CN113151041B

CN113151041B - Paenibacillus polymyxa and preparation and application of microbial inoculum thereof

Info

Publication number: CN113151041B
Application number: CN202110146365.5A
Authority: CN
Inventors: 洒荣波; 宋枚枚; 许乐; 王子强; 张爽; 邢倩; 刘丽娜
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2022-07-05
Anticipated expiration: 2041-02-03
Also published as: CN113151041A

Abstract

The invention discloses a paenibacillus polymyxa DS-R5 and preparation and application of a microbial inoculum thereof, wherein the strain is preserved in China center for type culture Collection in 2021, 1, 4 days, and the biological preservation number is as follows: CCTCC M2021005. The strain provided by the invention has an inhibiting effect on various pathogenic bacteria, and particularly has a strong antagonistic effect on fusarium solani, rhizoctonia solani and the like causing root rot. The strain can increase soil nutrient elements, improve the study on the microbial community structure of the salvia miltiorrhiza rhizosphere, inhibit the number of pathogenic bacteria in soil so as to interfere the impregnation of the pathogenic bacteria on the salvia miltiorrhiza roots, and achieve the double effects of preventing diseases and promoting growth. Meanwhile, the microbial fertilizer taking the strain as an active ingredient has low cost, high fertilizer efficiency and no pollution, and is the best substitute of the fertilizer and the pesticide which are frequently used in the prevention and the treatment of the root rot of the salvia miltiorrhiza; has guiding significance for developing new traditional Chinese medicine salvia miltiorrhiza resources, reducing salvia miltiorrhiza fungal diseases and improving the salvia miltiorrhiza yield.

Description

Paenibacillus polymyxa and preparation and application of microbial inoculum thereof

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to paenibacillus polymyxa and preparation and application of a microbial inoculum thereof.

Background

Salvia miltiorrhiza (Salvia militirhiza) is a perennial herb, is used as a medicine by root, has the effects of removing blood stasis, relieving pain, promoting blood circulation, regulating menstruation, nourishing heart, relieving restlessness and the like, and is widely used for treating cardiovascular and cerebrovascular diseases. The salvia miltiorrhiza is one of medicinal materials with larger cultivation area, and under the condition of artificial cultivation, due to poor diversity and low abundance of biological communities in the planting ecological environment and prominent dominant population of diseases and insect pests, serious diseases and insect pests often occur, the growth and development of salvia miltiorrhiza plants are affected, and the yield and the quality of the medicinal materials are reduced.

Root rot of salvia miltiorrhiza belongs to typical fungal infectious diseases, pathogenic spores can be latent in soil, the latent period is long, the root rot is difficult to cure, and the root rot becomes an initial infection source in the next year. The root rot of salvia miltiorrhiza is a soil-borne disease caused by Fusarium solani, the spread of the root rot of salvia miltiorrhiza is rapid in recent years, the infected salvia miltiorrhiza plants are weak in growth and are seriously withered at the upper part of the roots, the xylem of the roots is completely rotten into black brown, the yield is obviously reduced, and the appearance character and the quality of the root rot of salvia miltiorrhiza do not meet the medicinal requirements. The root rot of salvia miltiorrhiza is mainly prevented and treated by chemical pesticides at present, but the prevention effect is very little; the beneficial microbial agent for preventing and treating the fungal diseases of the salvia miltiorrhiza is the best choice for solving the soil-borne diseases of the traditional Chinese medicinal materials.

Endophytic bacteria refer to a class of microorganisms that colonize healthy plant tissues and establish a harmonious association with the plant. Endogenous bacteria often secrete active substances to promote plant growth and to combat pests. The bacteria have the characteristics of high growth speed, easy survival and the like, and the separation of the endophytic bacteria with antibacterial activity from the plants has important significance for preventing and treating plant diseases. For medicinal plants, the relationship between their endophytes and their "co-evolution" determines the ability of certain endophytes to produce the same or similar bioactive substances as the host plant. In order to develop a new traditional Chinese medicine salvia miltiorrhiza resource, reduce salvia miltiorrhiza fungal diseases and improve the yield of salvia miltiorrhiza, a special microbial fertilizer for preventing and controlling salvia miltiorrhiza root rot, which is prepared from endophytic bacteria, is needed, so that not only can soil nutrient elements be increased, but also the research on the rhizosphere microbial community structure of salvia miltiorrhiza can be improved, the number of pathogenic bacteria in soil can be inhibited, the impregnation of the pathogenic bacteria to the salvia miltiorrhiza roots is interfered, and the dual effects of preventing diseases and promoting growth are achieved. Meanwhile, the microbial fertilizer has low cost, high fertilizer efficiency and no pollution, so the microbial fertilizer is the best substitute for the chemical fertilizer and the pesticide which are frequently used in the prevention and treatment of the root rot of the salvia miltiorrhiza.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a paenibacillus polymyxa strain and preparation and application of a microbial inoculum thereof. The invention separates, purifies and cultures endophytic bacteria of salvia miltiorrhiza plants in a certain salvia miltiorrhiza planting base in Taian city of Shandong, takes fusarium solani, a pathogenic bacterium of salvia miltiorrhiza root rot, as a target bacterium, and separates a bacterium DS-R5 with strong bacteriostatic effect on fusarium solani, and the salvia miltiorrhiza root rot control special microbial fertilizer prepared by the endophytic bacteria can increase soil nutrient elements, improve the research of rhizosphere microbial community structure of salvia miltiorrhiza, inhibit the number of pathogenic bacteria in soil so as to interfere the dip-dyeing of the root of salvia miltiorrhiza, and play dual effects of preventing diseases and promoting growth.

In order to achieve the purpose, the invention adopts the following technical scheme:

in the first aspect of the invention, a Paenibacillus polymyxa (Paenibacillus polymyxa) DS-R5 is provided, and the biological preservation number is as follows: CCTCC NO: m2021005.

The preservation unit is as follows: china center for type culture Collection, Collection Unit Address: wuhan university, preservation date: 1/4/2021.

In a second aspect of the invention, a microbial agent is provided, which takes Paenibacillus polymyxa (Paenibacillus polymyxa) DS-R5 as an active ingredient.

Preferably, the microbial agent is a liquid microbial agent or a solid microbial agent.

In a third aspect of the present invention, there is provided a method for preparing the microbial agent, comprising the steps of:

(1) activating strains: streaking and inoculating the paenibacillus polymyxa DS-R5 on a solid LB culture medium plate, placing the solid LB culture medium plate in an incubator for culture, picking out a single colony after the single colony grows out, continuously streaking and inoculating the single colony on the solid LB culture medium plate, and culturing in the incubator to obtain activated DS-R5 lawn;

(2) preparing a first-level seed solution: inoculating the activated DS-R5 lawn in the step (1) into a liquid LB culture medium, and performing shake culture to obtain a first-level seed solution;

(3) preparing a secondary seed liquid: inoculating the primary seed liquid obtained in the step (2) according to the volume percentage of 2%, stirring, and fermenting for 16h to obtain a secondary seed liquid;

(4) preparing a liquid microbial inoculum: inoculating the secondary seed liquid obtained in the step (3) into a fermentation culture medium according to the volume percentage of 5%, stirring, and fermenting for 36 hours to obtain a liquid microbial agent;

(5) preparing a solid microbial inoculum: and (4) centrifuging the liquid microbial agent obtained in the step (4) by using a centrifugal machine, collecting thalli, transferring the thalli to a stirrer, adding corn flour accounting for 5% of the total weight as a carrier matrix, uniformly stirring, and performing spray drying to obtain the solid microbial agent.

Preferably, in the preparation of secondary seed liquid, the fermentation temperature is 37 deg.C, and the ventilation amount is 3m³The tank pressure is maintained at 0.05 MPa; in the preparation of the liquid microbial inoculum, the fermentation temperature is 37 ℃, and the ventilation volume is 30m³The pot pressure was maintained at 0.05 MPa.

Preferably, the fermentation medium formula is as follows: glucose 10.0g, peptone 20.0g, (NH)₄)₂SO₄ 2.0g，KH₂PO₄0.5g，MgSO₄1.0g, NaCl 1.5g and distilled water 1L; sterilizing at 121 deg.C under 0.1MPa for 20 min.

In a fourth aspect of the present invention, there is provided a method for using the microbial agent, comprising: uniformly mixing the liquid microbial agent and water according to the proportion of 1:100, and irrigating roots for application; the solid microbial inoculum is applied by root irrigation according to the dosage of 5 kg/mu.

In a fifth aspect of the present invention, there is provided the use of the above-mentioned Paenibacillus polymyxa DS-R5 or microbial agent in at least one of the following (1) to (4):

(1) inhibiting phytopathogens;

(2) preparing a product for inhibiting phytopathogens;

(3) preventing and treating root rot of medicinal plants caused by plant pathogenic bacteria;

(4) preparing a product for preventing and treating root rot of medicinal plants caused by plant pathogenic bacteria.

Preferably, the phytopathogens include fungi and bacteria; the fungus is Fusarium solani, Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium pseudograminearum, anthrax or Staphyloccocus botrytis; the bacteria is Staphylococcus aureus, Escherichia coli, Candida albicans, Patrinia scabiosaefolia, Candida albicans or bacterial Populus canker.

Preferably, the medicinal plants are salvia miltiorrhiza, polygonatum, astragalus or isatis roots; preferably, the medicinal plant is salvia miltiorrhiza.

The invention has the beneficial effects that:

1. the antagonistic bacterium DS-R5 has a wider antagonistic spectrum, and has different degrees of inhibition effects on common plant pathogenic fungi; the microbial inoculum prepared from the DS-R5 strain can effectively prevent and treat the soil-borne fungal disease of the root rot of salvia miltiorrhiza caused by fusarium solani, has antagonistic effect on common pathogenic bacteria, and can inhibit both fungi and bacteria. The antagonistic bacterium DS-R5 can be stably colonized at the rhizosphere of medicinal plants such as salvia miltiorrhiza, rhizoma polygonati, astragalus and the like; the growth of salvia miltiorrhiza can be promoted while the root rot disease of salvia miltiorrhiza is prevented and treated. The antagonistic bacterium DS-R5 disclosed by the invention also has the capability of quickly degrading cellulose, is beneficial to directly returning medicinal plant straws such as salvia miltiorrhiza, rhizoma polygonati and astragalus membranaceus to the field, and is beneficial to quickly decomposing the straws.

2. The microbial inoculum has simple production process, short period, good fermentation property of the strain DS-R5 and low cost, and is beneficial to industrial production and transportation. The microbial inoculum of the invention can increase soil nutrient elements, improve the study of the rhizosphere microbial community structure of salvia miltiorrhiza, inhibit the quantity of pathogenic bacteria in soil so as to interfere the dip dyeing of the root of salvia miltiorrhiza by the pathogenic bacteria, and play a role in preventing diseases and promoting growth. Meanwhile, the microbial inoculum has low cost, high fertilizer efficiency and no pollution, and is the best substitute of fertilizer and pesticide for controlling the root rot of salvia miltiorrhiza. The invention has guiding significance for developing new traditional Chinese medicine salvia miltiorrhiza resources, reducing salvia miltiorrhiza fungal diseases and improving the yield of salvia miltiorrhiza.

Drawings

FIG. 1A is the colony morphology of strain DS-R5 on NA medium; FIG. 1B shows the cell morphology of strain DS-R5 under an optical microscope; FIG. 1C shows the spore morphology of strain DS-R5 under an optical microscope; FIG. 1D shows the cell morphology of the strain DS-R5 under an electron microscope.

FIG. 2 is a phylogenetic tree constructed from the 16S rDNA sequence of strain DS-R5.

FIG. 3 shows the bacterial inhibition spectrum of strain DS-R5 on part of pathogenic fungi; a is antagonistic action of a strain DS-R5 on fusarium solani; b is antagonistic action of the strain DS-R5 on fusarium graminearum; c is antagonistic action of the strain DS-R5 on rhizoctonia solani; d is antagonistic action of the strain DS-R5 on anthrax.

FIG. 4 shows the bacterial inhibition spectrum of strain DS-R5 on a part of pathogenic bacteria; a is antagonistic action of the strain DS-R5 on staphylococcus aureus; b is the antagonistic action of the strain DS-R5 on the septicemia melanothorax; c is antagonistic action of the strain DS-R5 on Candida albicans; d is antagonistic action of the strain DS-R5 on bacterial canker of poplar; e is antagonistic action of the strain DS-R5 on cryptococcus neoformans; f is antagonistic action of the strain DS-R5 on Escherichia coli.

FIG. 5 shows the green fluorescence emitted by strain DS-R5 labeled with Green Fluorescent Protein (GFP) under a fluorescent microscope.

FIG. 6 shows the inhibitory effect of fermentation broth of strain DS-R5 on germination of Fusarium solani spores.

FIG. 7A is a transparent circle of hydrolyzed Congo red cellulose of strain DS-R5; FIG. 7B is the proteolytic clearing circle of strain DS-R5.

FIG. 8A shows a standard curve for determining the cellulase activity of strain DS-R5; FIG. 8B is a standard curve for protease activity assay of strain DS-R5.

FIG. 9A shows the control hyphae, which are bright, thin, long and uniform in thickness; FIG. 9B shows Fusarium solani inhibited by antagonistic strain DS-R5, which has mostly distorted mycelium, winding and breaking mycelium, and local beaded cells.

FIG. 10 shows the inhibitory effect of strain DS-R5 on Fusarium solani hyphae under an optical microscope; a represents a control group, no zone of inhibition was formed; c is the bacteriostasis condition after the fermentation stock solution is added; and B is the bacteriostasis condition of the diluted fermentation liquor added by 2 times.

FIG. 11 shows the inhibitory effect of strain DS-R5 on germination of Fusarium solani spores under an optical microscope; a is a control group, and a considerable amount of spores are already germinated; b is the treatment group, with only occasional spore germination.

FIG. 12 is a real-time fluorescent quantitative PCR standard curve of Fusarium solani.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available.

Example 1

1. Isolation of Strain DS-R5

Taking healthy salvia miltiorrhiza plants with good growth vigor, cleaning the plants by running water, and disinfecting the surface of a material according to the following flow: soaking in 75% ethanol for 1min, and washing with sterile water for 3 times; soaking in 0.1% mercuric chloride for 3min, and washing with sterile water for 3 times. Grinding the treated root, stem, leaf, etc. tissues of Salvia miltiorrhiza in a sterile operating table into slurry with a mortar, diluting with sterile water by 10 times, spreading on NA culture medium, and culturing at 37 deg.C for 3 d. When visible colonies grow on the culture medium, the colonies are picked in time and subjected to streaking separation for multiple times to obtain a pure culture.

Fusarium solani is used as target pathogenic bacteria to screen the endophytic antagonistic bacteria of the salvia miltiorrhiza. Carrying out pathogenic fungus antagonism performance test on the separated salvia miltiorrhiza endophytic bacteria by adopting a two-point plate antagonism method, taking fusarium solani cultured for 7d, punching by using a puncher with the diameter of 5mm, inoculating a fungus cake with the diameter of 5mm to the center of a PDA (personal digital assistant) plate, carrying out parallel streaking on the upper part and the lower part at a position 2.5cm away from the fungus cake, inoculating the separated salvia miltiorrhiza endophytic bacteria, and placing the salvia miltiorrhiza endophytic bacteria in an incubator at the constant temperature of 30 ℃ by taking a fungus plate which is not connected with antagonistic bacteria as a control. And when the pathogenic bacteria colonies of the control group grow to fill the bottom of the dish, measuring the width of the bacteriostatic belt of the treatment group.

And (3) carrying out a flat plate confrontation test on the obtained pure cultures, measuring the width of the antibacterial band, and screening to obtain the salvia miltiorrhiza endogenous antagonistic bacterium DS-R5 strain with wide antibacterial spectrum and strong antibacterial effect.

2. Identification of Strain DS-R5

(1) Morphology and colony characteristics of thallus

After the strain DS-R5 is cultured on the NA culture medium for 36 hours, the bacterial colony is smooth and moist, is round and is not easy to pick up; under an optical microscope, the thallus is rod-shaped, gram staining is positive, oval spores are produced, and the spores are generated; scanning electron microscopy revealed that the cell size of the cells was about 0.8. mu. m.times.2.8. mu.m (see FIG. 1).

(2) Physiological and biochemical characteristics

The strain DS-R5 can utilize glucose and glycerol to ferment and produce acid, starch hydrolysis, gelatin hydrolysis, anaerobic growth, catalase, nitrate reaction, V-P reaction, casein hydrolysis reaction and the like which are all positive, and oxidase reaction, citrate reaction, succinate reaction and H reaction₂S production reaction and the like were all negative and could not grow on a medium containing 5% NaCl (Table 1).

TABLE 1 part of the physio-biochemical characteristics of the strain DS-R5

(3) Strain DS-R5 based on 16S rDNA sequence analysis

The 16S rDNA sequence of the strain DS-R5 is shown in SEQ ID NO.1, the sequence is subjected to Blast analysis and comparison with the sequence in an NCBI database, 6 strains with higher similarity to the sequence of the strain DS-R5 are selected for phylogenetic analysis, and a Neighbor-Joining method is adopted to construct a phylogenetic tree based on the 16S rDNA sequence by utilizing Mega 7.0 software (see figure 2). The result shows that the strain DS-R5 and the Paenibacillus polymyxa are in the same branch, and the strain DS-R5 is identified as the Paenibacillus polymyxa by combining the culture characteristics and the physiological and biochemical characteristics of the strain DS-R5.

Deposit unit of strain DS-R5: china center for type culture Collection, Collection Unit Address: wuhan university, preservation date: 1, month and 4 days 2021, the biological preservation number is CCTCC NO: m2021005.

Example 2: determination of bacterial inhibition spectrum of strain DS-R5 on common plant pathogenic fungi

And (3) carrying out pathogenic fungus antagonistic performance test on the separated salvia miltiorrhiza endophytic bacteria DS-R5 by adopting a two-point plate confronting method. Respectively taking Fusarium solani, Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium oxysporum, Fusarium pseudograminearum, anthrax and Staphylomyces viticola which are cultured for 7d, punching a hole by using a puncher with the diameter of 5mm, inoculating a fungus cake with the diameter of 5mm to the center of a PDA (personal digital Assistant) flat plate, inoculating separated salvia miltiorrhiza endophytic bacteria at a position 2.5cm away from the fungus cake, and placing the fungus cake without the antagonistic bacteria as a contrast in an incubator at the constant temperature of 30 ℃. And when the pathogenic bacteria colonies of the control group grow to be full of the bottom of the dish, measuring the width of the bacteriostatic belt of the treatment group, and repeating the treatment for three times. The test result shows that the strain DS-R5 has antagonistic effects on 8 pathogenic fungi to be tested in different degrees, the inhibitory effects of the strain DS-R5 on the 8 pathogenic fungi are shown in Table 2, and the bacteriostatic spectrum is shown in FIG. 3.

TABLE 2 inhibitory Effect of the Strain DS-R5 on 8 pathogenic fungi

Note: the data in the table are mean ± standard deviation. Different lower case letters after the same column of data indicate significant differences at a level P <0.05 as tested by duncan novice.

Example 3: determination of bacterial inhibition spectrum of strain DS-R5 on common pathogenic bacteria

Respectively inoculating a strain of staphylococcus aureus, escherichia coli, candida albicans, septicemia melanothoracis, candida albicans and bacterial poplar canker pathogen into an LB culture medium for overnight culture, adding 20 mu L of the 6 tested pathogenic bacteria in logarithmic phase into 20mL of the unsolidified LB culture medium, uniformly mixing, and pouring into a sterile culture dish with the diameter of 7.5 cm. After solidification, the strain DS-R5 is spotted by a sterilized toothpick, and after 24 hours of culture at 37 ℃, the antagonistic effect of the strain DS-R5 on the pathogenic bacteria to be tested is observed. The results of the tests show that the strain DS-R5 has various degrees of inhibitory effect on 6 pathogenic bacteria tested (see FIG. 4).

Example 4

1. Determination of colonization ability of strain DS-R5 in rhizosphere of radix salviae miltiorrhizae, rhizoma polygonati and radix astragali

(1) Construction of Green Fluorescent Protein (GFP) tagged Strain DS-R5-GFP

Firstly, strain DS-R5 competent cells are prepared according to a conventional method for standby, 100 mu L of prepared competent cells are taken to be mixed with a proper amount of plasmid pHT01-P43GFPmut3a and then transferred to a precooled electric shock cup, the mixture is lightly blown and sucked by a gun head, and the ice-bath time is not more than 5 min. After being loaded into an electric shock instrument (Berle MicroPulser electroporator), the electric shock is carried out, and the electric shock conversion condition is as follows: voltage 1.5kv, resistance 300 omega, capacitance 2.5 muF. Immediately after the electric shock, 900. mu.L of Luria Bertani (LB) medium preheated at 30 ℃ was added and transferred to a test tube. After shaking at 200rpm for 3 hours at 30 ℃, the culture was spread on LB plates containing 5. mu.g/mL of chloramphenicol. Colonies grown on the resistant plates were observed under a fluorescent microscope to see if the bacteria were able to excite green fluorescence under blue light. As can be seen from FIG. 5, the constructed strain DS-R5-GFP can emit bright green fluorescence under blue light excitation, which indicates that the strain DS-R5 is successfully constructed as a GFP-labeled strain.

(2) Colonization test

Planting the salvia miltiorrhiza, astragalus and isatis root seedlings with consistent growth vigor into a flowerpot with the diameter of 30cm for cuttage propagation, filling sandy soil mixed by peat soil and perlite according to the proportion of 3:2 into the flowerpot, and watering and managing the seedlings in a normal mode. Inoculating strain DS-R5-GFP into LB liquid culture medium containing 5mg/L chloramphenicol, culturing for about 12h, centrifuging at 6000R/min for 10min, collecting thallus, adding appropriate amount of sterile water, shaking thoroughly to disperse thallus, taking 10mL bacterial liquid, and respectively applying Plumbum PreparatiumAnd (3) digging soil samples of roots of the seedlings of the ginseng, the astragalus membranaceus and the isatis root in 5 th, 10 th, 20 th, 40 th and 60 th days after treatment, and coating chloramphenicol resistant plates for counting after gradient dilution. The quantitative conditions of the strain DS-R5-GFP in the rhizosphere soil of the salvia miltiorrhiza, astragalus and isatis root seedlings are shown in figure 6. As can be seen from the figure, with the extension of the bacteria irrigation time, the colonization density of the strain DS-R5-GFP in the rhizosphere soil of the seedlings of the salvia miltiorrhiza, the astragalus and the isatis root shows the tendency of rising firstly and then falling. At 5d treated with the strain DS-R5-GFP, the colony counts recovered in the rhizosphere soil of the seedlings of the salvia miltiorrhiza, the astragalus and the isatis root are respectively 3.5 multiplied by 10⁶CFU/g、2.6×10⁶CFU/g、1.9×10⁶CFU/g; at the 20 th day treated by the strain DS-R5-GFP, the colony numbers recovered from the rhizosphere soil of the seedlings of the salvia miltiorrhiza, the astragalus and the isatis root all reach the highest value, and are respectively 6.4 multiplied by 10⁶CFU/g、5.4×10⁶CFU/g、4.5×10⁶CFU/g. After 20 days, the colony count begins to decrease, and 4.1X 10 colonies can be recovered respectively till 60 days⁶CFU/g、4.3×10⁶CFU/g、2.1×10⁶CFU/g of DS-R5-GFP. This fully demonstrates that the strain DS-R5-GFP can colonize in the rhizosphere soil of salvia miltiorrhiza, astragalus and isatis root seedlings for a long time.

Example 5: disease prevention mechanism of strain DS-R5 for root rot of salvia miltiorrhiza

(1) Antagonism is obtained. Antagonism is an important mechanism for antagonistic bacteria to prevent and treat diseases. Antagonistic bacteria inhibit or kill pathogenic microorganisms by synthesizing and secreting antibacterial substances to act on cell walls, cell membranes, protein synthesis systems, energy metabolism systems and the like of pathogenic fungi. Antibacterial substances mainly include two main classes: antibiotics (peptides, bacteriocins, etc.) and hydrolases (chitinase, protease, cellulolytic enzymes, dextranase, lysozyme, etc.).

The paenibacillus polymyxa DS-R5 provided by the invention can secrete cellulase and protease. Respectively dibbling the strain DS-R5 on a cellulose Congo red culture medium plate and a skim milk culture medium plate, carrying out inverted culture at 37 ℃ for 2-3d, and observing the result. The results of the tests are shown in FIGS. 7A and 7B, from which it can be seen that strain DS-R5 is capable of producing significant permeabilities on the cellulose congo red plates and skim milk medium plates, indicating that strain DS-R5 is capable of producing cellulase and protease. The enzyme activities of the cellulase and the protease generated by the fermentation liquor are respectively 46.7u/mL and 68.4u/mL by liquid fermentation of the strain DS-R5. The cellulase and protease activity determination method refers to agricultural industry standard NY/T1847-2010 of the people's republic of China, and the cellulase and protease activity determination standard curve is shown in figures 8A and 8B.

The media formulations required in the experiments were as follows:

cellulose congo red medium: 1.88g of microcrystalline cellulose, 0.2g of Congo red, MgSO40.25g, K2HPO40.5g, 2.0g of gelatin, 14.0g of Agar and 1L of distilled water.

Cellulase liquid fermentation medium: corn flour 40g, peptone 3.0g, (NH)₄)₂SO₄ 2.0g，Na₂HPO₄0.5g，MgSO₄ 0.2g，FeSO₄.7H₂O 0.1g，CaCO₃0.5g, and 1L of distilled water.

Protease liquid fermentation medium: glucose 5g, Na₂HPO₄0.1g, peptone 5g, Tween-801 mL, CaCl₂0.1g, distilled water 1L.

Skim milk culture medium: skim milk 10.0g, K₂HPO₄ 1.0g，MgSO₄.7H₂O 0.5g，KCl 5.0g，FeSO₄.7H₂0.1g of O, 20.0g of agar and 1L of distilled water.

(2) Inhibition effect of strain DS-R5 on hypha growth and spore germination of pathogenic fungi

Firstly, picking out normal growth hypha of pathogenic bacteria and fusarium solani hypha inhibited by a strain DS-R5 by using an aseptic toothpick, and slicing, and observing the influence of an antagonistic strain DS-R5 on the shape of the fusarium solani hypha under an optical microscope. The microscopic examination of an optical microscope shows that the strain DS-R5 has obvious destructive effect on the growth of fusarium solani hyphae. The control group mycelia had bright color, long, thin and uniform thickness (FIG. 9A), while the Fusarium solani mycelia inhibited by the antagonistic strain DS-R5 were mostly distorted, and the mycelia were entangled and broken, and beaded cells appeared locally (FIG. 9B).

② the inhibiting effect of the fermentation liquor of the strain DS-R5 on the spore germination of pathogenic fungi

Spore germination plate test: inoculating a loopful strain DS-R5 into LB culture medium, culturing overnight at 200R/min and 37 ℃, inoculating 2% of inoculum size into an antibacterial active substance fermentation culture medium, fermenting at 200R/min and 30 ℃ for 72h, centrifuging at 6000R/min, taking supernatant, and filtering with a 0.22 mu m sterile filter membrane to prepare sterile fermentation liquid. Fusarium solani spores are prepared into spore suspension in 2% glucose solution, and the concentration is 1 x 10⁶spores/mL are preferred. Sterilizing the prepared PDA culture medium at 121 deg.C and 0.1Mpa for 20min, cooling to 50 deg.C, and making into spore suspension: adding the prepared spore suspension into the PDA culture medium at a ratio of 1:100, uniformly mixing, pouring the mixture into a flat plate, uniformly placing 3 sterilized Oxford cups 2cm away from the center of the flat plate after the flat plate is solidified, respectively adding a fermentation liquid stock solution (shown in figure 10C) and a 2-time diluent solution (shown in figure 10B) into 2 Oxford cups, and adding 200 mu L of the uninoculated fermentation culture medium as a control. The test result is shown in the attached figure 10, and it can be seen from the figure that the addition of the fermentation liquid can inhibit fusarium solani spores from germinating into hyphae to form an obvious inhibition zone, and the formed inhibition zone becomes obviously smaller with the increase of the dilution multiple of the fermentation liquid, while the spores of the control group normally germinate into hyphae without any inhibition zone (figure 10A).

Microscopic examination test of spore germination: mixing the stock solution of the sterile fermentation broth and the spore suspension in equal volume, dripping 10 mu L of the mixture on a concave slide, covering a cover glass, placing the concave slide in a sterilized culture dish, performing moisture-preserving culture in an incubator at 30 ℃, observing the spore germination number every 8h by microscopic examination, and continuously observing for 32 h.

The calculation formula of the germination rate and the inhibition rate is as follows:

the test results are shown in figure 11, and spore germination microscopic tests show that the fermentation liquor of the strain DS-R5 can obviously inhibit germination of Fusarium solani spores, a great amount of spores begin to germinate in a control group (figure 11A), and only sporadic spores germinate in a treatment group (figure 11B). As shown in Table 3, after Fusarium solani spores are treated by the fermentation broth of the strain DS-R5, the spore germination number and germination rate of a treated group are far smaller than those of a control group: when 8 hours are spent, the germination number and the germination rate of the control group spores are 15 percent and 17.3 percent respectively; while the germination number and the germination rate of the spores in the treatment group are only 3.7 percent and 4.3 percent, and the spore germination inhibition rate is 75.1 percent; at 32 hours, the control group had spores completely germinated, while the treatment group had only 17.7% of spore germination and 82.9% of spore germination inhibition. The data in the table strongly indicate that the strain DS-R5 has a strong inhibitory effect on the germination of Fusarium solani spores.

TABLE 3 inhibitory Effect of the DS-R5 antibacterial active substance of the Strain on the Germination of Fusarium solani spores

Note: symbol "+ indicates significant difference between the treated group and the control group (P < 0.05); the difference was significant without the same letter (P <0.05) compared between columns.

Example 6: preparation of Paenibacillus polymyxa DS-R5 microbial inoculum

(1) Activating strains: and (2) streaking and inoculating the paenibacillus polymyxa DS-R5 on an LB solid culture medium plate, culturing for 24h in an incubator at 37 ℃, picking out a single colony after the single colony grows out, continuously streaking and inoculating on the LB solid culture medium plate, and culturing for 24h in the incubator at 37 ℃ for later use.

(2) Preparing a first-level seed solution:

scraping one ring of activated DS-R5 thallus Porphyrae with inoculating ring, inoculating into 250ml triangular flask containing 100ml liquid LB culture medium, and shake-culturing at 37 deg.C and 200R/min for 16h as first-stage seed solution. The liquid LB medium formulation is as follows: peptone 1%, sodium chloride 1%, yeast powder 0.5%; sterilizing at 121 deg.C and 0.1MPa for 20 min.

(3) Preparing a secondary seed liquid: mixing the first-stage seed liquidInoculating into 100L fermentation tank according to the inoculation amount of 2% by volume, rotating speed of stirring paddle 200r/min, fermentation temperature 37 deg.C, and ventilation amount 3m³And h, maintaining the tank pressure at about 0.05MPa, and culturing for 16h to obtain a secondary seed solution. The formula and sterilization conditions of the second-stage seed liquid culture medium are consistent with those of the first-stage seed liquid.

(4) Preparing a liquid microbial inoculum: inoculating the second-stage seed solution into a 2-ton fermentation tank filled with a fermentation culture medium according to the inoculation amount of 5% by volume, wherein the rotation speed of a stirring paddle is 120r/min, the fermentation temperature is 37 ℃, and the ventilation volume is 30m³The tank pressure is maintained at about 0.05MPa, the culture is carried out for 36 hours under the condition, the liquid microbial inoculum of the strain DS-R5 is obtained, and the spore yield of the liquid microbial inoculum>80％。

The fermentation medium formula comprises: glucose 10.0g, peptone 20.0g, (NH)₄)₂SO₄ 2.0g，KH₂PO₄ 0.5g，MgSO₄1.0g, NaCl 1.5g, and 1L of distilled water. Sterilizing at 121 deg.C under 0.1MPa for 20 min.

(5) Preparing a solid microbial inoculum: centrifuging the fermented DS-R5 liquid microbial inoculum by a centrifuge at 10000R/min for 20min, collecting the microbial inoculum, transferring the microbial inoculum to a stirrer, adding corn flour accounting for 5 percent of the total weight as a carrier matrix, uniformly stirring, and performing spray drying to obtain the strain DS-R5 solid microbial inoculum, wherein the number of the living microbial inoculum can reach 1200 hundred million/g.

Example 7: application of paenibacillus polymyxa DS-R5 microbial inoculum

(1) Pot test 1: biological control effect of strain DS-R5 on root rot of salvia miltiorrhiza

Selecting 40 potted salvia miltiorrhiza seedlings with good growth vigor, one seedling in each pot, dividing the seedlings into two groups, and 20 seedlings in each group. One group was treated by root irrigation with the microbial inoculum obtained in example 4 of the present invention, and the other group was a control group. The experimental group is that root irrigation of the microbial inoculum obtained in the example 4 is carried out to treat salvia miltiorrhiza seedlings, 30mL of the microbial inoculum is added to each salvia miltiorrhiza seedling, and after three days, root irrigation is carried out to inoculate 20mL of fusarium solani spore suspension, wherein the spore concentration is 1 multiplied by 10⁶Per mL; in the control group, 30mL of fermentation medium without inoculation is used for root irrigation to treat the salvia miltiorrhiza seedlings, and after three days, the root irrigation is used for inoculating 20mL of fusarium solani spore suspension, wherein the spore concentration is 1 multiplied by 10⁶one/mL. And counting and calculating the disease prevention effect of the strain DS-R5 on the root rot of the salvia miltiorrhiza after 120 days. The disease condition is classified into five grades according to the lesion area of the salvia miltiorrhiza seedlings. No scab is found in grade 1; grade 2 area of lesion<5 percent; grade 3, the area of the lesion is 5-20%; the 4-grade is that the area of the lesion spots is between 20 and 50 percent; grade 5 is the area of the lesion>50 percent. The total number of untreated plants at each stage was A, B, C, D, E, respectively, with disease index ═ 0 xa +1 xb +2 × 0C +3 × 1D +4 × E)/(4 × a +4 × B +4 × C +4 × D +4 × E) × 100; the disease prevention effect is 100% — (disease index of treatment group/disease index of control group) × 100%. Test results show that the effect of the microbial agent obtained in the example 6 on controlling root rot of salvia miltiorrhiza can reach 67.3%.

(2) Pot experiment 2: antagonistic action of strain DS-R5 liquid microbial inoculum on pathogenic fusarium solani of salvia miltiorrhiza root rot

Transplanting potted red sage seedlings with consistent growth into flowerpots with the diameter of 30cm, and carrying out fusarium solani root irrigation treatment in different groups. And (3) an empty control CK: respectively added at a concentration of 1 × 10⁶CFU/mL fusarium solani spore suspension and 20mL fermentation medium of missed DS-R5 strain; blank CK 1: respectively added at a concentration of 1 × 10⁶20mL of CFU/mL fusarium solani spore suspension and 20mL of inactivated DS-R5 strain fermentation culture solution; treatment group: respectively added at a concentration of 1 × 10⁶CFU/mL of Fusarium solani spore suspension and 20mL of fermentation culture solution of DS-R5 strain. The inhibition effect of the DS-R5 strain on Fusarium solani spores is monitored every 10 days after the strain addition treatment by adopting a real-time fluorescent quantitative PCR technology. The test results are shown in Table 4, and the real-time fluorescence quantitative PCR standard curve of Fusarium solani is shown in figure 12. As can be seen from Table 4, at 10d, 20d and 30d of the added bacteria treatment, the copy number of Fusarium solani in the control group CK showed a trend of descending and then ascending, and the number of Fusarium solani in the rhizosphere soil of Salvia miltiorrhiza at the time of sampling at 10d was 27.3X 10⁵copies/g, 20d down to 8.4X 10⁵copies/g, 30d, again up to 18.4X 10⁵copies/g. Compared with CK1, the change rule of the quantity of fusarium solani of the treatment group is the same, and the change rule is the trend of descending all the time; the number of Fusarium solani in CK1 and treatment groups at 10d was 20.7X 10⁵copiesG and 15.1X 10⁵The ratio of copies/g is respectively reduced to 6.3X 10 at 20d⁵copies/g and 2.2X 10⁵The Fusarium solani treated at 30 days of copies/g is only 3X 10³copies/g, much lower than 1.9X 10 of CK1⁴copies/g. The number change of the fusarium solani in the treated group fully illustrates the inhibition effect of the DS-R5 strain on the fusarium solani, and simultaneously proves that the DS-R5 strain can be stably colonized at the rhizosphere of the salvia miltiorrhiza.

TABLE 4 real-time fluorescent quantitation PCR monitoring of Fusarium solani number

(3) Pot experiment 3: growth promoting effect of paenibacillus polymyxa DS-R5 microbial inoculum on salvia miltiorrhiza

In order to verify the growth promotion effect of the strain DS-R5 as a microbial fertilizer in the planting production of the salvia miltiorrhiza, a salvia miltiorrhiza potting test is carried out. Transplanting the salvia miltiorrhiza seedlings with uniform growth into a flowerpot with the diameter of 30cm, and carrying out the following experimental design: the control group is prepared by inoculating inactivated strain DS-R5 liquid microbial inoculum into irrigated roots, and the concentration is 20 mL/basin; the treatment group is to irrigate roots and inoculate cultured strain DS-R5 liquid microbial inoculum with 20 mL/basin. After the salvia miltiorrhiza seedlings grow for 3 months, the salvia miltiorrhiza seedlings of the treatment group and the control group are taken out from the flower pots, 10 salvia miltiorrhiza seedlings are randomly selected respectively, soil samples attached to the salvia miltiorrhiza rhizosphere are carefully shaken off, the salvia miltiorrhiza rhizosphere is washed clean by tap water, growth indexes such as the root length, the salvia miltiorrhiza tissue dry weight, the fresh weight and the like of the salvia miltiorrhiza are measured after the salvia miltiorrhiza seedlings are dried, and test results are shown in table 5. The results show that compared with the control group, the wheat root system of the treatment group is long and has more branches, the average length of the root system is 23.6cm, the average fresh weight of the salvia miltiorrhiza seedlings is 1.55g, and the average dry weight is 0.57 g; the average root length of the salvia miltiorrhiza seedlings in the control group is 19.8cm, the average fresh weight of the salvia miltiorrhiza seedlings is 1.02g, and the average dry weight is 0.42 g. Data analysis results show that the 3 growth indicators of the treatment group and the control group, such as root length, average fresh weight of root tissues, average dry weight of root tissues and the like, have significant differences.

TABLE 5 measurement results of Salvia miltiorrhiza seedling growth index

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong first medical university (Shandong province medical science institute)

<120> Paenibacillus polymyxa and preparation and application of microbial inoculum thereof

<130> 2021

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1517

<212> DNA

<213> Strain DS-R5

<400> 1

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

ggggttaatt agaagcttgc ttctaattaa cctagcggcg gacgggtgag taacacgtag 120

gcaacctgcc cataagacag ggataactac cggaaacggt agctaatacc tgatacatcc 180

ttttcctgca tgggtgaagg aggaaaggcg gagcaatctg tcacttgtgg atgggcctgc 240

ggcgcattag ctagttggtg gggtaaaggc ctaccaaggc gacgatgcgt agccgacctg 300

agagggtgat cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag 360

tagggaatct tccgcaatgg gcgaaagcct gacggagcaa cgccgcgtga gtgatgaagg 420

ttttcggatc gtaaagctct gttgccaggg aagaacgtct tgtagagtaa ctgctacaag 480

agtgacggta cctgagaaga aagccccggc taactacgtg ccagcagccg cggtaatacg 540

tagggggcaa gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gctctttaag 600

tctggtgttt aatcccgagg ctcaacttcg ggtcgcactg gaaactgggg agcttgagtg 660

cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca 720

ccagtggcga aggcgactct ctgggctgta actgacgctg aggcgcgaaa gcgtggggag 780

caaacaggat tagataccct ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg 840

gtttcgatac ccttggtgcc gaagttaaca cattaagcat tccgcctggg gagtacggtc 900

gcaagactga aactcaaagg aattgacggg gacccgcaca agcagtggag tatgtggttt 960

aattcgaagc aacgcgaaga accttaccag gtcttgacat ccctctgacc gctgtagaga 1020

tatggctttc cttcgggaca gaggagacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080

tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta tgcttagttg ccagcaggtc 1140

aagctgggca ctctaagcag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200

aatcatcatg ccccttatga cctgggctac acacgtacta caatggccgg tacaacggga 1260

agcgaagccg cgaggtggag cgaatcctag aaaagccggt ctcagttcgg attgcaggct 1320

gcaactcgcc tgcatgaagt cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa 1380

tacgttcccg ggtcttgtac acaccgcccg tcacaccacg agagtttaca acacccgaag 1440

tcggtgaggt aaccgcaagg ggccagccgc cgaaggtggg gtagatgatt ggggtgaagt 1500

cgtaacaagg tagccgt 1517

Claims

1. A strain of Paenibacillus polymyxa (Paenibacillus polymyxa) DS-R5 is provided with the biological deposit number: CCTCC NO: m2021005.

2. A microbial agent characterized by comprising the Bacillus polymyxa (Paenibacillus polymyxa) DS-R5 of claim 1 as an active ingredient.

3. The microbial agent according to claim 2, wherein the microbial agent is a liquid microbial agent or a solid microbial agent.

4. The method for preparing a microbial agent according to claim 2 or 3, comprising the steps of:

5. The method according to claim 4, wherein the fermentation temperature in the secondary seed liquid preparation is 37 ℃ and the aeration rate is 3m³The tank pressure is maintained at 0.05 MPa; in the preparation of liquid microbial inoculum, the fermentation temperature is 37 ℃, and the ventilation volume is 30m³The pot pressure was maintained at 0.05 MPa.

6. The method of claim 4, wherein the fermentation medium formulation is: glucose 10.0g, peptone 20.0g, (NH)₄)₂SO₄ 2.0g，KH₂PO₄ 0.5g，MgSO₄1.0g, NaCl 1.5g and distilled water 1L; sterilizing at 121 deg.C under 0.1MPa for 20 min.

7. The use method of the microbial inoculum according to claim 2 or 3, characterized in that the liquid microbial inoculum and water are mixed evenly according to the proportion of 1:100 and applied by root irrigation; the solid microbial inoculum is applied by root irrigation according to the dosage of 5 kg/mu.

8. Use of the Paenibacillus polymyxa (Paenibacillus polymyxa) DS-R5 of claim 1 or the microbial agent of any one of claims 2-3 in at least one of the following (1) to (4):

(1) inhibiting phytopathogens;

(2) preparing a product for inhibiting phytopathogens;

(4) preparing a product for preventing and treating root rot of medicinal plants caused by plant pathogenic bacteria;

in the above application of (1) to (4), the plant pathogenic bacteria is fungi; the fungus is Fusarium solani (F.) (F. solani) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)RSolani), alternaria alternata (A. alternata) Fusarium graminearum (F.graminearum)F. graminearum) Fusarium oxysporum (F.), (F. oxysporum) Fusarium pseudograminearum (F.graminearum)F. pseudograminearum) Anthrax bacteria (b), anthrax bacteriaC. orbiculare) Staphylococcus aureus (Haptospira interrogans)B. ribis) Or Candida albicans (Candida alibicas).

9. The use of claim 8, wherein the medicinal plant is Salvia miltiorrhiza, Polygonatum sibiricum, Astragalus membranaceus, or Isatis indigotica root.

10. The use according to claim 9, wherein the medicinal plant is salvia miltiorrhiza.