CN110564637B - A compound inoculant for promoting wheat growth and its application - Google Patents

Abstract

The invention discloses a compound bacterial agent for promoting the growth of wheat and its application. The compound bacterial agent is prepared by mixing and fermenting Azotobacter chroococcum N24 bacterial liquid, Rhizobium radiobacter J16 bacterial liquid and Klebsiella quasivariicola P5 bacterial liquid. The present invention separates nitrogen-fixing bacteria N24, potassium-solubilizing bacteria J16 and phosphorus-dissolving bacteria P5 from the wheat rhizosphere soil. Significantly increase the content of available nitrogen, available phosphorus and available potassium in the soil, significantly increase the plant height, fresh weight, dry weight, nitrogen content, phosphorus content and potassium content, promote plant growth, reduce the use of chemical fertilizers, and increase crop yields , is of great significance in agricultural production.

Description

A compound inoculant for promoting wheat growth and its application

technical field

The invention relates to the technical field of plant rhizosphere growth-promoting bacteria, in particular, to a composite inoculant for promoting wheat growth and its application.

Background technique

Nitrogen, phosphorus and potassium are essential nutrients for plant growth and metabolic activities, and play a key role in the high yield of crops. Relevant literature shows that 95% of phosphorus in soil combines with calcium, aluminum, iron and other ions to form compounds that are difficult to dissolve; and potassium, sodium, calcium and other alkali metals also exist in the form of aluminosilicates, reducing the solubility of potassium. Therefore, there are less active phosphorus and potassium elements that plants can absorb and utilize in the soil. In addition, although a large amount of chemical fertilizers are put into farmland every year, the utilization rate of these fertilizers by plants is only about 35%, and the excess nitrogen fertilizer enters the groundwater system, causing certain damage to the environment. Therefore, reducing the use of chemical fertilizers and improving the efficient utilization of nitrogen, phosphorus, and potassium in the soil are of great significance for high crop yield and soil ecological maintenance.

Microorganisms can play an important role in the transformation of soil nutrients. For example, phosphorus-solubilizing bacteria can convert insoluble phosphorus into a form that can be used by plants, silicate bacteria can release potassium ions from potassium feldspar, and nitrogen-fixing bacteria can convert The nitrogen in the air is converted to ammonium. These plant probiotics not only enhance the absorption of nitrogen, phosphorus and potassium by plants by activating nutrients in the soil, but also some microorganisms can also synthesize and secrete substances that promote plant growth, such as indole-3-acetic acid (IAA ), siderophore and 1-aminocyclopropane-carboxylic acid (ACC) deaminase, etc., thereby promoting the growth and development of plants.

For example, Han Xiaoyang et al. (2018) collected soil from tea gardens and isolated a strain of Bacillus subtilis, which has a strong ability to transform and release soil potassium. After the isolated potassium-solubilizing bacteria were inoculated into the soil, it was found that the content of available potassium in the tea garden soil increased by about 28%. By measuring the bud weight of tea trees, it was found that the weight of 100 buds increased significantly after the use of potassium-solubilizing bacteria; at the same time, the content of tea polyphenols in tea was reduced, and the concentration of amino acids was significantly increased, indicating that potassium-solubilizing bacteria can not only increase the yield of tea, but also Improve the quality of tea. Although this technology can promote the growth of plants to a certain extent, due to the addition of only one type of potassium-solubilizing bacteria, although the available potassium content in the soil is increased, the phosphorus element still becomes a limiting factor for plant growth. Soil nutrient transformation and plant growth enhancement capacity is limited.

Another example is the Chinese patent with the application number CN201610155718.7, which discloses a kind of high-efficiency phosphate-solubilizing bacteria that can promote the growth of above-ground tissues of white clover and its application, specifically collecting the rhizosphere soil of white clover, using PKO to select medium from the sample. A phosphate-dissolving bacterium was isolated and purified, which was identified as Enterobacter cloacae, which could form a larger phosphate-dissolving circle on PKO medium. After inoculation of the bacteria on white clover, the biomass of the host plant was greatly increased. This technology contains only one type of phosphorus-solubilizing bacteria, which has a single function and can only increase the phosphorus content in the soil. Plant growth requires a large amount of nitrogen, phosphorus and potassium, and a single phosphorus-solubilizing bacteria cannot meet the needs of plant growth, and there are certain limitations in its promotion and use.

For another example, Reinhold et al. (1993) isolated Azoarus spp. from the pioneer plant Leptochlotusca in Pakistan's saline-alkali land, proving that carrageenan growing in saline-alkali soil without any nitrogen The biological yield can reach 20～40t/hm ² , which is closely related to the role of nitrogen-fixing Vibrio. Endophytic nitrogen-fixing bacteria that exist in some grasses and host plants form an efficient nitrogen-fixing system-endophytic nitrogen-fixing system in the long-term co-evolution process. Studies have shown that endophytic nitrogen-fixing bacteria avoid the inhibition of compound nitrogen and the competition of indigenous microorganisms, and are more conducive to fully exerting nitrogen-fixing efficiency, secreting nitrogen-fixing products and directly supplying plants for absorption, showing higher nitrogen-fixing efficiency. At the same time, it also has the functions of secreting auxin, dissolving phosphorus, enhancing plant disease resistance, stress resistance and other aspects of promoting plant growth. Since this technology contains only one nitrogen-fixing bacteria, the function is relatively single. Although it can play a role in nitrogen-fixing and certain phosphorus-dissolving role, it still cannot meet the needs of a large amount of nitrogen, phosphorus and potassium for plant growth.

Therefore, it is of great significance to screen related functional microorganisms and study their growth-promoting effects on plants to reduce the use of potassium fertilizers and improve crop yields.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a composite bacterial agent for promoting the growth of wheat in order to overcome the above-mentioned deficiencies of the prior art. The present invention separates nitrogen-fixing bacteria N24, potassium-solubilizing bacteria J16 and phosphorus-dissolving bacteria P5 from the wheat rhizosphere soil. The mixed bacterial liquid obtained by the mixed fermentation of bacterial liquid can significantly improve the growth performance of plants.

Another object of the present invention is to provide the application of the above-mentioned composite inoculant in improving plant growth performance.

In order to achieve the above object, the present invention is achieved through the following schemes:

In the present invention, a nitrogen-fixing bacteria N24 is isolated from the wheat rhizosphere soil, and its morphology, physiological and biochemical characteristics and genetics are identified, and the 16S rDNA identification results verify that it is a nitrogen-fixing bacteria (Azotobactersp.CCNWSX1904). The nitrogen-fixing bacteria N24 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019281. The deposit address is China. Wuhan. Wuhan University. By measuring its nitrogenase activity, the results show that the nitrogenase activity of this strain is relatively high, which is 152.56nmol/gh, which can effectively fix nitrogen in the atmosphere, greatly increase the content of available nitrogen in the soil, and improve the nitrogen content of plants. The utilization rate of elements, thereby promoting plant growth, reducing the use of chemical fertilizers, and increasing crop yields.

In the present invention, a potassium-solubilizing bacteria J16 is also isolated from the wheat rhizosphere soil, and its morphology, physiological and biochemical characteristics and genetics are identified, and the 16S rDNA identification results verify that it is a rhizobium (Rhizobium sp.CCNWSX1901) . The potassium-solubilizing bacteria J16 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019279. The deposit address is China. Wuhan. Wuhan University. By measuring the potassium content, the results show that the strain can effectively dissolve the insoluble potassium in the rhizosphere soil of plants, and can greatly increase the content of available potassium in the soil, improve the utilization rate of potassium in the soil by plants, and then promote plant growth. , reduce the use of chemical fertilizers and increase crop yields.

In the present invention, a phosphorus-dissolving bacteria P5 is also isolated from the wheat rhizosphere soil, and its morphology, physiological and biochemical characteristics and genetics are identified, and the 16S rDNA identification results verify that it is Klebsiella sp. .CCNWSX1902). The phosphorus-dissolving bacteria P5 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019280. The deposit address is China. Wuhan. Wuhan University. By measuring the phosphorus content, the results show that the strain can effectively dissolve the insoluble phosphorus in the rhizosphere soil of plants, and can greatly increase the content of available phosphorus in the soil, improve the utilization rate of phosphorus in the soil by plants, and then promote plant growth. , reduce the use of chemical fertilizers and increase crop yields.

The bacterial liquid of the above three bacteria is inoculated into the LB liquid medium according to a certain proportion, and the mixed bacterial liquid obtained after fermentation has the functions of nitrogen fixation, phosphorus dissolution and potassium dissolution at the same time, which can greatly increase the available nitrogen, available phosphorus and potassium in the soil. The content of available potassium can significantly increase the plant height, fresh weight, dry weight, nitrogen content, phosphorus content and potassium content, and has the effect of promoting plant growth.

Therefore, the present invention requires a composite bacterial agent for promoting the growth of wheat, which is composed of nitrogen-fixing bacteria (Azotobactersp.CCNWSX1904) N24 bacteria liquid, Rhizobium sp.CCNWSX1901) J16 bacteria liquid, Klebsiella sp.CCNWSX1902 ) P5 bacterial liquid mixed and fermented.

Preferably, the volume ratio of the nitrogen-fixing bacteria (Azotobacter sp. CCNWSX1904) N24 bacterial liquid, the Rhizobium (Rhizobium sp. CCNWSX1901) J16 bacterial liquid, and the Klebsiella (Klebsiella sp. CCNWSX1902) P5 bacterial liquid is (1～2 ): (1 to 2): (1 to 2).

More preferably, the volume ratio of described nitrogen-fixing bacteria (Azotobacter sp.CCNWSX1904) N24 bacterial liquid, Rhizobium (Rhizobium sp.CCNWSX1901) J16 bacterial liquid, Klebsiella (Klebsiella sp.CCNWSX1902) P5 bacterial liquid is 2: 1:1.

Preferably, the total effective viable count of the three bacteria in the compound bacterial preparation is 2×10 ⁸ to 9×10 ⁸ cells/mL.

Preferably, the nitrogen-fixing bacteria (Azotobacter sp. CCNWSX1904) N24 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019281.

Preferably, the Rhizobium sp. CCNWSX1901 J16 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019279.

Preferably, the Klebsiella sp. CCNWSX1902 P5 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019280.

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

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

Preferably, the culturing conditions of the fermentation are 25-30° C. and 150-180 rpm for 24-48 hours.

More preferably, the culture conditions of the fermentation are cultured at 18° C. and 180 rpm for 24-48 hours.

The present invention also claims to protect the application of the above-mentioned compound inoculants in improving plant growth performance.

Preferably, the improving plant growth performance includes promoting plant growth, increasing plant height, increasing plant dry weight, and increasing plant nitrogen content, potassium content, and phosphorus content.

More preferably, the plant is wheat.

The present invention also claims to protect the application of the above-mentioned compound bacterial agent in dissolving insoluble potassium and insoluble phosphorus in plant rhizosphere soil.

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

The present invention also claims to protect the application of the above-mentioned compound inoculant in increasing the content of available nitrogen, available potassium and available phosphorus in plant rhizosphere soil.

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

(1) The present invention separates nitrogen-fixing bacteria N24, potassium-solubilizing bacteria J16 and phosphorus-dissolving bacteria P5 from the wheat rhizosphere soil, and the three strains have good adaptability to wheat, can colonize the wheat rhizosphere and promote the growth of wheat .

(2) The compound inoculum prepared by the mixed fermentation of the three bacterial liquids has the functions of nitrogen fixation, phosphorus dissolution and potassium dissolution at the same time, which can greatly increase the content of available nitrogen, available phosphorus and available potassium in the soil, and significantly increase the plant height of the plant. , fresh weight, dry weight, nitrogen content, phosphorus content and potassium content, promote plant growth, reduce the use of chemical fertilizers, and improve crop yield, which is of great significance in agricultural production.

Description of drawings

Figure 1 shows the results of the enzyme activity assay of different nitrogen-fixing bacteria.

Figure 2 is a phylogenetic tree of strain N24.

Figure 3 shows the changes in plant height, dry weight and nitrogen content of wheat after inoculation with strain N24.

Figure 4 shows the potassium depletion circle formed by strain J16.

Figure 5 shows the results of colorimetric detection of IAA produced by strain J16.

Figure 6 is a phylogenetic tree of strain J16.

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

Figure 8 shows the phosphate solubilization circle formed by strain P5.

Figure 9 is a phylogenetic tree of strain P5.

Figure 10 shows the changes in plant height, dry weight, nitrogen content and phosphorus content of wheat after inoculation with strain P5.

Figure 11 shows the growth of wheat after applying the compound inoculant.

Detailed ways

The present invention will be further elaborated below in conjunction with the accompanying drawings and specific embodiments of the description, and the embodiments are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

Ashby medium: 10 g of glucose, 0.2 g of K ₂ HPO ₄ , 0.2 g of NaCl, 0.2 g of MgSO ₄ ·7H ₂ O 0.2 g, 0.2 g of K ₂ SO ₄ , 5 g of CaCO ₃ , 15 g of agar, and 1000 mL of distilled water.

Potassium solubilizing medium: sucrose 5g, Na ₂ HPO ₄ 2g, MgSO ₄ 7H ₂ O 0.5g, FeCl ₃ 0.005g, CaCO ₃ 0.1g, potassium feldspar powder 1g (soak in sterile water for 3d after passing through a 100-mesh sieve, remove Water-soluble potassium), agar 15g, distilled water 1000mL, pH7.0～7.5.

PKO inorganic phosphorus medium: glucose 10g, Ca ₃ (PO ₄ ) ₂ 5g, (NH ₄ )SO ₄ 0.5g, NaCl 0.2g, KCl 0.2g, MgSO ₄ ·7H ₂ O 0.3g, MnSO ₄ 0.03g, FeSO ₄ ·7H ₂ O 0.03g, yeast extract 0.5g, agar 15g, distilled water 1000ml, pH value 6.8～7.0.

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

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

Salkowski color developing solution: 0.5M FeCl ₃ 1mL, H ₂ SO ₄ 30mL, distilled water 50mL.

Montkina organophosphorus medium: glucose 10g, (NH ₄ ) ₂ SO ₄ 0.5g, NaCl 0.3g, KCl 0.3g, FeSO ₄ ·7H ₂ O 0.03g, MnSO ₄ ·4H ₂ O 0.03g, lecithin 0.2 g, CaCO ₃ 5g, yeast extract 0.4g, agar 15g, distilled water 1000mL, pH 7.0-7.2.

Example 1 Screening and identification of nitrogen-fixing bacteria

1. Screening of nitrogen-fixing bacteria

1. Preliminary screening of nitrogen-fixing bacteria

In September 2017, wheat plants that were about 7 months old were collected from the farmland in Yangling District, Shaanxi Province, and the loosely bound soil of the wheat roots was shaken off. Cut off the plant roots and put them into a 10 mL sterilized centrifuge tube, inject 5 mL of sterile water, and then perform ultrasonic treatment to dissolve the rhizosphere soil tightly bound to the plant roots in the water. The collected soil samples were yellow loam soil, with available phosphorus 24.03 mg/kg, available potassium 126.17 mg/kg, alkaline hydrolyzable nitrogen 56.28 mg/kg, organic matter 21.06 g/kg, pH=8.27.

A series of 10-fold dilutions of the aqueous solution were made, and 5 μL of soil suspensions with dilutions of 10 ^-5 , 10 ^-6 and 10 ^-7 were spread on Ashby medium and cultured at 28° C. for 5 days. The grown strains were transferred to LB solid medium for future use.

2. Determination of nitrogenase activity

The Ashby medium was injected into a 10 mL bottle with a rubber stopper to form a slant, and the 11 strains of bacteria grown on the Ashby medium were inoculated onto the slant, and cultured at 28° C. for 24 hours. 1 mL of air was drawn from the bottle with a syringe, and 1 mL of C ₂ H ₂ was injected with a syringe, and the reaction was carried out at 28° C. for 12 h. Then ₁ mL of gas was extracted from the bottle, and the peak areas of C ₂ H ₄ and C ₂ H _{2 were} measured using a Shimadzu GC _- 14C gas chromatograph _. Standard curve, the amount of ethylene generated is calculated from the standard curve, and the nitrogenase activity is calculated according to the formula: nitrogenase activity=C ₂ H ₄ moles (nmol)/[cell weight (g)*reaction time (h)] .

The test results showed that the nitrogenase activity of strain N24 (ie CCNWSX1904) was higher, which was 152.56 nmol/gh (as shown in Figure 1).

3. IAA standard curve production

Accurately weigh 10 mg of IAA, dissolve it with a small amount of ethanol, and then dilute to 10 mL with deionized water to obtain a standard stock solution of IAA with a concentration of 1 mg/mL. Accurately draw a certain amount of standard stock solution into a 10mL volumetric flask, and dilute the volume to the mark with deionized water, so that the standard concentration of IAA series standard solution is 0, 40, 80, 120, 160, 200 μg/mL. Light save. Take 1 mL of each concentration standard solution, add an equal volume of Salkowski colorimetric solution, react in the dark for 30 min, and detect the absorbance value at 530 nm with a UV-Vis spectrophotometer, draw the standard curve with the absorbance value as the ordinate and the IAA concentration as the abscissa.

4. Determination of the ability of N24 to produce IAA

The nitrogen-fixing strain N24 was inoculated into a 20 mL conical flask of TY liquid medium, and cultured with shaking at 150 r/min for 48 h at 28 °C. The culture solution was centrifuged at 10,000 rpm for 10 min, 1 mL of supernatant was aspirated, an equal volume of Salkowski chromogenic solution was added, and the reaction was performed in the dark for 30 min at room temperature, and the OD ₅₃₀ was measured by a UV spectrophotometer.

The results showed that strain N24 could produce IAA, and the concentration in its culture medium was 149.23 mg/L.

2. Identification of strain N24

1. Morphological characteristics

The N24 strain was prepared into a bacterial suspension, diluted and spread on Ashby medium, and the colony and cell morphology were observed after culturing at 28°C for 24 hours.

The results showed that the bacterium was negative for Gram staining, the cells were oval, produced capsules, but did not produce spores; after 24 hours of culture on Ashby medium, the colonies were round, with neat edges and translucent, and the colonies grew slightly in the later stage. Brown.

2. Physiological and biochemical characteristics

The physiological and biochemical characteristics of strain N24, such as VP reaction, carbon source utilization, and indole test, were determined with reference to the bacterial identification manual. The results showed that the strain could use glucose, citric acid and lactose as carbon sources for growth. The hydrogenase and contact enzyme reactions were positive, and the VP reaction and methyl red test were negative.

3. 16S rDNA sequencing

The genomic DNA of strain N24 was extracted with a bacterial genome extraction kit, and the 16S rDNA was amplified with bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-TACCTTGTTACGACTT). Compare. The 16S rDNA sequence of the strain is shown in SEQ ID NO:1.

Strain N24 16S rDNA sequence (SEQ ID NO: 1):

GTGAGTAATGCCTAGGAATCTGCCCGATAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGCTCTTCGGACCTCACGCTATCGGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCTGTAAGCGAATACCTTGCAGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGGTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGCCTGACTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGCTCCTTGAGAGCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACC CTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGTCGCGAGGCGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTATCAGTCGATCAGACCTTGTTCGCCCGCCCGACCGATCGATCGATCGACCGCGA

Identification results: Strain N24 belongs to nitrogen-fixing bacteria in classification, and the similarity with Azotobacter chroococcum is 99.92%. The phylogenetic tree of strain N24 is shown in Figure 2.

Azotobacter sp. CCNWSX1904 N24 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019281. The deposit address is China. Wuhan. Wuhan University.

3. Wheat growth promotion test

Pick full-grained wheat seeds, disinfect the surface with 70% ethanol, rinse with sterile water for at least 6 times, and apply the sterile water from the last rinse on beef extract peptone solid medium to check whether the surface disinfection of wheat is thorough. The sterilized wheat seeds were sown in pot soil, 6 seeds per pot, and the surface was covered with about 1 cm of soil. Two treatments were set up in the experiment. In treatment 1, each plant was inoculated with 1 mL of sterile medium, and in treatment 2, each plant was inoculated with 1 mL of N24 bacterial solution (about 10 ⁸ cells/mL), and each treatment was replicated 3 times. The flowerpots were placed in the greenhouse, and the culture conditions were set as light 16h/8h and temperature 25°C/18°C. After the wheat seedlings emerged, water was watered every 5 days, and the plant height, dry weight and nitrogen content were measured after 60 days.

The results are shown in Table 1 and Figure 3. It can be seen from Table 1 that the growth of the wheat inoculated with nitrogen-fixing bacteria is significantly better than that of the uninoculated control group, and the plant height, dry weight and nitrogen content of the inoculated plants are respectively higher than those of the blank control group. improved by 33.7%, 41.8% and 57.7%.

Table 1 Changes in plant height, dry weight and nitrogen content of wheat before and after inoculation with N24 bacterial solution

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

1. Screening of potassium-solubilizing bacteria

1. Primary screening of potassium-solubilizing bacteria

In September 2017, wheat plants that were about 7 months old were collected from the farmland in Yangling District, Shaanxi Province, and the loosely bound soil of the wheat roots was shaken off. Cut off the plant roots and put them into a 10 mL sterilized centrifuge tube, inject 5 mL of sterile water, and then perform ultrasonic treatment to dissolve the rhizosphere soil tightly bound to the plant roots in the water. The collected soil samples were yellow loam soil, with available phosphorus 24.03 mg/kg, available potassium 126.17 mg/kg, alkaline hydrolyzable nitrogen 56.28 mg/kg, organic matter 21.06 g/kg, pH=8.27.

A series of 10-fold dilutions of the aqueous solution were made, and 5 μL of soil suspensions with dilutions of 10 ^-5 , 10 ^-6 and 10 ^-7 were spread on potassium solubilizing medium, and cultured at 28° C. for 5 days. The strains capable of producing transparent potassium-solubilizing circles were transferred to LB solid medium for future use.

2. Determination of potassium solubilizing ability

The above-mentioned strains with potassium-solubilizing circle were purified and re-inoculated on the potassium-solubilizing medium, and the potassium-solubilizing circle diameter D and colony diameter d were determined. Pick colonies with a D/d greater than 2 in LB liquid medium and culture at 28°C for 24h. Pipette 1 mL of the above bacterial suspension into 50 mL of potassium feldspar liquid medium, and use 1 mL of LB medium to replace the bacterial suspension as a control, and each treatment has three replicates. After culturing at 28° C. and 200 rpm for 3 days, the content of available potassium in the supernatant of the potassium-solubilizing bacteria culture solution was measured by flame photometry.

The test results showed that the strain numbered J16 (ie CCNWSX1901) had a relatively large content of soluble and available potassium in the solution of potassium circle and the culture solution, which were 3.85 and 224.7 mg/L, respectively. The potassium solution circle formed by strain J16 is shown in Figure 4.

3. Ability to dissolve inorganic phosphorus

The above-mentioned J16 strain with potassium-dissolving circle was inoculated into LB liquid medium at 1%, and cultured at 28°C for 24h. Pipette 1 mL of the above bacterial suspension into 50 mL of phosphate-dissolving liquid medium, and use 1 mL of LB medium to replace the bacterial suspension as a control, and each treatment has three replicates. After culturing for 5 days at 28 °C and 200 rpm, 5 mL of the culture solution was centrifuged at 10,000 rpm, 1 mL of the above supernatant was taken and 5 mL of molybdenum-antimony anti-reagent was added, and the volume was adjusted to 50 mL with ultrapure water. OD ₆₅₀ and calculate phosphorus content.

The test results showed that the strain J16 had the ability to dissolve phosphorus, and the content of soluble phosphorus in the fermentation broth reached 156.89 mg/L.

4. Determination of the ability of J16 to produce IAA

The J16 strain with potassium-solubilizing ability was inoculated into a 20 mL conical flask of TY liquid medium, and cultured with shaking at 150 r/min for 48 h at 28 °C. Pipette 50 μL of culture solution onto a white ceramic plate, then add 50 μL of Salkowski chromogenic solution, use 50 μL of 50 mg/L IAA standard solution as a positive control, and observe the color after 30 min of reaction in the dark at room temperature.

The results are shown in Figure 5, indicating that the bacterial suspension of the strain J16 reacts with the chromogenic solution to turn red, indicating that the strain can produce IAA.

2. Identification of strain J16

1. Morphological characteristics

The J16 strain was prepared into a bacterial suspension, diluted and spread on LB solid medium, and the colony and bacterial morphology were observed after culturing at 28 °C for 24 h.

The results showed that the bacterium was Gram-negative, the cells were short rod-shaped and did not produce spores; after culturing on LB medium for 24 hours, the colonies were round, with neat edges and milky white colonies.

2. Physiological and biochemical characteristics

The physiological and biochemical characteristics of strain J16, such as VP reaction, carbon source utilization, and indole test, were determined with reference to the bacterial identification manual. The results showed that the strain could grow with glucose and citric acid as carbon sources, but could not use lactose and malonate. , VP reaction, indole test, methyl red test and starch hydrolysis test were negative, can produce urease.

3. 16S rDNA sequencing

The genomic DNA of strain J16 was extracted with a bacterial genome extraction kit, and the 16S rDNA was amplified with bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-TACCTTGTTACGACTT). Compare. The 16S rDNA sequence of strain J16 is shown in SEQ ID NO:2.

Strain J16 16S rDNA sequence (SEQ ID NO: 2):

GGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCATGCTGATCTGCGATTACTAGCGATTCCAACTTCATGCACTCGAGTTGCAGAGTGCAATCCGAACTGAGATGGCTTTTGGAGATTAGCTCGACATCGCTGTCTCGCTGCCCACTGTCACCACCATTGTAGCACGTGTGTAGCCCAGCCCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCGGCTTATCACCGGCAGTCCCCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTTCTGGGGCCAGCCTAACTGAAGGACATCGTCTCCAATGCCCATACCCCGAATGTCAAGAGCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGTTTAATGCGTTAGCTGCGCCACCGAACAGTATACTGCCCGACGGCTAACATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCAGTAATGGACCAGTAAGCCGCCTTCGCCACTGGTGTTCCTCCGAATATCTACGAATTTCACCTCTACACTCGGAATTCCACTTACCTCTTCCATACTCAAGATACCCAGTATCAAAGGCAGTTCCAGAGTTGAGCTCTGGGATTTCACCCCTGACTTAAATATCCGCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAACGCTAGCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCTTCTCCGGATACCGTCATTATCTTCTCCGGTGAAAGAGCTTTACAACCCTAAGGCCTTCATCACTCACGCGGCATGGCTGGA TCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCTGATCATCCTCTCAGACCAGCTATGGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCAACGCGGGCCAATCCTTCCCCGATAAATCTTTCCCCGTAGGGCGTATGCGGTATTAATTCCAGTTTCCCGGAGCTATTCCGCAGGAAAGGGT

Identification results: Strain J16 belongs to Rhizobium in classification, and the similarity with Rhizobium radiobacter is 99.68%. The phylogenetic tree of strain J16 is shown in Figure 6.

Rhizobium sp. CCNWSX1901 J16 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019279. The deposit address is China. Wuhan. Wuhan University.

3. Wheat growth promotion test

Pick full-grained wheat seeds, disinfect the surface with 70% ethanol, rinse with sterile water for at least 6 times, and apply the sterile water from the last rinse on beef extract peptone solid medium to check whether the surface disinfection of wheat is thorough. The sterilized wheat seeds were sown in pot soil, 6 seeds per pot, and the surface was covered with about 1 cm of soil. Two treatments were set up in the experiment. In treatment 1, each plant was inoculated with 1 mL of sterile medium, and in treatment 2, each plant was inoculated with 1 mL of J16 bacterial solution (about 10 ⁸ cells/mL), and each treatment was replicated 3 times. The flowerpots were placed in the greenhouse, and the culture conditions were set as light 16h/8h and temperature 25°C/18°C. After the wheat seedlings emerged, water was watered every 5 days, and the plant height, fresh weight, dry weight and NPK content were measured after 60 days.

The results are shown in Table 2 and Figure 7. It can be seen from Table 2 that the absorption of phosphorus and potassium by the wheat inoculated with potassium-solubilizing bacteria is significantly higher than that of the non-inoculated control group, and the growth of the plants is better. Compared with the blank control group, the plant height, dry weight, potassium content and phosphorus content of the inoculated plants were increased by 30.2%, 51.1%, 24.8% and 42.9%, respectively.

Table 2 Changes of plant height, dry weight, potassium content and phosphorus content before and after inoculation with J16 bacterial solution

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 phosphorus-dissolving bacteria

1. Screening of Phosphorus Dissolving Bacteria

1. Primary screening of phosphorus-dissolving bacteria

In September 2017, wheat plants that were about 7 months old were collected from the farmland in Yangling District, Shaanxi Province, and the loosely bound soil of the wheat roots was shaken off. Cut off the plant roots and put them into a 10 mL sterilized centrifuge tube, inject 5 mL of sterile water, and then perform ultrasonic treatment to dissolve the rhizosphere soil tightly bound to the plant roots in the water. The collected soil samples were yellow loam soil, with available phosphorus 24.03 mg/kg, available potassium 126.17 mg/kg, alkaline hydrolyzable nitrogen 56.28 mg/kg, organic matter 21.06 g/kg, pH=8.27.

A series of 10-fold dilutions of the aqueous solution were made, and 5 μL of soil suspensions with dilutions of 10 ^-5 , 10 ^-6 and 10 ^-7 were spread on PKO inorganic phosphorus medium and cultured at 28°C for 5 days. The strains that can produce transparent phosphate-solubilizing circles were transferred to LB solid medium for future use.

2. Determination of ability to dissolve inorganic phosphorus

The above-mentioned strains with phosphate-solubilizing circle were purified and re-inoculated on PKO inorganic phosphorus medium, and the phosphate-solubilizing circle diameter D and colony diameter d were determined. Pick colonies with a D/d greater than 2 in LB liquid medium and culture at 28°C for 24h. Pipette 1 mL of the above bacterial suspension into 50 mL of phosphate solubilization medium (NBRIP medium), and use 1 mL of LB medium to replace the bacterial suspension as a control, and each treatment has three replicates. After culturing for 5 days at 28 °C and 200 rpm, 5 mL of the culture solution was centrifuged at 10,000 rpm, 1 mL of the above supernatant was taken and 5 mL of molybdenum-antimony anti-reagent was added, and the volume was adjusted to 50 mL with ultrapure water. OD ₆₅₀ and calculate phosphorus content.

The test results showed that the strain numbered P5 (ie CCNWSX1902) had a larger content of soluble phosphorus in the phosphorus-dissolving circle and fermentation broth, which were 2.56 and 231.68 mg/L, respectively. The phosphorus-dissolving circle formed by strain P5 is shown in Figure 8.

3. Determination of the ability of P5 to produce IAA

The P5 strain with phosphate-dissolving ability was inoculated into a 20 mL LB liquid medium conical flask, and cultured with shaking at 150 r/min for 48 h at 28°C. Pipette 50 μL of culture solution onto a white ceramic plate, then add 50 μL of Salkowski chromogenic solution, use 50 μL of 50 mg/L IAA standard solution as a positive control, and observe the color after 30 min of reaction in the dark at room temperature.

The results showed that the bacterial suspension of strain P5 reacted with the chromogenic solution to turn red, indicating that the strain could produce IAA.

4. Dissolved organic phosphorus

The P5 strain was inoculated into LB liquid medium at 1% for shaking culture for 24 hours, and 5 μL of the bacterial suspension was added dropwise to Montkina organophosphorus medium, and cultured at 28° C. for 5 days.

The results showed that a transparent phosphorus-dissolving circle appeared around the colony, indicating that the strain P5 could also dissolve organic phosphorus.

5. Test of nitrogen fixation ability

The strain P5 was inoculated on LB medium for 24 hours, and the colonies were picked and dissolved in sterile water to prepare a P5 bacterial suspension. 5 μL of the bacterial suspension was added dropwise to Ashby medium with a pipette, and cultured at 28°C for 3 days.

The results showed that the strain could grow normally on Ashby nitrogen-free medium, indicating that the strain had certain nitrogen fixation ability.

2. Identification of strain P5

1. Morphological characteristics

The P5 strain was prepared into a bacterial suspension, diluted and spread on LB solid medium, and the colony and bacterial morphology were observed after culturing at 28°C for 24 hours.

The results showed that the bacterium's Gram staining was negative, the cells were short rod-shaped and did not produce spores; after culturing on LB medium for 24 hours, the colonies were round, with neat edges, 2-3 mm in diameter, milky white and slightly pale in the center. yellow.

2. Physiological and biochemical characteristics

The physiological and biochemical characteristics of strain P5, such as VP reaction, carbon source utilization, and indole test, were determined with reference to the bacterial identification manual. Indol test and methyl red test were negative, VP reaction was positive, can produce amylase and urease.

3. 16S rDNA sequencing

The genomic DNA of strain P5 was extracted with a bacterial genome extraction kit, and the 16S rDNA was amplified with bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-TACCTTGTTACGACTT). Compare. The 16S rDNA sequence of strain P5 is shown in SEQ ID NO:3.

Strain P5 16S rDNA sequence (SEQ ID NO: 3):

ACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTGGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTGAGGTTAATAACCTCATCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAAAGGCTAGATCGTTCGTTAGCCACCGTCGACGTTCGTCGCCCGCCCG

Identification results: Strain P5 belongs to Klebsiella in classification, and the similarity with Klebsiella quasivariicola is 99.85%. The phylogenetic tree of strain P5 is shown in Figure 9.

Klebsiella sp. CCNWSX1902 P5 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019280. The deposit address is China. Wuhan. Wuhan University.

3. Wheat growth promotion test

Pick full-grained wheat seeds, disinfect the surface with 70% ethanol, rinse with sterile water for at least 6 times, and apply the sterile water from the last rinse on beef extract peptone solid medium to check whether the surface disinfection of wheat is thorough. The sterilized wheat seeds were sown in pot soil, 6 seeds per pot, and the surface was covered with about 1 cm of soil. Two treatments were set up in the experiment. In treatment 1, each plant was inoculated with 1 mL of sterile medium, and in treatment 2, each plant was inoculated with 1 mL of P5 bacterial solution (about 10 ⁸ cells/mL), and each treatment was replicated 3 times. The flowerpots were placed in the greenhouse, and the cultivation conditions were set as light 16h/8h and temperature 25°C/18°C. After the wheat seedlings emerged, water was watered every 5 days, and the plant height, fresh weight, dry weight and NPK content were measured after 60 days.

The results are shown in Table 3 and Figure 10. It can be seen from Table 3 that the absorption of nitrogen and phosphorus by the wheat inoculated with phosphorus-dissolving bacteria was significantly higher than that of the uninoculated control group, and the plant growth was better. Compared with the blank control group, the plant height, dry weight, nitrogen content and phosphorus content of the inoculated plants were increased by 20.2%, 55.3%, 31.8% and 51.2%, respectively.

Table 3 Changes of plant height, dry weight, nitrogen content and phosphorus content before and after wheat inoculation with P5 bacterial solution

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

The screening of embodiment 4 compound bacterial agent

1. Preparation and ratio screening of compound bacterial agents

The three strains of N24 with strong nitrogen fixation ability, J16 with strong phosphorus dissolution ability and P5 with strong potassium solubilization ability were inoculated into LB liquid medium and cultured at 28°C and 180rpm for 24-48 hours. According to the N24:J16:P5 volume ratio of 1:1:1, 2:1:1, 1:2:1, 1:1:2 to combine, through the determination of phosphorus dissolution, potassium dissolution and nitrogen fixation ability, it is found that N24: J16: P5=2:1:1 combination had the best effect (Table 4).

Table 4 Effects of different ratios of compound inoculants on soil nitrogen, available phosphorus and potassium

N24: J16: P5 Soil alkaline hydrolyzable 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 inoculum:

The strain N24 was inoculated into the beef extract peptone liquid medium at a proportion of 1%, and cultured at 28° C. and 180 rpm for 36 hours to obtain the N24 inoculum.

Preparation of liquid P5 inoculum:

P5 was inoculated into beef extract peptone liquid medium at a ratio of 1%, and cultured at 28° C. and 180 rpm for 36 hours to obtain P5 inoculum.

Preparation of liquid J16 inoculum:

J16 was inoculated into beef extract peptone liquid medium at a ratio of 1%, and cultured at 28° C. and 180 rpm for 36 hours to obtain J16 inoculum.

Preparation of compound bacterial agent:

After culturing P5, N24 and J16 in beef extract peptone liquid medium for 36 hours, they were mixed in a ratio of 2:1:1, and the total effective viable count of the three bacteria in the mixture was 2×10 ⁸ ～ 9×10 ⁸ /mL, this mixed bacterial solution is the compound bacterial agent.

2. Wheat pot experiment

Pick full-grained wheat seeds, disinfect the surface with 70% ethanol, rinse with sterile water for at least 6 times, and apply the sterile water from the last rinse on beef extract peptone solid medium to check whether the surface disinfection of wheat is thorough. The sterilized wheat seeds were sown in the pot soil, and the surface was covered with about 1 cm of soil. Two treatments were set up in the experiment, treatment 1 was inoculated with 5 mL of sterile medium per pot, and treatment 2 was inoculated with 5 mL of compound bacterial agent per pot, and each treatment was replicated 3 times. The flowerpots were placed in the greenhouse, and the culture conditions were set as light 16h/8h and temperature 25/18°C. After the emergence of wheat, water was watered every 5 days, and soil available nitrogen, phosphorus, potassium, plant height, fresh weight, dry weight, and nitrogen, phosphorus, and potassium content were measured after 60 days. The results are shown in Table 5 and Table 6.

It can be seen from Table 5 that the transformation effect of the compound bacterial agent on the nutrient elements in the soil is significantly higher than that of the uninoculated control group. The total nitrogen, alkali-hydrolyzed nitrogen, available phosphorus, available potassium and organic matter in the soil inoculated with the compound bacterial agent increased by 18.4%, 81.2%, 99.5%, 19.4% and 55.7% respectively compared with the blank control group (Table 5).

Table 5 Effects of inoculation treatment on soil nutrients

It can be seen from Table 6 that the absorption of nitrogen, phosphorus and potassium by the wheat inoculated with the compound bacterial agent was significantly higher than that of the non-inoculated control group, and the growth of the plants was better. Compared with the blank control group, the plant height, dry weight, nitrogen content, potassium content and phosphorus content of the plants inoculated with the compound bacterial agent were increased by 59.8%, 105%, 65.6%, 50.3% and 75.1% respectively (Table 6).

Table 6 Effects of inoculation treatment on wheat growth

3. Use compound microbial inoculants to reduce the use of chemical fertilizers

Apply local conventional compound fertilizers or compound inoculants to the soil where wheat is grown to verify whether the application of compound inoculants can replace compound fertilizers and reduce the amount of chemical fertilizers used.

75% compound fertilizer was applied to the soil as a blank control group, and 100% compound fertilizer was applied as a control group. In addition, 75% compound fertilizer + compound bacterial agent was applied to the soil as the test group.

The results are shown in Figure 11. From Figure 11, it can be seen that reducing the use of chemical fertilizers resulted in poor wheat growth and yellow leaves. However, after adding compound microbial inoculants to the soil reduced by 25% of chemical fertilizers, the growth of wheat was significantly improved. Compared with the control group that completely used chemical fertilizers, the growth of wheat in the experimental group that reduced fertilization and added inoculants was even better than that of the control group. It shows that the compound microbial inoculant can reduce the usage of chemical fertilizer without affecting the growth of wheat.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Variations or changes in other different forms are not required and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

sequence listing

<110> Northwest A&F University

<120> A compound bacterial agent for promoting wheat growth and its application

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Claims (7)

1. a compound microbial inoculum promoting wheat growth, is characterized in that, described compound microbial inoculum is by nitrogen-fixing bacteria ( Azotobacter sp.) N24 bacterial liquid, Rhizobium ( Rhizobium sp.) J16 bacterial liquid and Klebsiella ( Klebsiella sp.) P5 bacterial liquid mixed and fermented; the nitrogen-fixing bacteria ( Azotobacter sp.) N24 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019281; the Rhizobium sp. J16 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019279; the Klebsiella sp. .) P5 was deposited in the China Center for Type Culture Collection (CCTCC) on April 22, 2019, and the deposit number is CCTCC NO: M 2019280. 2. according to the described compound bacterial agent of claim 1, it is characterized in that, described nitrogen-fixing bacteria ( Azotobacter sp.) N24 bacterial liquid, Rhizobium ( Rhizobium sp.) J16 bacterial liquid, Klebsiella ( Klebsiella sp.) The volume ratio of P5 bacterial liquid is 1～2:1～2:1～2. 3. according to the described compound bacterial agent of claim 2, it is characterized in that, described nitrogen-fixing bacteria ( Azotobacter sp.) N24 bacterial liquid, Rhizobium ( Rhizobium sp.) J16 bacterial liquid, Klebsiella ( Klebsiella sp.) The volume ratio of P5 bacterial solution was 2:1:1. 4 . The composite bacterial preparation according to claim 1 , wherein the total effective viable count of the three bacteria in the composite bacterial preparation is 2×10 ⁸ to 9×10 ⁸ cells/mL. 5 . 5. The compound bacterial preparation according to claim 1 is characterized in that, the fermented medium is LB liquid medium, and is made up of the following components: the final concentration is the yeast extract of 3～7g/L, and the final concentration is 8~12g/L tryptone, the final concentration is 5~15g/L NaCl, the balance is water. 6 . The compound bacterial agent according to claim 1 , characterized in that, the culture conditions of the fermentation are 25-30° C., 150-180 rpm for 24-48 hours. 7 . 7. The application of the composite inoculum of any one of claims 1 to 6 in improving plant growth performance.

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