Microbial complex microbial inoculant capable of increasing crop yield and application of microbial complex microbial inoculant

Abstract

The invention belongs to the field of microorganisms and organic fertilizers, and particularly relates to a compound microbial inoculant capable of increasing crop yield and application thereof. The bacillus amyloliquefaciens strain is prepared from bacillus amyloliquefaciens FH-1, gluconacetobacter Gluconacetobacter sp. QZR14, ochrobactrum sp. S112 and Brevundimonassp. NH1, and the bacillus amyloliquefaciens strain is prepared from bacillus amyloliquefaciens FH-1, gluconacetobacter Gluconacetobacter sp. QZR14, ochrobactrum sp. S112 and Brevundimonassp. NH1. The bacillus amyloliquefaciens strain is prepared from bacillus amyloliquefaciens FH-1, bacillus amyloliquefaciens Gluconacetobacter Gluconacetobacter Gluconacetobacter sp. The composite flora composition provided by the invention can promote the growth of crops and increase the yield of the crops.

Description

Technical Field

[0001] This invention belongs to the field of microorganisms and organic fertilizers, specifically relating to a microbial compound inoculant that can improve crop yield and its application. Background Technology

[0002] With the development and application of modern agricultural technology, the excessive use of chemical fertilizers and pesticides has not only caused enormous environmental pollution but also seriously affected the sustainable development of agriculture. Microbial inoculants can promote plant nutrient absorption, growth and development, and enhance crop resistance, making them an important pathway to achieving green and sustainable agricultural development. Despite their broad application prospects, the effectiveness and stability of microbial inoculants in field application are poor due to various influencing factors, severely hindering their large-scale application in mainstream agriculture. Microbial compound inoculants are microbial communities synthesized from two or more plant growth-promoting bacteria. Because of the diversity of species and functions in microbial compound inoculants, their application effects are more stable and their growth-promoting effects are stronger than those of single inoculants.

[0003] Rice is an important food crop in my country and the world, but there is currently no microbial compound inoculant specifically for rice farming, and the effect of microbial compound inoculants suitable for other crops in rice varieties is not stable. Summary of the Invention

[0004] The purpose of this invention is to provide a microbial compound inoculant that can improve crop yield, specifically a microbial compound inoculant suitable for rice cultivation.

[0005] The present invention first provides a compound microbial agent FNSQ, which is composed of Bacillus amyloliquefaciens FH-1 (F), Ochrobactrum sp. S112 (S), Gluconacetobacter sp. QZR14 (Q) and Brevundimonas sp. NH1 (N).

[0006] The *Bacillus amyloliquefaciens* FH-1 was obtained from Huangshan soil, with accession number CGMCC NO.17050. The *Bacillus cereus* S112 was obtained from Tianjin soil, with accession number CGMCC NO.27794. The *Staphylococcus aureus* QZR14 was obtained from Tieling soil, with accession number CGMCC NO.10983. The *Staphylococcus shorter thanatus* NH1 was obtained from Huangshan soil, with accession number CGMCC NO.27793.

[0007] Specifically, the compound microbial agent FNSQ is in powder or liquid form.

[0008] For the liquid form, the volume ratio of the fermentation liquid of Bacillus amyloliquefaciens FH-1, Bacillus cereus S112, Gluconobacterium tumefaciens QZR14, and Shortwave Monoclonal NH1 is 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5.

[0009] More specifically, each strain was inoculated into LB liquid medium or LB + 5-50% glucose liquid medium and cultured on a shaker at 25-35℃ for 18-30 hours. Then, it was transferred to a medium containing wheat bran and cultured on a shaker at 25-35℃ for 48-96 hours to obtain four single-strain microbial agents. These were then mixed in proportion to obtain the compound microbial agent FNSQ.

[0010] Furthermore, in powder form, it is obtained by drying from a liquid state.

[0011] Another aspect of this invention also protects the application of the provided compound microbial inoculant in the cultivation of plants, such as crops, like rice. Specifically, the application is to improve crop growth, such as dry weight or plant height; or to increase crop yield.

[0012] This invention utilizes a combination of fermentation broths from Bacillus amyloliquefaciens, Bacillus cereus, Staphylococcus aureus, and Bacillus shortwave diffusa to obtain a compound microbial inoculant. The compound microbial community provided by this invention can promote crop growth and increase crop yield.

[0013] Information on the preservation of biological materials:

[0014] The Bacillus amyloliquefaciens FH-1 was deposited on December 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Its classification name is Bacillus amyloliquefaciens, and its accession number is CGMCC NO.17050.

[0015] The Ochrobactrum sp. was deposited on July 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Its classification name is Ochrobactrum sp., and its accession number is CGMCC NO. 27794.

[0016] The Gluconacetobacter QZR14 was deposited on June 16, 2015, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Its classification name is Gluconacetobacter sp., and its accession number is CGMCC NO.10983.

[0017] The shortwave monoclonal bacterium NH1 was deposited on July 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Its classification name is: shortwave monoclonal bacterium Brevundimonas diminuta, and its accession number is CGMCC NO. 27793. Attached Figure Description

[0018] Figure 1 Interactions between strains on different culture media plates. (a) TSA medium plate, (b) nitrogen-fixing medium plate, (c) phosphate-solubilizing medium plate, (d) potassium-solubilizing medium plate. F, Bacillus amyloliquefaciens FH1; Q, Gluconobacterium glutamicum QZR14; N, Shortwave monoclonal bacteria NH1; S, Amyloliquefaciens S112. Different lowercase letters indicate significant differences between treatments (P<0.05).

[0019] Figure 2 The nitrogen-fixing capacity, phosphorus-solubilizing capacity, potassium-solubilizing capacity, indoleacetic acid-producing capacity, ACC deaminase-producing capacity, and siderophore-producing capacity of different microbial inoculants.

[0020] Figure 3 Effects of different microbial inoculants on dry weight (a) and plant height (b) of potted rice.

[0021] Figure 4 Effects of different microbial inoculants on the yield of field-grown rice (a) and fragrant rice (b). Detailed Implementation

[0022] The technical solution of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods; the materials and reagents used can be obtained commercially unless otherwise specified.

[0023] Example 1: Source of experimental strain

[0024] Our laboratory previously obtained the following highly efficient siderophore-producing strains: *Bacillus amyloliquefaciens* FH-1(F), accession number CGMCC NO.17050; *Ochrobactrum* sp. S112(S), accession number CGMCC NO.27794; *Gluconacetobacter* sp. QZR14(Q), accession number CGMCC NO.10983; and *Brevundimonas* sp. NH1(N), accession number CGMCC NO.27793.

[0025] Example 2: Determination of the growth-promoting properties of the strain

[0026] Strains F / S / Q / N were inoculated into 10 mL of LB liquid medium (1 L of medium contains 0.5 g tryptone, 0.25 g yeast extract, and 0.25 g sodium chloride) or 10 mL of LB + 10% glucose liquid medium (1 L of medium contains 0.5 g tryptone, 0.25 g yeast extract, 0.25 g sodium chloride, and 100 g glucose) and cultured overnight at 30°C with shaking. 1 mL of the bacterial suspension was inoculated into 50 mL of liquid LB medium, and after 24 h of shaking, the cells were washed twice with sterile deionized water and resuspended. The absorbance at 600 nm was measured, and the OD values ​​of all tested strains were adjusted to be similar using sterile deionized water before use.

[0027] 1.1 Nitrogen fixation capacity determination

[0028] Nitrogen-fixing ACCC55 solid medium (g / L): sucrose 10.0g, sodium chloride 0.2g, calcium carbonate 1.0g, potassium dihydrogen phosphate 0.5g, manganese sulfate heptahydrate 0.2g, pH 7.0-7.2, water 1000mL, agar powder 20.0g, sterilized at 121℃ for 30min.

[0029] Nitrogen fixation capacity determination: After each strain was cultured in LB liquid medium at 30℃ on a shaker for 24 h, 1.5 mL of bacterial suspension was pipetted and centrifuged at 10000 x g for 1 min. The suspension was then washed twice with 0.9% physiological saline by shaking and centrifugation before resuspending the cells. 5 μL of the resuspended suspension was inoculated onto nitrogen-fixing solid medium and incubated at 30℃ for 3 days. Colony growth and the formation of clear zones were then observed.

[0030] 1.2 Determination of Inorganic Phosphorus Solubility

[0031] Inorganic phosphorus IP medium g / L: Component 1: MgSO4 4.7H2O0.30g, KCl0.30g, NaCl0.30g, MnSO40.03g, CaPO45.00g, (NH4)2SO40.50g, FeSO 4. 0.03 g 7H2O, 20.00 g agar, 980 mL distilled water, pH 7.0-7.5.

[0032] Component 2: Dissolve 10g of glucose in 20mL of distilled water.

[0033] Sterilize components one and two at 121°C for 20 minutes, cool to about 50°C, and mix them in a clean bench.

[0034] Determination of the ability to break down inorganic phosphorus IP: Each plant growth-promoting bacterium was cultured in LB liquid medium at 30°C for 24 hours using a shaker. 1.5 mL of the bacterial suspension was pipetted and centrifuged at 10000g for 1 minute. The suspension was then washed twice with 0.9% physiological saline via shaking and centrifugation before resuspending the cells. 5 μL of the resuspended suspension was inoculated onto inorganic phosphorus solid medium and incubated at 30°C for 3 days. Colony growth and the formation of clear zones were then observed.

[0035] 1.3 Determination of potassium solubilization capacity

[0036] Potassium-solubilizing medium (g / L): sucrose 10.0g, potassium feldspar powder (washed 5 times with deionized water) 1.0g, Na2HPO4 2.0g, agar 15.0g, MgSO4·7H2O 0.5g, CaCO3 1.0g, yeast extract 0.5g, (NH4)2SO4 1.0g, distilled water 1000mL, pH 7.0-7.4.

[0037] Determination of potassium-solubilizing ability: After culturing each plant growth-promoting bacterium in LB liquid medium at 30℃ on a shaker for 24 h, 1.5 mL of bacterial suspension was pipetted and centrifuged at 10000 x g for 1 min. The suspension was then washed twice with 0.9% physiological saline by shaking and centrifugation, and the bacterial cells were resuspended. 5 μL of the resuspended suspension was inoculated onto potassium-solubilizing solid medium and incubated at 30℃ for 3 days. Colony growth and the formation of clear zones were observed.

[0038] 1.4 Determination of Iron Production Capacity

[0039] CAS medium: Each 100 mL contains 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein, 100 μL of 1 mmol / L CaCl2, 2 mL of 1 mmol / L MgSO4, and 2 g of agar. At approximately 60 °C, slowly add 5 mL each of phosphate buffer and CAS staining solution, mix thoroughly, and then pour into plates.

[0040] 20% sucrose solution: Dissolve 20g of sucrose in 100mL of deionized water.

[0041] 10% acid-hydrolyzed casein: Dissolve 10g of acid-hydrolyzed casein in 100mL of deionized water.

[0042] 1 mmol / L CaCl2: 0.0111 g calcium chloride dissolved in 100 mL deionized water.

[0043] 1mmol / L MgSO 4 0.012g magnesium sulfate dissolved in 100mL deionized water.

[0044] The CAS staining solution for siderophore detection is not sterile and must be completely dissolved by sonication before use: Dissolve 0.079g of CAS chromate azurite in 50mL of deionized water, then add 10mL of 1mmol / L FeCl3 solution (containing 12mmol / L HCl), solution A. Dissolve 0.069g of hexadecyltrimethylammonium bromide (HDTMA) in 40mL of deionized water, solution B. Slowly add solution A along the wall of a beaker to solution B, stirring until well mixed to obtain 100mL of CAS staining solution.

[0045] Preparation of 12 mmol / L HCl: Dissolve 0.1 mL of concentrated hydrochloric acid (12 mol / L) in 100 mL of deionized water.

[0046] Preparation of 1 mmol / L FeCl3: Dissolve 0.0162 g of ferric chloride in 100 mL of 12 mmol / L HCl.

[0047] 0.1 mol / L phosphate buffer, pH 6.8: Each 100 mL contains 2.427 g Na2HPO4·12H2O, 0.5905 g NaH2PO4·2H2O, 0.075 g KH2PO4, 0.250 g NH4Cl, and 0.125 g NaCl. Dilute 10 times before use.

[0048] Siderophore production capacity rating: Centrifuge 1 mL of activated bacterial suspension at 10000 x g for 1 min, wash twice with 0.9% physiological saline, and resuspend the bacterial cells. Spot 5 μL of the resuspended bacterial suspension onto a CAS plate and incubate at 30°C for 48 h. Three replicates were set up for each strain.

[0049] 1.5. Assay for ACC deaminase production capacity

[0050] Plotting the α-butanol acid standard curve: 0.0, 5.0, 10.0, 15.0, 20.0, 25.0, and 30.0 μL of prepared 1 μmol / 200 μL α-butanol acid solution were pipetted into 2 mL centrifuge tubes. Deionized water was added to bring the volume to 200 μL. The mixture was pipetted three times to ensure homogeneity. 300 μL of 2,4-dinitrophenylhydrazine solution was added, and the mixture was pipetted three times to ensure homogeneity. The mixture was incubated at 30°C for 30 min. 2 mL of 2 mol / L NaOH was added, and the mixture was pipetted three times to ensure homogeneity. 200 μL of this solution was added to a 96-well plate, and the absorbance at 540 nm was measured using a microplate reader. Each gradient was repeated three times. An α-butanol acid standard curve was constructed with α-butanol acid concentration on the x-axis and OD value on the y-axis.

[0051] Protein concentration standard curve construction: 0.0, 0.1, 0.2, 0.4, 0.6, and 0.8 mL of 100 μg / mL bovine serum albumin solution were pipetted into test tubes, and deionized water was added to a final volume of 1 mL. The mixture was then pipetted three times to ensure homogeneity. 5 mL of Coomassie Brilliant Blue G250 solution was added, and the mixture was pipetted three times to ensure homogeneity again. After incubation for 3 min, 200 μL of the solution was transferred to a 96-well plate. The absorbance of the solution at 595 nm was measured using a microplate reader. Each gradient was repeated three times. A protein concentration standard curve was constructed with protein concentration on the x-axis and OD value on the y-axis.

[0052] Sample preparation: Take 15 mL of culture medium grown in ADF liquid medium for 24 h, centrifuge at 6000 r / min for 10 min at 4 °C to collect bacterial cells, centrifuge at 2000 r / min for 5 min with 0.1 mol / L Tris-HCl buffer (pH 7.6) to collect bacterial cells, and wash the bacterial cells twice. Resuspend the bacterial cells in 600 μL of 0.1 mol / L Tris-HCl buffer (pH 8.5), add 30 μL of toluene, shake rapidly for 30 s to lyse the cells, and store at 4 °C.

[0053] Protein concentration determination (μg / mL): Take 100 μL of crude enzyme solution and cell lysate in a test tube, add 900 μL of distilled water and 5 mL of Coomassie Brilliant Blue G250 solution in sequence, mix well and react for 3 min, and measure the absorbance value at 595 nm. Use 1 mL of distilled water and 5 mL of Coomassie Brilliant Blue G250 as a control. Each sample is repeated three times.

[0054] Determination of methyl ethyl ketone (MEK) concentration in samples (μmol / μL): Take 200 μL of the above crude enzyme solution and cell lysate, add 20 μL of 0.5 mol / L LAC, mix well, and incubate in a 30℃ water bath for 15 min. Add 1 mL of 0.56 mol / L HCl to terminate the reaction, centrifuge at 12000 r / min for 5 min, take 1 mL of the supernatant, add 800 μL of 0.56 mol / L HCl and 300 μL of 0.2% 2,4-dinitrophenylhydrazine solution in 2 mol / L HCl to dissolve, and incubate at 30℃ for 30 min. Add 2 mL of 2 mol / L NaOH, mix well, and measure the absorbance at 540 nm. Replace the crude enzyme solution in the above solution with an equal volume of distilled water as a control. Repeat the experiment three times for each sample.

[0055] Formula for calculating the ability to produce ACC deaminase:

[0056]

[0057] 1.6 Results of the determination of the growth-promoting properties of the strain

[0058] like Figure 1 As shown in Table 1, among the four strains, *Bacillus amyloliquefaciens* FH1 exhibited the strongest siderophore-producing and indoleacetic acid-producing abilities, in addition to its ability to solubilize inorganic phosphorus and potassium. *Bacillus cereus* S112 showed the strongest potassium-solubilizing ability, and also possessed the ability to produce indoleacetic acid and ACC deaminase. *Gluconobacterium tumefaciens* QZR14 exhibited the strongest phosphorus-solubilizing ability, and also possessed nitrogen-fixing and indoleacetic acid-secreting abilities. *Bacillus shortwave monocytogenes* NH1 showed the strongest ACC deaminase-producing ability, and also possessed the ability to produce siderophores and indoleacetic acid.

[0059] Table 1. Growth-promoting properties of the strains

[0060]

[0061] Note: The data is the average of three plates. The value D / d indicates the strength of the growth-promoting ability. D is the diameter of the clear zone and d is the diameter of the colony. 1 means that the bacteria can grow on this plate and 0 means that it does not have the corresponding growth-promoting ability.

[0062] Example 2: Study on strain interactions

[0063] The interactions among the four strains were tested using a modified dual-plate assay. Each of the four strains was cultured separately in trypsin-soy broth (TSB) (per liter: 17.0 g tryptone, 3.0 g soy protein, 2.5 g glucose, 5.0 g NaCl, 2.5 g K₂HPO₄) (Doyle et al., 1968) at 30 °C with shaking at 250 rpm for 24 h. The suspensions were centrifuged at 8000 rpm at 4 °C and washed three times with phosphate-buffered saline (PBS) (per liter: 1.15 g Na₂HPO₄, 0.2 g KH₂PO₄, 8.0 g NaCl, 0.2 g KCl, 0.1 g CaCl₂, 0.1 g MgCl₂) (Perchetti et al., 2020) and adjusted to OD₆₀₀ = 0.1. Then, 3 μL of bacterial suspension was added dropwise to TSA (TSB with 15.0 g agar), nitrogen-fixing medium ACCC55 (per liter: 10.0 g sucrose, 0.5 g K2HPO4·3H2O, 0.2 g NaCl, 1.0 g CaCO3, 0.2 g MgSO4·7H2O, 15.0 g agar) (Gao et al., 2013), potassium-solubilizing medium (per liter: 10.0 g sucrose, 0.5 g yeast extract, 1.0 g (NH4)2SO4, 2.0 g Na2HPO4, 0.5 g MgSO4·7H2O, 1.0 g CaCO3, 1.0 g potassium feldspar powder, 15.0 g agar) (Liu Xiaolu, 2013), and phosphorus-solubilizing medium (per liter: 0.3 g MgSO4·7H2O, 0.3 g KCl, 0.3 g NaCl, 0.03 g... The interaction between bacteria was examined using a mixture of MnSO4, 5.0 g CaPO4, 0.5 g (NH4)2SO4, 0.03 g FeSO4·7H2O, and 20.0 g agar (Li et al., 2019). Any two bacteria were placed on plates at a 0.5 cm spacing and incubated at 30°C for 3 days. Three replicates (n=3) were set for each strain. The diameter of the clear zone or colony was measured.

[0064] Double-plate assays showed that *Bacillus amyloliquefaciens* FH1 (F) significantly inhibited the growth of *Acetobacter gluconate* QZR14 (Q), *Bacillus niger* NH1 (N), and *Bacillus cereus* S112 (S) on TSA agar plates, without being affected by them. There was no significant interaction between Q, N, and S, but N had a slight inhibitory effect on the growth of Q on TSA agar plates. Figure 1 (a)

[0065] Only Q could grow and produce zona pellucida on nitrogen-fixing medium plates. N significantly increased the diameter of the zona pellucida formed by Q on nitrogen-fixing medium plates by 22%. F significantly decreased the diameter of the zona pellucida formed by Q on nitrogen-fixing medium plates by 35.7%. S had no effect on the growth of Q on nitrogen-fixing medium plates or on zona pellucida formation. Figure 1 (b)

[0066] Only *Q* could grow and produce a clear band on phosphate-solubilized medium. *F* significantly inhibited the growth of *Q* and the formation of a clear band on phosphate-solubilized medium. *S* and *N* had no effect on the growth of *Q* and the formation of a clear band on phosphate-solubilized medium. Figure 1 (c)

[0067] Only S could grow on potassium-solubilizing medium plates and produce zona pellucida. F significantly reduced the diameter of the zona pellucida formed by S on potassium-solubilizing medium plates by 33.3%. Q and N had no effect on the growth of S on potassium-solubilizing medium plates or the formation of zona pellucida. Figure 1 (d).

[0068] Example 3: Construction and preparation of compound microbial inoculants

[0069] The function of synthetic microbial communities is determined by the functions of the species within them and the interactions between them. Example 2 shows that strains F / N / S / Q have different functions. Example 3 shows that strain F inhibits the growth of strains N / S / Q. However, considering multifunctionality, this invention attempts to account for the potentially different effects of compound microbial agents applied in open environments such as open fields, because higher-order interactions between species can lead to emergent microbial communities, and open, complex environments can provide more niches for different strains. Therefore, this invention designs compound microbial agents FNSQ containing four strains with different functions but antagonistic interactions between species, and NSQ containing three strains with different functions but no antagonistic interactions between species, to compare their effects.

[0070] Strains F / N / S / Q were inoculated into 100 mL of LB liquid medium (composition as in Example 2) or 100 mL of LB + 5-50% glucose liquid medium (composition as in Example 2), and cultured in a shaker at 30°C for 24 h. They were then transferred to 1.5 L of LB or LB + 5-50% glucose liquid medium at an inoculation ratio of 1-10% and cultured in a shaker at 30°C for 72 h to obtain four single-strain microbial agents (F / N / S / Q). The four activated strains (F / N / S / Q) were mixed at a ratio of 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5 to obtain the compound microbial agent FNSQ. The three activated strains (N / S / Q) were mixed at a ratio of 0.5-1.5:0.5-1.5:0.5-1.5 to obtain the compound microbial agent NSQ.

[0071] Example 4: Functions of Compound Microbial Agents

[0072] The phosphorus-solubilizing ability, potassium-solubilizing ability, indoleacetic acid production ability, siderophore production ability, and ACC deaminase production ability of four single bacterial agents and two compound microbial agents were determined according to the method in Example 2. The results showed that Q, SQN, and FSQN had nitrogen-fixing and phosphorus-solubilizing abilities (…). Figure 2 Among the various microbial agents (ab), Q has the highest nitrogen fixation and phosphorus solubility, SQN has the lowest nitrogen fixation, and FSQN has the lowest phosphorus solubility. Inoculants S, SQN, and FSQN all possess potassium-solubilizing ability, with S exhibiting the highest potassium-solubilizing ability and FSQN the lowest. Figure 2 (c) All microbial agents have the ability to produce indoleacetic acid, among which F and FSQN have a higher ability to produce indoleacetic acid (c). Figure 2 (d). All microbial agents have the ability to produce ACC deaminase, among which FSQN has the highest ability to produce ACC deaminase ( Figure 2 (e). All microbial agents have the ability to produce siderophores, among which N, F, and FSQN have higher siderophore production capabilities. Figure 2 (f) Compared with other microbial agents, the compound microbial agent FSQN has the most functions and the strongest ability to produce ACC deaminase, indicating that it may have strong stress resistance.

[0073] Example 4: Pot experiment of compound microbial inoculant

[0074] The experimental soil was saline-alkali soil from the Da'an Demonstration Area, which was air-dried and sieved through a 5mm sieve. The soil's physicochemical properties were: pH 8.44, electrical conductivity 1283.50 μS / cm, organic matter 14.75 g / kg, organic carbon 8.56 g / kg, total nitrogen 1.30 g / kg, total phosphorus 0.32 g / kg, total potassium 25.18 g / kg, available nitrogen 88.87 mg / kg, available phosphorus 33.15 mg / kg, and available potassium 377.29 mg / kg. The experimental plant was rice, variety Nei 5 You 8015, purchased from Zhejiang Lixin Seed Industry Co., Ltd. Rice seeds were spread evenly in a tray, covered with sterile water, sealed with plastic wrap, and incubated at 30℃. After 2 days, a large number of rice seeds began to sprout. After the rice seedlings show signs of sprouting, sow 10 rice seeds in each pot containing 240g of soil and add 100mL of tap water that has been left to stand overnight. On the 5th day of rice growth, retain 7 rice seedlings per pot to ensure that the growth of rice in each pot is basically uniform, thereby reducing differences caused by plant heterogeneity. Adjust the concentration of 6 different microbial inoculants (F / S / Q / N / SQN / FSQN) to 10. 6 CUF / mL: After ensuring uniform rice seedling growth, 50 mL of bacterial agent was added to each pot, while 50 mL of sterile water was added to the blank control. Seven replicates were set for each treatment. The temperature of the light incubator was set to 30℃, and the light duration was 16 hours per day. The plants were randomly moved every two days and watered with 50 mL of tap water. Samples were collected 16 days after sowing, and the plant height and dry weight were measured.

[0075] Depend on Figure 3 It is evident that the dry weight and plant height of rice in the FSQN and N treatment groups were superior to those in other treatments. The plant height of rice in the FSQN treatment group was significantly higher than that in the control group CK, indicating that FSQN can stably promote rice growth in saline-alkali soil.

[0076] Example 4: Field application of microbial inoculants

[0077] Four single-strain microbial inoculants F / S / Q / N and one compound microbial inoculant FSQN were applied in rice cultivation at the Da'an Alkali Land Ecological Experimental Station of the Chinese Academy of Sciences in Honggangzi Township, Anguang Town, Da'an City, Jilin Province. The microbial inoculants were applied after rice (Dongdao 122 variety) was sown in June 2023. Seven microbial inoculant treatment groups were set up: Treatment 1 was the conventional treatment (control group C); Treatment 2 was treated with commercial microbial inoculant O; Treatment 3 was treated with inoculant F; Treatment 4 was treated with inoculant N; Treatment 5 was treated with inoculant S; Treatment 6 was treated with inoculant Q; and Treatment 7 was treated with inoculant FSQN. Each treatment covered 140 square meters. The dosage of microbial inoculants was 1 L for all treatments. Other agronomic management practices were the same. Plot yield measurements were conducted after rice maturity.

[0078] The results showed that the rice yields treated with inoculants FSQN, Q, F, S, and N were significantly higher than those treated with the control group C. Figure 4(b) Among them, the microbial agent FSQN had the best yield-increasing effect, with a yield of 8225 kg / ha, which increased rice yield by 28.37% compared with the control C yield of 6407.5 kg / ha.

Claims

1. A compound microbial inoculant FNSQ, characterized in that, Bacillus amyloliquefaciens Bacillus amyloliquefaciens FH-1, Glucosamine Gluconacetobacter sp.QZR14, Paleobacterium Ochrobactrum sp. S112 and shortwave monocytogenes Brevundimonas Composition of sp.NH1, The Bacillus amyloliquefaciens FH-1 has the accession number CGMCC NO.17050; the Staphylococcus aureus QZR14 has the accession number CGMCC NO.10983; the Bacillus cereus S112 has the accession number CGMCC NO.27794; and the Shortwave Monoclonal bacteria NH1 has the accession number CGMCC NO.27793.

2. The compound microbial agent FNSQ as described in claim 1, characterized in that, It is in powder or liquid form.

3. The compound microbial agent FNSQ as described in claim 2, characterized in that, For the liquid form, the volume ratio of the fermentation liquid of Bacillus amyloliquefaciens FH-1, Gluconobacterium tumefaciens QZR14, Bacillus cereus S112, and Shortwave Monoclonal NH1 is 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.

5.

4. The compound microbial agent FNSQ as described in claim 2, characterized in that, Each strain was inoculated into LB liquid medium or LB + 5-150% glucose liquid medium and cultured on a shaker at 25-35℃ for 18-30 h. Then, it was transferred to bran medium and cultured on a shaker at 25-35℃ for 48-96 h to obtain 4 single microbial agents. These were then mixed in proportion to obtain the compound microbial agent FNSQ.

5. The compound microbial agent FNSQ as described in claim 4, characterized in that, The bran culture medium formula is as follows: 1 L of culture medium contains 10 g of bran, 30 g of glucose, 5 g of ammonium sulfate, and 0.5 g of manganese sulfate.

6. The compound microbial agent FNSQ as described in claim 3, characterized in that, For powder form, it is obtained by drying from a liquid state.

7. The application of the compound microbial agent FNSQ as described in any one of claims 1 to 5 in plant cultivation.

8. The application as described in claim 7, characterized in that, The plant in question is a crop, such as an agricultural crop.

9. The application as described in claim 8, characterized in that, It can improve crop growth, such as dry weight or plant height; or increase crop yield.

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