Composite microbial inoculant and use thereof

Abstract

The present invention relates to the technical field of bioengineering, and in particular to a composite microbial inoculant and a use thereof. The composite microbial inoculant comprises nitrogen-fixing Paenibacillus mucilaginosus MSSW01, potassium-decomposing Paenibacillus mucilaginosus MSSW02, and Paenibacillus mucilaginosus MSSW03. According to the present invention, by means of strain screening and plant growth promotion experiments, a composite microbial inoculant capable of effectively promoting growth of plants is obtained, and has important significance for improving the growth state of crops in the environment and the rhizosphere environment of crops and improving the effect of microbial fertilizers.

Description

A composite microbial agent and its application Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a composite microbial agent and application thereof. Background Art

[0002] Excessive use of chemical fertilizers and pesticides in agricultural production has caused a series of ecological pollution. Microbial inoculants, as green, environmentally friendly fertilizers, have the potential to increase soil fertility, reduce the use of chemical fertilizers and pesticides, purify and repair soil, reduce plant diseases, improve quality and yield, and enhance food safety.

[0003] Currently, most commercially available microbial agents are single-use, with limited functionality and poor yield-increasing effects. However, composite agents, made from a combination of different functional microbial strains, have similar biological characteristics and mechanisms of action. Their synergistic use can overcome the shortcomings of single agents and effectively improve crop yields, representing a growing trend in the development of microbial agents.

[0004] Therefore, how to provide a microbial agent containing multiple probiotics to solve the technical problem of single function of microbial agents of a single strain in the prior art is an urgent problem to be solved by those skilled in the art.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a composite microbial agent and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a composite microbial agent, comprising: nitrogen-fixing jelly-like Paenibacillus MSSW01, potassium-solubilizing jelly-like Paenibacillus MSSW02 and jelly-like Paenibacillus MSSW03.

[0009] Preferably, the volume ratio of the nitrogen-fixing Paenibacillus MSSW01, the potassium-solubilizing Paenibacillus MSSW02 and the Paenibacillus MSSW03 is 1-2:1-2:1-2.

[0010] Preferably, the bacterial contents of the nitrogen-fixing jelly-like Paenibacillus MSSW01, potassium-solubilizing jelly-like Paenibacillus MSSW02 and jelly-like Paenibacillus MSSW03 are respectively: 2 to 5×10 9 cfu·mL -1 .

[0011] Preferably, the bacterial content of the composite microbial agent is 2 to 5×10 9 cfu·mL -1 .

[0012] The present invention also provides application of the composite microbial agent in crop planting.

[0013] Preferably, the crop is wheat.

[0014] The present invention also provides the use of the composite microbial agent in soil nitrogen fixation, potassium solubilization or phosphorus solubilization.

[0015] The present invention mixes bacterial liquids of different strains and prepares a composite microbial agent, thereby providing the market with a composite microbial agent product with better growth-promoting effect.

[0016] The present invention obtains a composite microbial agent that can effectively promote plant growth through strain screening and plant growth promotion experiments. It is of great significance for improving the growth status of crops and the root zone environment of crops in the environment and enhancing the effect of microbial fertilizers.

[0017] Biological Deposit Description

[0018] The nitrogen-fixing jelly-like Paenibacillus MSSW01, whose Latin name is Paenibacillus mucilaginosus, is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is August 14, 2023, and the deposit number is CCTCC NO: M 20231471.

[0019] Paenibacillus mucilaginosus MSSW02, whose Latin name is Paenibacillus mucilaginosus, is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is August 14, 2023, and the deposit number is CCTCC NO: M 20231472.

[0020] Paenibacillus mucilaginosus MSSW03;

[0021] The strain was deposited in the China Center for Type Culture Collection: address: Wuhan University, the deposit date is August 14, 2023, and the deposit number is CCTCC NO: M 20231473. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] The jelly-like Paenibacillus sp. MSSW03 (viable bacteria count 3×10 9 cfu·mL -1 ), Paenibacillus sp. MSSW02 (viable count 3×10 9 cfu·mL -1 ), Paenibacillus nitrogenus MSSW01 (viable count 3×10 9 cfu·mL -1 ) The bacterial solutions of the three strains were mixed at volume ratios of 1:1:1, 1:2:1, 1:1:2, and 2:1:1 to produce liquid composite microbial inoculants, which were named M1, M2, M3, and M4, respectively, as shown in Table 1. Meanwhile, bacterial solutions of a single strain were retained and designated A1, A2, and A3 for use as single-strain inoculants.

[0025] Table 1 Composite microbial agents and single microbial agents

[0026] Example 2

[0027] 1. Crop cultivation experiment

[0028] 1) Taking Jinchun No. 6 wheat as an example, the growth-promoting effect of compound microbial agents on crops was explored, and the optimal volume ratio of compound microbial agents was determined based on the comprehensive effects.

[0029] In a 10cm long and wide plastic pot, add farmland soil and compact it to 1.5cm from the pot rim. Evenly place 25 wheat seeds of roughly the same size and cover with soil up to the pot rim. Add an appropriate amount of water to maintain a consistent soil moisture level in each pot, and water each pot the same amount daily. Water each pot every two days for the next step.

[0030] One week after the seedlings emerged, the composite microbial agent of Example 1 was inoculated by watering. When inoculating, 1 mL of each of the composite microbial agents M1, M2, M3, and M4 was taken, centrifuged at 12000 r / min for 2 min to collect the bacteria, and washed with sterile water to adjust the concentration to 2×10 8 CFU / ml. Suspend the bacteria in 9 mL of sterile distilled water and then inoculate 5 mL into the flower pot. Inoculate each flower pot once, and set 5 replicates. Use the potted plants without inoculation as the blank control group ck, other commercially available composite microbial inoculant products (Shandong Mairuisi Biotechnology Co., Ltd.; composite microbial inoculant EM bacteria) as the regular control group, and inoculate single strain bacterial liquid A1, A2, and A3 as the positive control group, inoculated in the same way, and set 5 replicates.

[0031] 2) Cultivation conditions: Cultivate outdoors, maintain appropriate humidity, irrigate with distilled water, and harvest after 30 days.

[0032] 3) 30 days after sowing, carefully pull out the entire wheat plant from the pot, shake off most of the soil stuck to the roots, rinse with tap water, and dry with newspaper. Spread the wheat plants flat on a table. Use a ruler to measure the length from the cotyledon node to the growing point as the wheat plant height. Use a ruler to measure the length of the third true cotyledon as the wheat leaf length.

[0033] Wheat roots are a fibrous root system consisting of seminal roots and secondary roots. Wash and dry the wheat plants. Count the number of roots extending from the seed embryo as the number of seminal roots. Use a ruler to measure the length from the rhizome junction to the longest root tip of the entire plant, i.e., the length of the longest seminal root in the seed embryo, as the maximum root length.

[0034] After washing and drying the wheat plants, weigh them directly on a scale (accurate to 0.01 g) to determine their fresh weight. Then, dry the wheat plants in an oven at 100°C until constant weight is reached, and weigh them to determine their dry weight. Record these data in Table 2.

[0035] Table 2 Effects of different treatments on wheat traits

[0036] After treating wheat seedlings with different bacterial suspensions, their growth was observed every day, and the growth of wheat plants was recorded on the 15th, 25th and 30th days. Differences began to appear in growth from the 15th day. Compared with the control group (ck), it can be clearly seen that the four bacterial agent treatments have a significant promoting effect on the growth of wheat. As shown in Table 2, the inoculation of composite bacterial agent M2 significantly improved the plant height, leaf length, maximum root length, fresh weight and dry weight of wheat compared with the blank control group, reaching 20.15%, 39.17%, 23.21%, 42.11% and 113.33% respectively. At the same time, compared with the inoculation of a single bacterial agent and other commercially available bacterial agents, the inoculation of composite microbial agent M2 has a better effect on improving the growth traits of wheat.

[0037] 2. Experiment on determination of soil physical and chemical properties

[0038] Thirty days after sowing in step 1, harvest the potted soil from each experimental group, air-dry it, sieve it, and package it in plastic bags. pH, organic matter, total nitrogen, available potassium, and available phosphorus were measured. Soil that had not been planted with wheat was used as the original soil group. Data are recorded and shown in Table 3.

[0039] Determination of total nitrogen content in soil: Refer to NY / T 53-1987, "Determination of Total Nitrogen in Soil (Semi-micro Kelvin Method)." In the presence of sodium thiosulfate, concentrated sulfuric acid, perchloric acid, and a catalyst, a redox reaction converts all nitrogen into ammonium. The digested solution is alkalized, and the distilled ammonia is absorbed by boric acid. Titration is then performed with standard hydrochloric acid solution, and the total nitrogen content in the soil is calculated based on the amount of standard hydrochloric acid solution used.

[0040] Determination of available potassium in soil: Refer to NY / T 889-2004, "Determination of available and slow-release potassium in soil." Weigh 5 g of air-dried soil sample that has passed a 1-µm sieve into a 100-mL Erlenmeyer flask. Add 50.0 mL of ammonium acetate solution (soil-to-liquid ratio of 1:10). Tightly cap the flask, shake at 150-180 rpm for 30 min at 20-25°C, and dry filter. Measure the filtrate directly using a flame photometer.

[0041] Determination of available phosphorus in soil: Referring to NY / T 1121.7-2014, "Soil Testing Part 7: Determination of Available Phosphorus in Soil," available phosphorus in the soil was extracted using a 0.5 mol / L sodium bicarbonate solution (pH 8.5). The phosphorus in the extract reacted with a molybdenum-antimony anti-colorant to produce phosphomolybdenum blue, the absorbance of which was measured at a wavelength of 880 nm. Within a certain concentration range, the phosphorus content and absorbance values ​​conformed to the Lambert-Beer law.

[0042] Table 3 Effects of inoculation of composite microbial agents on soil fertility

[0043] The experimental results are shown in Table 3. The total nitrogen, available phosphorus, available potassium, and organic matter contents in the soil inoculated with the composite inoculum were significantly higher than those in the uninoculated control (CK). The maximum increases in each of these were 185.2% (M4) for total nitrogen, 186.1% (M2) for available potassium, 244.9% (M3) for available phosphorus, and 67.54% (M2) for organic matter. The soil improvement effect of the composite inoculum-inoculated experimental group was higher than that of the blank and conventional controls, indicating that the synergistic effect of the different functional microbial strains in the composite inoculum can effectively compensate for the shortcomings of a single inoculum compared to soil inoculated with a single strain.

[0044] Taking into account the effects on improving wheat growth traits and soil improvement, the compound microbial agent M2 with a composition ratio of MSSW01:MSSW02:MSSW03=1:2:1 has the best effect.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A composite microbial inoculant, characterized in that, It is composed of Paenibacillus mucilaginosus MSSW01 for nitrogen fixation, Paenibacillus mucilaginosus MSSW02 for potassium solubilization, and Paenibacillus mucilaginosus MSSW03; The Paenibacillus mucilaginosus MSSW01 has the Latin name Paenibacillus mucilaginosus and the deposit number CCTCC NO: M 2023147; The Paenibacillus mucilaginosus MSSW02 for potassium solubilization has the Latin name Paenibacillus mucilaginosus and the deposit number CCTCC NO: M 20231472; The Paenibacillus mucilaginosus MSSW03 has the Latin name Paenibacillus mucilaginosus and the deposit number CCTCC NO: M 20231473.

2. The compound microbial inoculum according to claim 1, characterized in that, The preparation method of the composite microbial inoculum is as follows: The bacterial liquid of Paenibacillus mucilaginosus MSSW01 with a bacterial content of 2 - 5×10 9 cfu·mL -1 , the bacterial liquid of Paenibacillus mucilaginosus MSSW02 with a bacterial content of 2 - 5×10 9 cfu·mL -1 , and the bacterial liquid of Paenibacillus mucilaginosus MSSW03 with a bacterial content of 2 - 5×10 9 cfu·mL -1 are mixed according to a volume ratio of 1 - 2:1 - 2:1 - 2.

3. A composite microbial inoculum according to claim 2, characterized in that The viable count of the composite microbial inoculum is 2 - 5×10 9 cfu·mL -1 .

4. Application of a composite microbial inoculant according to any one of claims 1 to 3 in crop planting.

5. The application according to claim 4, wherein The crop is wheat.

6. Application of a composite microbial inoculant according to any one of claims 1 to 3 in soil nitrogen fixation, potassium solubilization or phosphorus solubilization.

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