A microbial composite bacterium agent, its preparation method and application

Abstract

The present invention provides a microbial complex bacterium agent, a preparation method thereof and an application thereof. The complex bacterium agent comprises Pseudomonas orientalis, methylotrophic bacteria, Massilia, Penicillium fructicola, Paecilomyces lilacinus and Streptomyces enniatinus. The present invention also provides an application of the above complex bacterium agent in promoting the formation of a plant rhizosheath structure. By applying the above microorganisms to the soil at the roots of plants, it significantly promotes the formation of the plant rhizosheath, can help improve the ability of plants to tolerate drought stress, can improve the soil fertility, and effectively improves the soil properties.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically relates to a microbial composite bacterium agent, a preparation method thereof, and an application thereof. Background Art

[0002] A sand sheath is a sheath-like structure with a certain thickness and a length of about 15 cm to 30 cm, formed by a large number of soil particles adhering to the surface of plant roots. The formation of the sand sheath has important ecological functions and significance for arid and semi-arid ecosystems. The sand sheath can improve the ability of roots to prevent wind and fix sand and resist surface high temperatures, help fix sand dunes, reduce soil erosion, improve the lodging resistance of plants, and stabilize the soil structure. The existence of the sand sheath structure is also conducive to the information exchange and the exchange of water and nutrients at the plant root-soil interface.

[0003] In recent years, the limited research on this feature has mainly focused on its structure, formation, and function. Its formation and composition are complex and related to many factors, including the length, density, and morphology of root hairs, the mucus of roots and microorganisms, soil water content, soil texture, mycorrhizal fungi, and active bacteria. The formation of the sand sheath is the product of the interaction between plant roots, soil, and microorganisms. Soil microorganisms are important factors affecting the formation and stability of the sand sheath structure. The secretions of microorganisms can bond sand grains together with root exudates, promote the formation and stability of the sand sheath, and can also promote the cyclic metabolism between the soil and plants, helping to improve the distribution of soil nutrients.

[0004] Many major cereals (wheat, corn, barley, oats, rye, and sorghum) form sand sheaths. Understanding the genetics behind this underground trait may provide genetic markers for plant breeders to improve the ability of other crop species to cope with environmental variability and abiotic stresses. The research on microorganisms in the sand sheath structure plays an important role in promoting the formation of the sand sheath structure and stabilizing the sand sheath structure. By studying the richness and species of microorganisms in the sand sheath structure, it can help solve the problem of soil desertification in recent years and some adverse effects brought by agricultural production to human production and life.

[0005] At present, there are few reports on specific strains dedicated to promoting the formation of the plant sand sheath structure, and there is also little research on composite bacterium agents for promoting the formation of the sand sheath structure. The research of this application is of great significance for improving the ability of plants to tolerate drought stress through the sand sheath structure and the ability of crop species to cope with environmental variability and abiotic stresses. Summary of the Invention

[0006] The purpose of the present invention is to provide a microbial composite bacterium agent, a preparation method thereof, and an application thereof, so as to solve the problem that there is a lack of a composite bacterium agent for promoting the formation of the sand sheath structure in the prior art.

[0007] The technical solution adopted by the present invention is as follows: a microbial composite bacterium agent, the composite bacterium agent includes Pseudomonas, Methylotrophs, Massilia, Penicillium, Paecilomyces lilacinus and Streptomyces enniatinus; the viable count ratio of Pseudomonas, Methylotrophs, Massilia, Penicillium, Paecilomyces lilacinus and Streptomyces enniatinus in the composite bacterium agent is 1-2:1-2:1-2:1-2:1-2:1-2.

[0008] The viable count ratio of Pseudomonas, Methylotrophs, Massilia, Penicillium, Paecilomyces lilacinus and Streptomyces enniatinus in the composite bacterium agent is 2:2:2:2:1:1.

[0009] The total viable count of the composite bacterium agent is 10 9 ~10 10 CFU / g or 10 8 ~10 9 CFU / mL.

[0010] The preparation method of the above-mentioned composite bacterium agent is to inoculate Pseudomonas, Methylotrophs, Massilia, Penicillium, Paecilomyces lilacinus and Streptomyces enniatinus into a liquid medium for fermentation respectively. After fermentation, the fermentation broths are mixed according to the volume ratio of the fermentation broths of Pseudomonas, Methylotrophs, Massilia, Penicillium, Paecilomyces lilacinus and Streptomyces enniatinus of 1-2:1-2:1-2:1-2:1-2:1-2 to obtain a liquid composite bacterium agent.

[0011] The liquid composite bacterium agent is subjected to vacuum freeze-drying to obtain a solid composite bacterium agent.

[0012] The application of the above-mentioned composite bacterium agent in promoting the formation of plant rhizosheaths.

[0013] The composite bacterium agent is applied to the root soil of plants and evenly mixed with the soil around the plants.

[0014] The plant is a gramineous plant that can produce rhizosheaths.

[0015] The application of the above-mentioned composite bacterium agent in improving the properties of plant rhizosheath soil.

[0016] The soil properties refer to soil pH, electrical conductivity, and the contents of organic matter, available phosphorus, and alkaline hydrolyzable nitrogen.

[0017] Beneficial effects:

[0018] 1. The compound microbial agent of the present invention is based on metagenomic analysis and uses various microorganisms as raw materials. It can promote the formation of the rhizosheath structure of plants, improve the micro-ecological environment of the soil, and enhance soil fertility.

[0019] 2. Pseudomonas can produce various antibiotics to improve plant nutrition and promote plant growth, and can also degrade toxic substances in the soil, playing a significant role in promoting plant growth and preventing diseases; methylotrophic bacteria can convert methanol into various valuable compounds and produce extracellular polysaccharides, amino acids, enzymes and other substances; Massilia can synthesize various secondary metabolites and enzymes, dissolve phosphorus, tolerate heavy metals, and carry out soil remediation; Paecilomyces lilacinus can produce natural vitamin A and vitamin E, and also has the function of killing nematodes; Penicillium and Actinomycetes can produce hyphae to help bind the soil. The microbial compound microbial agent prepared by compounding Pseudomonas orientalis, methylotrophic bacteria, Massilia, Penicillium fructiforme, Paecilomyces lilacinus and Streptomyces enniatin and applying it to the soil of plant roots further enhances the role of promoting rhizosheath formation.

[0020] 3. The microorganisms in the compound microbial agent of the present invention have a high viable count, which is beneficial for colonization in the soil. In addition, the formula design of the compound microbial agent provided by the present invention is reasonable and easy to produce and manufacture. Description of the Drawings

[0021] Figure 1 It is a partial rhizosheath structure formation result diagram when different microbial agents are applied in Example 2 of the present invention. Among them, a: no microbial agent is applied; b: Pseudomonas orientalis; c: methylotrophic bacteria; d: Massilia; e: Streptomyces enniatin; f: Penicillium fructiforme; g: Paecilomyces lilacinus.

[0022] Figure 2 Partial rhizosheath structure formation result diagram when different compound microbial agents are applied in Example 3 of the present invention. Detailed Embodiments

[0023] The following combines specific embodiments to detail the present invention. Reagents and operations not mentioned in the embodiments are implemented according to the conventional operations in the art.

[0024] Example 1 Metagenomic Analysis of the Rhizosheath Microorganisms of Agropyron cristatum

[0025] Collect the rhizosheath of the roots of Agropyron cristatum at the late fruiting stage in Yudaokou, Chengde City, Hebei Province as a sample. Select an area without other plants around, and collect 3 independent repeated samples. Send the collected samples to Shanghai Personal Biotechnology Co., Ltd. for metagenomic analysis. This includes genomic DNA extraction, sequence quality control and screening filtration, gene assembly and splicing, gene prediction and data analysis, etc. Conduct a diversity analysis on the soil microorganisms of the roots of Agropyron cristatum, and obtain that the dominant genera in the rhizosheath of Agropyron cristatum are Sphingomonas, Streptomyces, Pseudomonas, Bacillus, Penicillium, etc.

[0026] Screening of Strains in Example 2

[0027] 1. According to the dominant genera obtained from the metagenomic analysis in Example 1, 29 strains belonging to the dominant genera were purchased from the existing strain bank and further screened. The 29 strains were tested in batches, fermented using the corresponding culture medium, and the fermentation broth was applied around the roots of Agropyron cristatum. Strains that could promote the formation of sand sleeves were selected by comparing the weights of the sand sleeves. The results are shown in Tables 1 and 2.

[0028] Determination of the weight of the sand sleeve: Separate the sample at the root, and without damaging the integrity of the root system, gently shake the root system until no clumps of soil fall off. Transfer the root system to a 250 mL beaker containing 150 mL of distilled water, and then place it in an ultrasonic cleaner and shake for 20 min. Wait until the sand sleeve is completely peeled off from the root system, and finally weigh to obtain the weight of the sand sleeve soil. Here, the weight of the sand sleeve is directly represented by the weight of the sand sleeve soil. The unit of the weight of the sand sleeve soil is g.

[0029] Table 1 Effects of 29 Strains of Solid Bacterial Agents (I)

[0030]

[0031]

[0032] Table 2 Effects of 29 Strains of Solid Bacterial Agents (II)

[0033]

[0034] The strains that can significantly promote the formation of sand sleeves after comparison are strains 5, 6, 10, 15, 17, and 18, namely Pseudomonas orientalis (purchased from China Center for Type Culture Collection, preservation number CCTCC S2013634), Methylotrophs (purchased from ATCC American Type Culture Collection, preservation number ATCC BAA - 2500), Massilia sp. (purchased from China Center for Type Culture Collection, preservation number CCTCC AB 2012441), Penicillium fructigenum (purchased from China General Microbiological Culture Collection Center, preservation number CGMCC 3.10065), Paecilomyces lilacinus (purchased from China Center for Type Culture Collection, preservation number CCTCC NF20082457), and Streptomyces enniatinus (purchased from China General Microbiological Culture Collection Center, preservation number CGMCC4.6279).

[0035] 2. Effects of Strains on the Physical and Chemical Properties of Sand Sleeve Soil

[0036] The physical and chemical indicators of the soils that significantly promoted the growth of sand traps were measured. pH was determined using the potentiometric method with a water-soil ratio of 1:1; conductivity was determined using the leaching method with a water-soil ratio of 5:1; organic matter content was determined using the muffle furnace ignition method; available phosphorus content was determined using the molybdenum-antimony colorimetric method; and alkaline nitrogen content was determined using the alkaline diffusion method.

[0037] Table 3 Effects of six strains on the physical and chemical properties of sand soil

[0038]

[0039] By measuring the physical and chemical indicators of the soil, it was found that the application of the bacterial strain reduced the pH of the sandy soil and increased the electrical conductivity, organic matter, available phosphorus, and alkaline nitrogen content of the sandy soil compared to the untreated soil. This shows that the bacterial strain can effectively improve the properties of the soil.

[0040] 3. Preservation of strains

[0041] For short-term use, the bacteria can be stored in a 4°C refrigerator. For long-term storage, use the glycerol preservation method: take 1 mL of bacterial liquid sample into a sterile 2 mL centrifuge tube, then add 1 mL of 60% glycerol, mix thoroughly, and then place in a -80°C low-temperature refrigerator.

[0042] Example 3 Preparation of composite bacterial agent

[0043] 1. Strain selection: Combined with metagenomic analysis data, six strains selected from the dominant bacterial community were cultured and used to prepare a composite bacterial agent. These included Pseudomonas orientalis (purchased from the China Center for Type Culture Collection, with the accession number CCTCCS2013634), Methylotrophic Bacteria (purchased from the ATCC American Collection, with the accession number ATCC BAA-2500), Marseilles (purchased from the China Center for Type Culture Collection, with the accession number CCTCC AB 2012441), Penicillium fruticosum (purchased from the China General Microorganism Culture Collection, with the accession number CGMCC 3.10065), Paecilomyces lilacinus (purchased from the China Center for Type Culture Collection, with the accession number CCTCC NF 20082457), and Streptomyces octadecene (purchased from the China General Microorganism Culture Collection, with the accession number CGMCC 4.6279).

[0044] 2. Bacterial culture: The six strains were inoculated into a seed culture medium (LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL distilled water) and cultured at 37°C for 24 hours. The cultured strains were then inoculated into a fermentation medium (LB medium) at a 1% inoculum rate and fermented in a shake flask at 37°C for 48 hours to obtain fermentation broths of the six strains. The viable counts in the fermentation broths were 1.2 × 10 9 CFU / mL, 1.6×109 CFU / mL, 2×10 9 CFU / mL, 2.1×10 9 CFU / mL, 1.3×10 9 CFU / mL, 1.2×10 9 CFU / mL, 1.7×10 9 CFU / mL.

[0045] 3. Ratio and Preparation of Bacterial Agents: The obtained fermentation broth was mixed in different ratios. A) No bacterial liquid was applied; B) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia was 1:1:1; C) The viable cell count ratio of Penicillium fructigenum: Paecilomyces lilacinus: Streptomyces enomycin was 1:1:1; D) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 1:1:1:1:1:1; E) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 2:2:2:1:1:1; F) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 1:1:1:2:2:2; G) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 2:1:1:1:1:1; H) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 2:2:1:1:1:1; I) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 2:2:2:2:1:1; J) The viable cell count ratio of Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructigenum, Paecilomyces lilacinus: Streptomyces enomycin was 2:2:2:2:2:1. A liquid compound fermentation broth was obtained by mixing according to the above ratios.

[0046] Example 4

[0047] The fermentation broth obtained in Example 3 was mixed according to the viable cell count ratio of Pseudomonas orientalis, Methylobacterium, Massilia, Penicillium fructigenum, Paecilomyces lilacinus and Streptomyces enomycin of 1:1:1 to obtain a liquid compound fermentation broth, and then vacuum freeze-drying was carried out: First, it was pre-frozen in a liquid nitrogen tank at -196°C for 3 min; then, under a vacuum pressure of 0.01 mbar, it was vacuum frozen at -85°C for 15 h to obtain a solid compound bacterial agent.

[0048] Example 5

[0049] The fermented liquid obtained in Example 3 was mixed according to the ratio of viable counts of Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 1:1:1 to obtain a liquid composite fermented liquid, and then vacuum freeze-dried: First, it was pre-frozen in a liquid nitrogen tank at -196°C for 3 min; then, it was vacuum frozen at -85°C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0050] Example 6

[0051] The fermented liquid obtained in Example 3 was mixed according to the ratio of viable counts of Pseudomonas orientalis, Methylotroph, Massilia, Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 1:1:1:1:1:1 to obtain a liquid composite fermented liquid, and then vacuum freeze-dried: First, it was pre-frozen in a liquid nitrogen tank at -196°C for 3 min; then, it was vacuum frozen at -85°C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0052] Example 7

[0053] The fermented liquid obtained in Example 3 was mixed according to the ratio of viable counts of Pseudomonas orientalis, Methylotroph, Massilia, Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 2∶2:2:1∶1:1 to obtain a liquid composite fermented liquid, and then vacuum freeze-dried: First, it was pre-frozen in a liquid nitrogen tank at -196°C for 3 min; then, it was vacuum frozen at -85°C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0054] Example 8

[0055] The fermented liquid obtained in Example 3 was mixed according to the ratio of viable counts of Pseudomonas orientalis, Methylotroph, Massilia, Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 2∶2:2:1∶1:1 to obtain a liquid composite fermented liquid, and then vacuum freeze-dried: First, it was pre-frozen in a liquid nitrogen tank at -196°C for 3 min; then, it was vacuum frozen at -85°C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0056] Example 9

[0057] The fermented liquid obtained in Example 3 was mixed according to the ratio of viable counts of Pseudomonas orientalis, Methylotroph, Massilia, Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 1:1:1:2:2:2 to obtain a liquid composite fermented liquid, and then vacuum freeze-dried: First, it was pre-frozen in a liquid nitrogen tank at -196°C for 3 min; then, it was vacuum frozen at -85°C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0058] Example 10

[0059] The fermentation broth obtained in Example 3 was mixed according to the ratio of viable cell counts of Pseudomonas orientalis, methylotrophic bacteria, Massilia sp., Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 2:1:1:1:1:1 to obtain a liquid composite fermentation broth, and then vacuum freeze-drying was carried out: First, it was pre-frozen in a liquid nitrogen tank at -196 °C for 3 min; then, it was vacuum frozen at -85 °C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0060] Example 11

[0061] The fermentation broth obtained in Example 3 was mixed according to the ratio of viable cell counts of Pseudomonas orientalis, methylotrophic bacteria, Massilia sp., Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 2:2:1:1:1:1 to obtain a liquid composite fermentation broth, and then vacuum freeze-drying was carried out: First, it was pre-frozen in a liquid nitrogen tank at -196 °C for 3 min; then, it was vacuum frozen at -85 °C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0062] Example 12

[0063] The fermentation broth obtained in Example 3 was mixed according to the ratio of viable cell counts of Pseudomonas orientalis, methylotrophic bacteria, Massilia sp., Penicillium fructigenum, Paecilomyces lilacinus, and Streptomyces enniatinus being 2:2:2:2:1:1 to obtain a liquid composite fermentation broth, and then vacuum freeze-drying was carried out: First, it was pre-frozen in a liquid nitrogen tank at -196 °C for 3 min; then, it was vacuum frozen at -85 °C for 15 h under a vacuum pressure of 0.01 mbar to obtain a solid composite microbial agent.

[0064] Example 13

[0065] The solid composite microbial agents obtained in Examples 4 - 12 were evenly spread on the Agropyron cristatum soil and uniformly mixed with the soil around the plants. The plants were placed in a suitable light position and appropriate moisture and nutrients were maintained. The formation of the rhizosheath structure of the plants with and without the application of the composite microbial agent was observed for comparison, and the weight of the rhizosheath was statistically analyzed. The results are shown in Table 4.

[0066] Table 4 Effects of different composite microbial agents and no composite microbial agent on rhizosheath formation

[0067]

[0068] As can be seen from Table 4, the compound microbial agent can significantly promote the formation of the plant rhizosheath structure. Compared with the control, the weight of the rhizosheath has increased by more than 2 times. Among them, Treatment I has the most significant promotion effect, which is more than 5 times higher than the control. The compound microbial agent is Pseudomonas orientalis: Methylobacterium: Massilia: Penicillium fructiforme, Paecilomyces lilacinus: Streptomyces enniatin with the viable cell number ratio of 2:2:2:2:1:1, and it is superior to other mixed treatments.

Claims

1. A microbial composite agent, characterized in that: The composite bacterial agent is composed of Pseudomonas orientalis with a preservation number of CCTCC S2013634, Marseilles with a preservation number of CCTCC AB 2012441, a methylotrophic bacterium with a preservation number of ATCC BAA-2500, Penicillium fructiferum with a preservation number of CGMCC 3.10065, Paecilomyces lilacinus with a preservation number of CCTCC NF 20082457 and Streptomyces enemycin with a preservation number of CGMCC 4.6279; the composite bacterial agent comprises Pseudomonas orientalis with a preservation number of CCTCCS2013634, a methylotrophic bacterium with a preservation number of ATCC BAA-2500, a Marseilles with a preservation number of CGMCC AB2012441, Penicillium fructiferum with a preservation number of CGMCC 3.10065, The ratio of the viable cell counts of Paecilomyces lilacinus with the deposit number of 20082457 and Streptomyces enemycin with the deposit number of CGMCC 4.6279 is 1-2:1-2:1-2:1-2:1-2:1-2.

2. The microbial composite agent according to claim 1, characterized in that: The ratio of the number of viable bacteria in the composite bacterial agent of Pseudomonas orientalis with a preservation number of CCTCC S2013634, the methylotrophic bacteria with a preservation number of ATCC BAA-2500, the Marseilles with a preservation number of CCTCC AB 2012441, the Penicillium fructificum with a preservation number of CGMCC 3.10065, the Paecilomyces lilacinus with a preservation number of CCTCC NF20082457 and the Streptomyces enemycin with a preservation number of CGMCC 4.6279 is 2:2:2:2:1:

1.

3. The microbial composite agent according to claim 1, characterized in that: The total number of viable bacteria in the composite bacterial agent is 10 9 ~10 10 CFU / g or 10 8 ~10 9 CFU / mL.

4. The method for preparing the composite bacterial agent according to any one of claims 1 to 3, characterized in that: Pseudomonas orientalis with a preservation number of CCTCCS2013634, a methylotrophic bacteria with a preservation number of ATCC BAA-2500, a Marseillaria with a preservation number of CCTCC AB2012441, a Penicillium fructiferum with a preservation number of CGMCC 3.10065, a Paecilomyces lilacinus with a preservation number of CCTCC NF 20082457, and a Streptomyces enemycinus with a preservation number of CGMCC 4.6279 are respectively inoculated into a liquid culture medium for fermentation. After the fermentation is completed, the fermentation broths are mixed according to the fermentation broth volume ratio of Pseudomonas orientalis, the methylotrophic bacteria, the Marseillaria, the Penicillium fructiferum, the Paecilomyces lilacinus, and the Streptomyces enemycinus of 1-2:1-2:1-2:1-2:1-2:1-2 to obtain a liquid composite bacterial agent.

5. The method for preparing the composite bacterial agent according to claim 4, wherein the liquid composite bacterial agent is subjected to vacuum freeze-drying to obtain a solid composite bacterial agent.

6. Use of the composite bacterial agent according to any one of claims 1 to 3 in promoting the formation of sand sheath structures at the roots of wheatgrass.

7. The use according to claim 6, characterized in that: Apply the compound fungal agent to the soil at the roots of the ice grass and mix it evenly with the soil around the ice grass.

8. Use of the composite bacterial agent according to any one of claims 1 to 3 in improving the properties of the sandy soil around the roots of wheatgrass.

9. The use according to claim 8, characterized in that: The soil properties refer to soil pH, electrical conductivity, and the contents of organic matter, available phosphorus, and alkaline-hydrolyzable nitrogen.

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