A polymyxa bacillus agent, preparation method and application thereof

Abstract

The present invention proposes a polymyxa bacillus bacterial agent, a preparation method and an application thereof, and relates to the field of microbial technology. The polymyxa bacillus Cs12 can effectively inhibit the growth of Fusarium oxysporum, thereby fundamentally reducing the incidence of saffron corm rot; in addition, the present invention also proposes an application of polymyxa bacillus in the preparation of a drug for preventing and treating saffron corm rot, and the bacterial agent prepared by the polymyxa bacillus can effectively inhibit the growth of Fusarium oxysporum, thereby reducing the incidence of saffron corm rot, and has an obvious prevention and treatment effect on saffron corm rot, and does not produce problems such as pesticide residues and environmental pollution.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Paenibacillus polymyxa bacterial agent, a preparation method and application thereof. Background Art

[0002] Saffron (Crocus sativus L.), also known as saffron, is only used as medicine for the three red stigmas on the upper part of the pistil. It has a low yield and is known as "red gold". It has the effects of promoting blood circulation and removing blood stasis, cooling blood and detoxifying, relieving depression and calming the mind. It is a traditional precious Chinese medicinal material and the most precious spice and dye in the world. Saffron is an allotriploid, which only blooms but does not produce seeds. It can only be produced by asexual reproduction of bulbs. Therefore, the yield and quality of bulbs directly affect the yield and quality of saffron. Saffron originated in the Mediterranean region. Iran is now the main production area, and its output accounts for more than 90% of the world's total output. my country successfully introduced it in Chongming Island, Shanghai in 1986. At present, Zhejiang is the main production area, with a planting area of ​​about 6,000 mu, accounting for more than 50% of the country. In 2018, saffron was rated as the new "Zhejiang Eight Flavors". However, unlike the Mediterranean climate in the main producing areas, the summer in Jiangsu and Zhejiang in my country is warm and humid, and the underground bulbs are easily infected by pathogens and become sick and rotten. Therefore, when the new bulbs mature in early May, they are dug out and placed indoors for half a year until they bloom. After the indoor flowers are harvested in December, the bulbs are propagated in the fields in a "two-stage" cultivation method. However, even during the indoor period, saffron rot disease still often occurs. The average annual reproduction rate of saffron bulbs is only 1.2. In the more than 30 years since the introduction of the species, the planting area in Zhejiang is only about 4,000 acres. The prevention and control of saffron bulb rot disease is a bottleneck in the development of my country's saffron planting industry.

[0003] Therefore, bulb rot has become the most critical destructive factor hindering the development of the saffron industry. Bulb rot is mainly caused by a soil-borne pathogenic fungus, Fusarium oxysporum (F.oxysporum), which uses soil as a transmission medium, infects bulbs from the roots, consumes host nutrients, and causes plants to wilt and die. It can occur in all cultivation periods, especially in late June to late August when the temperature is high in summer. The incidence rate can be as high as 50% in high-incidence years in Jiande, Zhejiang, the main production area in my country, which seriously hinders the development of domestic saffron planting industry. Therefore, 80% of domestic saffron still needs to be imported. At present, farmers mainly use chemical pesticides for prevention and control, but the effect is not good, and the harmful effects of these chemicals on the environment and humans have been fully confirmed, resulting in serious pesticide residues, changes in the physical and chemical properties of the soil, and the continuous enhancement of pathogen resistance, which will also affect the beneficial microbial community associated with plants.

[0004] Therefore, there is an urgent need to find new and effective methods to prevent and control saffron bulb rot, which is of great significance to promoting the healthy development of my country's ecological environment and saffron industry. Summary of the invention

[0005] The object of the present invention is to provide a polymyxa bacillus, which can effectively inhibit the growth of Fusarium oxysporum, thereby fundamentally reducing the incidence of saffron bulb rot.

[0006] The second object of the present invention is to provide a use of Paenibacillus polymyxa in the preparation of a medicament for preventing and treating saffron bulb rot. The Paenibacillus polymyxa can effectively inhibit the growth of Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot, has a significant effect on the prevention and treatment of saffron bulb rot, and does not produce pesticide residues, environmental pollution and other problems.

[0007] The third object of the present invention is to provide a bacterial agent comprising the polymyxa bacillus, which can effectively inhibit the growth of Fusarium oxysporum, thereby effectively reducing the incidence of saffron bulb rot caused by Fusarium oxysporum, and thus improving the survival rate of saffron.

[0008] The fourth purpose of the present invention is to provide a method for using a microbial agent, which specifies the amount and number of uses of the microbial agent each time, thereby ensuring that the microbial agent can successfully inhibit the growth and development of Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot.

[0009] The fifth object of the present invention is to provide a method for preparing a bacterial agent comprising the polymyxa bacillus, which method is simple and easy to operate and does not cause harm to the environment.

[0010] The present invention solves the technical problem by adopting the following technical solutions.

[0011] The embodiment of the present invention provides a polymyxa bacillus, named polymyxa bacillus Cs12, the deposit number of the polymyxa bacillus Cs12 is CGMCC No. 22612, the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is May 26, 2021. Soil microbial communities are of great significance to plant health. The healthy growth of plants is closely related to the balance of rhizosphere soil microbial communities, and the imbalance of rhizosphere soil microecological systems is the main cause of soil-borne diseases. Saffron is intercropped in the soil where grapes have been planted for 10 years. The developed grape root system is closely intertwined with the saffron root system. The underground root system of the grape introduces new microorganisms to the saffron root system, shaping the saffron rhizosphere microecology with unique intercropping advantages, which has a significant effect on the prevention and treatment of saffron bulb rot. The present invention utilizes the main pathogenic bacteria of saffron corm rot, Fusarium oxysporum, to efficiently antagonize and separate the rhizosphere soil microorganisms of saffron intercropped in a grape field, and obtains a bacterium with a significant antagonistic effect of Fusarium oxysporum, namely, Paenibacillus polymyxa Cs12. The Paenibacillus polymyxa is separated from the soil of grape-saffron intercropping by an efficient 96-well plate confrontation test and further separated and purified by in vivo disease resistance tests, etc. It can effectively inhibit the growth of Fusarium oxysporum, the pathogen causing saffron corm rot, thereby fundamentally reducing the incidence rate of saffron corm rot; and the Paenibacillus polymyxa Cs12 will not cause damage and harm to the soil and the environment, so that it can be used with confidence without worrying about pollution problems. Bacillus polymyxa exists in the form of spores, has good temperature resistance, stable quality, and can "treat bacteria with bacteria" with long-lasting efficacy. It can "act as both medicine and fertilizer" and is not only the most ideal agent for preventing and treating soil-borne diseases such as bacterial wilt, Fusarium wilt, root rot, and soft rot, but also has obvious effects in promoting growth, increasing yield, and improving quality. It does not have any drug damage or residue, and is the preferred microbial preparation for pollution-free and green production.

[0012] The embodiment of the present invention also provides an application of Paenibacillus polymyxa in the preparation of a drug for preventing and treating saffron bulb rot. The Paenibacillus polymyxa can effectively inhibit the growth of Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot, has a significant effect on the prevention and treatment of saffron bulb rot, and does not produce problems such as pesticide residues and environmental pollution.

[0013] The embodiment of the present invention also provides a bacterial agent prepared by the above-mentioned Bacillus polymyxa Cs12. The bacterial agent prepared by the Bacillus polymyxa Cs12 can be applied to the roots of saffron infected with Fusarium oxysporum by root irrigation or injection, thereby inhibiting the growth of Fusarium oxysporum in the roots of saffron, thereby reducing the incidence of saffron bulb rot, thereby improving the survival rate of saffron; and the bacterial agent is harmless and pollution-free, will not burden the soil and the environment, and is convenient for long-term use.

[0014] The present invention also provides a method for using the above-mentioned bacterial agent, comprising the following steps: applying the bacterial agent once every 3 days, 5 mL each time, for 21-30 days. The method of use limits the amount of the bacterial agent used each time and the number of uses to ensure that the bacterial agent can effectively inhibit Fusarium oxysporum.

[0015] The embodiment of the present invention also provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: under sterile conditions, picking a single colony of Paenibacillus polymyxa Cs12 and inoculating it in NA or PDA medium, dark culturing at 28-37°C and 75% RH for 24-48 hours to obtain a purified colony; under sterile conditions, picking a purified colony and inoculating it in NB or PDB medium, shaking and culturing at 28°C and 180rpm for 24 hours to obtain a seed bacteria; inoculating the seed bacteria in NB or PDB medium, fermenting at 28°C and 180rpm for 48 hours to form a fermentation liquid, and obtaining a bacterial agent. The method is simple and convenient to operate, and a single colony and a seed bacteria are obtained by separating and purifying the strain, and the bacterial liquid is obtained after the seed bacteria of the colony are fermented.

[0016] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:

[0017] 1. An embodiment of the present invention provides a polymyxa bacillus, which is named polymyxa bacillus Cs12, isolated from the soil of grape-saffron intercropping, and can effectively inhibit the growth and development of Fusarium oxysporum in the soil, thereby reducing the incidence of saffron bulb rot.

[0018] 2. An embodiment of the present invention also provides an application of Bacillus polymyxa in the preparation of a drug for preventing and treating saffron bulb rot. The Bacillus polymyxa can effectively inhibit the growth of Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot, has obvious preventive and control effects on saffron bulb rot, and does not cause problems such as pesticide residues and environmental pollution.

[0019] 3. An embodiment of the present invention further provides a bacterial agent, which is prepared from Bacillus polymyxa Cs12. The bacterial agent can be absorbed by the roots of saffron, thereby inhibiting the growth of Fusarium oxysporum in the roots of saffron. Since Fusarium oxysporum is the main pathogenic bacteria of saffron bulb rot, the incidence of saffron bulb rot can be reduced by inhibiting the growth of Fusarium oxysporum, which is of great significance to the development of saffron in my country.

[0020] 4. An embodiment of the present invention also provides a method for using a microbial agent, which ensures that the microbial agent can effectively and reasonably inhibit the growth and development of Fusarium oxysporum by using a reasonable dosage and method, thereby ensuring that the microbial agent has a significant inhibitory effect.

[0021] 5. An embodiment of the present invention also provides a method for preparing a microbial agent, which comprises isolating and purifying the polymyxa bacillus Cs12 to obtain a single colony, subsequently isolating and purifying the single colony to obtain a seed fungus, and finally fermenting the seed fungus to obtain the microbial agent. The preparation method is simple, convenient and easy to operate, and the prepared microbial agent has a good inhibitory effect on Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is a diagram showing the inhibitory effect of Paenibacillus polymyxa (P. polymyxa) Cs12 on Fusarium oxysporum, the pathogen of saffron bulb rot, in Example 1 of the present invention (A is Fusarium oxysporum, and B is the confrontation between Cs12 and F. oxysporum);

[0024] Figure 2 This is a gel electrophoresis diagram of Paenibacillus polymyxa Cs12 amplified based on 16SrDNA primers in Example 1 of the present invention;

[0025] Figure 3 The phylogenetic tree of Paenibacillus polymyxa Cs12 based on 16S rDNA obtained by the neighbor-joining method in Example 2 of the present invention;

[0026] Figure 4 This is a colony morphology diagram of Paenibacillus polymyxa (P. polymyxa) on NA solid culture medium in Example 2 of the present invention;

[0027] Figure 5 This is a graph showing the growth of the corms 28 days after administration of Fusarium oxysporum in Example 2 of the present invention (where A is the blank control group, B is the model group, and C is the probiotic group). DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0029] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0030] The embodiment of the present invention provides a polymyxa bacillus, named polymyxa bacillus Cs12, the deposit number of polymyxa bacillus Cs12 is CGMCC No. 22612, the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is May 26, 2021. Polymyxa bacillus has a strong colonization ability in plants such as roots, stems, and leaves, and can prevent pathogens from infecting plants through site competition; at the same time, the broad-spectrum antibacterial substances continuously secreted by polymyxa bacillus can inhibit or kill pathogens; in addition, polymyxa bacillus can also induce plants to produce disease resistance. Soil microbial communities are of great significance to plant health. The healthy growth of plants is closely related to the balance of rhizosphere soil microbial communities. The imbalance of the rhizosphere soil microecological system is the main cause of soil-borne diseases. Saffron is intercropped in the soil where grapes have been planted for 10 years. The developed grape root system is closely intertwined with the saffron root system. The underground root system of the grape introduces new microorganisms to the saffron root system, shaping the saffron rhizosphere microecology with unique intercropping advantages, which has a significant effect on the prevention and treatment of saffron bulb rot. The present invention uses the main pathogenic bacteria of saffron bulb rot, Fusarium oxysporum, to efficiently antagonize and separate the saffron rhizosphere soil microorganisms intercropped in the grape field, and obtains a bacterium with a significant anti-Fusarium oxysporum effect, namely, Paenibacillus polymyxa Cs12. The polymyxa bacillus Cs12 mainly colonizes in the roots of saffron, thereby preventing Fusarium oxysporum from infecting the saffron corm through site competition, thereby cutting off the nutrient source of Fusarium oxysporum, thereby inhibiting the growth of Fusarium oxysporum and reducing the incidence of saffron corm rot; and the polymyxa bacillus Cs12 will not pollute the soil and the environment, and is therefore convenient for long-term use.

[0031] In some embodiments of the present invention, the 16SrDNA sequence of the above-mentioned Paenibacillus polymyxa is shown as SEQ ID NO.1.

[0032] On the other hand, an embodiment of the present invention also provides a use of Paenibacillus polymyxa in the preparation of a drug for preventing and treating saffron bulb rot. Since the Paenibacillus polymyxa Cs12 can effectively inhibit the growth of Fusarium oxysporum, which is the main pathogenic bacteria of saffron bulb rot, the drug prepared using the strain can effectively reduce the incidence of saffron bulb rot by inhibiting the growth of Fusarium oxysporum.

[0033] On the other hand, an embodiment of the present invention further provides a bacterial agent, which is prepared from the above-mentioned Paenibacillus polymyxa Cs12. The bacterial agent prepared by using the Paenibacillus polymyxa Cs12 can be applied to the roots of saffron infected with Fusarium oxysporum, thereby inhibiting the growth of Fusarium oxysporum in the roots of saffron, thereby reducing the incidence of saffron bulb rot, and ultimately improving the survival rate of saffron; and the bacterial agent is pollution-free, pollution-free, does not burden the soil and the environment, and is convenient for long-term use.

[0034] In some embodiments of the present invention, the concentration of Bacillus polymyxa Cs12 in the above-mentioned bacterial agent is 10 7 -10 8 CFU / mL. This concentration range of Paenibacillus polymyxa Cs12 can ensure that the bacterial agent can effectively exert the control effect while avoiding the influence of Paenibacillus polymyxa Cs12 on the growth and development of saffron itself.

[0035] In some embodiments of the present invention, the above-mentioned bacterial agent is applied once every 3 days, 5 mL each time, for 21-30 days. This is the method of using the bacterial agent, and the application amount and number of applications are used to ensure that the polymyxa bacillus Cs12 can stably inhibit the growth of Fusarium oxysporum and avoid excessive accumulation of the polymyxa bacillus Cs12 in the roots of saffron to affect the growth and development of saffron itself.

[0036] In some embodiments of the present invention, the method of application is root irrigation or injection. The bacterial agent is used to irrigate the roots of saffron infected with saffron rot or directly inject the roots to inhibit the growth of Fusarium oxysporum in the infected plants, thereby reducing the incidence of saffron rot.

[0037] On the other hand, an embodiment of the present invention also provides a method for preparing the above-mentioned bacterial agent, which comprises the following steps: under sterile conditions, picking a single colony of Bacillus polymyxa Cs12 and inoculating it into a sterilized NA or PDA culture medium, and dark culturing it at 28-37°C and 75% RH for 24-48 hours to obtain a purified colony; under sterile conditions, picking a purified colony and inoculating it into a sterilized NB or PDB culture medium, and shaking and culturing it at 28°C and 180rpm for 24 hours to obtain a seed bacteria; inoculating the seed bacteria into the NB or PDB culture medium, and fermenting it at 28°C and 180rpm for 48 hours to form a fermentation liquid to obtain a bacterial agent. The polymyxa bacillus Cs12 is first cultured in NA medium for a period of time to obtain a purified single colony of the polymyxa bacillus Cs12, and the single colony is picked and inoculated into NB or PDB medium to obtain a seed culture medium for subsequent fermentation. Finally, the seed culture medium is inoculated into NB or PDB medium for fermentation, so that the polymyxa bacillus Cs12 can be multiplied in large quantities in the NB medium to obtain a bacterial agent.

[0038] In some embodiments of the present invention, the NA medium includes 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride and 20 g of agar, and the pH of the NA medium is 7.0-7.4; the NB medium includes 10 g of peptone, 3 g of beef extract and 5 g of sodium chloride, and the pH of the NB medium is 7.0-7.4. The PDA medium includes 200 g of potato decoction, 20 g of glucose and 15 g of agar; the PDB medium includes 200 g of potato decoction and 20 g of glucose, and the pH of the PDB medium is 7.0-7.4.

[0039] The above-mentioned raw materials are the formulas of the NA or PDA culture medium and the NB or PDB culture medium used, which provide basic nutrients and raw materials for the growth and metabolism of Bacillus polymyxa Cs12, which can be beneficial to the growth and metabolism of Bacillus polymyxa Cs12, so that the metabolic rate of Bacillus polymyxa Cs12 is at a higher level, thereby being beneficial to the large-scale growth and reproduction of Bacillus polymyxa Cs12 and the formation of seed bacteria, etc.

[0040] In some embodiments of the present invention, the above-mentioned bacterial agent is inoculated at an inoculum amount of 1:(10-100) of seed bacterial solution:NB or PDB culture medium volume ratio. The seed bacteria in this inoculum amount range can proliferate and develop in large quantities in the fermentation liquid, thereby improving the disease resistance effect of the final bacterial agent.

[0041] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0042] Example 1

[0043] Screening, isolation, purification and identification of Paenibacillus polymyxa Cs12:

[0044] (1) Healthy saffron rhizosphere soil was collected from the grape and saffron intercropping field in the Nan Taihu Modern Agriculture Demonstration Park in Huzhou, Zhejiang Province on August 15, 2020. 0.5 g of soil was weighed and added to 2 mL of sterile water. The suspension was shaken at 180 rpm / min for 20 min to obtain a suspension. The suspension was automatically spotted on a 96-well plate using the RoToR HDA colony high-throughput screening workstation (SINGER, UK). Fusarium oxysporum was in the middle and four soil suspensions were spotted in squares at equal distances around it. After spotting, the plate was sealed with a sealing film and cultured in the dark at 37°C for 2-3 days. Colonies with obvious inhibition zones were selected for isolation and purification.

[0045] (2) Pick colonies with obvious inhibition zones and inoculate them onto NA medium by streaking method for purification. Pick a purified single colony and inoculate it into 30 mL (50 mL conical flask) of NB liquid medium. Incubate it in a constant temperature shaking incubator at 28°C and 180 rpm for 12-48 h. Use a disposable Pasteur pipette to draw a small amount of bacterial agent onto a blood cell counting plate in a clean bench. Count the spores under a Zeiss upright fluorescence microscope and dilute the spore suspension to a concentration of 10 with sterile water. 7 -10 8 CFU / mL.

[0046] (3) The pathogen of saffron bulb rot, Fusarium oxysporum (isolated from the rhizosphere soil of the diseased plants in the same plot), was inoculated onto the PDA medium and cultured in a dark environment at 26±2℃ for 5-7 days. A sterile cork punch with a diameter of 6mm was used to make a bacterial block, which was connected to the center of a new PDA medium. Three sterile filter papers with a diameter of 6mm were placed at an equal distance of 2cm from the center of the circle. 3μL of the spore suspension was aspirated with a pipette and placed on the filter paper. An equal volume of NB culture medium was used as a negative control, and each treatment was repeated 3 times. The culture was placed in an incubator and cultured in a dark environment at 26±2℃ for 5-7 days. The results of the confrontation culture were tested. The results were as follows. Figure 1 As shown, according to Figure 1 Results The relative inhibition rate was calculated. Relative inhibition rate (%) = (control colony radius - treated colony radius) / (control colony radius - bacterial cake radius) × 100%, and strains with a relative inhibition rate greater than 85% were selected for molecular identification.

[0047] The above strains with an inhibition rate of more than 85% against Fusarium oxysporum were inoculated into NB liquid culture medium respectively, and oscillated at a constant temperature of 30°C at a speed of 180rpm / min in a shaker for 24-48h. 2mL was drawn into a centrifuge tube, and the strain DNA was extracted according to the steps of Tiangen DP302 Bacterial Genomic DNA Extraction Kit (Beijing Tiangen Biotechnology Co., Ltd.). The bacterial universal primers were used to amplify the isolated bacterial 16SrDNA. The sequence of the forward primer 27F used is shown in SEQ ID NO.2, and the sequence of the reverse primer 1492R used is shown in SEQ ID NO.3. The amplification conditions are as follows: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, 30 cycles; finally, extension at 72°C for 5min, termination of the reaction, and the amplified sequence of the polymyxa bacillus Cs12 obtained is shown in Table 1. The PCR product after agarose gel electrophoresis was sent to Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. for sequence determination. Figure 2 The sequence obtained from the PCR product was used as the target sequence, and the homologous sequence was searched in the GenBank database in NCBI. The reference sequence most similar to the morphological sequence was downloaded, and the neighbor-joining method (NJ) was used for phylogenetic analysis to determine the phylogenetic position of the strain to be identified. The results are shown in Figure 3 Through morphological, physiological and biochemical analysis (see Table 2, where + is available or positive, - is unavailable or negative) and 16SrRNA molecular biological identification, it was determined that the rhizosphere bacterial strain of the present invention is Paenibacillus polymyxa Cs12, and the deposit number is CGMCC No.22612.

[0048] Table 1 Sequence table of 16S rDNA sequence of Paenibacillus polymyxa Cs12 and primers used

[0049]

[0050]

[0051] Table 2 Physiological and biochemical characteristics of Paenibacillus polymyxa Cs12

[0052] index Expression index Expression glucose + 3% NaCl Hydrogen Sulfide - Xylose + Sodium malonate - Arabinose + Urease + maltose + Catalase - Sorbitol - Gelatin hydrolysis - Mannitol + MP - Nitrate Broth + VP +

[0053] Example 2

[0054] Disease resistance experiment of the bacterial agent of Bacillus polymyxa Cs12:

[0055] (1) Take the Fusarium oxysporum strain stored on the slant, and under sterile conditions, pick up a small amount of mycelium with an inoculation needle and inoculate it onto the PDA medium for activation. Incubate it in the dark at 26±2℃ for 5-7 days; use a sterile punch with a diameter of 6mm to make a bacterial block, inoculate it into the PDB liquid medium for culture, and inoculate 1 bacterial block for every 10ml PDB. Cultivate at 30℃ and 180rpm / min for 4-5 days. Filter the cultured Fusarium oxysporum fermentation liquid with two layers of sterile gauze in a clean bench. Pipette 100μL of spore suspension, count the number of spores with a hemocytometer, and adjust to F.oxysporum 10 5 -10 6 spores / mL suspension.

[0056] (2) Cs12 strain was frozen and stored in 25% glycerol. 100 μL was spread on NA medium under sterile conditions and cultured in the dark at 28-37°C and 75% humidity for 24-48 hours. The culture characteristics were observed. The results were as follows: Figure 4 As shown; pick a single colony of the activated strain with an inoculation needle and inoculate it into 25mL NB medium, and shake culture it at 28℃, 180rpm / min for 24h to prepare seed bacteria; inoculate the seed bacteria into new NB medium at a rate of 1-10%, and ferment it at 28℃, 180rpm for 48h to form a bacterial agent. The cultured bacterial agent is diluted with sterile water, and the OD600 of the bacterial agent is adjusted to 0.4-0.6.

[0057] (3) Saffron bulbs entered the dormant period. Saffron bulbs with no disease spots and a single weight of 25±1g were purchased from the Sandu Saffron Professional Cooperative in Jiande City. At the end of July, when the bulbs were about to end their dormancy, the outer skin of the bulbs was removed and surface sterilized using a three-step disinfection method (75% ethanol for 60 seconds, 2.5% sodium hypochlorite for 5 minutes, and 75% ethanol for 30 seconds). The bulbs were then rinsed with sterile water several times and dried. They were used for the experiment.

[0058] (4) The corms were randomly divided into 3 groups (blank control group: Control; model group: Fusarium oxysporum + sterile water; probiotic group: Cs12 + Fusarium oxysporum), with 42 in each group and 3 biological replicates in each group. They were placed in a 43cm×33cm×28cm seedling box for cultivation. Both pathogens and probiotics were inoculated by acupuncture. The Cs12 group was injected with 10μL of Cs12 at 3 locations on the top of the corm 3 days in advance, and the blank group and model group were inoculated with an equal volume of sterile water. The injection was performed every 3 days. During the second inoculation, the model group and the Cs12 group were inoculated with Fusarium oxysporum at the same time, which lasted for 28 days. The growth of the corms after 28 days was as shown in the figure. Figure 5As shown, the lesion area was counted using AutoCAD2019 software to calculate the disease index. Disease grading standard: 0, no lesions; 1, the area of ​​corm lesions is less than 10%; 2, the area of ​​corm lesions is 10%-30%; 3, the area of ​​corm lesions is 30%-50%; 4, the area of ​​corm lesions is 50%-70%; 5, the area of ​​corm lesions is greater than 70%. Disease index (DI) = [Σ (number of diseased plants × representative value) / total number of plants] × 100. The disease index of each group is shown in Table 3.

[0059] Table 3 Disease index of blank control group, model group and probiotic group

[0060] Group Blank control group Model Group Probiotics group Disease index 67±10.2 439±48.3 102±19.2

[0061] According to the results in Table 3 and the formula control effect (%) = (model group disease index - probiotic group disease index) / model group disease index × 100%, it was calculated that the control effect of Cs12 was 76.7%. Therefore, it can be concluded that the polymyxa bacillus Cs12 has a significant antibacterial effect on Fusarium oxysporum and can effectively reduce the incidence of saffron bulb rot.

[0062] Example 3

[0063] Field experiment of using the bacterial agent of Paenibacillus polymyxa Cs12 to control saffron bulb rot:

[0064] A saffron plot that had been planted continuously for 5 years was selected and divided into three plots of equal size. Two parallel plots (20m×4m) were set up, which were the control group and the Cs12 group. The Cs12 group was given the inoculant in Example 2 by root irrigation, 5mL for each corm, and the conventional cultivation management method was used as the control group. During the dormant period of the corms, the corms were dug, the incidence of the corms was counted, and the prevention and control effect was calculated. The results showed that the incidence of corm rot in the plots where polymyxa bacillus Cs12 was applied was below 5%, which was significantly lower than that of the control group where it was not applied, indicating that the inoculant prepared by the polymyxa bacillus Cs12 can effectively reduce the incidence of saffron corm rot.

[0065] In summary, the embodiments of the present invention provide a polymyxa bacillus and its application, a bacterial agent including the polymyxa bacillus and its preparation method:

[0066] 1. An embodiment of the present invention provides a polymyxa bacillus, which is named polymyxa bacillus Cs12, isolated from the soil of grape-saffron intercropping, and can effectively inhibit the growth and development of Fusarium oxysporum in the soil, thereby reducing the incidence of saffron bulb rot.

[0067] 2. An embodiment of the present invention also provides an application of Bacillus polymyxa in the preparation of a drug for preventing and treating saffron bulb rot. The Bacillus polymyxa can effectively inhibit the growth of Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot, has obvious preventive and control effects on saffron bulb rot, and does not cause problems such as pesticide residues and environmental pollution.

[0068] 3. An embodiment of the present invention further provides a bacterial agent, which is made from Bacillus polymyxa Cs12. The bacterial agent can be absorbed by the roots of saffron, thereby inhibiting the growth of Fusarium oxysporum in the roots of saffron. Since Fusarium oxysporum is the main pathogenic bacteria of saffron bulb rot, the incidence of saffron bulb rot can be reduced by inhibiting the growth of Fusarium oxysporum, which is of great significance to the development of saffron in my country.

[0069] 4. An embodiment of the present invention also provides a method for using a microbial agent, which ensures that the microbial agent can effectively and reasonably inhibit the growth and development of Fusarium oxysporum by using a reasonable dosage and method, thereby ensuring that the microbial agent has an excellent inhibitory effect.

[0070] 5. An embodiment of the present invention also provides a method for preparing a microbial agent, which comprises isolating and purifying the polymyxa bacillus Cs12 to obtain a single colony, subsequently isolating and purifying the single colony to obtain a seed fungus, and finally fermenting the seed fungus to obtain the microbial agent. The preparation method is simple, convenient and easy to operate, and the prepared microbial agent has a good inhibitory effect on Fusarium oxysporum, thereby reducing the incidence of saffron bulb rot.

[0071] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Sequence Listing <110> Huzhou Central Hospital <120> A polymyxa bacillus agent, preparation method and application thereof <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1461 <212> DNA <213> Artificial Sequence <400> 1 accggtggcg gcgtgcctaa tacatgcagt cgagcggggt tagttagaag cttgcttcta 60 actaacctag cggcggacgg gtgagtaaca cgtaggcaac ctgcccacaa gacagggata 120 actaccggaa acggtagcta atacccgata catccttttc ctgcatggga gaggaggaa 180 aggcggagca atctgtcact tgtggatggg cctgcggcgc attagctagt tggtggggta 240 aaggcctacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac 300 tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc aatgggcgaa 360 agcctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaaa gctctgttgc 420 cagggaagaa cgtcttatag agtaactgct cttgaagtga cggtacctga gaaaagcc 480 ccggctaact acgtgccagc agccgcggta atacgtaggg ggcaagcgtt gtccggaatt 540 attgggcgta aagcgcgcgc aggcggctct ttaagtctgg tgtttaatcc cgaggctcaa 600 cttcgggtcg cactggaaac tggggagctt gagtgcagaa gaggagagtg gaattccacg 660 tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggg 720 ctgtaactga cgctgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag 780 tccacgccgt aaacgatgaa tgctaggtgt taggggtttc gatacccttg gtgccgaagt 840 taacacatta agcattccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg 900 acggggaccc gcacaagcag tggagtatgt ggtttaattc gaagcaacgc gaagaacctt 960 accaggtctt gacatccctt tgaccggtct agagataggc ctttccttcg ggagagga 1020 gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttatgctt agttgccagc aggtcaagct gggcactcta agcagactgc 1140 cggtgaaaa ccgggagaag gtgggggatga cgtcaaatca tcatgcccct tatgacctgg 1200 gctacacacg tactacaatg gccggtacaa cgggaagcga aggagcgagg tggagccaat 1260 cctagaaaag ccggtctcag ttcggattgt aggctgcaac tcgcctacat gaagtcggaa 1320 ttgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggtct tgtacacacc 1380 gcccgtcaca ccacgagagt ttacaacacc cgaagtcggt gaggtaccg caaggagcca 1440 gccgccgaaa gggttggttt a 1461 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 ggttaccttg ttacgactt 19

Claims

1. A Paenibacillus polymyxa Cs12, characterized in that: The deposit number is CGMCC No.22612, and it is deposited in the China General Microbiological Culture Collection Center. The deposit date is May 26, 2021, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The polymyxa bacillus according to claim 1, characterized in that Its 16SrDNA sequence is shown in SEQ ID NO.

1.

3. Use of the polymyxa bacillus as claimed in claim 1 or 2 in the preparation of a medicament for preventing and treating saffron bulb rot.

4. A bacterial agent, characterized in that The invention is prepared by the polymyxa bacillus according to claim 1 or 2.

5. The bacterial agent according to claim 4, characterized in that The concentration of Bacillus polymyxa in the bacterial agent is 10 7 -10 8 CFU / mL.

6. A method for using the bacterial agent according to claim 4 or 5, characterized in that: The method comprises the following steps: the bacterial agent is applied once every 3 days, 5 mL each time, for 21-30 days.

7. The method of use according to claim 6, characterized in that: The method of application is root irrigation or injection.

8. A method for preparing the bacterial agent according to claim 4 or 5, characterized in that: The method comprises the following steps: picking a single colony of the polymyxa bacillus under sterile conditions and inoculating it into a NA medium or a PDA medium, and culturing it in the dark at 28-37° C. and 75% RH for 12-48 hours to obtain a purified colony; Under sterile conditions, the purified colonies were picked and inoculated into NB medium or PDB medium, and cultured at 28° C. and 180 rpm for 24 h to obtain seed bacteria; The seed bacteria are inoculated into NB medium or PDB medium, and fermented at 28° C. and 180 rpm for 48 hours to form a fermentation liquid, thereby obtaining the bacterial agent.

9. The method for preparing the bacterial agent according to claim 8, characterized in that: The NA medium includes 10g of peptone, 3g of beef extract, 5g of sodium chloride and 20g of agar, and the pH of the NA medium is 7.0-7.4; the NB medium includes 10g of peptone, 3g of beef extract and 5g of sodium chloride, and the pH of the NB medium is 7.0-7.4; the PDA medium includes a decoction of 200g of potatoes, 20g of glucose and 15g of agar; the PDB medium includes a decoction of 200g of potatoes and 20g of glucose, and the pH of the PDB medium is 7.0-7.

4.

10. The method for preparing the bacterial agent according to claim 8, characterized in that: Inoculate at a volume ratio of seed solution to NB culture medium of 1:(10-100).

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