Paenibacillus polymyxa NK-1 as well as preparation method and application of biological agent of paenibacillus polymyxa NK-1

Abstract

The invention belongs to the field of prevention and treatment of potato dry rot, and particularly relates to paenibacillus polymyxa NK-1 as well as a preparation method and application of a biological agent of the paenibacillus polymyxa NK-1. The paenibacillus polymyxa NK-1 is preserved in the China General Microbiological Culture Collection Center on May 21, 2024, and the preservation number of the paenibacillus polymyxa NK-1 is CGMCC (China General Microbiological Culture Collection Center) No. 30715. The preparation of the biological agent comprises the steps of activating strains, preparing fermentation mother liquor, preparing fermentation liquor and preparing the biological agent. The paenibacillus polymyxa NK-1 can be used for preventing and treating the potato dry rot, and the morbidity of the potato dry rot during potato storage can be effectively reduced by spraying and soaking the paenibacillus polymyxa NK-1 biological agent.

Description

Technical Field

[0001] The present invention belongs to the field of prevention and control of potato dry rot, and specifically relates to a Paenibacillus polymyxa NK-1, a preparation method and application of its biological bactericide. Background Art

[0002] Potato, as a staple food, has a planting area of about 4.8 million hm 2 , ranking first in the world. Potato production is of great significance in ensuring food security and nutritional balance in China and the world, promoting farmers' income increase and regional economic development. In recent years, in order to pursue high yield and high efficiency of potatoes, problems such as continuous cropping obstacles and difficult crop rotation in the production process have become increasingly serious, resulting in insufficient soil nutrients, deterioration of the rhizosphere microecological environment, weakening of potato plants, hindered growth and development, and increased soil-borne diseases. Potato dry rot is one of the main diseases among many soil-borne diseases, and its main pathogenic bacterium is Fusarium, which can survive in the soil for 5 to 6 years, and some can survive for more than 10 years. Fusarium will infect through the damaged epidermis of tubers, and the degree of infection will continue to be severe during storage, resulting in a high incidence of potato rot in production. At present, how to reduce the potato rot loss caused by dry rot has become an urgent problem to be solved in the industrial development.

[0003] Potato dry rot is a major post-harvest disease of potatoes caused by Fusarium infection of potato tubers and continuously aggravated during storage. It belongs to a typical soil-borne disease. Fusarium infects tubers through mechanical injuries on the surface of potato tubers during harvesting. Generally, the incidence rate in production is 20% - 30% or even higher. Since potato tubers cannot be treated with pesticides or chemical agents harmful to the human body as food and food processing raw materials, potato dry rot has always been a production technical problem that has not been effectively solved after potato harvest. At present, biological control has been widely recognized because of its advantages such as safety, non-toxicity, pollution-free, no residue, and not easy to produce drug resistance. Therefore, the use of biocontrol bacteria and their metabolites to control plant diseases has been widely recognized. Paenibacillus polymyxa ( Paenibacillus polymyxa ) is used as a biocontrol bacterium because of its significant antibiotic and competitive effects, which can inhibit the growth or infection of Fusarium. However, in practical applications, the application of Paenibacillus polymyxa ( Paenibacillus polymyxa ) in controlling potato dry rot is less. The secondary metabolites, culture conditions, etc. of Paenibacillus polymyxa from different sources vary greatly, and the control effects are uneven. Summary of the Invention

[0004] To solve the above problems, the present invention provides a Paenibacillus polymyxa NK-1, a preparation method and application of its biological bactericide.

[0005] The technical solution of the present invention is as follows: Paenibacillus polymyxa ( Paenibacillus polymyxa)NK-1, which was deposited on May 21, 2024 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms with the deposit number CGMCC No. 30715.

[0006] Application of Paenibacillus polymyxa ( Paenibacillus polymyxa )NK-1 in controlling potato dry rot.

[0007] Method for preparing a biological agent of Paenibacillus polymyxa ( Paenibacillus polymyxa )NK-1, comprising the following steps: Step 1, strain activation: Inoculate Paenibacillus polymyxa NK-1 into NA medium and place it in an incubator at 28 °C for 3 days of incubation. Wait until the strain grows for later use; the formula of NA medium is: beef extract 3 g, peptone 5 g, glucose 2.5 g, agar powder 20 g, distilled water 1000 mL, pH value 7.0 ± 0.2; Step 2, preparation of fermentation mother liquor: Pick a single colony cultured in Step 1 and inoculate it into NB culture solution. Place it in a constant temperature shaker at a temperature of 26 - 28 °C and a rotation speed of 170 - 190 r / min for 24 - 26 h of oscillating culture to obtain the NK-1 fermentation mother liquor; the formula of NB culture solution is: beef extract 3 g, peptone 5 g, glucose 2.5 g, distilled water 1000 mL, pH value 7.0 ± 0.2; Step 3, preparation of fermentation broth: Inoculate the fermentation mother liquor prepared in Step 2 into the optimized culture solution at a mass ratio of 2.5% and mix well. Place it in a constant temperature shaker at a temperature of 26 - 28 °C and a rotation speed of 170 - 190 r / min for 48 - 50 h of oscillating culture to obtain the NK-1 fermentation broth. The viable count of Paenibacillus polymyxa NK-1 in the fermentation broth is not less than 10 8 CFU / mL; the formula of the optimized culture solution is: sucrose 10 g, beef extract 70 g, sodium chloride 1 g, magnesium sulfate 0.5 g, distilled water 1000 mL, pH value 8.0 ± 0.2; Step 4, preparation of biological agent: Inoculate the fermentation broth prepared in Step 3 into the liquid biological fermentation medium at a mass ratio of 12% and mix well. Place it in a constant temperature shaker at a temperature of 26 - 28 °C and a rotation speed of 170 - 190 r / min for 48 - 50 h of oscillating culture to prepare the biological agent; the components and their mass percentages of the liquid biological fermentation medium are: 15% natamycin 0.4 mg / L, 1.5% arabic gum, 1% maltodextrin, 0.5% sucrose, 0.5% Tween 80, 0.2% dipotassium hydrogen phosphate, 0.02% ascorbic acid, 0.01% tocopherol, and the balance is water. Adjust the pH value to 8.0 ± 0.2.

[0008] Potato storage method using the biological agent, comprising the following steps: Step 1. Storage facility treatment: Before storing potatoes, dilute the Paenibacillus polymyxa NK-1 biological fungicide 100 times and spray it on the walls and floor of the storage facility. Spray once every 20 days for a total of 3 times. Step 2. Potato tuber treatment: After harvesting potatoes from the field, pack them into mesh bags and transport them to the laboratory. Use a potato dry cleaner to remove the sediment on the surface, soak them in the NK-1 biological fungicide diluted 200 times for 3 minutes, and then air-dry them naturally and transfer them into the storage facility.

[0009] The present invention has the following beneficial effects: The Paenibacillus polymyxa ( Paenibacillus polymyxa ) NK-1 of the present invention has a good antibacterial effect on the pathogen of potato dry rot. Description of the Drawings

[0010] Figure 1 Colony morphology of strain NK-1 on NA medium; Figure 2 Colony morphology of strain NK-1 on BPA medium; Figure 3 Colony morphology of strain NK-1 on AYDA medium; Figure 4 Colony morphology of strain NK-1 on BPY medium; Figure 5 Colony morphology of strain NK-1 on LB medium; Figure 6 Microscopic morphology of strain NK-1; Figure 7 Phylogenetic tree of strain NK-1; Figure 8 Antibacterial rates of different concentrations of natural antibacterial components against Fusarium sambucinum ... Detailed Embodiments

[0011] The biocontrol bacterium is Paenibacillus polymyxa ( Paenibacillus polymyxa ) NK-1. This strain was deposited at the China General Microbiological Culture Collection Center (abbreviated as CGMCC) on May 21, 2024, with the deposit number CGMCC No. 30715, and the deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

[0012] Paenibacillus polymyxa ( Paenibacillus polymyxa ) NK-1 was isolated and purified from the soil in the courtyard of the Shanxi Academy of Agricultural Sciences in September 2023. The strain was cultured at a constant temperature of 28 °C for 72 hours on NA medium. The colonies were semi-transparent, milky white, sticky on the surface, with irregular edges and close to circular ( Figure 1 ); on BPA medium, the colonies were beige with irregular edges ( Figure 2); It grows on AYDA medium, with milky white colonies and spreading bacterial lawns ( Figure 3 ); It grows on BPY medium, with spherical, foamy colonies with small holes in the center and a milky white color ( Figure 4 ); On LB medium, the colonies are milky white with a flower-shaped edge ( Figure 5 ). The microscopic morphology is short rod-shaped, and the spores are oval in the later stage of cultivation ( Figure 6 ).

[0013] Extract the genome of strain NK-1, use the universal primers 27F and 1492R to perform PCR amplification on the 16S rDNA sequence, and obtain a sequence length of 1426 bp. Download relevant sequences from the GenBank database to construct a phylogenetic tree. The results show that strain NK-1 and Paenibacillus polymyxa Paenibacillus polymyxa are on the same branch of the phylogenetic tree. Therefore, strain NK-1 was identified as Paenibacillus polymyxa Paenibacillus polymyxa ( Figure 7 ).

[0014] The preparation method of Paenibacillus polymyxa ( Paenibacillus polymyxa ) NK-1 biological inoculant includes the following steps: (1) Strain activation: Inoculate Paenibacillus polymyxa NK-1 into NA medium and place it in a constant temperature incubator at 28 °C for 3 d, and wait for the strain to grow before use.

[0015] The formula of NA medium is: beef extract 3 g, peptone 5 g, glucose 2.5 g, agar powder 20 g, distilled water 1000 mL, pH value 7.0 ± 0.2; (2) Preparation of fermentation mother liquor: Pick the single colonies cultured in step (1) and inoculate them into NB culture solution, and place them in a constant temperature shaker at a temperature of 26-28 °C and a rotation speed of 170-190 r / min for oscillation culture for 24-26 h to obtain the NK-1 fermentation mother liquor.

[0016] The formula of NB culture solution is: beef extract 3 g, peptone 5 g, glucose 2.5 g, distilled water 1000 mL, pH value 7.0 ± 0.2; (3) Preparation of fermentation broth: Inoculate the fermentation mother liquor prepared in step (2) into the optimized culture solution at a mass ratio of 2.5% and mix well, and place it in a constant temperature shaker at a temperature of 26-28 °C and a rotation speed of 170-190 r / min for oscillation culture for 48-50 h to obtain the NK-1 fermentation broth. The viable count of Paenibacillus polymyxa NK-1 in the fermentation broth is not less than 10 8 CFU / mL.

[0017] The formula of the optimized culture solution is: sucrose 10 g, beef extract 70 g, sodium chloride 1 g, magnesium sulfate 0.5 g, distilled water 1000 mL, pH value 8.0 ± 0.2; (4) Preparation of biological agents The fermentation liquid prepared in step (3) was inoculated into the liquid biological fermentation medium at a mass ratio of 12% and mixed evenly, and then placed in a constant temperature shaker at a temperature of 26-28°C and a rotation speed of 170-190 r / min for oscillation culture for 48-50 hours to prepare a biological agent.

[0018] The components and their mass percentages of the liquid biological fermentation medium are: 15% 0.4 mg / L natamycin, 1.5% gum arabic, 1% maltodextrin, 0.5% sucrose, 0.5% Tween 80, 0.2% dipotassium hydrogen phosphate, 0.02% ascorbic acid, 0.01% tocopherol, and the balance is water, and the pH value is adjusted to 8.0±0.2.

[0019] Paenibacillus polymyxa ( Paenibacillus polymyxa )The usage of NK-1 biological agent is: (1) Storage warehouse treatment: Before storing potatoes, dilute the polymyxa bacillus NK-1 biological agent 100 times and spray it on the walls and ground of the storage warehouse once every 20 days, for a total of 3 times; (2) Potato tuber processing: After the potatoes are harvested from the field, they are packed into mesh bags and transported to the laboratory. A potato cleaning machine is used to remove the surface mud and sand. They are then soaked in 200-fold diluted NK-1 biological bacteria agent for 3 minutes. After natural air drying, they are moved to a storage warehouse.

[0020] Antibacterial test and control effect test of Paenibacillus polymyxa NK-1 1. Screening and identification of biocontrol bacteria NK-1 (1) Test method Take 10g of soil and add it to a 250mL conical flask containing 90mL of sterile water. Add sterile glass beads to fully disperse it, place it in a shaker at 28℃ and 120r / min and fully shake it for 30min to make a soil stock solution. Use the dilution plate method to absorb 100μL of the upper layer of the soil stock solution and add it to a centrifuge tube containing 900μL and mix it well. Then dilute it 10 times to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ; 200 µL was applied to PDA culture medium respectively, and cultured in a constant temperature incubator at 28℃ for 1-3 days; single colonies of different sizes, shapes and colors were picked out for streaking pure culture, and stored at 4℃ for future use; the strains were inoculated into NA, BPA, AYDA, BPY and LB culture medium respectively, and cultured at 28℃ for 3 days, and the colony morphology was observed; the microscopic morphology was observed using a BX51 microscope (×100) and an oil objective.

[0021] (2)Test Results Six antagonistic strains were isolated from soil samples. In the preliminary test, it was found that NK-1 had a good antibacterial effect on the pathogen of potato dry rot.

[0022] Morphological characteristics of strain NK-1: Cultured at 28 °C for 72 h on NA medium, the colony was semi-transparent, milky white, with a viscous surface, irregular edges, and was nearly circular ( Figure 1 ); on BPA medium, the colony was beige with irregular edges ( Figure 2 ); growing on AYDA medium, the colony was milky white and the bacterial lawn spread ( Figure 3 ); growing on BPY medium, the colony was spherical, foamy, with a small hole in the center and was milky white in color ( Figure 4 ); on LB medium, the colony was milky white with a flower-shaped edge. ( Figure 5 ) The microscopic morphology was short rod-shaped, and the endospores were oval in the later stage of cultivation ( Figure 6 ).

[0023] Molecular biological characteristics: The length of the 16S rDNA gene sequence of NK-1 was 1426 bp. Relevant sequences were downloaded from the GenBank database to construct a phylogenetic tree. The results showed that strain NK-1 and Paenibacillus polymyxa Paenibacillus polymyxa were on the same branch of the phylogenetic tree. Therefore, strain NK-1 was identified as Paenibacillus polymyxa Paenibacillus polymyxa Figure 7 ).

[0024] 2. Test on the inhibition of 5 potato dry rot pathogens by Paenibacillus polymyxa NK-1 (1)Test method Determination of antibacterial effect: Paenibacillus polymyxa NK-1 was cultured at 28 °C for 3 d. The mycelial discs of 5 potato dry rot pathogens ( Fusarium sambucinum Fusarium sambucinum Fusarium solani Fusarium solani Fusarium oxysporum Fusarium oxysporum Fusarium acuminatum Fusarium acuminatum Fusarium dimerum and Fusarium dimerum

[0025] )were inoculated in the center of PDA, and Paenibacillus polymyxa NK-1 was inoculated 2 cm around it. Only inoculating the pathogen was used as the control. It was cultured in a constant temperature incubator at 28 °C. After 7 d, the colony diameter was observed and measured, and the inhibition rate was calculated. Inhibition rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100. When Paenibacillus polymyxa NK-1 was co-cultured with 5 potato dry rot pathogens, an antibacterial zone was produced at the junction with the 5 pathogens, indicating that strain NK-1 could inhibit F. sambucinum , F. solani, F. oxysporum , F. acuminatum and F. dimerum colony and mycelial growth. The bacteriostatic rates of Paenibacillus polymyxa NK-1 against F. sambucinum , F. solani , F. oxysporum , F. acuminatum and F. dimerum were 91.08%, 48.20%, 47.31%, 70.55% and 86.28% in turn (Table 1). Among the 5 pathogenic bacteria, the bacteriostatic rates against F. sambucinum and F. dimerum were significantly higher than those against the others, being 91.08% and 86.28% respectively.

[0026] Table 1 Inhibitory effects of Paenibacillus polymyxa NK-1 against 5 potato dry rot pathogens Note: The lowercase letters after the bacteriostatic rate are p <0.05 significance analysis.

[0027] 3. Optimization test of Paenibacillus polymyxa NK-1 (1) Test method Prepare the optimized medium: Weigh 10 g of sucrose, 70 g of beef extract, 1 g of sodium chloride, and 0.5 g of magnesium sulfate, and dissolve them thoroughly in 1000 mL of distilled water. Adjust the pH to 8 with 1 mol / L NaOH, dispense it into 250 mL conical flasks, with a filling volume of 50 mL, and sterilize it at 121 °C under high-pressure steam for 30 min. Inoculate 2.5% of the strain NK-1 fermentation mother liquor into the optimized medium and mix well, and place it in a constant temperature shaker at 26 - 28 °C and 170 - 190 r / min for 48 - 50 h; The test shows that the viable count of Paenibacillus polymyxa NK-1 on the optimized fermentation medium is about 3.0×10 8 CFU / mL, which is significantly higher than that on other media. (Table 2) Table 2 Viable counts of Paenibacillus polymyxa NK-1 on different media Prepare the biological bactericide: Add 1.5% gum arabic, 1% maltodextrin, 0.5% sucrose, 0.5% Tween 80, 0.2% dipotassium hydrogen phosphate, 0.02% ascorbic acid, and 0.01% tocopherol by volume ratio, with a filling volume of 50 mL / 250 mL, and sterilize it at 121 °C under high pressure for 30 min. After it cools down, add 15% 0.4 mg / L natamycin and mix well. Inoculate 12% of the strain NK-1 fermentation mother liquor into the liquid biological fermentation medium and mix well, and place it in an environment at 26 - 28 °C and 170 - 190 r / min for 48 - 50 h to make the biological bactericide.

[0028] (2)Test Results The test shows that the production capacity of Paenibacillus polymyxa NK-1 can be significantly improved on the liquid biological fermentation medium, and the viable cell count is about 5.5×10 8 CFU / mL, which is significantly higher than that of other media. (Table 2) 4. Screening of Auxiliaries and Optimization of Their Contents (1)Test Method Screening of natural antibacterial components: The mycelial growth rate method was used to detect the antibacterial effects of chitosan, citral, curcumin, gallic acid, geraniol, piperine, and natamycin on Fusarium sambucinum Fuckel Fusarium sambucinum ). The above reagents were respectively sucked and dissolved with 1% glacial acetic acid to prepare a stock solution of 20 g / L. The stock solution was added to PDA to make its mass concentration 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, and 1.0 mg / L respectively, and the PDA without adding reagents was used as the control. Fusarium sambucinum Fuckel was inoculated in the center of the plate and cultured at 28 °C for 7 d to observe the colony diameter, and the antibacterial rate was calculated. Antibacterial rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100. ( Figure 8 ) The effects of different natural antibacterial components on strain NK-1 were judged by detecting the viable cell count through the dilution plating method. The above natural antibacterial components were configured into drug-containing LB plates with a concentration of 0.5 mg / L, and the LB plate added with an equal amount of sterile water was used as the control. The fermentation broth of strain NK-1 cultured for 48 h was spread on the plate and cultured at 28 °C for 3 d to calculate the viable cell count.

[0029] Screening of other auxiliaries: Different contents of liquid media were configured according to Table 3 by the dilution plating method. After sterilization at 121 °C for 30 min and cooling, the fermentation mother liquor of strain NK-1 was transferred to each liquid culture medium at an inoculation amount of 12%, and an equal amount of sterile water was added as the control. It was placed in a shaker with parameters set at 28 °C and 180 r / min for constant temperature culture for 48 h. 100 μL of appropriate gradient dilution was sucked and spread for detecting the viable cell count, and the test was repeated 3 times.

[0030] Table 3 Types and Contents of Processing Auxiliaries (2)Test Results The test shows that the biocompatibility of the natural antibacterial component natamycin and strain NK-1 is good, and the inhibition rate of 0.4 mg / L natamycin against the pathogen of potato dry rot F. sambucinum is 98.02%.

[0031] The liquid biological fermentation medium comprises active ingredients, thickeners, emulsifiers, stabilizers and anti-ultraviolet agents. The components and their mass percentages are as follows: 0.4 mg / L natamycin at 15%, gum arabic at 1.5%, maltodextrin at 1%, sucrose at 0.5%, Tween 80 at 0.5%, dipotassium hydrogen phosphate at 0.2%, ascorbic acid at 0.02%, tocopherol at 0.01%, with the balance being water, and the pH value is adjusted to 8.0 ± 0.2. The number of viable bacteria in the liquid biological fermentation medium is 1.5 times higher than that in the optimized culture solution, and the number of viable bacteria of strain NK-1 is 5.50×10 8 CFU / mL.

[0032] 5. Antibacterial test of the Paenibacillus polymyxa NK-1 biological agent (1) Test method Determination of antibacterial effect: The mycelial growth rate method was used to determine the antibacterial effect of the Paenibacillus polymyxa NK-1 biological agent against 5 Fusarium species causing potato dry rot. 5% (volume fraction) of the NK-1 biological agent was added to the sterilized and cooled PDA medium, and after culturing for 5 - 7 days, the diameter of the fungal cake was measured and the antibacterial rate was calculated. Adding sterile water was used as the control, and each treatment was repeated 3 times. Antibacterial rate (%) = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group × 100.

[0033] (2) Test results The Paenibacillus polymyxa NK-1 biological agent has inhibitory effects on all 5 kinds of potato dry rot pathogens, and the size of the pathogen fungal cake is significantly inhibited. The inhibitory effects of the Paenibacillus polymyxa NK-1 biological agent on F. sambucinum , F. solani , F. oxysporum , F. acuminatum and F. dimerum are 86.82%, 39.89%, 40.86%, 65.35% and 76.38% in sequence (Table 4).

[0034] Table 4 Inhibitory effects of the optimized Paenibacillus polymyxa NK-1 on 5 potato dry rot pathogens Note: The lowercase letters after the antibacterial rate are for p <0.05 significant analysis.

[0035] 6. Control effect test of the Paenibacillus polymyxa NK-1 biological agent (1) Test method Treatment of potato storage Select 6 potato storage warehouses with consistent specifications as test warehouses, numbered 1, 2, 3, 4, 5, and 6 respectively, with a relative humidity of 80% - 90%; two months before storage, after cleaning the 6 storage warehouses, the following treatments were carried out: no treatment was done in Warehouse No. 1 as a control; in Warehouses No. 2, 3, 4, 5, and 6, the walls and floors were evenly sprayed with a bio-bacterial agent diluted 100 times, sprayed once every 20 days, and sprayed a total of 3 times.

[0036] Treatment of potato tubers After harvesting potatoes in the field, they were put into mesh bags and transported to the laboratory, and a potato dry cleaner was used to remove the dust on the surface of the tubers. The potatoes were soaked in a bio-bacterial agent diluted 200 times for 3 minutes, and after natural air drying, they were placed in the above 6 storage warehouses.

[0037] After 3 months of storage, 500 potato tubers were randomly selected, the number of tubers with dry rot was investigated and counted, and the incidence rate was calculated. Incidence rate (%) = number of tubers with dry rot / 500 × 100, control effect (%) = (incidence rate of the control group - incidence rate of the experimental group) / incidence rate of the control group.

[0038] (2) Test results The bio-bacterial agent Paenibacillus polymyxa NK-1 has a significant control effect on potato dry rot during storage. The test results show that compared with the control (Warehouse No. 1, with an incidence rate of 26.1%), the incidence rate of potato dry rot in Warehouses No. 2, 3, 4, 5, and 6 decreased, ranging from 8.6% to 11.3%, and the control effect of the bio-bacterial agent on dry rot was 56.7% - 67.1% (Table 5).

[0039] Table 5 Control effect of Paenibacillus polymyxa NK-1 bio-bacterial agent on potato dry rot Storage kiln Incidence Control effect No. 1 26.1% — No. 2 11.3% 56.7% No. 3 8.6% 67.1% No. 4 9.2% 64.8% No. 5 10.9% 58.2% No. 6 9.4% 64.0%

Claims

1. Paenibacillus polymyxa ( Paenibacilluspolymyxa )NK-1, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 21, 2024, with the deposit number CGMCC No.30715.

2. The polymyxa bacillus according to claim 1 ( Paenibacilluspolymyxa ) Application of NK-1 in preventing and controlling potato dry rot.

3. Using the polymyxa bacillus described in claim 1 ( Paenibacilluspolymyxa ) A method for preparing a biological bacterial agent of NK-1, characterized in that: The steps include: Step 1, strain activation: inoculate Paenibacillus polymyxa NK-1 into NA medium, place it in a constant temperature incubator at 28°C for 3 days, and wait for the strain to grow for use; the formula of the NA medium is: 3g beef extract, 5g peptone, 2.5g glucose, 20g agar powder, 1000mL distilled water, pH 7.0±0.2; Step 2, prepare the fermentation yeast solution: pick the single colony cultured in step 1 and inoculate it into NB culture solution, place it in a constant temperature shaker at a temperature of 26-28°C and a speed of 170-190r / min for shaking culture for 24-26h, that is, NK-1 fermentation yeast solution; the formula of the NB culture solution is: 3g beef extract, 5g peptone, 2.5g glucose, 1000mL distilled water, pH value 7.0±0.2; Step 3, prepare fermentation broth: inoculate the fermentation broth with 2.5% of the mass of the yeast solution prepared in step 2 into the optimized culture broth and mix well, place in a constant temperature shaker at a temperature of 26-28°C and a speed of 170-190 r / min for 48-50 hours, and obtain NK-1 fermentation broth, in which the number of viable Bacillus polymyxa NK-1 in the fermentation broth is not less than 10 8 CFU / mL; the optimized culture medium formula is: sucrose 10g, beef extract 70g, sodium chloride 1g, magnesium sulfate 0.5g, distilled water 1000mL, pH value 8.0±0.2; Step 4, preparing a biological agent: inoculate 12% of the fermentation liquid prepared in step 3 into a liquid biological fermentation medium and mix well, place it in a constant temperature shaker at a temperature of 26-28°C and a rotation speed of 170-190 r / min for shaking culture for 48-50 hours to prepare a biological agent; the components of the liquid biological fermentation medium and their mass percentages are: 15% 0.4 mg / L natamycin, 1.5% gum arabic, 1% maltodextrin, 0.5% sucrose, 0.5% Tween 80, 0.2% dipotassium hydrogen phosphate, 0.02% ascorbic acid, 0.01% tocopherol, and the balance is water, and the pH value is adjusted to 8.0±0.

2.

4. A method for storing potatoes using the biological agent as claimed in claim 3, characterized in that: The following steps are included: Step 1, storage warehouse treatment: before storing potatoes, dilute the polymyxa bacillus NK-1 biological agent 100 times and spray it on the walls and ground of the storage warehouse, spraying once every 20 days, for a total of 3 times; Step 2: Potato tuber processing: After the potatoes are harvested from the field, they are put into mesh bags and transported to the laboratory. A potato dry cleaner is used to remove the surface mud and sand. They are then soaked in 200-fold diluted NK-1 biological bacteria agent for 3 minutes. After natural air drying, they are moved to a storage warehouse.

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