Paenibacillus chibensis space mutant strain and application thereof in preparation of nano-selenium

Abstract

The application discloses a strain of Paenibacillus kribbensis spaceflight mutant and application thereof in preparation of nano selenium. The application obtains a strain of Paenibacillus kribbensis △PS04-2 through spaceflight mutation, the strain has high reduction capacity for selenite, can tolerate high-concentration selenite, and can tolerate a concentration of more than 1000 mM; the strain also has the capacity of efficiently converting sodium selenite into nano selenium, the synthesized nano selenium has high conversion rate, the nano selenium particles are uniform, the preparation method is simple and convenient, the cost is low, the product has high purity, and the method is environment-friendly. The nano selenium synthesized by the strain of Paenibacillus kribbensis △PS04-2 has good antagonistic effect on various plant pathogenic fungi, can prevent and treat various plant diseases, and can develop more selenium-rich microbial preparations. Meanwhile, the nano selenium synthesized by the strain of Paenibacillus kribbensis △PS04-2 is used in sesame production, can promote the growth of sesame, and can obtain selenium-rich sesame, thereby providing more effective methods for improving the selenium content of crops.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganism synthesis of nanomaterials. More specifically, it relates to a strain of Paenibacillus kribbensis spaceflight mutant and its application in the preparation of nanometer selenium. BACKGROUND

[0002] Selenium is one of the essential trace elements for the human body, and is crucial for maintaining normal physiological functions and health. It is involved in the activity of various enzymes, including glutathione peroxidase (GPx), which helps protect cells from oxidative stress. Selenium is a component of some antioxidant enzymes that can protect cells from damage by free radicals, reducing oxidative stress. Selenium is essential for the normal functioning of the immune system, as it helps regulate immune responses and enhance the activity of immune cells. Selenium plays an important role in the synthesis and metabolism of thyroid hormones, as it is involved in the metabolism of iodine and the synthesis of thyroid hormones. Some studies suggest that selenium may have anticancer effects, particularly in preventing certain types of cancer such as prostate, colon, and lung cancer. While selenium is an essential trace element, excessive intake of selenium can also lead to selenium poisoning, with symptoms such as hair loss, nail deformation, and skin damage. Selenium is unevenly distributed on Earth, with higher selenium content in the soil of some areas and lower content in others. This affects the selenium content in the local food chain, which in turn affects the selenium intake of residents. The main sources of selenium include Brazil nuts, meat, fish, grains, and some vegetables. The selenium content in different foods varies greatly, depending on the selenium content in the soil where they grow.

[0003] Selenite is the most common form of selenium in nature, however, due to its high toxicity and high mobility, areas with excessive selenite concentration often face significant ecological risks. Elemental nanometer selenium is the least toxic form of selenium discovered, and the reduction of high-toxicity selenite to low-toxicity elemental nanometer selenium is an important means for selenium pollution treatment and selenium resource recovery. Traditional selenite removal and nanometer selenium preparation usually use chemical methods, such as adding vitamin C reducing agent and bovine serum albumin stabilizer to prepare nanometer selenium, but the process has the disadvantages of high cost, low purity of nanometer selenium product, uneven particle size distribution, and poor stability, which limits the application of nanometer selenium.

[0004] With more and more research on microorganisms, at present, selenium nanoparticles can be synthesized by reducing selenite through microbial strains, such as Bacillus sp., Bacillus amyloliquefaciens, Escherichia coli, Rhodospirillum molisch and Pseudomonas fluorescens. The biological selenium nanoparticles synthesized by microbial strains have stable properties, high activity and simple preparation method. However, due to the great difference in properties of different strains, the tolerance of different strains to sodium selenite is not high, the synthesis efficiency is low, and at present, there is also a lack of excellent strain resources that can synthesize selenium nanoparticles to efficiently convert sodium selenite into selenium nanoparticles. Therefore, screening and developing more microbial strains that can tolerate higher concentration of sodium selenite are of great significance for promoting the synthesis of biological selenium nanoparticles. SUMMARY

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

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

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

[0008] The third object of the present application is to provide a kind of selenium nanoparticles.

[0009] The fourth object of the present application is to provide a preparation method of selenium nanoparticles.

[0010] The fifth object of the present application is to provide the application of selenium nanoparticles.

[0011] The sixth object of the present application is to provide a kind of selenium nanoparticles.

[0012] The seventh object of the present application is to provide a composite microbial inoculant containing △PS04-2 strain.

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

[0014] The above objects of the present application are achieved by the following technical solutions:

[0015] The present application provides a Kribbensis Paenibacillus strain △PS04-2, which has been preserved in Guangdong Microbial Culture Collection Center on September 25, 2024, and the preservation number is GDMCC No:65207.

[0016] The application obtains a strain of Paenibacillus kurstaki △PS04-2 from a mutant library of the wild type strain PS04 of Paenibacillus kurstaki by space mutation, which has high reduction ability of selenite and can tolerate high concentration of selenite, and can tolerate a concentration of more than 1000 mM; and has the ability of high-efficiency conversion of sodium selenite into nano-selenium, and can convert inorganic selenium into nano-selenium for preparing nano-selenium, and the content of nano-selenium reaches 513.05 mg / kg; the method for efficiently producing extracellular nano-selenium by reducing sodium selenite to synthesize nano-selenium has mild reaction conditions and simple operation, improves the conversion efficiency of nano-selenium, and the method for preparing nano-selenium from the strain of △PS04-2 has low requirements for equipment, simple process, low cost, high purity of product and environmental friendliness, and provides more strain resources for microbial synthesis of nano-selenium.

[0017] Therefore, the application protects the application of the strain of △PS04-2 in converting inorganic selenium into nano-selenium or in preparing nano-selenium.

[0018] The application provides a kind of nano-selenium, which is prepared from the strain of △PS04-2.

[0019] The application provides a method for preparing nano-selenium, which comprises the following steps: activating and culturing the strain of △PS04-2 to obtain a seed culture solution; inoculating the seed culture solution into a liquid fermentation medium containing sodium selenite; and performing oscillation culture, centrifuging the fermentation liquid, discarding the supernatant, washing the precipitate, and obtaining nano-selenium after purification; wherein the liquid fermentation medium containing sodium selenite comprises the following components: 3.0-5.0 g / L of sodium nitrate, 30-60 g / L of sucrose, 1.0-3.0 g / L of potassium phosphate dibasic, 0.5-1.0 g / L of magnesium sulfate heptahydrate, 0.5-1.0 g / L of potassium chloride, 0.01-0.02 g / L of ferrous sulfate heptahydrate, and 0.5-10 mM of sodium selenite, and the pH is 6.5-7.5.

[0020] As the most preferred preparation method, the application provides a specific method for converting inorganic selenium into nano-selenium by the strain of △PS04-2:

[0021] The strain of △PS04-2 is inoculated into a solid culture medium for constant temperature culture and activation; the activated strain of △PS04-2 is inoculated into a liquid culture medium for oscillation culture to obtain a seed culture solution; a liquid fermentation medium is prepared, and the concentration of sodium selenite in the liquid fermentation medium is 1×10 6The seed liquid of 1.5 mL is inoculated into a liquid fermentation medium containing sodium selenite at an inoculation amount of 1%, and is cultured under the condition of 28 DEG C and a shaking speed of 150 rpm for 2 days, and then the fermentation liquid is centrifuged at 12000 rpm for 5-10 min, the supernatant is discarded, and the purification is performed by washing twice with sterile water, and then the ultrasonic crushing is performed by adding sterile water in an ice bath, and the ultrasonic condition is set as follows: power 450 W, ultrasonic operation 5 s and pause 5 s, and the total operation time is 40 min, and after the ultrasonic operation, the centrifugation is performed at 12000 rpm for 10 min, the precipitate is washed three times with 1.5 mol / L Tris / HCl buffer (pH 8.3) in sequence, the precipitate is collected by centrifugation and is resuspended in 4 mL ultrapure water, 2 mL n-octanol is added, and the solution is shaken for 5 min, and then the centrifugation is performed at 3000 rpm for 5 min, and the solution is placed in a refrigerator at 4 DEG C for 24 h, so that the solution is obviously layered, and after the red nano-selenium precipitate is at the bottom of the centrifuge tube, the cell crushing between the two phases is discarded, the precipitate is washed twice with sterile water, and finally the nano-selenium particles are resuspended in 10 mL ultrapure water, and the pure nano-selenium particles are obtained by freeze-drying.

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

[0023] Meanwhile, the nano-selenium synthesized by the △PS04-2 strain has good antagonistic effect on various plant pathogenic fungi, and can be used for preventing and treating strawberry root rot, pepper anthracnose, wheat scab, wheat foot rot, sesame bacterial wilt and other plant diseases, and has good development and application potential in the field of selenium-rich microbial preparation of microbial agents.

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

[0025] Further, the pathogenic fungi are one or more of Neopestalotiopsis clavispora, Colletotrichum fructicola, Fusarium graminearum, Fusarium pesudograminearum and Ralstonia solanacearum, and the plant diseases are one or more of strawberry root rot, pepper anthracnose, wheat scab, wheat foot rot, rice sheath blight and bacterial wilt.

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

[0027] The application provides application of nano selenium in preparation of antibacterial products or growth promoting products.

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

[0029] The application provides a microbial nano selenium microbial agent containing the △PS04-2 strain.

[0030] The application provides a method for producing selenium-rich sesame, and the microbial nano selenium microbial agent is sprayed on sesame plants.

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

[0032] More preferably, the microbial nano selenium microbial agent is diluted 200 times, and then sprayed on the sesame four times during the growth period of the sesame, with an interval of 15 days, so that the yield is increased to reach the standard of selenium-rich sesame.

[0033] The application has the following beneficial effects:

[0034] The application obtains a strain of Paenibacillus chibensis △PS04-2 through spaceflight mutation, the strain has high reduction capacity for selenite, can tolerate high-concentration selenite, and can tolerate a concentration of more than 1000 mM; the strain also has high conversion capacity for selenite into nano selenium, the strain synthesizes nano selenium with high conversion rate, uniform nano selenium particles, and a nano selenium content of 513.05 mg / kg; the provided preparation method is simple, convenient, low in cost, high in product purity, and friendly to the environment. The nano selenium synthesized by the strain of Paenibacillus chibensis △PS04-2 has good antagonistic effect on various plant pathogenic fungi, can prevent and treat various plant diseases, and can develop more selenium-rich microbial agents. Meanwhile, the nano selenium synthesized by the strain of Paenibacillus chibensis △PS04-2 is used for sesame production, can promote the growth of sesame, and can obtain selenium-rich sesame, thereby providing more effective methods for increasing the selenium content of crops. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A result graph of growth of different strains in a 10 mM sodium selenite-containing medium for 12 h (A1 in the figure is a wild-type strain of Paenibacillus chibensis PS04; A2-D6 are 23 different spaceflight mutant strains in sequence).

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

[0037] Figure 3 A scanning electron microscope observation graph of the strain of Paenibacillus chibensis △PS04-2.

[0038] Figure 4 Figure 1 is a scanning electron microscope observation diagram of ΔPS04-2 and extracellular nano-selenium.

[0039] Figure 5 Figure 2 is a scanning electron microscope observation diagram of purified ΔPS04-2 synthesized nano-selenium particles.

[0040] Figure 6 Figure 3 is an EDS analysis diagram of the elemental composition of the purified nano-selenium particles.

[0041] Figure 7 Figure 4 is a transmission electron microscope observation diagram of nano-selenium particles.

[0042] Figure 8 Figure 5 is a control group's prevention effect on plant pathogenic fungi (A is the pathogenic fungus of strawberry root rot; B is the pathogenic fungus of anthracnose; C is the pathogenic fungus of wheat scab; D is the pathogenic fungus of wheat foot rot).

[0043] Figure 9 Figure 6 is the prevention effect of ΔPS04-2 synthesized nano-selenium on plant pathogenic fungi (A is the pathogenic fungus of strawberry root rot; B is the pathogenic fungus of anthracnose; C is the pathogenic fungus of wheat scab; D is the pathogenic fungus of wheat foot rot).

[0044] Figure 10 Figure 7 is the inhibition effect of ΔPS04-2 synthesized nano-selenium on sesame bacterial wilt.

[0045] Figure 11 Figure 8 is the prevention effect on pepper anthracnose (A is the control group inoculated with pathogenic fungi only; B is the treatment group inoculated with pathogenic fungi after spraying nano-selenium solution). DETAILED DESCRIPTION

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

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

[0048] Example 1 Isolation and identification of Klebsiella-like Bacillus ΔPS04-2

[0049] 1. Obtaining of strain mutant library

[0050] The wild type strain PS04 of Paenibacillus kribbensis was divided into two parts, one was left on the ground for routine preservation as a control sample; the other was launched into space in March 2020 at the Hainan Wenchang Launch Site, and a mutant library of the wild type strain PS04 of Paenibacillus kribbensis was obtained after a series of space condition mutagenesis (for specific mutagenesis method conditions, see the published text CN114480222A).

[0051] 2. Screening of selenite-reducing bacteria

[0052] Taking the wild type strain PS04 of Paenibacillus kribbensis as a control, the ability of different space mutagenized strains to convert selenite into nanoselenium was compared in the mutant library, and a mutant strain with high selenite reduction ability was screened.

[0053] The wild type strain PS04 of Paenibacillus kribbensis and 23 space mutant strains were activated, and seed liquid with a concentration of 1×10 6 cuf / mL was prepared, and was inoculated into Czapek's culture solution containing 10 mM of selenium salt at an inoculation amount of 1%, and the Czapek's culture solution without sodium selenite was used as a control treatment; each treatment had 3 replicates, and the liquid volume of each bottle was 100 mL; the culture was carried out at 28℃ and a shaking speed of 180 rpm for 2 days, and the color change of the solution was observed at 12 h, 24 h, 36 h and 48 h, respectively.

[0054] The results are shown in Table 1. Figure 1 As shown in Table 1, the strain that turned red first at 12 h had high selenite reduction ability, and was named as △PS04-2 strain; the wild type strain PS04 of Paenibacillus kribbensis did not turn red within 48 h, indicating that the space mutant strain had the ability to reduce sodium selenite to nanoselenium compared with the wild type strain PS04 of Paenibacillus kribbensis.

[0055] 3. Classification and identification of the strain

[0056] Further classification and identification of the strain were conducted. Specific primers were used to amplify the 16S rDNA of the strain, and sequence alignment analysis was performed. The results showed that the 16S rDNA sequence of strain △PS04-2 is shown in SEQ ID NO.1. According to taxonomic identification results, strain △PS04-2 shares more than 99% homology with *Paenibacillus kribbensis*. It was ultimately identified as *Paenibacillus kribbensis* and named strain △PS04-2. It was deposited on September 25, 2024, at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No:65207, located at the Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

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

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

[0059] 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, 0.01 g / L ferrous sulfate heptahydrate, and sodium selenite (0–1000 mM) were prepared. Seed culture of strain △PS04-2 was then added at a 1% inoculum to the liquid culture media with final concentrations of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mM sodium selenite. The culture temperature was 28℃, the rotation speed was 150 r / min, and the culture time was 48 h.

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

[0061] 2. Conversion capacity of △PS04-2 for sodium selenite

[0062] The strain was first activated to a concentration of 1×10⁻⁶. 6The seed liquid of 1 mL was inoculated into Chai's culture medium containing selenium salt at concentrations of 0.5, 1, 2, 3, 4, 5 and 6 mM at an inoculation amount of 1%, and Chai's culture medium without selenium salt was used as a control treatment. Each treatment had 3 replicates, and the liquid volume in each bottle was 150 mL. The fermentation was carried out at 28°C and a shaking speed of 150 rpm for 2 days, and then the fermentation liquid containing selenium was centrifuged at 12000 rpm for 5 min. The supernatant was taken, and the sodium selenite content was determined by Agilent 5800 inductively coupled plasma emission spectrometry.

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

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

[0065]

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

[0067] 1. Separation and purification of nano selenium

[0068] A liquid medium containing sodium nitrate 3.0 g / L, sucrose 30 g / L, potassium phosphate dibasic 1.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L was prepared, and the seed culture liquid of △PS04-2 strain was added to the liquid medium at a final concentration of 5 mM sodium selenite at an inoculation amount of 1%. The culture temperature was 28°C, the rotation speed was 150 rpm, and the culture time was 48 h. The fermentation liquid obtained after the culture was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitated cells were washed twice with sterile water. Sterile water was added to adjust the volume of the precipitated cells to not less than 1 / 2 of the original sample volume. The cells were broken by ice bath ultrasonication under the following conditions: power 450 W, ultrasonic operation 5 s and pause 5 s, and total operation time 40 min. After ultrasonication, the cells were centrifuged at 12000 rpm for 10 min. The precipitate was washed with 1.5 mol / L Tris / HCl buffer (pH 8.3) for 3 times, and the nano selenium precipitate of the culture was obtained by centrifugation.

[0069] The precipitate was resuspended in 4 mL ultrapure water, 2 mL n-octanol was added, and the solution was shaken for 5 min at 3000 rpm. The solution was allowed to stand in a 4°C refrigerator for 24 h to allow the solution to separate into layers. The red nano selenium precipitate was at the bottom of the centrifuge tube, and the cell fragments between the two phases were discarded. The precipitate was washed twice with sterile water. Finally, the nano selenium particles were resuspended in 10 mL ultrapure water, freeze-dried, and pure nano selenium particles were obtained.

[0070] 2. Identification of nano selenium

[0071] The seed culture solution of the ΔPSO4-2 strain was added to a liquid fermentation medium containing sodium nitrate 3.0 g / L, sucrose 30 g / L, potassium phosphate dibasic 1.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium chloride 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, and sodium selenite 5 mM at a volume ratio of 1% inoculation amount, and the culture conditions were 30°C, 150 rpm, and 48 h of culture. At the same time, a group of liquid medium containing sodium nitrate 3.0 g / L, sucrose 30 g / L, potassium phosphate dibasic 1.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium chloride 0.5 g / L, and ferrous sulfate heptahydrate 0.01 g / L was set as a control group, and the culture was carried out under the same culture conditions.

[0072] After the culture was completed, the sample was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was added to 2.5% glutaraldehyde solution for fixation at 4°C overnight. The sample was taken out from the glutaraldehyde, and gradient dehydration was carried out with 50%, 70%, and 100% ethanol, each for about 15 minutes, then taken out and placed in a supercritical drying machine for drying. After drying for about 1 hour, it was taken out, fixed on a sample stage, gold sprayed, and tested. Imaging and photographing were carried out using a Hitachi SU8010 scanning electron microscope, and EDS analysis of the elemental composition of the nanose particles was carried out by scanning electron microscope observation.

[0073] The electron microscope photograph showed that the nanose synthesized by the bacterial cells was distributed in and outside the cells. In the bacterial culture without the addition of selenite, the synthesis of nanose was not observed. The scanning electron microscope photograph of the blank control group is shown in Figure 3 , and in the bacterial culture without the addition of selenite, the synthesis of nanose was not observed. The scanning electron microscope photograph of the experimental group is shown in Figure 4 , showing the production of spherical nanose; and the scanning electron microscope photograph of the purified nanose particles is shown in Figure 5 , where the nanose particles are indicated by arrows. The nanose particle size was determined to be 100-300 nm, and the nanose particle elemental energy spectrum is shown in Figure 6 , which shows the characteristic peak of Se, indicating that the prepared nanoparticles are nanose particles, and the nanose content is determined to be 513.05 mg / kg.

[0074] In order to more clearly understand the characteristics of the synthesis of nanometer selenium by the △PS04-2 strain, the sample after the end of the culture was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, the precipitate was added with 2.5% glutaraldehyde solution and fixed at 4°C overnight, the fixing solution was poured out, the sample was rinsed with 0.1M phosphate buffer solution for three times, each time for 15 min; the sample was fixed with 1% osmium acid solution for 1-2 h; the osmium acid waste liquid was taken out, the sample was rinsed with 0.1M phosphate buffer solution for three times, each time for 15 min; the sample was dehydrated with gradient concentration (including 30%, 50%, 70%, 80%, 90% and 95% five concentrations) ethanol solution, each concentration for 15 min; then the sample was treated with 100% ethanol for 20 min; finally, the sample was excessively treated with pure acetone for 20 min; the sample was infiltrated, the sample was treated with embedding agent:acetone = 1:1 for 1 h, the sample was treated with the mixture of embedding agent:acetone = 3:1 for 3 h, and the sample was treated with pure embedding agent overnight; the sample after the infiltration treatment was embedded, and the embedded sample was obtained after heating at 70°C overnight; the sample was sliced in a LEICA EMUC7 ultrathin microtome, and a slice with a thickness of 70-90 nm was obtained; after the slice was dyed with lead citrate solution and acetic acid hydrogen peroxide solution for 5 min respectively, the slice was dried, and was observed under a microscope.

[0075] The results are shown in Table 1. Figure 7 As shown in Table 1, the position of the nanometer selenium particles can be clearly identified by transmission electron microscopy, and the electron micrograph shows that the nanometer selenium synthesized by the bacterial cells is distributed in and out of the cells, and it is inferred that the △PS04-2 strain belongs to intracellular selenium production.

[0076] Example 4: Prevention and treatment effect of nanometer selenium synthesized by △PS04-2 on pathogenic bacteria

[0077] The prevention and treatment effect of the nanometer selenium synthesized by the △PS04-2 strain on various common plant fungal disease pathogens was determined, and the pathogenic bacteria were strawberry root rot pathogen (Neopestalotiopsis clavispora), fruit anthracnose pathogen (Colletotrichum fructicola), wheat scab pathogen (Fusarium graminearum), and wheat basal stem rot pathogen (Fusarium pesudograminearum).

[0078] Inhibition activity of pathogenic fungi growth: plate confrontation culture method was adopted, 5mm corresponding pathogenic fungal cake was inoculated in the center of PDA plate, then 20μL nanometer selenium solution was dropped on four points 2.5cm away from the cake by using a pipette gun, the culture plate inoculated with pathogenic fungi only was used as a control, and the plates were placed in a constant temperature incubator at 28°C, and after 5d of confrontation culture, the colony diameter was measured and the inhibition rate was calculated.

[0079] Bacteriostatic rate = (control colony diameter - treatment colony diameter) / (control colony diameter - bacterial cake diameter) x 100%.

[0080] The results show 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 the four pathogenic fungi of strawberry root rot, anthracnose, wheat scab and wheat foot rot, as shown in Figure 9 , and the bacteriostatic rates are 73%, 96%, 86% and 73%, respectively.

[0081] Meanwhile, the inhibitory effect of the nano selenium synthesized by the △PS04-2 strain on Ralstonia solanacearum was also determined. The Ralstonia solanacearum was inoculated in a Ralstonia solanacearum liquid medium and cultured at 37°C and 180r / min for 48h. After the LB medium was heated, the temperature was reduced to about 50°C, 1% (OD 600 =1) of the Ralstonia solanacearum liquid was added, and after the medium was solidified, 5μL of the nano selenium synthesized by the △PS04-2 strain was added to the center of the plate. 5μL of the Czapek liquid medium was added as a blank group, each treatment was repeated three times, the plate was sealed and placed in a 37°C bacterial incubator for 2d, the strain growth was observed and the inhibition zone was recorded, and the inhibition zone diameter was measured by the cross method. As shown in Figure 10 , the nano selenium synthesized by the △PS04-2 strain also has a good inhibitory effect on Ralstonia solanacearum, and the inhibition zone is as high as 30mm.

[0082] Example 5 Prevention and treatment effect of the nano selenium synthesized by the △PS04-2 on pepper anthracnose

[0083] The nano selenium solution synthesized by the Paenibacillus campinasensis △PS04-2 strain was prepared by the method of Example 3. Uniformly sized bell pepper fruits were cleaned and disinfected by wiping with 75% alcohol, and then dried for standby. The bell pepper fruits were pricked with sterile toothpicks, the treatment group was first sprayed with the nano selenium solution synthesized by the △PS04-2 strain and then dried, and fresh bell pepper fruit anthracnose fungus (Colletotrichum fructicola) blocks (8mm in diameter) were inoculated at the pricked position, covered with sterilized cotton and moisturized with sterile water, and then stored in a 28°C, 85% humidity incubator. The bell pepper fruits sprayed with sterile water were used as a control. Each treatment was set with 3 repeats, and each repeat had 5 fruits. The relative inhibition rate was calculated by measuring the lesion area of the bell pepper fruits and recording the release of the bell pepper lesion.

[0084] The results are shown in Figure 11 . The average lesion area of the control group was 3.23cm 2 , and the average lesion area of the treatment group was 0.07cm 2, indicating that the nano-selenium synthesized by △PS04-2 strain has a good control effect on pepper anthracnose, and the control effect reaches 97.6%.

[0085] Example 6 Application of nano-selenium synthesized by △PS04-2 in the production of selenium-rich sesame

[0086] A field test was carried out in Qianfang Town, Jinxian County, Nanchang City, Jiangxi Province from May to August 2024. The crop planted was sesame, and the variety was Gan Zhi No. 7. In late May, 45% Stanley potassium sulfate compound fertilizer (15-15-15) 30 kg was applied per mu of land, and after application, the land was plowed and leveled using a farm machine. In early June, take the ridge and make the plot, each plot area is 30 square meters (4m x 7.5m), and each plot uses strip planting, 12 rows per plot, and about 300g of seeds per mu is used. After sowing, 720g / L metamifop 90mL is mixed with 15kg of water per mu to spray and close the weeds.

[0087] The plots were divided into a control group (CK) and a treatment group (nano-selenium synthesized by △PS04-2), the CK group was conventionally fertilized, and the nano-selenium group was conventionally fertilized + nano-selenium, that is, on the basis of conventional fertilization, nano-selenium bacterial agent was applied, and the time and amount of conventional fertilization were consistent; and the two groups were consistent in field management except for whether to apply elemental selenium bacterial agent.

[0088] The elemental selenium bacterial agent group started on June 27, with a 15-day interval, and used 200mL of microbial nano-selenium bacterial agent mixed with 15kg of water to root drenching once; starting from July 27, with a 15-day interval, 200mL of elemental selenium bacterial agent was mixed with 15kg of water to spray the leaves. During the test period, no other agents were used for disease and pest control in the plots. When the sesame was harvested, a 3-point random sampling method was used to measure the yield, and the biological properties of sesame were measured and counted.

[0089] The statistical results of the biological properties of sesame are shown in Table 2. After being treated with selenium-rich bacterial agent for four times, the growth of sesame was significantly promoted; through detection, the selenium content of sesame seeds reached 0.29±0.20mg / kg, reaching the standard of selenium-rich sesame.

[0090] Table 2 Properties of selenium-rich sesame

[0091]

[0092] In conclusion, the application obtains a strain of Paenibacillus koreensis space mutant △PS04-2, the strain has high ability of reducing selenite and can tolerate high concentration of selenite, and has high efficiency of transforming sodium selenite into nano selenium, and can be used for synthesizing nano selenium, the content of the nano selenium reaches 513.05 mg / kg, the preparation method has low requirement on equipment, simple process, low cost, high purity of product and environmental friendliness. The nano selenium synthesized by the strain △PS04-2 has good antagonistic effect on various plant pathogenic fungi, and can be used for preventing and treating strawberry root rot, anthracnose, wheat scab, wheat foot rot, bacterial wilt and other plant diseases, and has good development and application potential in the field of selenium-rich microbial preparation of fungicide. Meanwhile, the nano selenium synthesized by the strain can be used in sesame production, not only can promote the growth of sesame, but also can obtain selenium-rich sesame, and provides more effective methods for improving the selenium content of crops.

[0093] The above examples are the preferred embodiments of the application, but the embodiments of the application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the application shall be equivalent replacement methods, and all shall be included in the protection scope of the application.

Claims

1. A strain of Cribbella ( Paenibacillus kribbensis ) △PS04-2 strain, characterized in that, This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 25, 2024, with accession number GDMCC No:65207.

2. The application of the △PS04-2 strain according to claim 1 in the preparation of nano-selenium.

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

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

5.

5. The application of the nano-selenium according to claim 3 in inhibiting plant pathogens or in preventing and controlling plant diseases caused by pathogens, characterized in that, The pathogen is *Neoplasmosis rubescens* ( ). Neopestalotio psisclavispora Fruit anthracnose fungus ( Colletotrichum fructicola Fusarium graminearum ( ), Fusarium graminearum ( Fusarium graminearum ), Fusarium graminearum ( Fusarium pesudograminearum ), bacterial wilt ( Ralstonia solanacearum One or more of the following: strawberry root rot, pepper anthracnose, wheat scab, wheat stem base rot, rice sheath blight, and sesame bacterial wilt.

6. The application of the nano-selenium as described in claim 3 in promoting sesame growth and increasing the selenium content of sesame.

7. A product characterized in that, Contains the nano-selenium as described in claim 3.

8. A microbial inoculant containing the △PS04-2 strain as described in claim 1.

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

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