Preparation method and application of compound microbial agent

Abstract

The invention relates to the technical field of soil fertilizers, and mainly relates to application of a tobacco planting soil improvement compound microbial agent. The compound microbial agent disclosed by the invention is composed of microorganisms with different functions. The compound microbial agent is used for replacing part of common chemical fertilizer and is matched with tobacco base fertilizer for co-application, and a new thought is provided for reducing application of chemical fertilizer in the tobacco growing period. In addition, the compound microbial agent provided by the invention has an improvement effect on the microbial community structure in soil and the quality and yield of flue-cured tobacco, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of fertilizers, and in particular to a novel soil improving microbial agent and a preparation method and application thereof. Background Art

[0002] Tobacco is an important economic crop in my country. In order to obtain higher yields, tobacco farmers have been using chemical fertilizers for a long time. Although the residual and toxic side effects of chemical fertilizers are not large, they have accumulated over time and have now reached a critical point that the soil cannot bear. Various problems continue to arise, such as compaction, poor air permeability, soil acidification, a large number of soil-borne diseases, and imbalance of the ecological environment of soil and soil microorganisms. Therefore, it is necessary to improve soil fertility and restore the ecological balance of tobacco-growing soil.

[0003] Microbial organic fertilizers can improve soil quality, increase soil organic carbon input and increase the number of microorganisms in the soil, and have a positive effect on crop production; soil microorganisms can promote crop growth and enhance the ability of crops to resist biological and non-biological stresses, and play an important role in crop protection and soil improvement. As a functional fertilizer that is harmless to the soil environment and has a long-lasting fertilizer effect, the application of microbial organic fertilizers can promote the effective components in the soil to be fully utilized by tobacco plants.

[0004] The application of microbial organic fertilizer can promote the spread of microbial communities, and the communities with significant characteristics in the soil at different growth stages of tobacco plants change. After the application of microbial organic fertilizer, the yield and quality of tobacco leaves increased, and the content of nicotine and nitrogen in tobacco leaves was reduced. In summary, the application of microbial organic fertilizer can improve the physical and chemical properties of tobacco soil and increase the quality and yield of tobacco leaves. Summary of the invention

[0005] The present invention aims to provide a preparation method of a novel microbial organic fertilizer. The purpose of the present invention is to provide a composite microbial organic fertilizer and application thereof. The composite microbial organic fertilizer is a mixed compound bacterial agent of Bacillus Velez, Rhodopseudomonas palustris and yeast. The fermentation cost is low, the photosynthetic carbon fixation capacity of soil microorganisms can be improved, the input of soil available potassium is enhanced, the soil quality is improved, and a sufficient source of soil nutrient elements is provided for realizing "potassium extraction" of tobacco leaves.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: The present invention provides a new type of composite microbial agent, the classification names of which are Bacillus Velezii ( Bacillus velezensis ) is deposited with the number CGMCC No.24342; Rhodopseudomonas palustris ( Rhodopseudomonaspalustris) is deposited as CGMCC No.1.8929; the deposited number of yeast (Saccharomyces) is CGMCC No. 2.5692. The composite microbial agent is prepared by expanding the mixed bacterial species seed solution; the mixed bacterial species seed solution includes Bacillus Velez seed solution, Rhodopseudomonas palustris seed solution and yeast seed solution.

[0007] Furthermore, the required ratio of Bacillus velez seed solution, Rhodopseudomonas palustris seed solution and yeast seed solution is: 1.5~2.0:1.0~2.0:1.0.

[0008] Furthermore, the application method of the composite microbial agent is: dilute the composite microbial agent 10 times, and apply it by soil flushing or plant root irrigation; or add the composite microbial agent to the basic fertilizer at a volume-to-mass ratio of 1-5:1000 to make a biological fertilizer, and apply it by hole application or ring application.

[0009] Compared with the prior art, the present invention has the following beneficial technical effects: 1. The composite microbial agent of the present invention contains three microbial strains and has the advantages of easy operation, no pollution, low cost and high activity. In addition, the composite microbial agent can replace part of conventional fertilizers, reduce the planting costs of tobacco farmers, and improve the economic benefits of tobacco leaves.

[0010] 2. The composite microbial agent of the present invention can improve the microbial diversity of tobacco-growing soil, enhance the soil fermentation and nitrate reduction functions during the tobacco leaf growth and maturity stages, and significantly increase the number of soil microorganisms at all tobacco growth and development stages.

[0011] 3. The composite microbial agent of the present invention can improve the quality of flue-cured tobacco, which is specifically reflected in the growth of tobacco leaves and the quality of flue-cured tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a graph showing the degree of yellowing of tobacco leaves.

[0013] Figure 2 This is a Venn diagram of species composition in tobacco-growing soil at different stages.

[0014] Figure 3 This is the functional prediction map of the FAPROTAX database. DETAILED DESCRIPTION

[0015] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] The technical solution provided by the present invention is: a method for preparing a composite microbial agent, which comprises the following steps: mixing three agents according to a certain proportion.

[0017] Specifically, the flora includes Rhodopseudomonas palustris ( Rhodopseudomonas palustris ), Bacillus Velez Bacillus velezensis ) and yeast (Saccharomyces).

[0018] Preferably, the deposit number of Rhodopseudomonas palustris is CGMCC No.1.8929; the deposit number of Bacillus velezensis is CGMCC No.24342; the deposit number of yeast is CGMCC No. 2.5692. The deposit number of Bacillus velezensis is CGMCC No.24342, the deposit date is January 19, 2022, the deposit unit is: China General Microbiology Center (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0019] The preparation steps of the composite microbial agent are as follows: Step 1: Under sterile conditions, inoculate the palustris Rhodopseudomonas stored at -80°C in a PM plate culture medium, add a carbon source, and culture at 30°C for 48 hours. Pick a single colony and transfer it to the PM culture medium, add a carbon source, and culture it at 30°C under light anaerobically to obtain the palustris Rhodopseudomonas bacterial liquid seed liquid.

[0020] Under sterile conditions, Bacillus Velez preserved at -80°C was inoculated into LB plate culture medium and cultured at 38°C for 48 h to obtain Bacillus Velez bacterial liquid seed solution.

[0021] Under sterile conditions, yeast stored at -80°C was inoculated into a YPD plate culture concentrate and cultured at 30°C for 48 h to obtain a yeast culture seed solution.

[0022] The culture medium used in the present invention is as follows: LB liquid culture medium (1000 mL): peptone 10.0 g, sodium chloride 5.0 g, yeast extract 10.0 g, distilled water 1000 mL, pH 7.2. Sterilize at 0.15 MPa, 121°C for 30 min.

[0023] PM liquid medium (1000 mL): 1.701125 g potassium dihydrogen phosphate, 1 g ammonium sulfate, 1.7745 g disodium hydrogen phosphate, 1 mL concentrate, 100 μL 0.1 mol / L sodium thiosulfate pentahydrate, 1000 mL distilled water. Sterilize at 0.15 MPa, 121°C for 30 min and set aside. The concentrate (100 mL) formula is: 2 g aminotriacetic acid, 2.89 g anhydrous magnesium sulfate, 5.03 g calcium chloride, 0.0198 g ferrous sulfate heptahydrate. Sterilize at 121°C for 20 min.

[0024] YPD medium (1000 mL): peptone 20 g, anhydrous glucose 20 g, yeast extract 10 g, sterilized at 121°C for 20 min.

[0025] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0026] Example 1

[0027] Experiment on the effect of compound microbial agent on tobacco plant growth

[0028] 1. Field experiment design

[0029] The selected experimental site is located in Changjiang Village, Renyi Town, Guiyang County, Chenzhou City, Hunan Province, China. The selected tobacco fields are tobacco-rice rotation fields. Composite microbial agents and tobacco growth basal fertilizers were added to the experimental group (MOF) field; tobacco growth basal fertilizers were added to the control group (CK) field. The rest of the farming operations were the same. Each treatment was repeated 3 times. A field plot experiment was adopted, and the experiment was arranged in random blocks. Each plot had 5 rows, each row was 10 m long, and the area was 65 m 2 A 0.5 m wide walkway was designed between cells to facilitate investigation and sampling, and protection rows were designed for the test plots.

[0030] 2. Tobacco plant growth

[0031] The growth of tobacco leaves is evaluated based on the agronomic traits of tobacco leaves such as plant height, stem girth, number of leaves, leaf length and width during the flat-top period. The agronomic trait table is shown below:

[0032] Plant height (cm) Stem circumference (cm) Number of blades Upper leaf length (cm) Upper leaf width (cm) Middle leaf length (cm) Middle leaf width (cm) CK 79.8±1.2 10.2±0.1 15.2±0.2 73.7±1.7 25.9±0.5 74.9±1.0 29.7±0.8 MOF 87.8±2.8 10.2±0.3 15.3±0.3 75.9±4.6 27.1±0.6 78.3±6.5 30.3±3.3

[0033] It can be seen from the above table that the plant height and upper and middle leaf size of tobacco in the flat-top stage in the MOF treatment group with the addition of the composite microbial agent were significantly higher than those in the control group CK. The plant height increased by 10%, and the leaf size increased by about 2% to 4.5%, indicating that the addition of the composite microbial agent has a certain promoting effect on tobacco growth.

[0034] 3. Chlorophyll content of tobacco leaves at maturity

[0035] The SPAD value of tobacco leaves and the degree of yellowing of tobacco leaves are shown in the following table. Figure 1 Shown

[0036] deal with Upper leaves Middle leaf Lower leaves CK 39.8±2.3a 35.9±1.0a 30.2±1.1a MOF 41.1±1.3a 34.0±0.9a 28.1±2.5a

[0037] From the above table and Figure 1 It can be seen that the chlorophyll content of the middle and lower leaves of the MOF treatment group with the addition of the composite microbial agent is lower than that of the control group CK, the chlorophyll content of the middle leaves is reduced by 5.3%; the chlorophyll content of the lower leaves is reduced by 7%. This shows that this composite microbial fertilizer is better than the control group in promoting the yellowing process of the middle and lower leaves, and plays a certain role in promoting the yellowing of plants.

[0038] 4. Chemical composition of flue-cured tobacco

[0039] The chemical composition of flue-cured tobacco is shown in the following table

[0040] Location deal with Nicotine (%) Total sugar (%) Reducing sugar (%) Total nitrogen (%) Potassium (%) chlorine(%) upper part CK 2.42±0.27a 25.25±0.10a 21.32±2.52a 1.88±0.20a 2.05±0.11a 0.48±0.10a upper part MOF 2.39±0.27a 26.42±0.17a 21.39±1.01a 1.80±0.08a 2.10±0.15a 0.42±0.00a Central CK 1.65±0.00a 29.35±0.87a 23.29±2.38a 1.51±0.06a 2.45±0.14a 0.44±0.11a Central MOF 1.64±0.31a 28.64±2.72a 23.21±2.87a 1.56±0.22a 2.58±0.10a 0.44±0.08a lower part CK 1.56±0.20a 26.51±1.99ab 19.54±0.85ab 1.64±0.01ab 2.73±0.01b 0.430.05±a lower part MOF 1.42±.19a 25.12±3.74ab 18.68±0.66ab 1.68±0.17ab 2.81±0.11ab 0.43±0.04a

[0041] From the table above, it can be concluded that the nicotine content of the upper and lower flue-cured tobacco leaves in the MOF treatment group with the addition of composite microbial agents was lower than that in the control group. The nicotine content of the upper leaves was reduced by 1.2% compared with the control group; the nicotine content of the middle leaves was reduced by 0.6% compared with the control group; and the nicotine content of the lower leaves was reduced by 8.9% compared with the control group. The potassium content increased, and the potassium-chloride ratio increased. The potassium content of the upper leaves increased by 2.4% compared with the control group; the potassium content of the middle leaves increased by 5.3% compared with the control group; and the potassium content of the lower leaves increased by 2.9% compared with the control group. It can be seen that the addition of composite microbial agents has a certain improvement in the quality of flue-cured tobacco.

[0042] 5. Microbial diversity in tobacco-growing soil

[0043] Figure 2 It was shown that the addition of compound microbial fertilizer had an effect on the microbial composition of rhizosphere soil in the transplanting period (Mck, Mma), clustering period (Rck, Rma), vigorous growth period (Pck, Pma) and mature period (Tck, Tma) of tobacco. During the transplanting period, the number of unique bacterial orders in the experimental group was 657, and the number of unique bacteria in the control group was 423. During the clustering period, the number of unique bacterial orders in the experimental group was 1151, and the number of unique bacteria in the control group was 744. During the vigorous growth period, the number of unique bacterial orders in the experimental group was 972, and the number of unique bacteria in the control group was 1604. During the mature period, the number of unique bacterial orders in the experimental group was 916, and the number of unique bacteria in the control group was 558. Figure 3It can be concluded that the fermentation and nitrate_reduction functions of rhizosphere soil bacterial communities after microbial organic fertilizers were improved in both the granulation and maturity stages. The improvement of soil microbial fermentation function can increase soil microbial activity and increase bacterial abundance; nitrate reduction of soil microbial communities can drive iron oxidation and improve the utilization rate of free ammonia. Therefore, the addition of composite microbial agents can significantly enhance the abundance of tobacco-growing soil microorganisms and enhance the function of tobacco-growing soil microorganisms.

[0044] In summary, adding microbial organic fertilizer composed of composite microbial agents to ordinary tobacco base fertilizer can significantly improve the abundance and function of microbial flora in tobacco-growing soil. At the same time, it can promote the growth and development of tobacco leaves and improve the yellowing effect in the later period to a certain extent, reduce the nicotine content of tobacco leaves, increase the potassium-chlorine ratio, improve the quality of tobacco leaves, and ultimately achieve the effect of "improving quality and increasing efficiency".

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing a composite microbial agent, characterized in that: The composite microbial agent is prepared by expanding the bacterial seed liquid; the mixed bacterial seed liquid includes Bacillus Velezii ( Bacillus velezensis )、Rhodopseudomonas palustris( Rhodopseudomonaspalustris ) and yeast ( Saccharomyces ).

2. The composite microbial fertilizer according to claim 1, characterized in that The volume ratio of the Bacillus Velez, Rhodopseudomonas palustris and yeast bacterial liquid seed liquid is 1:1:

1.

3. The composite microbial agent according to claim 1, characterized in that: The deposit number of the Velez Bacillus is CGMCC No. 24342, and its effective viable count is greater than 2.0×10 10 cfu / mL; Bacillus Velezii was activated and injected into a fermentation bottle containing LB medium and cultured in a shaking incubator at 37°C for 48 h to obtain a fermentation broth.

4. The composite microbial agent according to claim 1, characterized in that: The deposit number of the Rhodopseudomonas palustris is CGMCC No.1.8929, and its effective viable count is greater than 2.0×10 10 cfu / mL; Rhodopseudomonas palustris was activated, and the activated strain was added into the anaerobic bottle containing PM medium with a syringe, and cultured in a light incubator at 30°C for 48 h to obtain the fermentation liquid.

5. The composite microbial agent according to claim 1, characterized in that: The yeast has a preservation number of CGMCC No. 2.5692, and its effective viable count is greater than 2.0×10 10 cfu / mL; the activated yeast strain was added to the YPD medium and cultured in a light incubator at 30°C for 48 h to obtain the fermentation broth.

6. The preparation method according to claim 1, characterized in that The method comprises the following steps: (1) inoculating activated Bacillus velez, Rhodopseudomonas palustris and yeast in a liquid culture medium for cultivation respectively, so as to prepare Bacillus velez seed liquid, Rhodopseudomonas palustris seed liquid and yeast seed liquid; (2) inoculating the Bacillus seed liquid, Rhodopseudomonas palustris seed liquid and yeast seed liquid in step (1) into a fermentation culture medium at a volume ratio of 1:100-150, so as to obtain a microbial composite bacterial liquid; (3) mixing the Bacillus fermentation liquid, Rhodopseudomonas palustris fermentation liquid and yeast fermentation liquid in step (2) at a volume ratio of 1.5-2.0:1.0-2.0:1.0, so as to obtain a composite microbial fertilizer.

7. The composite microbial agent according to any one of claims 1 to 5 has certain effects in improving soil structure, enhancing soil fertility, enriching the microbial diversity of tobacco-growing soil and promoting vigorous growth of tobacco plants.

8. The use according to claim 7, characterized in that: The method for using the composite microbial agent is as follows: dilute the composite microbial fertilizer by 10 times and apply it by soil flushing or plant root irrigation; or add the composite microbial agent to basic fertilizer at a volume-to-mass ratio of 1-5:1000 to prepare a biological organic fertilizer, and apply it by hole application or ring application.

9. The use of the composite microbial agent according to claim 8, characterized in that: The microbial organic fertilizer can be used to increase the richness and diversity of soil microorganisms, while improving the physical and chemical properties of the soil, ultimately achieving the purpose of "improving the quality and efficiency" of flue-cured tobacco leaves.

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