Selenium-enriched Paenibacillus splendidus strain FZY-10 and its application

Abstract

The present invention belongs to the field of microorganisms, and relates to a selenium-rich brilliant bacillus strain FZY-10 and an application thereof. The selenium-rich brilliant bacillus FZY-10 is named as brilliant bacillus Paenibacillus lautus, and the selenium-rich brilliant bacillus FZY-10 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 17, 2023, and the deposit number is CGMCC No. 26833. The present invention provides a selenium-rich brilliant bacillus strain FZY-10 and an application thereof.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and relates to a Paenibacillus splendens and applications thereof, and in particular to a selenium-enriched Paenibacillus splendens strain FZY-10 and applications thereof. Background Art

[0002] Selenium is an essential trace element for life. It is an essential component of selenocysteine ​​and selenoenzymes. It plays an important role in anti-cancer and anti-oxidation. Appropriate selenium supplementation helps improve human immunity, while selenium deficiency can cause many diseases, such as Keshan disease. Selenium has three biological functions: nutrition, toxicity and detoxification. It plays an important role in regulating normal human metabolism. Studies have shown that more than 40 human diseases such as cancer, heart disease, and diabetes are directly related to selenium deficiency in the body. Experts from the Institute of Oncology, Chinese Academy of Medical Sciences collected 1,458 blood samples from 24 cities in 8 provinces and measured the selenium content. The results showed that the mortality rates of gastric cancer, esophageal cancer, and liver cancer in low-selenium areas were significantly higher than those in high- and medium-selenium areas.

[0003] Like most countries in the world, my country is also a selenium-deficient region. There are more than 20 provinces and cities in my country that are selenium-deficient, and the selenium content in the soil is about 0.03-0.1 mg / kg. In the food chain, selenium mainly comes from plants, and supplementing selenium to the soil is an effective way to obtain selenium-rich agricultural products (food). Over the years, people have used raw materials such as selenium-rich ores and selenium-rich coal gangue to obtain a small amount (20%) of soluble inorganic selenium through crushing and alkali treatment, and mixed it with inorganic salts or organic matter such as nitrogen, phosphorus, potassium, copper, zinc, iron, magnesium, and boron to form selenium-rich fertilizers, or prepared selenium into sodium selenate and sodium selenite, and then mixed its solution with inorganic or organic fertilizers to form selenium-rich fertilizers, which are sprayed on crop leaves or directly applied to the soil. Although these selenium-rich fertilizers can produce selenium-rich agricultural products and selenium-rich foods, the utilization rate of inorganic selenium in fertilizers for crops is only 10%-15%, which is still very low. Applying organic selenium may be a way to improve the utilization rate of selenium in crops.

[0004] For example, a selenium-rich combined nitrogen-fixing bacterial agent invented by patent CN200710010871 adds soluble selenium to the preparation of the bacterial agent, and uses nitrogen-fixing bacteria to transform inorganic selenium, but only nitrogen-fixing bacteria are used in its preparation, and it is a selenium-rich microbial fertilizer with only a single nitrogen-fixing physiological function. Another example is the invention disclosure with publication number CN102618469A, which provides a capsule red bacterium and its application, and discloses a capsule red bacterium AYGHJ-16, which belongs to photosynthetic bacteria, uses light energy to metabolize substances, promotes the transformation and absorption of inorganic selenium, and has nitrogen fixation and carbon fixation. Another example is the invention with authorization announcement number 100512666C, which discloses a selenium-containing microbial feed additive and its preparation method, and discloses the use of live microbial bacteria such as red pseudomonas, natto bacillus and yeast for liquid fermentation and solid fermentation to achieve microbial transformation of inorganic selenium. In addition, the invention patent with the authorization announcement number CN 115261257B discloses a selenium-enriched Bacillus subtilis L11 derived from silage and its application. The Bacillus subtilis is isolated from silage and has obvious antagonistic effects on common mold and other diseases, but the Bacillus subtilis does not have any effect on promoting the growth of crops. From the search results, the current bacterial agents use a single strain or similar strains for the conversion of selenium. At the same time, no Bacillus, especially Paenibacillus splendidus, has been found to be used in selenium enrichment. Summary of the invention

[0005] In order to solve the above technical problems existing in the background technology, the present invention provides a selenium-enriched Paenibacillus splendens strain FZY-10 and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A selenium-rich brilliant Bacillus FZY-10, characterized in that: the selenium-rich brilliant Bacillus FZY-10 is named as brilliant Bacillus Paenibacillus lautus, and the selenium-rich brilliant Bacillus FZY-10 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on March 17, 2023, and the deposit number is CGMCC No. 26833.

[0008] According to the application of selenium-rich brilliant Bacillus FZY-10 in promoting plant growth as described above.

[0009] According to the application of selenium-enriched brilliant Bacillus FZY-10 to promote plant growth under the condition of selenium enrichment as described above.

[0010] According to the application of selenium-enriched Paenibacillus splendens FZY-10 to promote plant growth in selenium-enriched soil as described above.

[0011] According to the application of selenium-rich brilliant Bacillus FZY-10 in promoting crop growth as described above.

[0012] According to the application of selenium-rich brilliant Bacillus FZY-10 in promoting corn growth as described above.

[0013] The advantages of the present invention are:

[0014] The invention provides a selenium-enriched Paenibacillus splendens FZY-10. The strain can be used to prepare a selenium-enriched bacterial agent. The preparation method can be prepared using a traditional general fermentation process, the operation method is simple, and the production cost is low. The prepared bacterial agent has the characteristics of high selenium resistance, and has multiple functions such as selenium enrichment, growth promotion, and regulating plant rhizosphere health for crops. Soluble selenium can promote the expression of corresponding differential genes of crops after further regulation by microorganisms, which is more conducive to the absorption and utilization of selenium by crops. Therefore, after application, the yield of crops can be increased, the quality of agricultural products can be improved, the comprehensive selenium content of agricultural products is increased by 47.42%, the selenium-enriched effect of crops is enhanced, and the quality standard of selenium-enriched products is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The colony morphology of the selenium-enriched Paenibacillus lautus strain FZY-10 provided by the present invention (A) and the colony morphology of the selenium-enriched culture (B);

[0016] Figure 2 This is a diagram showing the growth effect of corn after applying the selenium-rich Paenibacillus splendens FZY-10 bacterial agent;

[0017] Figure 3 This is the growth curve of the selenium-rich Paenibacillus lautus strain FZY-10;

[0018] Figure 4 The electron microscope scanning image of the selenium-enriched Paenibacillus lautus strain FZY-10 (A) and the electron microscope image of selenium enrichment distribution in the bacteria (B);

[0019] Figure 5 This is the energy spectrum analysis diagram of the selenium-rich Paenibacillus lautus strain FZY-10;

[0020] Figure 6 The effect of using selenium-rich Paenibacillus lautus strain FZY-10 to promote root dry weight growth in corn;

[0021] Figure 7The effect of using selenium-rich Paenibacillus lautus strain FZY-10 to promote the aboveground dry weight of corn;

[0022] Figure 8 The effect diagram of promoting root fresh weight of corn using selenium-rich Paenibacillus lautus strain FZY-10;

[0023] Fig. 9 The effect diagram of using selenium-rich Paenibacillus lautus strain FZY-10 to promote aboveground fresh weight of corn;

[0024] Fig.10 The effect of using selenium-rich Paenibacillus lautus strain FZY-10 to promote stem thickness in corn;

[0025] Fig.11 The growth-promoting effect of using selenium-rich Paenibacillus lautus strain FZY-10 on corn;

[0026] Fig.12 To evaluate the selenium-enriched effect of applying selenium-enriched Paenibacillus splendidus FZY-10 to corn;

[0027] Fig.13 This is the phylogenetic tree of the selenium-enriched Paenibacillus lautus strain FZY-10. DETAILED DESCRIPTION

[0028] Example 1 Isolation and identification of selenium-enriched functional strains

[0029] From the mountain meadow soil in Shuang'an Town, Ziyang County, Ankang City, Shaanxi Province, located at the northern foot of Daba Mountain (31°16′N, 108°57′E), a strain FZY-10 was screened out by enrichment, separation and purification in a beef extract peptone medium prepared with 3.0g beef extract, 10.0g peptone, 15.0g agar, 3.0g NaCl, 0.2g NaOH, 80mg / L sodium selenite, and 1L distilled water. The specific operation method is as follows:

[0030] (1) Isolation of bacterial strains

[0031] Weigh 10.0 g of rhizosphere soil sample and put it into a sterile fermentation tank containing 90 mL of sterile water. Fix it on a shaker and shake it at 200 r / min for 10 min. Take it out and draw 1 mL on a sterile operating table and add it to a test tube containing 9 mL of sterile water. And so on. 0 , 10-1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Take 10% of the above dilution -2 , 10 -4 , 10 -6 The soil suspension was inoculated into the bacterial culture medium for dilution and coating, and then placed in a constant temperature incubator at 30°C for culture. When complete colonies were formed in the culture medium, colonies of different forms were picked out by visual inspection with sterilized bamboo sticks and inoculated on new plates to continue culturing for one cycle. Monoclonal colonies were then obtained by continuous streaking culture. The strains were preserved on solid slant and 20% sterile glycerol for subsequent experiments on the tolerance of the strains to selenite and the conversion rate.

[0032] (2) Bacterial Activation

[0033] In order to improve the activity of the strain, the preserved FZY-10 strain was inoculated from the test tube into the solid culture medium and cultured in a constant temperature incubator at 32°C for more than 2 days until a single colony grew on the plate. A single colony was picked from the plate and inoculated into a test tube containing liquid culture medium and cultured at 32°C with shaking at 180 rpm to obtain the activated strain.

[0034] (3) Initial screening of strains

[0035] The activated strains were cultured in a solid selective medium containing sodium selenite, with a sodium selenite concentration of 100 mg / L. Since the reduction of selenite is accompanied by the generation of red elemental selenium, the selenium-producing strains were preliminarily identified by visual observation. If the plate colonies were red, it indicated that biological organic selenium was generated, and if they were not red, it indicated that no biological organic selenium was generated. The strains that could reduce sodium selenite to generate biological organic selenium were preliminarily screened out.

[0036] (4) Rescreening of strains with high tolerance to selenite

[0037] In order to screen out strains with higher conversion rates, strains with sodium selenite reduction ability were rescreened. Strains were picked from the activation plate and transferred to a liquid culture medium with a sodium selenite concentration of 500 μg / mL to observe whether red appeared. If red appeared, it indicated that the strain could grow in a culture medium with a sodium selenite concentration of 500 μg / mL, and a sodium selenite reduction reaction occurred, which could produce biological organic selenium.

[0038] (5) Identification of selenium-rich microorganisms

[0039] Finally, the obtained strains were identified by molecular biology using the bacterial DNA extraction method.

[0040] The total DNA of the bacterial strain was extracted by the CTAB method, and the universal primers 27F (5'-TCCGTAGGTGAACCTGCGG-3') (see sequence list NO.2) and 1492R (5'-TCCTC-CGCTTATTGATATGC-3') (see sequence list NO.3) were used for PCR amplification, with genomic DNA as the template. The PCR reaction system (30 μL) was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 58℃ annealing for 1 min, 72℃ extension for 1.5 min, a total of 36 cycles; 72℃ extension for 10 min, PCR products were detected by 1% agarose gel electrophoresis, and the PCR products were sequenced by Qingke Bioengineering Co., Ltd. The obtained 16SrDNA sequence was compared with the spliced ​​sequence (removing the inaccurate bases at the 2 ends) in the NCBI database (blast.ncbi.nlm.nih.gov) and EZbiocloud.

[0041] The determination results of the 16SrRNA gene sequence of the strain (see sequence list No. 1 for details) are shown in the attached sequence list. The specific method and operation are as follows:

[0042] Based on the cell morphology, biochemical test results, and gene sequence determination results, strain FZY-10 was identified as Paenibacillus lautus.

[0043] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC for short; address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; Postal Code: 100101) on March 17, 2023, with the deposit number CGMCC No.26833.

[0044] Example 2 Domestication of Selenium-enriched Paenibacillus splendens FZY-10 strain

[0045] The strain FZY-10 was aspirated from a culture medium with a sodium selenite concentration of 500 μg / mL, transferred to a liquid culture medium with a sodium selenite concentration of 1000 μg / mL, and observed for growth. If it grew, it was inoculated into liquid culture medium with sodium selenite concentrations of 2000 μg / mL, 3000 μg / mL, 4000 μg / mL, 5000 μg / mL, 6000 μg / mL, 8000 μg / mL, 10000 μg / mL, 12000 μg / mL, 14000 μg / mL, 16000 μg / mL, 18000 μg / mL, 20000 μg / mL, 22000 μg / mL, 24000 μg / mL, and 26000 μg / mL in sequence to be acclimated under a selenium concentration gradient. Each gradient was cultured at 32° C. for 24 h to 96 h. The growth of the FZY-10 colony was observed, and it was finally found that FZY-10 Paenibacillus splendens could still grow normally in a liquid culture medium of 15000 μg / mL, indicating that the FZY-10 bacteria provided by the present invention have potential advantages in application in the selenium-rich industry.

[0046] Example 3 Determination of the growth curve of selenium-rich Paenibacillus splendens FZY-10 strain

[0047] The FZY-10 strain was cultured under the maximum selenium concentration conditions determined in Example 2 above, and at OD 540 1 mL of FZY-10 bacterial solution was added to the primary seed culture medium with a pH of 7.5 and shaken well. The primary seed culture medium without bacterial solution was used as a blank and the OD was measured three times in parallel. 540 The culture medium was cultured at 35°C and samples were taken every 4 hours within 24 hours to measure the OD 540 nm absorbance. Finally, OD 540 The nm value data is taken as the ordinate and the time is taken as the abscissa to plot the OD 540 The growth curve of the bacterial solution in the experimental group treated with nm was drawn simultaneously with the blank control of the CK group as a reference.

[0048] The growth curve was measured as Figure 3 As shown, it can be seen that under the conditions of maximum selenium concentration, the growth curve trend of FZY-10 bacteria plus selenium is consistent with the growth trend of normal bacteria. The changes in different growth periods are clear and the growth rates are slightly different, indicating that the growth activity of FZY-10 strain is not affected by high-concentration selenium stress. This bacterium has the potential to develop high-concentration selenium-rich active bacterial agent products.

[0049] like Figure 3The growth curve of the experimental group in the stable period was OD 0.05. The growth curve of the experimental group in the stable period was OD 0.05. The growth curve of the experimental group in the stable period was OD 0.05. The growth curve of the experimental group in the stable period was OD 0.05. 540 nm is around 0.4-0.5, indicating that inorganic selenium has no obvious effect on the growth of FZY-10 bacteria under this condition. After adding sodium selenite, it began to turn red, indicating that part of sodium selenite was converted into elemental selenium by Bacillus.

[0050] Example 4 Selenium-enriched brilliant bacillus FZY-10 bacterial agent

[0051] The frozen FZY-10 bacteria were streaked on the plate, and after culturing for 24 hours, a single colony was picked and placed in a liquid culture medium. The culture medium composition was: 3.0 g / L beef extract, 10.0 g / L peptone, 3 g / L NaCl, 20.2 g / L NaOH, and the pH was adjusted to 6.2. 540 When nm is close to 0.00, sodium selenite is added to a final concentration of 100 mg / L. After continued static culture at 32°C for 48 h, the selenium-rich brilliant Bacillus FZY-10 bacterial agent is obtained. After appropriate concentration and dilution of the bacterial solution, it can be prepared to 5.0×10 10 CFU / mL or so.

[0052] Example 5 Application of Selenium-enriched Paenibacillus Brilliant FZY-10 Bacterial Agent in Plant Growth Promotion

[0053] Use the isolated and identified Paenibacillus to culture on beef extract peptone medium for 5 days, add sterile saline and use a sterile applicator to scrape the surface mycelium, and filter through 4 layers of sterile gauze to obtain the conidia filtrate. Calculate the concentration of the conidia solution with a hemocytometer, and adjust the solution to a concentration of 1×10 by centrifugal concentration or dilution with sterile water. 6 A conidial suspension with spores / mL was prepared for use. Two treatments were set up: ①CK; ②FZY-10 seed suspension mixed with soil; each treatment was repeated with 40 pots, and the seed suspension of Paenibacillus was mixed into the sieved soil of group ② in proportion, with 3kg of soil per pot (Lou soil, collect the same type of healthy soil 0-20cm tillage layer soil, air-dry, crush and mix through a 10mm sieve), and 1mg / kg of sodium selenite was applied to each pot plant, that is, 1mg was added per kilogram of culture medium soil, and arranged in random blocks. After 7 days of seedling acclimatization, the root irrigation concentration for each plant was 1×10 620mL of the spore suspension of the developed fungicide was added 2-3 times according to the growth status of corn, and sprayed on the leaves and petioles of corn plants. Sampling and measurement were carried out on the 10th day after the last inoculation of the fungicide. The plants were dug out completely, and the aboveground part was separated from the underground part. The stem thickness and plant height of the corn plants were measured, and the fresh weight of the two parts was weighed separately, and the dry weight was obtained by drying and weighing. Data analysis was performed using Origin2017 software, and the non-parametric test method was used to test the significance of the differences in the obtained data.

[0054] The physiological indexes of the test plants were measured, and the growth-promoting effect of the strain on corn growth was investigated after the potted plants were inoculated with the FZY-10 strain. The results (Table 1) showed that compared with the control group CK, the average dry weight and fresh weight of the corn plant roots in the treatment group inoculated with the FZY-10 strain were 0.37g and 4.08g, respectively, and after treatment with the FZY-10 strain, they were 0.43g and 4.17g, respectively, an increase of 16.22% and 2.21%, respectively; the average fresh weight of the aboveground part of the corn plants treated with the strain was 24.15g, and the fresh weight of the aboveground part of the corn plants not treated with the strain was 18.0 0g, the fresh weight of the aboveground part of the corn plants inoculated with FZY-10 strain increased by 34.17% compared with the group without strain application; the average stem diameter of the corn plants in the treatment group was 6.36mm, and the stem diameter of the corn plants in the control group was 5.80mm. The stem diameter of the corn plants inoculated with FZY-10 strain increased by 9.66% compared with the group without strain application; the average plant height of the corn plants in the control group was 58.32cm, and the average plant height of the corn plants in the treatment group was 68.33cm. The plant height of the corn plants inoculated with FZY-10 strain increased by 17.16% compared with the blank group, and there was a significant difference.

[0055] Specific results such as Figure 6 to Figure 11 , maize root dry weight ( Figure 6 ), root fresh weight ( Figure 8 ), fresh weight of above-ground parts ( Fig. 9 )、Stem Thickness( Fig.10 ) and plant height ( Fig.11 ) all had significant growth-promoting effects, only the aboveground dry weight ( Figure 7 ) The growth-promoting effect was not obvious, which was considered to be a systematic error caused by the large root crown of the crop in the seedling stage.

[0056] Table 1 Determination of physiological indexes of corn plants

[0057]

[0058] Different lowercase letters in the same column indicate significant differences (P<0.05).

[0059] Example 6 Application of Selenium-enriched Paenibacillus Brilliant FZY-10 Bacterial Agent in Selenium-enriched Plant Agricultural Products

[0060] The strains and the basic treatment methods and data analysis methods of the test plants in Example 5 were used to verify the application effect of selenium-enriched plant agricultural products. The selenium-enriched content of corn leaves and roots was measured. The selenium-enriched content of the plants was calculated by atomic fluorescence spectrophotometry for the results of the two different treatment groups, and the selenium-enriched effect was measured. The specific method is based on the isolation, screening, domestication and selenium-enriched research of selenium-enriched lactic acid bacteria in Song Zhaojun, Wang Shuning, Pan Runshu, et al. [J]. China Brewing, 2004, 23 (11): 4-7.

[0061] To evaluate the selenium-enrichment effect of the FZY-10 strain, a potted experiment was conducted on corn in the summer of 2022 to implement the above-prepared FZY-10 Bacillus: 1 mg / kg of sodium selenite was applied to each potted plant, that is, 1 mg was added per kilogram of culture soil, and 5×10 10 CFU / ml, the results showed that the average total selenium concentration in the roots of corn plants using the seed suspension was 5.26 mg / kg, and the average total selenium concentration in the leaves was 4.02 mg / kg ( Fig.12 ), the average total selenium concentration in the roots of unused corn plants was 3.68 mg / kg, and the average total selenium concentration in the leaves was 2.61 mg / kg ( Fig.12 ). In summary, the selenium content in the roots of corn plants treated with FZY-10 Bacillus seed suspension increased by 42.76% and the selenium content in the leaves increased by 54.00% compared with the plants not treated with seed suspension. This indicates that the application of the Bacillus seed suspension increased the comprehensive selenium content of the target agricultural products by 47.42%, enhanced the selenium enrichment effect of crops, and improved the quality of agricultural products.

[0062] Table 2 Selenium enrichment in roots and leaves of corn plants

[0063]

[0064] Different lowercase letters in the same column indicate significant differences (P<0.05).

Claims

1. A selenium-rich brilliant bacillus ( Paenibacillus lautus )FZY-10, characterized by: The selenium-rich brilliant Bacillus FZY-10 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 17, 2023, and the deposit number is CGMCC No. 26833.

2. Use of the selenium-rich brilliant Bacillus FZY-10 according to claim 1 in promoting corn growth.

3. The use of the selenium-enriched Paenibacillus splendens FZY-10 in promoting corn growth according to claim 2, wherein the application condition is under selenium enrichment.

4. The use of the selenium-enriched Paenibacillus splendens FZY-10 in promoting corn growth according to claim 3, wherein the application condition is in soil enriched with selenium.

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