Paenibacillus kribbensis spaceflight mutant strain and application thereof in preparation of nano-selenium

Abstract

The invention discloses a paenibacillus kribbensis spaceflight mutant strain and application of the paenibacillus kribbensis spaceflight mutant strain in preparation of nano-selenium. According to the invention, a paenibacillus kribbensis PS04-2 strain is obtained through space mutation, and the strain has high selenite reducing capacity and can tolerate high-concentration selenite, and the tolerable concentration exceeds 1000mM; the method has the capacity of efficiently converting sodium selenite to generate nano-selenium, the synthesized nano-selenium is high in conversion rate and uniform in nano-selenium particle, and the provided preparation method is simple, convenient, low in cost, high in product purity and environmentally friendly. The nano-selenium synthesized by the PS04-2 strain has a good antagonistic effect on various plant pathogenic fungi, can prevent and treat various plant diseases, and can be used for developing more selenium-enriched microbial preparations. Meanwhile, nano-selenium synthesized by the PS04-2 strain is used for sesame production, so that sesame growth can be promoted, selenium-rich sesame can be obtained, and more effective methods are provided for increasing the selenium content of crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial synthesis of nanomaterials, and more specifically, relates to a space mutant strain of Paenibacillus cribenbergii and its application in the preparation of nano-selenium. Background Art

[0002] Selenium is one of the essential trace elements for the human body and is essential for maintaining normal physiological functions and health. It is involved in the activity of a variety of enzymes, including glutathione peroxidase (GPx), which helps protect cells from damage caused by oxidative stress. Selenium is a component of some antioxidant enzymes, which protect cells from damage caused by free radicals and reduce oxidative stress. Selenium is essential for the normal function of the immune system, helping to regulate immune responses and enhancing the activity of immune cells. Selenium plays an important role in the synthesis and metabolism of thyroid hormones, and it is involved in the metabolism of iodine and the synthesis of thyroid hormones. Some studies have shown that selenium may have anti-cancer effects, especially in preventing certain types of cancer, such as prostate cancer, colon cancer, and lung cancer. Although selenium is an essential trace element, excessive intake of selenium may also lead to selenium poisoning, which manifests as symptoms such as hair loss, nail deformation, and skin damage. Selenium is unevenly distributed on the earth, with soils in some areas having higher selenium content and other areas having lower selenium content. This affects the selenium content in the local food chain, which in turn affects the selenium intake of residents. The main food sources of selenium include Brazil nuts, meat, fish, grains, and certain vegetables. The selenium content of different foods varies greatly, depending on the selenium content of the soil in which they are grown.

[0003] Selenite is the most common form of selenium in nature. However, due to its high toxicity and high mobility, selenium-contaminated areas with excessive selenite concentrations often face huge ecological risks. Elemental nano-selenium is the least toxic form of selenium discovered. Reducing highly toxic selenite to low-toxic elemental nano-selenium is an important means of treating selenium pollutants and recycling selenium resources. Traditional selenite removal and nano-selenium preparation usually use chemical methods, such as adding vitamin C reducing agent and bovine serum albumin stabilizer to selenite for preparation. However, the high cost of this process, low purity of nano-selenium products, uneven particle size distribution and poor stability limit the application of nano-selenium.

[0004] With more and more research on microorganisms, nano-selenium can be synthesized by reducing selenite through microbial strains, such as Bacillus sp., Bacillus amyloliquefaciens, Escherichia coli, Rhodospirillum molisch, Pseudomonasfluorescens, etc. The biological nano-selenium synthesized by microbial strains is stable, highly active, and has a simple preparation method. Due to the large differences in the properties of different strains, the tolerance to sodium selenite is not high and the synthesis efficiency is low. At present, there is a great lack of excellent bacterial strains that can synthesize nano-selenium to efficiently convert sodium selenite to synthesize nano-selenium. Therefore, screening and developing more microbial strains that can tolerate higher concentrations of sodium selenite is of great significance for promoting the synthesis of biological nano-selenium. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the shortage of existing microbial strain resources for synthesizing nano-selenium, and to provide a space mutant strain of Paenibacillus kribensis and its application in preparing nano-selenium.

[0006] The first object of the present invention is to provide a strain of Paenibacillus cribenbergii.

[0007] The second object of the present invention is to provide the application of Paenibacillus cribenbergii.

[0008] The third object of the present invention is to provide nano-selenium.

[0009] The fourth object of the present invention is to provide a method for preparing nano-selenium.

[0010] The fifth object of the present invention is to provide applications of nano-selenium.

[0011] The sixth object of the present invention is to provide nano-selenium.

[0012] The seventh object of the present invention is to provide a composite microbial agent containing the ΔPS04-2 strain.

[0013] The eighth object of the present invention is to provide a method for producing selenium-enriched sesame.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] The present invention provides a Paenibacillus kribbensis △PS04-2 strain, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 25, 2024, with a deposit number of GDMCC No: 65207.

[0016] The invention obtains a Paenibacillus cribernoe △PS04-2 strain from a mutant library of a wild-type strain PS04 of aerospace mutant Paenibacillus cribernoe. The strain has high selenite reduction ability and can tolerate high concentration of selenite, and can tolerate a concentration exceeding 1000mM. The strain also has the ability to efficiently convert sodium selenite to generate nano-selenium, and converts inorganic selenium into nano-selenium for preparing nano-selenium, and the nano-selenium content reaches 513.05mg / kg. The extracellular nano-selenium is efficiently produced by reducing sodium selenite to synthesize nano-selenium, and the reaction conditions are mild, the operation is simple, and the conversion efficiency of nano-selenium is improved. The method for preparing nano-selenium by using the △PS04-2 strain has low equipment requirements, simple process, low cost, high product purity, and is environmentally friendly, and provides more bacterial species resources for microbial synthesis of nano-selenium.

[0017] Therefore, the present invention protects the use of the ΔPS04-2 strain in converting inorganic selenium into nano-selenium or in preparing nano-selenium.

[0018] The invention provides nano-selenium, which is prepared by △PS04-2 strain.

[0019] The invention provides a method for preparing nano-selenium. The method comprises the following steps: activating and culturing a △PS04-2 strain to obtain a seed culture solution, inoculating the seed culture solution into a liquid fermentation medium containing sodium selenite, oscillating and culturing the culture solution, collecting the fermentation solution and centrifuging the solution, discarding the supernatant, washing the precipitate, and purifying the solution to obtain nano-selenium. The liquid fermentation medium containing sodium selenite comprises: 3.0-5.0 g / L sodium nitrate, 30-60 g / L sucrose, 1.0-3.0 g / L dipotassium hydrogen phosphate, 0.5-1.0 g / L magnesium sulfate heptahydrate, 0.5-1.0 g / L potassium chloride, 0.01-0.02 g / L ferrous sulfate heptahydrate and 0.5-10 mM sodium selenite, and the pH value is 6.5-7.5.

[0020] As the most preferred preparation method, the present invention provides a specific method for converting inorganic selenium into nano-selenium by the ΔPS04-2 strain:

[0021] △PS04-2 was inoculated into a solid culture medium and cultured at a constant temperature for activation; the activated △PS04-2 was inoculated into a liquid culture medium for shaking culture to obtain a seed culture solution; the concentration of the prepared solution was 1×10 6cuf / mL seed liquid was inoculated into a liquid fermentation medium containing sodium selenite at an inoculum size of 1%, and cultured under shaking conditions at 28°C and a shaker speed of 150 rpm for 2 days. The fermentation liquid was centrifuged at 12000 rpm for 5-10 min, the supernatant was discarded, and the liquid was washed twice with sterile water for purification. Sterile water was added for ultrasonic crushing in an ice bath. The ultrasonic conditions were set to 450 W power, 5 s ultrasonic operation and 5 s pause, and a total operation time of 40 min. After the ultrasonication, the liquid was centrifuged at 12000 rpm for 10 min, and 1.5 mol / L Tris / HCl buffer (pH 8.3) Wash the precipitate three times continuously, collect the precipitate by centrifugation and resuspend it in 4 mL of ultrapure water, add 2 mL of n-octanol, shake on an oscillator for 5 min, centrifuge at 3000 rpm for 5 min, and place it in a 4°C refrigerator for 24 h to allow the solution to be clearly separated. Wait for the red nano-selenium to precipitate at the bottom of the centrifuge tube, discard the cell fragments between the two phases, wash the precipitate twice with sterile water, and finally resuspend the nano-selenium particles in 10 mL of ultrapure water, freeze-dry, and obtain pure nano-selenium particles.

[0022] Preferably, the above method for purifying nano-selenium can also be carried out by other conventional methods in the art.

[0023] At the same time, the nano-selenium synthesized by the △PS04-2 strain has a good antagonistic effect on a variety of plant pathogenic fungi, and can be used to prevent and treat a variety of plant diseases such as strawberry root rot, pepper anthracnose, wheat fusarium head blight, wheat stem base rot, sesame bacterial wilt, etc., and has good development and application potential in selenium-enriched microbial preparations for biocontrol agents. At the same time, the use of the synthesized nano-selenium in sesame production can not only promote sesame growth, but also obtain selenium-enriched sesame, providing more effective methods for increasing the selenium content of crops.

[0024] The invention provides application of nano-selenium in inhibiting plant pathogenic fungi or in preventing and controlling plant diseases caused by pathogenic bacteria.

[0025] Furthermore, the pathogenic fungus is one or more of Neopestalotiopsis clavispora, Colletotrichum fructicola, Fusarium graminearum, Fusarium pesudograminearum, and Ralstonia solanacearum; the plant disease is one or more of strawberry root rot, pepper anthracnose, wheat fusarium fusarium, wheat stem base rot, rice sheath blight, and bacterial wilt.

[0026] The invention provides application of nano-selenium in promoting growth of sesame and increasing selenium content of sesame.

[0027] The present invention provides application of nano-selenium in preparing antibacterial products or growth-promoting products.

[0028] The invention provides a product containing the nano-selenium.

[0029] The invention provides a microbial nano-selenium bacterial agent containing a ΔPS04-2 strain.

[0030] The invention provides a method for producing selenium-enriched sesame, which comprises spraying sesame plants with a microbial nano-selenium agent.

[0031] Preferably, the microbial nano-selenium agent is diluted 200 times and then sprayed 4 times during the growth period of sesame.

[0032] More preferably, the sesame is sprayed four times during the growing period with an interval of 15 days to increase the yield to the standard of selenium-enriched sesame.

[0033] The present invention has the following beneficial effects:

[0034] The present invention obtains a strain of Paenibacillus cribenensis △PS04-2 through aerospace mutation. The strain has high selenite reduction ability and can tolerate high concentrations of selenite, and can tolerate concentrations exceeding 1000mM; it also has the ability to efficiently convert sodium selenite to generate nano-selenium, and synthesizes it into nano-selenium with high conversion rate, uniform nano-selenium particles, and nano-selenium content of 513.05mg / kg; the preparation method provided is simple and convenient, low in cost, high in product purity, and environmentally friendly. The nano-selenium synthesized by the △PS04-2 strain has good antagonistic effects on a variety of plant pathogenic fungi, can prevent and treat a variety of plant diseases, and can develop more selenium-rich microbial preparations. At the same time, the nano-selenium synthesized by the △PS04-2 strain is used in sesame production, which can not only promote sesame growth, but also obtain selenium-rich sesame, providing more effective methods for increasing the selenium content of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results of different strains growing in a medium containing 10 mM sodium selenite for 12 hours (A1 in the figure is the wild-type Paenibacillus cribenbergii PS04; A2-D6 are 23 different space mutant strains respectively).

[0036] Figure 2 The graph shows the growth result of △PS04-2 strain in culture medium containing different concentrations of sodium selenite for 48 hours (A1-A5 in the graph are culture medium containing 100, 200, 300, 400 and 500 mM sodium selenite; B1-B5 are culture medium containing 600, 700, 800, 900 and 1000 mM sodium selenite).

[0037] Figure 3 This is the scanning electron microscope observation image of △PS04-2.

[0038] Figure 4 This is a scanning electron microscope observation of △PS04-2 and extracellular nano-selenium.

[0039] Figure 5 This is the scanning electron microscope observation image of the purified △PS04-2 synthetic nano-selenium particles.

[0040] Figure 6 This is the EDS analysis diagram of the elemental composition of purified nano-selenium particles.

[0041] Figure 7 This is a diagram of the position of nano-selenium particles observed by transmission electron microscopy.

[0042] Figure 8 This is the control effect of the control group on plant pathogenic fungi (in the figure, A is the pathogen of strawberry root rot; B is the pathogen of anthracnose; C is the pathogen of wheat fusarium rust; D is the pathogen of wheat stem base rot).

[0043] Fig. 9 This is the control effect of △PS04-2 synthesized nano-selenium on plant pathogenic fungi (in the figure, A is the pathogen of strawberry root rot; B is the pathogen of anthracnose; C is the pathogen of wheat fusarium rust; D is the pathogen of wheat stem base rot).

[0044] Fig.10 This is the inhibitory effect of △PS04-2 synthesized nano-selenium on sesame bacterial wilt pathogen.

[0045] Fig.11 The lesion area statistics of the control effect of pepper anthracnose (A in the figure is the control group inoculated with pathogens only; B is the treatment group sprayed with nano-selenium solution first and then inoculated with pathogens). DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Example 1 Isolation and Identification of Paenibacillus Cribbensis ΔPS04-2

[0049] 1. Obtaining strain mutant library

[0050] The wild-type strain PS04 of Paenibacillus kribbensis was divided into two parts, one of which was kept on the ground for routine storage as a control sample; the other was launched into space from the Wenchang Launch Center in Hainan in March 2020. After a series of mutagenesis under space conditions, a mutant library of the wild-type strain PS04 of Paenibacillus kribbensis was obtained (for specific mutagenesis method conditions, see the public text CN114480222A).

[0051] 2. Screening of selenite-reducing bacteria

[0052] Using the wild-type strain PS04 of Paenibacillus kribensis as a control, the ability of different space-induced strains to convert selenite into nano-selenium was compared in the mutant library, and mutants with high selenite reduction ability were screened.

[0053] The wild-type strain PS04 and 23 spaceflight mutant strains of Paenibacillus cribensis were activated and prepared at a concentration of 1×10 6 cuf / mL of seed liquid was inoculated into Czaper's culture medium with a selenium salt concentration of 10mM at an inoculum size of 1%, and Czaper's culture medium without sodium selenite was used as a control treatment; 3 replicates were set for each treatment, and the liquid volume in each bottle was 100mL; the culture was carried out at 28°C and a shaker speed of 180rpm for 2d, and the color change of the solution was observed at 12h, 24h, 36h and 48h respectively.

[0054] The results are as follows Figure 1 As shown, the strain that turned red first at 12 hours had a high ability to reduce selenite and was named △PS04-2 strain; the wild-type strain PS04 of Paenibacillus cribenbergii did not turn red within 48 hours, indicating that the aerospace mutant strain had the ability to reduce sodium selenite and convert it into nano-selenium compared with the wild-type strain PS04 of Paenibacillus cribenbergii.

[0055] 3. Classification and identification of strains

[0056] The strain was further classified and identified, and the 16S rDNA of the strain was amplified using specific primers and sequence comparison analysis was performed. The results showed that the 16S rDNA sequence of strain △PS04-2 was as shown in SEQ ID NO.1. According to the taxonomic identification results, the strain △PS04-2 had a homology of more than 99% with Paenibacillus kribbensis. It was finally identified as Paenibacillus kribbensis and named △PS04-2 strain. It was preserved in Guangdong Provincial Microbiological Culture Collection Center on September 25, 2024, with the preservation number GDMCC No: 65207, and the preservation address is: Guangdong Provincial Institute of Scientific Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0057] Example 2 Determination of selenium resistance and selenium conversion ability of strain △PS04-2

[0058] 1. Tolerance of △PS04-2 to selenite

[0059] Prepare a liquid culture medium containing 3.0 g / L sodium nitrate, 30 g / L sucrose, 1.0 g / L potassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate heptahydrate, and 0-1000 mM sodium selenite, respectively; then add the seed culture solution of the △PS04-2 strain at a 1% inoculation amount to the liquid culture medium with final concentrations of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mM sodium selenite, respectively; the culture temperature is 28°C, the rotation speed is 150 r / min, and the culture time is 48 h.

[0060] The results are as follows Figure 2 As shown, the △PS04-2 strain can grow well in 100-1000mM sodium selenite medium and can reduce sodium selenite to red nano-selenium, indicating that the △PS04-2 strain has a strong tolerance to selenite and can tolerate concentrations exceeding 1000mM.

[0061] 2. The conversion ability of △PS04-2 to sodium selenite

[0062] The strain was activated and prepared to a concentration of 1×10 6cuf / mL seed liquid was inoculated into Czapek's culture medium with selenium salt concentrations of 0.5, 1, 2, 3, 4, 5 and 6 mM at an inoculum size of 1%, and Czapek's culture medium without selenium salt was used as a control treatment; 3 replicates were set for each treatment, and the liquid volume in each bottle was 150 mL; the culture was carried out at 28°C and a shaker speed of 150 rpm for 2 days, and then the fermentation liquid treated with selenium was centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken, and the sodium selenite content was determined by Agilent 5800 inductively coupled plasma emission spectrometry.

[0063] The test results are shown in Table 1, which show that the content of inorganic selenium in the total selenium of the fermentation systems treated with all different sodium selenite concentrations is less than 1%, indicating that more than 99% of the selenium salts are converted into elemental selenium and bacterial organic selenium.

[0064] Table 1 Conversion rate of strains to different sodium selenite concentrations

[0065]

[0066] Example 3 Separation, purification and identification of nano-selenium

[0067] 1. Separation and purification of nano-selenium

[0068] The preparation method comprises 3.0 g / L sodium nitrate, 30 g / L sucrose, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium chloride, and 0.01 g / L ferrous sulfate heptahydrate, and then the seed culture solution of the △PS04-2 strain is added to the liquid culture medium with a final concentration of 5 mM sodium selenite at a 1% inoculation amount, and the culture temperature is 28°C, the rotation speed is 150 rpm, and the culture time is 48 hours. The fermentation liquid obtained after the culture is centrifuged at 12000 rpm for 10 minutes, the supernatant is discarded, and the cell volume of the precipitate is adjusted by adding sterile water to make it not less than 1 / 2 of the original sample volume, and the cell volume is crushed by ultrasonication in an ice bath, and the ultrasonic conditions are set to 450 W power, ultrasonic operation 5s pause 5s, and the total operation time is 40 minutes. After the ultrasonication, the cell is centrifuged at 12000 rpm for 10 minutes, and the precipitate is washed continuously 3 times with 1.5 mol / LTris / HCl buffer (pH 8.3), and the nano-selenium precipitate of the culture is obtained by centrifugation.

[0069] The precipitate was resuspended in 4 mL of ultrapure water, 2 mL of n-octanol was added, and the mixture was shaken on an oscillator for 5 min. The mixture was centrifuged at 3000 rpm for 5 min and placed in a 4°C refrigerator for 24 h to allow the solution to be clearly separated. The red nano-selenium was precipitated at the bottom of the centrifuge tube, and the cells between the two phases were discarded. The precipitate was washed twice with sterile water. Finally, the nano-selenium particles were resuspended in 10 mL of ultrapure water and freeze-dried to obtain pure nano-selenium particles.

[0070] 2. Identification of Nano-Selenium

[0071] The seed culture fluid of the △PS04-2 strain was added at a volume ratio of 1% inoculation to a liquid fermentation medium containing 3.0 g / L sodium nitrate, 30 g / L sucrose, 1.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate heptahydrate and 5 mM sodium selenite, and the culture conditions were 30°C, 150 rpm, and cultured for 48 hours. At the same time, a group of liquid culture media containing 3.0 g / L sodium nitrate, 30 g / L sucrose, 1.0 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L potassium chloride, and 0.01 g / L ferrous sulfate heptahydrate was set up as a control group and cultured under the same culture conditions.

[0072] After the culture, the sample was centrifuged at 12000rpm for 10min, the supernatant was discarded, and the precipitate was added to 2.5% glutaraldehyde solution and fixed at 4°C overnight. The sample was removed from the glutaraldehyde and dehydrated in a gradient of 50%, 70%, and 100% ethanol, with each replacement lasting about 15 minutes. Then the sample was removed and placed in a supercritical dryer for drying. After drying for about 1 hour, the sample was removed, fixed on the sample stage, sprayed with gold, and tested. A Hitachi SU8010 scanning electron microscope was used for imaging and photography, and the elemental composition of the nano-selenium particles was observed and analyzed by EDS using a scanning electron microscope.

[0073] Electron microscopic images show that the nano-selenium synthesized by bacterial cells is distributed both inside and outside the cells. In bacterial cultures without the addition of selenite, no synthesis of nano-selenium was observed. The scanning electron microscopic image of the blank control group is shown in Figure 2. Figure 3 As shown in Figure 2, in the bacterial culture without the addition of selenite, no synthesis of nano-selenium was observed. Figure 4 As shown in the figure, spherical nano-selenium is produced; and the scanning electron microscopy image of the purified nano-selenium particles is as follows Figure 5 As shown in the figure, the arrow indicates the nano-selenium particles. The particle size of the nano-selenium particles is 100-300nm. The energy spectrum of the nano-selenium particles is as follows: Figure 6 As shown, the figure shows the characteristic peak of Se, indicating that the prepared nanoparticles are nano-selenium particles, and the nano-selenium content is determined to be 513.05 mg / kg.

[0074] In order to more clearly understand the characteristics of the synthesis of nano-selenium by the △PS04-2 strain, the samples after the culture were centrifuged at 12000rpm for 10min, the supernatant was discarded, and the precipitate was added to a 2.5% glutaraldehyde solution and fixed overnight at 4°C. The fixative was poured out and the samples were rinsed three times with 0.1M phosphate buffer for 15min each time; the samples were fixed with 1% osmium acid solution for 1-2h; the osmium acid waste liquid was taken out and the samples were rinsed three times with 0.1M phosphate buffer for 15min each time; gradient concentrations (including 30%, 5 The samples were dehydrated with ethanol solutions of five concentrations (0%, 70%, 80%, 90% and 95%), and each concentration was treated for 15 minutes; then treated with 100% ethanol for 20 minutes; finally, transitioned to pure acetone for 20 minutes; infiltration, the samples were treated with embedding agent: acetone = 1:1 for 1 hour, the samples were treated with a mixture of embedding agent: acetone 3:1 for 3 hours, and the samples were treated with pure embedding agent overnight; the infiltration-treated samples were embedded and heated at 70℃ overnight to obtain embedded samples; the samples were sliced ​​in a LEICA EMUC7 ultrathin microtome to obtain 70-90nm slices; the slices were stained with lead citrate stain and dioxygen acetate stain for 5 minutes each, then dried and observed on a microscope.

[0075] The results are as follows Figure 7 As shown, the location of nano-selenium particles can be clearly identified through transmission electron microscopy observation. Electron microscopic photos show that nano-selenium synthesized by bacterial cells is distributed both inside and outside the cells, thus inferring that the △PS04-2 strain produces selenium intracellularly.

[0076] Example 4 △ PS04-2 Synthetic Nano-Selenium's Control Effect on Pathogens

[0077] The control effect of nano-selenium synthesized by the △PS04-2 strain on various common plant fungal disease pathogens was determined, including the strawberry root rot pathogen (Neopestalotio psisclavispora), fruit anthracnose pathogen (Colletotrichum fructicola), wheat fusarium graminearum, and wheat stem rot pathogen (Fusarium pesudograminearum).

[0078] Inhibitory activity on the growth of pathogenic fungi: The plate confrontation culture method was used. A 5 mm cake of the corresponding pathogenic fungi was inoculated in the center of the PDA plate. Then 20 μL of the nano-selenium solution was dripped on 4 points 2.5 cm away from the cake using a pipette. The culture plate inoculated only with pathogenic fungi was used as a control and cultured at a constant temperature of 28°C. After 5 days of confrontation culture, the colony diameter was measured and the inhibition rate was calculated.

[0079] Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0080] The results showed that compared with the control group ( Figure 8 ), the nano-selenium synthesized by the △PS04-2 strain has a good inhibitory effect on the growth of four pathogenic fungi: strawberry root rot, anthracnose, wheat fusarium rust, and wheat stem base rot. Fig. 9 As shown, the antibacterial rates were 73%, 96%, 86% and 73% respectively.

[0081] At the same time, the inhibitory effect of nano-selenium synthesized by △PS04-2 strain on Ralstonia solanacearum was also determined. The plate diffusion method was used to inoculate Ralstonia solanacearum in liquid culture medium and cultured in a shaking incubator at 37°C and 180 rpm for 48 h. The LB culture medium was heated and the temperature was lowered to about 50°C. 1% (OD 600 =1) of Ralstonia solanacearum culture medium, and after the culture medium solidified, 5 μL of nano-selenium synthesized by △PS04-2 strain was added to the center of the plate, and 5 μL of Czapek liquid medium was added as the blank group. Each treatment was repeated 3 times, and the plate was sealed and placed in a 37°C bacterial incubator for 2 days. The growth of the strain was observed and the inhibition zone was recorded. The diameter of the inhibition zone was measured by the cross method. Fig.10 As shown, nano-selenium synthesized by the △PS04-2 strain also has a good inhibitory effect on Ralstonia solanacearum, showing that its inhibition zone is as high as 30mm.

[0082] Example 5 △ PS04-2 Synthetic Nano-Selenium Control Effect on Pepper Anthracnose

[0083] The nano-selenium solution synthesized by the strain △PS04-2 of Paenibacillus Cribbenensis was prepared by the method of Example 3. Select pepper fruits of uniform size, clean them, wipe them with 75% alcohol for disinfection, and dry them for use. The pepper fruits were stabbed with a sterile toothpick, and the treatment group was first fully and evenly sprayed with the nano-selenium solution synthesized by the △PS04-2 strain and then dried. Fresh pepper fruit anthracnose bacteria (Colletotrichum fructicola) blocks (8 mm in diameter) were inoculated at the stabbing position, covered with sterilized cotton and moisturized with sterile water, and stored in an incubator at 28°C and 85% humidity. The pepper fruits sprayed with sterile water were used as the control. Each treatment was repeated 3 times, with 5 fruits in each repeat. By measuring the area of ​​pepper fruit lesions, the release of pepper lesions was recorded and the relative inhibition rate was calculated.

[0084] The results are as follows Fig.11 As shown in the figure, the average lesion area of ​​the control group was 3.23 cm 2 The average lesion area of ​​the treatment group was 0.07 cm 2, indicating that the nano-selenium synthesized by the △PS04-2 strain has a good preventive and therapeutic effect on pepper anthracnose, with a prevention efficiency of 97.6%.

[0085] Example 6 Application of PS04-2 Synthetic Nano-Selenium in Selenium-enriched Sesame Production

[0086] From May to August 2024, field trials were conducted in the agricultural land of Qianfang Town, Jinxian County, Nanchang City, Jiangxi Province. The crop planted was sesame, and the variety was Ganzhi No. 7. In late May, 30 kg of 45% Stanley potassium sulfate compound fertilizer (15-15-15) was applied per mu of land, and agricultural machinery was used for rotary tillage after application. In early June, ridge plots were taken, each plot was 30 square meters (4m×7.5m), and row sowing was adopted in each plot, with 12 rows in each plot, and the amount of seeds used per mu was about 300g. After sowing, 90 mL of 720g / L isopropylamine was used per mu, and 15 kg of water was added for spraying for closed weed control.

[0087] The plot was divided into two groups: a control group (CK) and a treatment group (△PS04-2 synthetic nano-selenium). The CK group was fertilized conventionally, and the nano-selenium group was fertilized conventionally + nano-selenium, that is, nano-selenium bacteria were applied on the basis of conventional fertilization. The time and amount of conventional fertilization were the same. Except for the difference in whether or not to apply elemental selenium bacteria, the other field management of the two groups was the same.

[0088] The single-substance selenium bacteria agent group used 200mL of microbial nano-selenium bacteria agent diluted with 15kg of water to drench the roots once every 15 days starting from June 27; and used 200mL of single-substance selenium bacteria agent diluted with 15kg of water to spray the leaves every 15 days starting from July 27. No other pesticides were used in the plot to prevent and control pests and diseases during the trial. The yield was measured by a three-point random sampling method when sesame was harvested, and the biological traits of sesame were measured and counted.

[0089] The statistical results of sesame biological traits are shown in Table 2. After four times of spraying with selenium-enriched bacteria, the growth of sesame was significantly promoted. After testing, the selenium content of sesame seeds reached 0.29±0.20 mg / kg, reaching the standard of selenium-enriched sesame.

[0090] Table 2 Characteristics of selenium-enriched sesame

[0091]

[0092] In summary, the present invention obtains a strain of Paenibacillus cribensis aerospace mutant △PS04-2 strain, which has high selenite reduction ability and can tolerate high concentrations of selenite, and has strong tolerance; it also has the ability to efficiently convert sodium selenite to generate nano-selenium, and can be used to synthesize nano-selenium, and the nano-selenium content reaches 513.05mg / kg, and its preparation method has low equipment requirements, simple process, low cost, high product purity, and environmental friendliness. The nano-selenium synthesized by the △PS04-2 strain has a good antagonistic effect on a variety of plant pathogenic fungi, can be used to prevent and treat strawberry root rot, anthrax, wheat fusarium rust, wheat stem base rot, bacterial wilt and other plant diseases, and has good development and application potential in biocontrol agent selenium-enriched microbial preparations. At the same time, the synthesized nano-selenium is used in sesame production, which can not only promote sesame growth, but also obtain selenium-enriched sesame, which provides more effective methods for increasing the selenium content of crops.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Paenibacillus kribbensis ΔPS04-2 strain, characterized in that: This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 25, 2024, with the deposit number GDMCC No: 65207.

2. Use of the ΔPS04-2 strain according to claim 1 in converting inorganic selenium into nano-selenium or in preparing nano-selenium.

3. A nano-selenium, characterized in that: It is prepared by the △PS04-2 strain described in claim 1.

4. The method for preparing nano-selenium according to claim 3, characterized in that: The △PS04-2 strain described in claim 1 is activated and cultured to obtain a seed culture solution, and then the seed culture solution is inoculated into a liquid fermentation medium containing sodium selenite, cultured by shaking, the fermentation solution is collected and centrifuged, the supernatant is discarded, the precipitate is washed, and nano-selenium is obtained after purification; The liquid fermentation medium containing sodium selenite comprises: 3.0-5.0 g / L sodium nitrate, 30-60 g / L sucrose, 1.0-3 g / L dipotassium hydrogen phosphate, 0.5-1 g / L magnesium sulfate heptahydrate, 0.5-1 g / L potassium chloride, 0.01-0.02 g / L ferrous sulfate heptahydrate and 0.5-10 mM sodium selenite, with a pH of 6.5-7.

5.

5. The use of nano-selenium according to claim 3 in inhibiting plant pathogens or in preventing and treating plant diseases caused by pathogens, characterized in that: The pathogen is one or more of Neopestalotio psisclavispora, Colletotrichum fructicola, Fusarium graminearum, Fusarium pesudograminearum, and Ralstonia solanacearum; the plant disease is one or more of strawberry root rot, pepper anthracnose, wheat fusarium fusarium, wheat stem base rot, rice sheath blight, and sesame bacterial wilt.

6. Use of the nano-selenium according to claim 3 in promoting the growth of sesame and increasing the selenium content of sesame.

7. Use of the nano-selenium according to claim 3 in the preparation of antibacterial products or growth-promoting products.

8. A product, characterized in that Containing the nano-selenium as claimed in claim 3.

9. A composite microbial agent containing the ΔPS04-2 strain according to claim 1.

10. A method for producing selenium-enriched sesame, characterized in that: The nano-selenium described in claim 3 is used to spray sesame plants.

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