CN113151117A

CN113151117A - Paenibacillus polymyxa for preventing and treating paris polyphylla gray mold and application thereof

Info

Publication number: CN113151117A
Application number: CN202110612244.5A
Authority: CN
Inventors: 游景茂; 唐涛; 王帆帆; 段媛媛; 郭杰; 郭晓亮
Original assignee: INSTITUTE OF CHINESE HERBAL MEDICINES HUBEI ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: INSTITUTE OF CHINESE HERBAL MEDICINES HUBEI ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2021-07-23

Abstract

The invention belongs to the technical field of plant disease and insect pest control, and particularly relates to paenibacillus polymyxa for controlling paris polyphylla gray mold and application thereof. The paenibacillus polymyxa provided by the invention is from the interior of the paris polyphylla stems, and has good compatibility with a paris polyphylla self circulation system; the microbial inoculum is simple to prepare, low in cost, non-toxic, harmless and free of environmental pollution, is beneficial to safely and effectively controlling the gray mold of the paris polyphylla, improves the yield and the quality of the paris polyphylla, is beneficial to green cultivation of the paris polyphylla, protects a farmland ecosystem and promotes the sustainable development of the paris polyphylla industry. The microbial inoculum is beneficial to improving the planting soil of the paris polyphylla and increasing the diversity of microorganisms in the soil.

Description

Paenibacillus polymyxa for preventing and treating paris polyphylla gray mold and application thereof

Technical Field

The invention belongs to the technical field of plant disease and insect pest control, and particularly relates to paenibacillus polymyxa for controlling paris polyphylla gray mold and application thereof.

Background

Paris polyphylla (Paris polyphylla) is a plant of Paris of Liliaceae, also known as Paris polyphylla, widely distributed in Sichuan, Yunnan, Guizhou, Hunan, Hubei and other places in China, has the effects of inhibiting bacteria, clearing heat and detoxicating, reducing swelling and relieving pain and the like, and can be clinically used for calming and relieving pain, treating venomous snake bite, treating convulsion and tetany, resisting cancer, protecting cardiovascular system and the like. In addition, rhizoma paridis is the main raw material of Chinese patent medicines such as Yunnan white drug powder, GONGXUENING, Qudesheng snake drug, etc., and the main active ingredients include pariphyllin, dioscin, steroid and alkaloid, etc. Due to slow growth of the paris polyphylla, and the fact that people abused and disorderly dig for a long time, wild paris polyphylla resources are exhausted and cannot meet market demands, market price of the paris polyphylla rises year by year, and the benefit per mu is nearly 20 ten thousand yuan in recent years. With the attention of governments at all levels to the biological medicine and health industry, the traditional Chinese medicine paris polyphylla becomes the main industry of the genuine production area because of various efficacies. In recent years, the artificial planting area of the paris polyphylla is enlarged year by year, the problem of diseases is obvious, and the gray mold of the paris polyphylla (Botrytis cinerea) in Hubei, Hunan, Sichuan and the like shows an outbreak trend, wherein the average incidence rate of diseases of various bases in Hubei province reaches about 55 percent, the serious disease can reach 100 percent, the humidity of the paris polyphylla in each year is 3-5 months, the field humidity of the paris polyphylla is high, and the temperature is proper, so that the period of the most serious gray mold of the paris polyphylla is provided.

The prevention and control of the gray mold are mainly chemical prevention and control at present, and for example, fenhexamid, fludioxonil and the like are common agents for preventing and controlling the gray mold. Because the registered traditional Chinese medicinal materials are few in medicament and the phenomenon of no medicament is serious, the frequent use of the pesticide is caused. The abuse of pesticide and the long-term use of a single agent in the actual production cause the problems of pesticide residue exceeding the standard, environmental pollution and drug resistance of pathogenic bacteria to be prominent, and seriously threaten the quality and ecological environment of the paris polyphylla.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a paenibacillus polymyxa for preventing and treating paris polyphylla gray mold and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention is realized in such a way that a paenibacillus polymyxa strain for preventing and treating paris polyphylla gray mold is preserved in a China center for type culture collection in 2017, 9 months and 20 days, and the preservation number is as follows: CCTCC NO: m2017521, name Paenibacillus polymyxa PJ10(Paenibacillus polymyxa PJ 10). Address: wuhan university in Wuhan, China.

The invention also provides application of the paenibacillus polymyxa for preventing and treating the gray mold of the paris polyphylla in preparing a gray mold preventing and treating microbial inoculum with rifampicin resistance or a rifampicin resistance preparation.

The invention also provides a gray mold control microbial inoculum with rifampicin resistance, which comprises the paenibacillus polymyxa.

The invention also provides a preparation method of the grey mould control microbial inoculum with rifampicin resistance, which comprises the steps of inoculating paenibacillus polymyxa in a PDB liquid culture medium, and carrying out aerobic fermentation at 28 ℃ to obtain bacteriostatic fermentation liquor.

Further, the method comprises the steps of centrifuging the bacteriostatic fermentation liquor, collecting supernatant, filtering with a filter membrane, and mixing the filtrate with PDA.

Further, the preparation method comprises the steps of coating paenibacillus polymyxa on a PDA culture medium for activation; inoculating the activated paenibacillus polymyxa into a PDB liquid culture medium for aerobic fermentation to prepare a fermentation seed solution; then inoculating the fermentation seed liquid into a PDB liquid culture medium for aerobic fermentation to obtain the bacteriostatic fermentation liquid.

Furthermore, the concentration of viable bacteria in the bacteriostatic fermentation liquor reaches 1 × 10⁸CFU/ml above.

Furthermore, the concentration of viable bacteria in the bacteriostatic fermentation liquor reaches 1 × 10¹⁰CFU/ml above.

The invention also provides application of the paenibacillus polymyxa in preventing and treating paris polyphylla gray mold.

The invention also provides application of the paenibacillus polymyxa in regulating and controlling the yield of the paris polyphylla and/or the bacterial community structure of paris polyphylla leaves.

Further, the rhizoma paridis yield includes at least one of fresh weight of single plant root, rhizoma paridis saponin I content and rhizoma paridis saponin II content.

The biological control has good popularization value and ecological benefit for preventing and treating the gray mold of the paris polyphylla, and no report is found in the research of preventing and treating the gray mold of the paris polyphylla by utilizing Paenibacillus polymyxa at home and abroad at present. The strain provided by the invention can be used for preventing and treating the gray mold of the paris polyphylla and has rifampicin resistance. The strain is verified to have obvious inhibition effect on the gray mold hyphae of the paris polyphylla, and can cause the gray mold hyphae of the paris polyphylla to be expanded and deformed, deficient and protoplasm to leak. When the concentration of the strain reaches 1X 10¹⁰And when CFU/mL is adopted, the control effect reaches 78.08 percent.

In summary, the advantages and positive effects of the invention are:

1. the target strain is from the interior of the paris polyphylla stems, and has good compatibility with a paris polyphylla self-circulation system.

2. The microbial inoculum is simple to prepare, low in cost, non-toxic, harmless and free of environmental pollution, and is beneficial to safely and effectively controlling the gray mold of the paris polyphylla.

3. The microbial inoculum is beneficial to increasing the microbial diversity in the leaves of the paris polyphylla.

4. The microbial inoculum is a biological preparation, has no defects of chemical pesticides, can not cause pathogenic bacteria to generate drug resistance, a grower can not use or reduce the use frequency of other chemical pesticides, and the biocontrol strain preparation has the functions of increasing yield and efficiency and can increase income of farmers.

The plate antagonism experiment and the antagonism experiment of fermentation supernatant with different concentrations show that: the target strain has an obvious inhibiting effect on the botrytis cinerea hyphae, and can cause the botrytis cinerea hyphae to expand and deform, be exhausted and cause protoplasm leakage. The field control experiment proves that when the concentration of the thallus reaches 1 multiplied by 10, the target microbial inoculum is on the overground part of the sprayed plant¹⁰The control effect of CFU/mL reaches 78.08%, compared with control, the tuber yield of rhizoma paridis can be increased by about 2.75%, and the control can be increased by 1.38% respectively for rhizoma paridis saponin I and rhizoma paridis saponin IIAnd 1.10%.

Drawings

FIG. 1 shows the effect of target bacteria on the growth of gray mold hyphae of Paris polyphylla; in the figure: the left is a confrontation graph of target bacteria and paris polyphylla botrytis; the right part is the comparison of Botrytis cinerea.

FIG. 2 is the effect of target bacteria on Gray mold bacteria hyphae during co-cultivation; in the figure: a is the hypha form of botrytis cinerea in the opposite area of co-culture; b is a comparison;

FIG. 3 is a graph showing the effect of different concentrations of target bacteria fermentation filtrates on the growth of Gray mold bacteria;

FIG. 4 shows the control effect of target bacteria for field application;

FIG. 5 is the colonization of the leaves by the target bacteria;

FIG. 6 is a table of microbial diversity indices for different treatment populations;

FIG. 7 is a statistical table of populations of different treated microorganisms;

FIG. 8 is a heat map of the genus Paris polyphylla leaf bacteria classification.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.

The invention discloses a paenibacillus polymyxa for preventing and treating paris polyphylla gray mold and application thereof. The details are shown in the following examples.

Example 1 isolation, screening and identification of target strains

Separating target strain from the stem of Paris polyphylla in Enshi city of Hubei province, taking the stem of healthy Paris polyphylla, sterilizing with 75% alcohol for 1min, washing with sterile water for 3 times, air drying under sterile condition, removing epidermis, grinding with sterile water, absorbing mixed solution, diluting, coating on PDA plate, culturing at 28 deg.C for 72 hr, and selecting single colony for purification and culture. And 3cm away from botrytis cinerea, placing the separated endophytes of the stems of the rhizoma paridis according to a cross method, after 3 days, measuring the distance from the outer edge of a bacterial colony to botrytis cinerea hyphae in a full dish, repeating for 3 times, and selecting the bacteria with the largest distance for later research.

The target strain is gram-positive bacteria, the strain is cultured on a solid PDA culture medium, the colony color is white, the periphery of the colony is higher than the middle part of the colony, the colony is glossy, and gram staining is positive; the bacterium produces spores and is oval; the smell is fragrant and presents the taste of roasted sweet potatoes. The organic phosphorus-removing agent has the capability of removing organic phosphorus, and can generate IAA, and the experiments of methyl red, anaerobic growth, carbon source utilization, hydrogen sulfide, citrate, catalase, starch hydrolysis and gelatin liquefaction show positive. Growth was not observed at a NaCl concentration of 5%.

Taking 20 mu L of fermentation liquor of a target strain to be coated on a PDA plate containing rifampicin at 10 mu g/mL, culturing for 2d at 28 ℃, picking a single colony, inoculating the single colony into 100mL of PDB culture medium containing rifampicin at 10 mu g/mL, culturing for 2d at 28 ℃, 200r/min, picking the single colony, transferring the single colony to the next concentration culture, sequentially inducing in PDB culture media containing rifampicin at 20, 40, 80, 160, 200 and 300 mu g/mL until a mutant strain resisting rifampicin at 300 mu g/mL is screened, continuously passaging the rifampicin strain on the PDB culture medium without antibiotics for 5 times, and then inoculating the rifampicin strain into the rifampicin-containing culture medium for detection to ensure the genetic stability of the drug resistance. The bacterial colony morphology, physiological and biochemical characteristics and antagonistic action on pathogenic bacteria of the strain are kept stable.

The strain is identified as Paenibacillus polymyxa PJ10(Paenibacillus polymyxa PJ10) by physiological, biochemical and sequencing analysis. The strain is preserved in the China center for type culture Collection in 2017, 9 and 20 months, and the preservation number is as follows: CCTCC NO: and M2017521. Address: wuhan university in Wuhan, China.

Example 2 preparation of microbial inoculum

S1, taking a loop of the endophytic bacterium paenibacillus polymyxa by using an inoculating loop, then spreading the loop on a PDA culture medium, and culturing for 72h at 28 ℃. The formula of the PDA solid culture medium is as follows: sterilizing potato 200 g, glucose 20 g, agar 15-20 g, distilled water 1000 ml, pH 7.2-7.4 with high pressure steam at 121 deg.C for 30 min.

S2, transferring the activated single colony obtained in the step S1 into a 250ml conical flask containing 100ml of PDB liquid culture medium, and culturing at 200rmp and 28 ℃ for 72 hours to obtain a fermented seed solution. The PDB liquid culture medium formula is as follows: taking 200 g of potato, 20 g of glucose and 1000 ml of distilled water, and sterilizing with high-pressure steam at 121 ℃ for 30min at pH 7.2-7.4 to obtain the potato food.

S3, inoculating the fermentation seed liquid in the step S2 into a conical flask containing 100ml of PDB according to the ratio of 1:150(V: V), placing the conical flask into a vibrator, fermenting at 28 ℃ and 200rmp for 72 hours to ensure that the concentration of viable bacteria reaches 1 x 10¹⁰CFU/ml or above, and then after productionThe microbial inoculum is stored in a refrigerator at 4 ℃.

Plate confrontation antagonism test

According to a plate confronting culture test method in the formula of 'plant disease research method', the inhibition effect of target bacteria on botrytis cinerea is evaluated, and meanwhile, the bacteria hyphae of a sterilized distilled water control treatment group are counted when the plates are full of bacteria hyphae, and the inhibition rate (%) is (the diameter of the control group pathogenic bacteria colony-the diameter of the treatment group pathogenic bacteria colony)/the diameter of the control group pathogenic bacteria colony is multiplied by 100%.

The result proves that the hypha growth inhibition rate of the target strain on the botrytis cinerea is 76.47% (shown in figure 1).

Microscopic observation of botrytis cinerea hyphae in confrontation area

And observing the hypha form of the botrytis cinerea by using a microscope in a confronting area of the target strain and the botrytis cinerea hypha. The results showed slow growth of hyphae, thin hyphae, malformation, and leakage of protoplasts of Gray mold of Paris polyphylla in the confronting areas (as shown in FIG. 2).

Example 3 Effect of fermentation supernatant of target Strain on Gray mold of Paris polyphylla

And (3) injecting 500 mu L of activated target bacterial liquid into 100mL of PDB, and culturing at 28 ℃ and 180r/min for 72h to obtain a fermentation stock solution. Centrifuging the fermentation stock solution at 12000r/min for 10min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain fermentation filtrate. Mixing the filtrate with PDA at about 45 deg.C to give final concentration of 5% and 10%, cooling, inoculating Botrytis cinerea mycelium block (diameter 0.5cm) at the center of the plate, setting blank control, repeating the treatment three times, and culturing at 21 deg.C in incubator. After the control was plated over (4 days), the diameter of the colony was measured and the inhibition rate was calculated. The inhibition ratio (%) - (control group pathogen colony diameter-treatment group pathogen colony diameter)/control group pathogen colony diameter × 100%.

The results prove that the fermentation filtrate of the target strain can obviously inhibit the growth of botrytis cinerea at both concentrations of 5% and 10%, the inhibition rates of the fermentation filtrate and the target strain are respectively 29.41% and 47.06%, and the inhibition rates of the fermentation filtrate and the target strain are obviously different (as shown in figure 3).

Example 4 field control of the target Strain against Gray mold of Paris polyphylla

The test point is arranged in a Paris polyphylla resource garden in a new pond, countryside and China medicinal plant garden in Enshi City of Hubei province. At an altitude of 1600m, gray mold occurs in the last year, the separated pathogenic bacteria are mainly botrytis cinerea, all operations are consistent in the planting process, other chemical agents are not used, and the paris polyphylla is a 4-year-old plant and can normally bloom and bear fruits. The test time is 3 months and 10 days in 2018, the weather is sunny, and the rate of emergence of the paris polyphylla is 95 percent.

Diluting the fermentation stock solution of the target bacteria to 1 × 10¹⁰CFU/mL、1×10⁹CFU/mL and 1X 10⁸CFU/mL is reserved, 1000 times of liquid of 50% iprodione wettable powder serving as a chemical agent, 800 times of liquid of 1000 hundred million spores/gram bacillus subtilis wettable powder serving as a biological agent serve as positive control, and clear water serves as negative control. Spraying the whole plant and the soil surface according to the recommended concentration of each medicament at 9 noon of 2018, 3-month 10, 3-month 16, 3-month 22 and 3-month 28, and continuously applying for 4 times, wherein no rain exists during the application period and 12 hours after the application. And investigating the influence of the treatment on the paris polyphylla in 2018, 4 and 17 days, sampling five points in each cell, taking 5 plants in each point, counting the fresh weight of rhizomes and the contents of paris polyphylla saponin I and paris polyphylla saponin II, and calculating the average weight and content of the rhizomes. And calculating the control effect of each treatment according to the grading standard. Grading standard: grade 0, no disease; grade 1, the affected area accounts for less than 25% of the total area of the fruit; grade 3, the affected area accounts for 25-50% of the total area of the fruit; grade 5, the affected area accounts for 50-75% of the total area of the fruit; grade 7, the affected area accounts for more than 75% of the total area of the fruit (Zhang J et al, Mycoscience,2010,51(6): 421.). The disease index is 100 × Σ (number of diseased plants at each stage × representative value at each stage)/(total number of plants × highest representative value at each stage); control effect (%) < 100 × (control disease index-treatment disease index)/control disease index.

The experimental result proves that at 10 days after the drug administration, the concentration of the endophytic target strain in the paris polyphylla is 1 multiplied by 10 in three concentrations¹⁰The control effect of CFU/mL treatment reaches 78.08 percent and is 1 multiplied by 10⁹The treatment control effect of CFU/mL is 70.00 percent, 1 is multiplied by 10⁸The treatment control effect of CFU/mL is 63.08% (shown in figure 4), wherein the control effect of the commercial microbial agent Bacillus subtilis is 59.04%. The control effect of the chemical agent iprodione in all treatments is the highest and reaches 86.73 percent. Of the target strains in the influence of the respective treatments on the rhizome weight of Paris polyphylla1×10¹⁰The fresh weight of CFU/mL treatment was 18.13g at the highest, followed by 1X 10⁹The fresh weight of the CFU/mL treatment is 18.05g, and the yield is increased by 2.75 percent and 2.31 percent respectively compared with the control. In each treatment content detection, 1X 10 of the target strain¹⁰The content of the rhizoma paridis saponin I and the rhizoma paridis saponin II is 0.1815% and 0.4339% respectively by CFU/mL treatment, which are higher than those of the control treatment, the content of the rhizoma paridis saponin I and the content of the rhizoma paridis saponin II are 1.38% and 1.10% respectively higher than that of the control treatment, and the difference reaches a very significant level, namely 1 multiplied by 10 of the target strain⁹In the CFU/mL treatment, the contents of the paris polyphylla saponin I and the paris polyphylla saponin II are respectively 0.1792% and 0.4311%, which are also higher than a control, but the difference is not very significant. The yield can be improved to a certain extent after the target microbial inoculum is applied, and the contents of the rhizoma paridis saponin I and the rhizoma paridis saponin II are improved. Specific experimental data are shown in table 1.

TABLE 1 field control effect of different treatments on gray mold of Paris polyphylla

In the table, different lower case letters in the same column of data indicate that the difference is significant and P is less than 0.05

Example 5 colonization of the leaves by the target Strain

3.10 and 3.16 of 2018, 1X 10¹⁰CFU/mL resistant target strain fermentation liquor is uniformly sprayed on leaves of the paris polyphylla for 2 times, the sprayed leaves are taken after inoculation for 1, 3, 5, 7, 10, 15, 20, 30, 40, 50 and 60d respectively, each sample is washed in sterile water for 6 times, 0.5g of the sample is taken, sterilized by 75% alcohol for 30S, sterilized by 1% sodium hypochlorite for 3min, washed by sterile water for 5 times, dried in an aseptic operation table, ground by adding 1mL of water into an aseptic mortar, stood for 10min, 100 mu L of supernatant is sucked, diluted by 10, 100 and 1000 times and then sequentially coated on an NA plate of 300 mu g/mL rifampicin, the steps are repeated for 3 times, the culture is carried out at 28 ℃ for 48h, the number of colonies is counted, and the number of bacteria per gram of tissue (CFU/g) is calculated according to the number of the colonies appearing on each dish. Meanwhile, randomly picking out the grown bacterial colony for morphological, physiological and biochemical analysis and 16SrDNA identification, and confirming that the bacterial colony is Paenibacillus polymyxa (Paenibacillu)s polymyxa)。

Through statistics, the target strain can still be detected on the leaves of the paris polyphylla till 60d, a colonization peak appears 7 days after spraying, and the bacterial quantity reaches 4.33 multiplied by 10⁵CFU/g, followed by a downward trend, about 1X 10 leaf bacterial load at day 60³CFU/g, it can be seen that the target strain can be well colonized on the leaves of the Paris polyphylla. (see FIG. 5 for a detailed profile).

Example 6 Effect of target strains on the bacterial colony Structure of Paris polyphylla leaves

Fermenting the target strain with 1 × 10¹⁰ Sampling 10 days after CFU/mL spraying of paris polyphylla leaves, and detecting the bacterial flora structure of the leaves by using an Illumina MiSeq sequencing platform in 2017 in 5 months. The sequencing results demonstrated that the Chao1 index and Shannon index of the paris polyphylla leaves after the target bacteria were used was 3 times higher than the control (shown in figure 6). Meanwhile, after the target strains are treated, the levels of phyla, class, order, family, genus and the like are higher than those of a blank control, other index controls are relatively higher (shown in figure 7), and heat map analysis is carried out on the levels of the genera aiming at different treatments, so that the treatment of the target strains of Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Brucella is higher than that of the clear water control treatment. In the displayed heat map of the first 50 genera of bacteria, the target strains were 32 dominant, accounting for 64%, and it was seen that the target strains had the property of promoting the population of certain bacteria (FIG. 8).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The paenibacillus polymyxa for preventing and treating the gray mold of the paris polyphylla has the following preservation number: CCTCC NO: m2017521, name Paenibacillus polymyxa PJ10(Paenibacillus polymyxa PJ 10).

2. The use of the paenibacillus polymyxa for controlling gray mold of paris polyphylla as claimed in claim 1 in the preparation of a gray mold control bacterial agent with rifampicin resistance or a rifampicin resistance preparation.

3. A gray mold control microbial inoculum with rifampicin resistance is characterized in that: comprising the Paenibacillus polymyxa as described in claim 1.

4. The method for preparing a gray mold control bacterial agent with rifampicin resistance as claimed in claim 3, characterized by: comprises inoculating Paenibacillus polymyxa in a PDB liquid culture medium, and aerobically fermenting at 28 deg.C to obtain antibacterial fermentation liquid.

5. The method for preparing a gray mold control bacterial agent with rifampicin resistance as claimed in claim 4, further comprising the steps of centrifuging bacteriostatic fermentation broth, collecting supernatant, filtering with filter membrane, and mixing filtrate with PDA.

6. The method for preparing a gray mold control bacterial agent with rifampicin resistance as claimed in claim 4, wherein the said method comprises activating Paenibacillus polymyxa by spreading it on PDA medium; inoculating the activated paenibacillus polymyxa into a PDB liquid culture medium for aerobic fermentation to prepare a fermentation seed solution; then inoculating the fermentation seed liquid into a PDB liquid culture medium for aerobic fermentation to obtain the bacteriostatic fermentation liquid.

7. The method for preparing a gray mold control bacterial agent with rifampicin resistance as claimed in claim 4, wherein the viable bacteria concentration in the bacteriostatic fermentation broth reaches 1 x 10⁸CFU/ml above.

8. The method for preparing a gray mold control bacterial agent with rifampicin resistance as claimed in claim 7, wherein the viable bacteria concentration in the bacteriostatic fermentation broth reaches 1 x 10¹⁰CFU/ml above.

9. Use of paenibacillus polymyxa as defined in claim 1 for the control of paris polyphylla gray mold.

10. Use of paenibacillus polymyxa as defined in claim 1 for regulating the production of paris polyphylla and/or the bacterial community structure of paris polyphylla leaves.