaa 660

gcgtggggag caaacaggat tagataccct ggtagtccac gctgtaaacg atgtcgattt 720

ggaggttgtg cccttgaggc gtggcttccg gagctaacgc gttaaatcga ccgcctgggg 780

agtacggccg caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc 840

atgtggttta attcgatgca acgcgaagaa ccttacctgg tcttgacatc cacagaactt 900

tccagagatg gattggtgcc ttcgggaact gtgagacagg tgctgcatgg ctgtcgtcag 960

ctcgtgttgt gaaatgttgg gttaagtccc gcaacgagcg caacccttat cctttgttgc 1020

cagcggttag gccgggaact caaaggagac tgccagtgat aaactggagg aaggtgggga 1080

tgacgtcaag tcatcatggc ccttacgacc agggctacac acgtgctaca atggcatata 1140

caaagagaag cgacctcgcg agagcaagcg gacctcataa agtatgtcgt agtccggatt 1200

ggagtctgca actcgactcc atgaagtcgg aatcgctagt aatcgtagat cagaatgcta 1260

cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga gtg 1313

Claims

1. The composite microbial inoculum for promoting the growth of wheat is characterized by comprising azotobacter (A)Azotobactersp.) bacterial liquid N24, rhizobia (Rhizobium: (Rhizobiumsp.) J16 bacterial liquid and Klebsiella (L.), (L.) B.Klebsiellasp.) P5 bacterial liquid is prepared after mixing and fermenting; nitrogen-fixing bacteria (A), (B)Azotobactersp.) N24 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: m2019281; the rhizobia bacterium (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Rhizobiumsp.) J16 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: m2019279; the bacterium Klebsiella (Klebsiella pneumoniae) (ii)Klebsiellasp.) P5 is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 22 months, with the preservation number of CCTCC NO: m2019280.

2. The complex microbial inoculant according to claim 1, wherein the nitrogen-fixing bacteria (A), (B), (C) and (D)Azotobactersp.) bacterial liquid N24, rhizobia (Rhizobium: (Rhizobiumsp.) J16 bacterial liquid, Klebsiella bacterium (Klebsiella sp.) (Klebsiellasp.) the volume ratio of P5 bacterial liquid is 1-2: 1-2: 1 to 2.

3. The composite microbial inoculum according to claim 2, wherein the azotobacter (a), (b), (c), or (c)Azotobactersp.) bacterial liquid N24, rhizobia (Rhizobium: (Rhizobiumsp.) J16 bacterial liquid, Klebsiella bacterium (Klebsiella sp.) (Klebsiellasp.) the volume ratio of P5 bacterial liquid is 2:1: 1.

4. the composite bacterial agent of claim 1, wherein the total effective viable count of three bacteria in the composite bacterial agent is 2 x 10⁸～9×10⁸One per mL.

5. The complex microbial inoculant according to claim 1, wherein the fermentation medium is an LB liquid medium and consists of the following components: yeast extract with the final concentration of 3-7 g/L, tryptone with the final concentration of 8-12 g/L, NaCl with the final concentration of 5-15 g/L and the balance of water.

6. The complex microbial inoculant according to claim 1, wherein the fermentation is carried out at a temperature of 25-30 ℃ and at a speed of 150-180 rpm for 24-48 h.

7. Use of the complex microbial inoculum of any one of claims 1 to 6 in improving the growth performance of plants.