Complex microbial inoculant for onions and preparation method of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for onions and a preparation method of the complex microbial inoculant. The complex microbial inoculant comprises acinetobacter JQ-MY-41 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.32731, pantoea JQ-MY-42 with the preservation number of CGMCC NO.32732 and alcaligenes YC-342 with the preservation number of CGMCC NO.32733, and the optimal mass ratio of the acinetobacter JQ-MY-41 to the pantoea JQ-MY-42 to the alcaligenes YC-342 to the alcaligenes YC-342 is 30: 25: 30. The preparation method comprises the steps of strain activation, strain culture, thallus collection, strain compounding, carrier addition, drying and the like. The three strains achieve synergistic growth promotion through different mechanisms such as phosphorus solubilization, nitrogen fixation and IAA production, and compared with a control group, the onion plant height is increased by 37.5%, the stem diameter is increased by 39.0%, the total biomass is increased by 103.7%, and the total chlorophyll amount is increased by 69.5%; the problems that an existing microbial agent is single in function and limited in growth promoting effect are solved, and technical support is provided for green and efficient cultivation of onions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial inoculants, in particular to a compound microbial inoculant for onions and a preparation method thereof. BACKGROUND

[0002] In the traditional cultivation process of onions, there are problems such as low soil nutrient utilization rate, poor root development, poor stress resistance, etc. Although the large use of chemical fertilizers can improve crop yield to a certain extent, long-term use will lead to soil acidification, structure deterioration, and reduction of beneficial microorganisms. As a kind of beneficial microorganism, plant growth promoting bacteria (PGPB) can promote plant growth through mechanisms such as nitrogen fixation, phosphorus solubilization, production of plant hormones, and antagonism of pathogenic bacteria, and has become an important direction of biological fertilizer research and development.

[0003] However, the existing microbial inoculants are mostly single strains or simple compounds, which have problems such as single function, poor stability, and limited growth-promoting effect. Acinetobacter, Pantoea and Alcaligenes are common growth-promoting bacteria genera in soil, which have different growth-promoting mechanisms and functional characteristics. Acinetobacter mainly has the ability to solubilize phosphorus, which can release the fixed phosphorus in the soil. Pantoea has multiple functions of nitrogen fixation, phosphorus solubilization and IAA production. Alcaligenes can regulate soil pH and antagonize pathogenic bacteria. Although the use of these strains alone has certain growth-promoting effect, it cannot fully exert their synergistic advantages.

[0004] Therefore, it is urgent to develop a compound microbial inoculant with multiple strains synergistic effect, which realizes functional complementation and synergistic effect among strains through scientific compounding, and provides more efficient and stable growth-promoting effect for onions and other crops. SUMMARY

[0005] The present application establishes a scientific strain ratio system by screening three dominant strains with different growth-promoting functions such as phosphorus solubilization, nitrogen fixation and IAA production, and develops a compound microbial inoculant for onions, which realizes the synergistic effect of multiple growth-promoting mechanisms and significantly improves the growth and development level and physiological activity of onions, providing technical support for green and efficient cultivation of onions.

[0006] On the one hand, the present application provides a compound microbial inoculant for onions, which comprises: Acinetobacter JQ-MY-41 with a preservation number of CGMCC NO.32731, Pantoea JQ-MY-42 with a preservation number of CGMCC NO.32732, and Alcaligenes YC-342 with a preservation number of CGMCC NO.32733.

[0007] Preferably, the complex microbial agent comprises, by mass fraction: Acinetobacter JQ-MY-41: 20-40 parts, Pantoea JQ-MY-42: 15-35 parts, Alcaligenes YC-342: 20-40 parts.

[0008] Preferably, the complex microbial agent further comprises a carrier material, by mass fraction: 40-80 parts, and the carrier material is composed of sodium alginate and trehalose at a weight ratio of 20:1.

[0009] Preferably, the complex microbial agent has an effective viable bacterial count of not less than 1x10 8 CFU / g, and a moisture content of not more than 8%.

[0010] The growth promotion mechanism of the complex microbial agent for onions is mainly based on the synergistic effect of the three strains. Acinetobacter JQ-MY-41 acts as a phosphorus-dissolving bacterium and can secrete various organic acids such as citric acid and malic acid. These organic acids can chelate iron and aluminum ions in the soil, release fixed phosphoric acid, and at the same time, reduce the rhizosphere pH and improve the solubility of phosphorus. In addition, the strain can also secrete plant growth promoting substances to directly stimulate plant root development.

[0011] Pantoea JQ-MY-42 has multiple growth promotion functions, including nitrogen fixation, phosphorus dissolution, and IAA production. The nitrogen fixation mechanism mainly reduces atmospheric nitrogen to ammonia through nitrogenase, providing nitrogen source nutrition for plants. At the same time, the strain can secrete phosphatase to hydrolyze organic phosphorus compounds and release available phosphoric acid. In terms of IAA synthesis, Pantoea JQ-MY-42 synthesizes IAA through the tryptophan pathway, regulates plant hormone levels, and promotes root elongation and lateral root development.

[0012] The main role of Alcaligenes YC-342 is to regulate the rhizosphere pH environment and antagonize soil-borne pathogens. The strain can produce alkaline substances to neutralize soil acidity and create a suitable living environment for other beneficial bacteria. At the same time, Alcaligenes can also secrete antibacterial substances to inhibit the growth of plant pathogens, playing a role in biological control.

[0013] The synergistic growth promotion mechanism of the three strains is reflected in multiple aspects: first, nutritional complementation, different strains have different utilization characteristics of carbon, nitrogen and phosphorus, which can fully utilize various nutrients in the environment; second, functional synergy, phosphorus-dissolving, nitrogen-fixing, and hormone-producing functions promote each other to form a positive feedback loop; third, niche differentiation, the three strains occupy different ecological niches, reducing competition and improving overall efficiency; and finally, environmental regulation, through pH regulation and antibacterial substance secretion, to create a suitable microenvironment for the complex microbial community.

[0014] On the other hand, the present application provides a preparation method of a complex microbial agent for onions, comprising the following steps:

[0015] Strain activation: the preserved Acinetobacter JQ-MY-41, Pantoea JQ-MY-42 and Alcaligenes YC-342 were inoculated into LB medium, nutrient agar medium and alkaline peptone medium respectively, and cultured at 28-32℃ for 18-24 hours.

[0016] Strain culture: the activated strains were inoculated into fermentation medium at 1-3%, and cultured at 28-32℃, 180-220rpm for 36-48 hours.

[0017] Strain collection: the culture solution was centrifuged at 4000-6000rpm for 10-15 minutes, and the strain was collected and washed with sterile normal saline for 2-3 times.

[0018] Strain compounding: the strain concentration of each strain culture solution was adjusted to 1×10 9 -5×10 9 CFU / mL, and the three strains were mixed into a strain suspension.

[0019] Carrier addition: the carrier material was dissolved in sterile water to prepare a 2-5% solution, and mixed with the strain suspension to prepare a compound microbial agent mixture.

[0020] Drying: the prepared compound microbial agent mixture was hot air dried at 40-60℃ for 8-16 hours until the moisture content was not more than 8%.

[0021] Preferably, the fermentation medium comprises: peptone 10-15g / L, yeast extract 5-8g / L, sodium chloride 5-10g / L, glucose 10-20g / L, potassium dihydrogen phosphate 1-3g / L, magnesium sulfate 0.2-0.5g / L, and the pH value is adjusted to 7.0-7.5.

[0022] Preferably, the compound microbial agent comprises, by mass fraction: Acinetobacter JQ-MY-41: 20-40 parts, Pantoea JQ-MY-42: 15-35 parts, Alcaligenes YC-342: 20-40 parts, carrier material: 40-80 parts.

[0023] The use concentration of the compound microbial agent is 1×10 7 -1×10 8 CFU / mL, and the application amount is 20mL / plant, and it is applied every 5-7 days, for 8-12 times continuously.

[0024] The synergistic mechanism of the three strains is analyzed as follows:

[0025] JQ-MY-41 secretes low molecular organic acids such as citric acid and oxalic acid, and the pH value is reduced by 0.5-1.0 units. The organic acids react with Fe 3+ , Al3+ It forms chelates, releasing the fixed phosphates; it secretes acid phosphatase, catalyzing the hydrolysis of organic phosphorus into inorganic phosphorus, increasing the phosphorus concentration in the rhizosphere by 5-10 times, and directly promoting phosphorus absorption.

[0026] JQ-MY-42 promotes the nitrogenase complex-catalyzed N2→NH3 reaction: N2 + 8H2O + +8e - +16ATP→2NH3+H2+16ADP+16Pi; It can fix 15-20 nmol of nitrogen per hour, which is equivalent to providing plants with 0.3-0.4 mg of nitrogen per day; The fixed nitrogen is released in the form of amino acids and directly absorbed by plant roots; It reduces the plant's dependence on soil nitrogen and improves nitrogen use efficiency.

[0027] The biosynthetic pathway of IAA is as follows: tryptophan → indolepyruvate → IAA; tryptophan → tryptamine → IAA; tryptophan → indoleethanol → IAA. The physiological functions of IAA include: activating cell wall relaxase, promoting cell elongation growth, inducing adventitious root formation, increasing the number of root branches, regulating apical dominance, promoting lateral bud germination, promoting vascular bundle differentiation, and improving nutrient transport.

[0028] YC-342 secretes alkaline substances such as ammonium bicarbonate and ammonia, neutralizing soil acidity and enhancing nitrate reductase activity: NO3 - +8H + +8e - →NH4 + +2H₂O+OH - Maintaining the rhizosphere pH at 6.5-7.5 provides a suitable environment for enzyme activity and improves the bioavailability of nutrients such as phosphorus, iron, and zinc.

[0029] Microbial agents improve iron and magnesium nutrition, provide raw materials for chlorophyll synthesis, regulate the expression of chlorophyll synthase genes, optimize nitrogen supply, ensure chlorophyll protein synthesis, improve root development, and enhance water and nutrient absorption.

[0030] JQ-MY-41 secretes organic acids, providing a carbon source for JQ-MY-42; JQ-MY-42 fixes nitrogen, providing a nitrogen source for YC-342; YC-342 regulates pH, creating a suitable environment for JQ-MY-41 to dissolve phosphorus.

[0031] JQ-MY-41 is mainly distributed in the root hair zone and plays a role in phosphorus solubilization. JQ-MY-42 is concentrated in the rhizosphere soil and carries out nitrogen fixation activities. YC-342 is distributed on the rhizosphere surface and forms a protective barrier.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] The three strains of the application realize the effect of 1+1+1>3 through the synergistic effect of different mechanisms such as phosphorus dissolution, nitrogen fixation and IAA production, compared with the control group, the plant height of onion is increased by 37.5%, the stem diameter is increased by 39.0%, and the total biomass is increased by 103.7%, and the problem of single strain function limitation is avoided; the preparation process is simple, the conditions are mild, and the industrial production is easy; by improving the yield and quality of onion and reducing the amount of chemical fertilizer, the application can bring significant economic benefits to the growers, and has good ecological environmental benefits. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The result graph of the flat plate interaction test of the application. DETAILED DESCRIPTION

[0035] The application will be further described below in combination with the drawings. The following examples are only used to more clearly illustrate the technical solutions of the application, and cannot be used to limit the protection scope of the application.

[0036] Biological material sample preservation: Acinetobacter YC-342 with the preservation number of CGMCC NO.32733 was preserved in China General Microbiological Culture Collection Center (CGMCC) on November 21, 2024; the preservation address is No.3, Yitian West Road, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, Latin name: Acinetobacter sp; Alcaligenes JQ-MY-41 with the preservation number of CGMCC NO.32731 was preserved in China General Microbiological Culture Collection Center (CGMCC) on November 21, 2024; the preservation address is No.3, Yitian West Road, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, Latin name: Alcaligenes sp; Pantoea JQ-MY-42 with the preservation number of CGMCC NO.32732 was preserved in China General Microbiological Culture Collection Center (CGMCC) on November 21, 2024; the preservation address is No.3, Yitian West Road, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, Latin name: Pantoea sp.

[0038] The following examples are referred to as JQ-MY-41, JQ-MY-42 and YC-342 for simplicity.

[0039] In the growth of onions, the amount of phosphate-solubilizing, nitrogen-fixing, and IAA-producing strains in the soil is an important growth-promoting indicator. In this example, three strains of phosphate-solubilizing, nitrogen-fixing, and IAA-producing strains were selected and activated on LB plates for plate interaction tests. The cross-intersection method was used to determine the antagonistic properties between strains. The test was conducted in three groups, with each group consisting of two strains. The results of the plate interaction test are shown in Table 1. Figure 1 As shown in Table 1, there was no break in the cross-intersection of the three groups of strains, indicating that there was no inhibition between the strains.

[0040] To explore the optimal formula of the complex microbial agent for promoting the growth of onions, the following examples and comparative examples were set up. The materials, specific steps, and experimental analysis are as follows:

[0041] LB medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, and 15 g / L of agar (solid medium).

[0042] Pikovskaya medium: 10 g / L of glucose, 5 g / L of Ca3(PO4)2, 0.5 g / L of (NH4)2SO4, 0.2 g / L of NaCl, 0.1 g / L of MgSO4·7H2O, 0.2 g / L of KCl, 0.5 g / L of yeast extract, 0.002 g / L of MnSO4, 0.002 g / L of FeSO4, and pH 7.0.

[0043] Burk nitrogen-free medium: 20 g / L of glucose, 0.8 g / L of K2HPO4, 0.2 g / L of KH2PO4, 0.2 g / L of MgSO4·7H2O, 0.1 g / L of NaCl, 0.13 g / L of CaSO4, 5 mg / L of FeSO4, 2 mg / L of Na2MoO4, and pH 7.2.

[0044] LB medium containing tryptophan: 100 mg / L of L-tryptophan was added to the LB medium.

[0045] Main reagents: ammonium molybdate, ascorbic acid, sulfuric acid, Salkowski reagent (0.5 M FeCl3 solution in 35% HCIO4), acetylene gas, L-tryptophan, sterile physiological saline, etc.

[0046] Main equipment: constant temperature shaking incubator, centrifuge, ultraviolet spectrophotometer, gas chromatograph, pH meter, clean bench, high-pressure sterilization pot, etc.

[0047] The preserved three strains were taken out from the -80℃ ultra-low temperature refrigerator, and activated according to the following steps: Acinetobacter JQ-MY-41 was inoculated in LB medium, Pantoea JQ-MY-42 was inoculated in nutrient agar medium, and Alcaligenes YC-342 was inoculated in alkaline peptone medium. All strains were cultured at 28℃ for 18-24 hours for activation. After activation, single colonies were inoculated into the corresponding liquid medium and cultured at 28℃, 180 rpm for 18-24 hours until the OD600 reached 0.8-1.0. 600 0.8-1.0, as seed liquid for standby.

[0048] Example 1

[0049] The composite microbial agent was 20 parts of JQ-MY-41, 15 parts of JQ-MY-42, and 20 parts of YC-342 by mass.

[0050] Example 2

[0051] The composite microbial agent was 20 parts of JQ-MY-41, 25 parts of JQ-MY-42, and 30 parts of YC-342 by mass.

[0052] Example 3

[0053] The composite microbial agent was 20 parts of JQ-MY-41, 35 parts of JQ-MY-42, and 40 parts of YC-342 by mass.

[0054] Example 4

[0055] The composite microbial agent was 30 parts of JQ-MY-41, 35 parts of JQ-MY-42, and 40 parts of YC-342 by mass.

[0056] Example 5

[0057] The composite microbial agent was 30 parts of JQ-MY-41, 25 parts of JQ-MY-42, and 30 parts of YC-342 by mass.

[0058] Example 6

[0059] The composite microbial agent was 40 parts of JQ-MY-41, 15 parts of JQ-MY-42, and 20 parts of YC-342 by mass.

[0060] Example 7

[0061] The composite microbial agent was 40 parts of JQ-MY-41, 35 parts of JQ-MY-42, and 40 parts of YC-342 by mass.

[0062] Example 8

[0063] The complex microbial agent is prepared according to the mass ratio: JQ-MY-41: 30 parts, JQ-MY-42: 15 parts, YC-342: 40 parts.

[0064] Example 9

[0065] The complex microbial agent is prepared according to the mass ratio: JQ-MY-41: 30 parts, JQ-MY-42: 15 parts, YC-342: 40 parts.

[0066] According to the different ratio requirements of examples 1-9, the suspension of each strain was prepared. The seed liquid of each strain was inoculated into 50 mL fermentation medium at a ratio of 1:100, and cultured at 28°C, 200 rpm for 36 hours. After the culture was completed, the bacterial cells were collected by centrifugation at 4°C, 5000 rpm for 15 minutes, washed twice with sterile physiological saline, and finally resuspended in sterile physiological saline. The concentration of each strain was adjusted to 1×10 9 CFU / mL.

[0067] According to the mass ratio specified in examples 1-9, the suspension of each strain with adjusted concentration was mixed. For example, in example 1, 20 mL of JQ-MY-41 bacterial suspension, 15 mL of JQ-MY-42 bacterial suspension, and 20 mL of YC-342 bacterial suspension were mixed evenly to prepare a complex bacterial suspension. The volume of the complex bacterial suspension prepared in each example was 100 mL, and the final bacterial concentration was maintained at 1×10 8 CFU / mL or more. The prepared complex bacterial suspension was stored at 4°C, and all determination experiments were completed within 24 hours.

[0068] Pikovskaya liquid medium was used for phosphorus solubilization capacity determination. The sterilized Pikovskaya medium was divided into 250 mL conical flasks, 100 mL per flask. According to the inoculation amount of 2%, the complex bacterial suspension prepared in each example was inoculated into the medium, and a blank control group without inoculation was set. All culture bottles were cultured at 28°C, 150 rpm for 7 days.

[0069] After the culture was completed, the culture solution was centrifuged at 8000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter to remove bacteria. The soluble phosphorus content was determined by molybdenum blue colorimetry: 1 mL of filtrate was added with 4 mL of ammonium molybdate solution, and after standing for 10 minutes, 1 mL of ascorbic acid solution was added. After mixing well, the reaction was carried out in a 40°C water bath for 30 minutes. After the reaction was completed, the solution was cooled to room temperature, and the absorbance value was measured at 650 nm wavelength by ultraviolet spectrophotometer. The soluble phosphorus concentration was calculated according to the phosphorus standard curve, and the phosphorus solubilization amount was expressed in mg / L. Three parallel samples were set for each example, and the average value was taken as the final result.

[0070] Nitrogenase activity was determined using Burk's nitrogen-free liquid medium. Sterilized Burk's medium was dispensed into 100 mL serum bottles, 50 mL per bottle, and sealed with a butyl rubber stopper. Each complex bacterial suspension was inoculated into the medium at a 2% inoculum, and an uninoculated blank control was set up. Immediately after inoculation, 20% of the bottle volume of air was withdrawn and replaced with an equal volume of acetylene gas to achieve a 10% acetylene concentration in the bottle. All serum bottles were incubated at 28°C for 48 hours.

[0071] After incubation, 1 mL of gas sample was withdrawn from the headspace of the serum bottle using a gas-tight syringe and immediately injected into a gas chromatograph for analysis. The gas chromatograph conditions were: hydrogen flame ionization detector (FID), nitrogen carrier gas at 30 mL / min, column temperature 80°C, and detector temperature 150°C. The amount of ethylene produced was calculated based on the ethylene peak area and a standard curve. Nitrogenase activity was expressed as nmol C2H4-h -1 ·mL -1 and reflects the nitrogen fixation ability of the complex bacterial agent. Three replicates were set up for each example, and the average value was taken.

[0072] IAA secretion was determined in LB medium containing L-tryptophan. Sterilized tryptophan-containing LB medium was dispensed into 250 mL conical flasks, 100 mL per flask. Each complex bacterial suspension was inoculated into the medium at a 2% inoculum, and an uninoculated blank control group was set up. All culture flasks were incubated at 28°C, 180 rpm for 72 hours.

[0073] After the end of the culture, centrifuge at 8000 rpm for 10 minutes, take the supernatant through 0.22 μm filter membrane. IAA determination uses Salkowski colorimetric method: take 2 mL of filtrate, add 1 mL of Salkowski reagent, mix thoroughly, and react for 30 minutes in the dark at room temperature. After the reaction, measure the absorbance value at 530 nm wavelength with a UV spectrophotometer. According to the IAA standard curve, calculate the IAA concentration, expressed in μg / mL. Set three parallels for each example, and take the average as the final result; while measuring the phosphorus solubilizing, nitrogen fixing and IAA secreting ability, measure the pH change of each culture system. Use a calibrated pH meter to measure the initial pH value of each medium before inoculation, and measure the final pH value after the end of the culture. When measuring, first wash the electrode with distilled water, immerse the pH meter electrode completely in the measured liquid, and record the pH value after the reading is stable. Before each measurement, calibrate the pH meter with a standard buffer to ensure accuracy; for the phosphorus solubilizing culture system, measure the pH change of Pikovskaya medium before and after the culture; for the nitrogen fixing culture system, measure the pH change of Burk medium; for the IAA secreting system, measure the pH change of tryptophan-containing LB medium. The pH change is expressed as ΔpH, which is the difference between the final pH and the initial pH. A positive value indicates an increase in pH, and a negative value indicates a decrease in pH.

[0074] The results of the experiment are shown in Table 1:

[0075] Table 1 Measurement results of phosphorus solubilizing, nitrogen fixing and IAA secreting amount of composite microbial agent examples 1-9

[0076]

[0077] Through comprehensive analysis of the 9 different ratio examples, it can be found that the strain ratio has a significant effect on the growth promoting performance of the composite microbial agent. In terms of phosphorus solubilizing ability, example 5 performs best, with a phosphorus solubilizing amount of 61.3 mg / L, which is significantly higher than other combinations. This is mainly because under this ratio, the three strains can synergistically play a role in phosphorus solubilizing. Acinetobacter JQ-MY-41 and Alcaligenes YC-342 have strong organic acid secretion ability, which can effectively dissolve calcium phosphate in the medium, and Pantoea JQ-MY-42 can secrete phosphatase to further improve the biological availability of phosphorus.

[0078] The nitrogenase activity determination results show that example 5 also has the highest nitrogen fixing ability, with a nitrogenase activity of 19.6 nmol C2H4·h -1 ·mL -1This is because the nitrogen fixation process requires a large amount of ATP consumption, and the synergistic effect of the three strains can provide sufficient energy supply for nitrogenase. Acinetobacter JQ-MY-41 has strong sugar metabolism ability, which can provide carbon source and energy for nitrogen fixation process; Pantoea JQ-MY-42 can improve the oxygen environment to create a suitable anaerobic microenvironment for nitrogenase; Alcaligenes YC-342 can maintain a suitable pH environment to ensure the activity of nitrogenase.

[0079] The IAA secretion amount determination results show that the IAA content of Example 5 is the highest, reaching 14.2 μg / mL. IAA is an important plant growth regulator that can promote root development and nutrient absorption. The three strains all have the ability to synthesize IAA from tryptophan, among which Pantoea JQ-MY-42 has the strongest IAA synthesis ability, followed by Acinetobacter JQ-MY-41, and Alcaligenes YC-342 is relatively weak. Under the ratio of Example 5, each strain can promote the synthesis of IAA, which may be achieved by secreting promoting factors or providing synthesis precursors.

[0080] The pH change value reflects the influence of strain metabolism on the culture environment. Most of the examples result in a slight increase in the pH value of the culture medium, which is mainly due to the decomposition of organic nitrogen compounds by the strains to produce ammonia and the alkali-producing property of Alcaligenes YC-342 itself. The pH change value of Example 5 is +0.6, indicating that the strain combination under this ratio can effectively regulate the pH of the rhizosphere microenvironment, which is of great significance for the activation and absorption of nutrients.

[0081] From the comprehensive performance of various indicators, Example 5 (JQ-MY-41: 30 parts, JQ-MY-42: 25 parts, YC-342: 30 parts) shows the best synergistic effect in phosphorus dissolution, nitrogen fixation, and IAA production, and has the strongest pH regulation ability. This ratio achieves the best balance of the functions of the three strains, ensuring the quantitative advantage of each strain and avoiding the competition inhibition phenomenon caused by the over dominance of a certain strain.

[0082] Next, the control experiment of the plant growth effect of the compound microbial agent for onions was continued to reveal the synergistic effect of the three strains.

[0083] Example 10

[0084] The blank control group (without applying any microbial agent) was only irrigated with pure water instead of the microbial agent.

[0085] Example 11

[0086] The microbial agent was composed of a single strain JQ-MY-41, concentration: 1 x 10 8 CFU / mL.

[0087] Example 12

[0088] The bacterial agent consists of single strain JQ-MY-42, concentration: 1 x 10 8 CFU / mL.

[0089] Example 13

[0090] The bacterial agent consists of single strain YC-342, concentration: 1 x 10 8 CFU / mL.

[0091] Example 14

[0092] The bacterial agent consists of double strains JQ-MY-41 + JQ-MY-42, JQ-MY-41: 50 parts, JQ-MY-42: 50 parts, total concentration: 1 x 10 8 CFU / mL.

[0093] Example 15

[0094] The bacterial agent consists of double strains JQ-MY-41 + YC-342, JQ-MY-41: 50 parts, YC-342: 50 parts, total concentration: 1 x 10 8 CFU / mL.

[0095] Example 16

[0096] The bacterial agent consists of double strains JQ-MY-42 + YC-342, JQ-MY-42: 50 parts, YC-342: 50 parts, total concentration: 1 x 10 8 CFU / mL.

[0097] The three-strain compound treatment group of Example 5 is used as a control group, and the onion seedlings are purple onion seedlings with a seedling age of 30 days. The cultivation substrate is: sterilized peat soil: vermiculite: perlite = 6:2:2. The cultivation container is a 15 cm diameter plastic pot, each pot containing 2 kg of substrate. The greenhouse temperature is 25±3℃, the relative humidity is 65-75%, and the natural light is used.

[0098] Uniform size onion seedlings are selected and transplanted into the potting substrate. Strong light is avoided within 3 days after transplanting, and the substrate is kept moist by watering every day to maintain a moisture content of 60-70%.

[0099] The bacterial agent is applied 7 days after transplanting, with rhizosphere irrigation, 20 mL of bacterial agent suspension per plant, applied every 7 days, a total of 8 times. The blank control group (Example 10) is applied with an equal amount of pure water

[0100] The 8 treatment groups (Examples 5, 10-16) each have 15 plants, randomly arranged, and sampled for testing 7 days after the last application of bacteria.

[0101] The distance from the soil surface to the top of the highest leaf was measured with a ruler, unit: cm; the stem diameter was measured at 2 cm above the stem base, 3 times per plant, and the average value was taken, unit: mm; Table 2 shows the measurement results of morphological indicators.

[0102] Table 2 Measurement results of morphological indicators

[0103] Example Plant height (cm) Stem diameter (mm) 10 28.5±1.2 8.2±0.4 11 32.1±1.4 9.1±0.5 12 33.8±1.6 9.6±0.4 13 31.2±1.3 8.9±0.5 14 36.4±1.8 10.3±0.6 15 34.9±1.5 9.8±0.5 16 35.7±1.7 10.1±0.6 5 39.2±2.1 11.4±0.7

[0104] The above-ground part and the underground part were separated, and the above-ground part (leaves + stems) and the underground part (roots + bulbs) were collected separately, and the fresh weight was weighed immediately after separation; the dry weight determination was performed by drying the sample with a fresh weight in an oven at 105°C for 30 minutes to fix the color, and then transferring to an oven at 80°C to continue drying until a constant weight (the difference between the adjacent two weighings was <0.001 g), and then weighing with an analytical balance after cooling to room temperature, unit: g / plant. Table 3 shows the measurement results of biomass indicators.

[0105] Table 3 Measurement results of biomass indicators

[0106] Example Aboveground fresh weight (g) Underground fresh weight (g) Aboveground dry weight (g) Underground dry weight (g) 10 12.3±0.8 8.9±0.6 1.85±0.12 1.42±0.09 11 15.1±1.0 11.2±0.7 2.28±0.15 1.79±0.11 12 16.8±1.2 12.8±0.9 2.52±0.18 2.05±0.14 13 14.6±0.9 10.8±0.8 2.19±0.14 1.73±0.12 14 19.2±1.4 15.6±1.1 2.88±0.21 2.49±0.18 15 17.8±1.3 14.3±1.0 2.67±0.19 2.29±0.16 16 18.5±1.2 15.1±1.1 2.78±0.18 2.41±0.17 5 22.6±1.6 18.4±1.3 3.39±0.24 2.94±0.21

[0107] The photosynthetic pigment content was determined by taking the 3rd-4th fully expanded functional leaves, taking 0.2 g of leaves per plant, and grinding into powder under light-proof conditions with liquid nitrogen; the extraction solvent was 95% ethanol solution, the leaf powder was added to 10 mL of extraction solvent, and the extraction was performed at 4°C in the dark for 24 hours, and then centrifuged at 5000 rpm for 10 minutes, and the supernatant was taken and diluted to 25 mL.

[0108] Table 4 Measurement results of photosynthetic pigment content

[0109] Example Chlorophyll a (mg / g) Chlorophyll b (mg / g) Carotenoid (mg / g) 10 1.42±0.08 0.58±0.04 0.31±0.02 11 1.63±0.09 0.67±0.05 0.36±0.03 12 1.71±0.11 0.72±0.06 0.39±0.03 13 1.58±0.08 0.64±0.04 0.34±0.02 14 1.89±0.12 0.81±0.07 0.45±0.04 15 1.78±0.10 0.76±0.06 0.42±0.03 16 1.84±0.11 0.79±0.06 0.44±0.03 5 2.05±0.14 0.89±0.08 0.51±0.04

[0110] As shown in Table 4, the spectral determination wavelength includes: chlorophyll a: 663 nm; chlorophyll b: 645 nm; carotenoid: 470 nm; a UV spectrophotometer was used; and the Arnon formula was used to calculate the content of each pigment:

[0111] Chlorophyll a content (mg / g): Chl a = (12.7 x A 663 -2.69 x A 645 ) x V / (1000 x W);

[0112] Chlorophyll b content (mg / g): Chl b = (22.9 x A 645 -4.68 x A 663 ) x V / (1000 x W);

[0113] Carotenoid content (mg / g): Car = (1000 x A 470- (2.05 x Chl a - 114.8 x Chl b) / 245; wherein: A is absorbance value, V is the volume of extract (mL), and W is the fresh weight of sample (g).

[0114] From the measurement results, it can be seen that the three single-strain treatments can significantly promote the growth of onions, but the effects are different: JQ-MY-42 (Pantoea) has the best effect, with a 18.6% increase in plant height, a 17.1% increase in stem diameter, a 36.6% increase in aboveground fresh weight, a 43.8% increase in underground fresh weight, and a significant increase in chlorophyll content, indicating an increase in photosynthesis; JQ-MY-41 (Acinetobacter) has a moderate effect: mainly promoting root development and improving phosphorus nutrition, with a 26.4% increase in underground biomass, which is better than the increase in aboveground biomass; YC-342 (Alcaligenes) has a relatively weak effect: mainly promoting growth indirectly through pH adjustment and disease resistance, with limited direct promotion of plant height and biomass.

[0115] The combination of two strains shows obvious synergistic effect, better than single strain: JQ-MY-41 + JQ-MY-42 combination is the best: combining the dual advantages of phosphorus dissolution and nitrogen fixation, the aboveground fresh weight is further increased by 14.3% compared with JQ-MY-42 alone, and the photosynthetic pigment content is 1.1-1.2 times that of single strain. JQ-MY-42 + YC-342: nitrogen fixation + pH adjustment, with good root development; JQ-MY-41 + YC-342: phosphorus dissolution + disease resistance, with high plant robustness.

[0116] Example 5 (three-strain complex) performs best in all indicators, with a 37.5% increase in plant height, a 39.0% increase in stem diameter, a 103.7% increase in total biomass (dry weight), and a 69.5% increase in total chlorophyll content.

[0117] Through systematic control experiment analysis, the following conclusions can be drawn:

[0118] 1. The three strains have growth-promoting effects, with the effect ranking as JQ-MY-42 > JQ-MY-41 > YC-342.

[0119] 2. Any combination of two strains shows significant synergistic effect, with the JQ-MY-41 + JQ-MY-42 combination being the best.

[0120] 3. The three-strain complex (Example 5) maximizes the growth-promoting effect, with all indicators significantly better than other treatments, and the growth-promoting effect is due to the comprehensive action of nutrient supply, hormone regulation, environmental improvement, and photosynthetic enhancement.

[0121] 4. The three-strain complex inoculant is the best choice for onion growth promotion, and is recommended for application in production.

[0122] The study provides a theoretical basis for scientific combination of microbial inoculants and technical support for green and efficient cultivation of onion and other crops.

[0123] The above detailed description of the embodiments of the present application further explains the purposes, technical solutions and advantages of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A complex microbial inoculant for onions, characterized in that, The complex bacterial agent comprises Acinetobacter JQ-MY-41 with a preservation number of CGMCC NO.32731, Pantoea JQ-MY-42 with a preservation number of CGMCC NO.32732, and Alcaligenes YC-342 with a preservation number of CGMCC NO.32733.

2. The complex microbial inoculum for onions according to claim 1, characterized by, The complex bacterial agent comprises, in terms of mass parts, 20-40 parts of Acinetobacter JQ-MY-41, 15-35 parts of Pantoea JQ-MY-42, and 20-40 parts of Alcaligenes YC-342.

3. The complex microbial inoculum for onions according to claim 2, characterized by the fact that, The complex bacterial agent further comprises a carrier material, which is composed of sodium alginate and trehalose at a weight ratio of 20:1, and the carrier material is 40-80 parts in terms of mass parts.

4. The complex microbial inoculum for onions according to claim 3, characterized by the fact that, The effective viable cell number in the complex microbial agent is not less than 1×10 8 CFU / g, and the moisture content is not more than 8%.

5. A method for preparing a complex microbial agent for onions, for preparing the complex microbial agent according to any one of claims 1 to 4, characterized in that, The preparation method comprises the following steps: Strain activation: inoculate the preserved Acinetobacter JQ-MY-41, Pantoea JQ-MY-42, and Alcaligenes YC-342 into LB culture medium, nutrient agar culture medium, and alkaline peptone culture medium respectively, and perform vibration culture at 28-32℃ for 18-24 hours for activation; Strain culture: inoculate the activated strains into fermentation culture medium at an inoculation amount of 1-3%, and perform vibration culture at 28-32℃ and 180-220 rpm for 36-48 hours; Bacterial cell collection: centrifuge the culture solution at 4000-6000 rpm for 10-15 minutes, collect the bacterial cells, and wash the bacterial cells with sterile normal saline for 2-3 times; Strain compounding: the bacterial concentration of each strain culture solution was adjusted to 1 x 10 9 -5 x 10 9 CFU / mL, and three bacterial suspensions were mixed together. Carrier addition: dissolve the carrier material in sterile water to prepare a 2-5% solution, and mix the solution with the bacterial cell suspension to prepare a complex bacterial agent mixture; Drying: hot air dry the prepared complex bacterial agent mixture at 40-60℃ for 8-16 hours until the water content is not more than 8%.

6. The preparation method of the complex bacterial agent according to claim 5, characterized in that, The fermentation culture medium comprises 10-15 g / L of peptone, 5-8 g / L of yeast extract, 5-10 g / L of sodium chloride, 10-20 g / L of glucose, 1-3 g / L of potassium dihydrogen phosphate, 0.2-0.5 g / L of magnesium sulfate, and the pH value is adjusted to 7.0-7.

5.

7. The preparation method of the complex bacterial agent according to claim 5, characterized in that, The complex bacterial agent comprises, in terms of mass parts, 20-40 parts of Acinetobacter JQ-MY-41, 15-35 parts of Pantoea JQ-MY-42, and 20-40 parts of Alcaligenes YC-342, and 40-80 parts of a carrier material. The complex bacterial agent comprises, in terms of mass parts, 20-40 parts of Acinetobacter JQ-MY-41, 15-35 parts of Pantoea JQ-MY-42, and 20-40 parts of Alcaligenes YC-342, and 40-80 parts of a carrier material.

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