A strain of Aeromonas intermedius CNBG-PGPR-19 and its application

Abstract

The present invention discloses a strain of Aeromonas intermedia CNBG‑PGPR‑19 and its application, which belong to the field of microbial technology. The strain of Aeromonas intermedia CNBG‑PGPR‑19 disclosed in the present invention has been deposited in the China General Microbiological Culture Collection Center, with a deposit number of CGMCC No. 23675, a deposit date of October 28, 2021, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The results of the embodiments of the present invention show that the strain CNBG‑PGPR‑19 reduces inorganic selenium to red elemental nano-selenium; the strain promotes the increase in the total fresh weight of green vegetables, and promotes the growth of the root system and above-ground parts of green vegetables; the application of CNBG‑PGPR‑19 bacterial solution promotes the increase in the total selenium and organic selenium content in green vegetables, and has an inhibitory effect on the content of inorganic selenium that is toxic to the human body.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Aeromonas intermedius CNBG-PGPR-19 and an application thereof. Background Art

[0002] Selenium (Se), as a trace element closely related to human health, is an important component of various enzymes and proteins in organisms, and has many biological functions such as improving human immunity, anti-aging, and preventing cancer. The human body mainly supplements selenium by eating exogenous selenium-containing foods, and improving the selenium-enriching capacity of plants and producing selenium-enriched products is a key way to meet the selenium needs of the human body.

[0003] About 70% of the soil in my country is selenium-deficient. In agricultural production, inorganic selenium is generally applied directly to increase the selenium content in plants. However, inorganic selenium is highly toxic. Excessive application will inhibit plant growth, and inorganic selenium remaining in plants will also be toxic to the human body. In addition, the irrational application of inorganic selenium sources will also cause waste of selenium nutrition and environmental pollution.

[0004] Selenium exists in four valence states in nature: −II, 0, +IV, and +VI. Microorganisms are important factors in regulating the form of selenium. They participate in the transformation of selenium through reduction, oxidation, assimilation, methylation, etc., which can promote the absorption and utilization of selenium by plants and make crops reach selenium-rich levels. Among the transformation pathways of selenium by microorganisms, research on selenium reduction has received more and more attention. Studies have shown that microorganisms can efficiently reduce inorganic selenium to elemental nano-selenium, which, as a safe and low-toxic form of selenium, can be efficiently absorbed by plants. Therefore, converting inorganic selenium into safe and green elemental nano-selenium to produce selenium-rich products through the reduction of microorganisms is a path with great application prospects and market potential.

[0005] Aeromonas intermedius is a type of Gram-negative bacteria. Chinese Patent 2021111394863 discloses that Aeromonas intermedius has the effect of enhancing plant stress resistance. Chinese Patent 2021102680550 discloses that Aeromonas intermedius has the effect of removing chloramphenicol from water. There are also related reports that Aeromonas intermedius has the effect of promoting plant growth.

[0006] However, there are currently no reports on the transformation of selenium by Aeromonas intermedius and its impact on the selenium content of crops. Summary of the invention

[0007] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a strain of Aeromonas intermedius. Another problem to be solved by the present invention is to provide an application of a strain of Aeromonas intermedius to produce and prepare single-substance nano-selenium and selenium-enriched bacterial agents, and then produce selenium-enriched crops and products.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A strain of Aeromonas media CNBG-PGPR-19, whose classification name is Aeromonas media CNBG-PGPR-19, has been deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 23675, the deposit date: October 28, 2021, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Application of Aeromonas intermedius CNBG-PGPR-19 in reduction and transformation of inorganic selenium.

[0011] Application of Aeromonas intermedius CNBG-PGPR-19 in regulating the growth of green vegetables.

[0012] The green vegetable growth regulation is to promote the increase of total fresh weight of green vegetables, promote the growth of green vegetable root system, and promote the growth of above-ground part of green vegetables.

[0013] Application of Aeromonas intermedius CNBG-PGPR-19 in regulating selenium content in green vegetables.

[0014] The regulation of selenium content in green vegetables is to promote the increase of total selenium content in green vegetables, promote the increase of organic selenium content in green vegetables, and promote the decrease of inorganic selenium content in green vegetables.

[0015] Application of bacterial culture of Aeromonas intermedius CNBG-PGPR-19 in regulating the growth of green vegetables.

[0016] Application of bacterial culture of Aeromonas intermedius CNBG-PGPR-19 in regulating selenium content in green vegetables.

[0017] Application of Aeromonas intermedius CNBG-PGPR-19 in the preparation of selenium-enriched bacterial agent.

[0018] Application of Aeromonas intermedius CNBG-PGPR-19 in the production and preparation of elemental nano-selenium.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The present invention discloses a strain of Aeromonas media CNBG-PGPR-19, which is classified and named Aeromonas media CNBG-PGPR-19, and has been deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 23675, a deposit date of October 28, 2021, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0021] 2) The results of the examples of the present invention show that the red color of the culture medium of the strain CNBG-PGPR-19 in a medium with a selenium concentration of 20 mM gradually deepens with the increase of culture time, indicating that the strain reduces inorganic selenium to red elemental nano-selenium.

[0022] 3) The results of the examples of the present invention show that the selenium content in the supernatant of the culture medium of the strain CNBG-PGPR-19 shows a significant decreasing trend over time; the selenium reduction amount of the strain gradually increases over time. The results show that the strain CNBG-PGPR-19 has a very prominent effect on the reduction and conversion of inorganic selenium.

[0023] 4) The results of the examples of the present invention show that after applying CNBG-PGPR-19 bacterial solution to the soil, the roots, aboveground parts and total fresh weight of the green vegetable plants were significantly higher than those of the control group. Applying CNBG-PGPR-19 bacterial solution promotes the increase of total selenium and organic selenium content in green vegetables, and inhibits the content of inorganic selenium that is toxic to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the colony morphology of strain CNBG-PGPR-19 in NA medium;

[0025] Figure 2 The bacterial morphology of strain CNBG-PGPR-19 in NB medium;

[0026] Figure 3 is the phylogenetic tree diagram of strain CNBG-PGPR-19;

[0027] Figure 4 The color change diagram of strain CNBG-PGPR-19 after being cultured in selenium-containing NB medium (20 mM sodium selenite) for different time periods;

[0028] Figure 5 The color change diagram of strain CNBG-PGPR-19 after culture in NB medium with different selenium concentrations (0, 20, 60, 100 mM sodium selenite);

[0029] Figure 6The growth curve of strain CNBG-PGPR-19 in NB medium with different selenium concentrations (0, 20, 60, 100 mM sodium selenite);

[0030] Figure 7 The graph shows the change trend of selenium concentration and selenium reduction amount in selenium-containing NB medium (20 mM sodium selenite) over time after adding strain CNBG-PGPR-19;

[0031] Figure 8 This is a graph showing the effect of strain CNBG-PGPR-19 on the fresh weight of green vegetables;

[0032] Fig. 9 This is a graph showing the effect of strain CNBG-PGPR-19 on the selenium content of green vegetables. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0034] The formula of NA medium is: 15 g agar powder, 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L distilled water, pH 7.2-7.5, and sterilization at 121 °C for 15 min.

[0035] The formula of NB medium is: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L distilled water, pH 7.2~7.5.

[0036] The preparation method of sodium selenite solution is as follows: prepare a stock sodium selenite solution with a concentration of 100 mg / mL, filter it using a sterile filter (0.22 μm), and store it away from light; chemicals such as sodium selenite (Na2SeO3, AR 98%) and phosphate buffered saline (PBS) are all domestically produced products.

[0037] Example 1

[0038] 1. Strain isolation and purification

[0039] Selenium-rich soil (selenium content of 1.12 mg / kg) was collected from Yixing City, Jiangsu Province. 10 g of soil sample was weighed and added to 90 mL of sterile PBS pre-filled with 10 glass beads, shaken at 30°C and 200 r / min for 30 min, and allowed to stand for 5 min. The soil suspension was collected and centrifuged at 5000 r / min for 15 min. The precipitate was suspended in 10 mL of PBS and stored at 4°C for later use. 2 mL of the prepared sample was added to 100 mL of liquid culture medium with a sodium selenite concentration of 100 μg / mL, and cultured at 30°C and 200 r / min for 2 days. 10 were prepared by gradient dilution method. -2 ~10 -6 For the serial dilutions, take 100 μL and spread them on Nutrient Agar (NA) solid culture medium plates, prepare two copies in parallel, invert them, and culture them in a 30°C constant temperature incubator for 24-36 hours. Select the dilution plates with obvious single colonies and moderate numbers, and purify the colonies with different morphologies on the plates until single colonies appear, and then repeat the streaking culture for at least 5 times.

[0040] 2. Colony and strain morphology

[0041] The morphological observation of the isolated and purified CNBG-PGPR-19 colonies showed that the colonies were round, raised in the middle, light yellow, and smooth, moist and opaque on the surface ( Figure 1 ).

[0042] Pick a single colony to NB liquid culture medium, activate it for 2 generations at a 5% inoculum, draw 10 μL of culture solution onto a glass slide, dry the water with an alcohol lamp, add a drop of safranin dye, wash off the excess dye with pure water after staining for 30 s, cover with a coverslip and observe the strain morphology under a microscope. The results showed that the CNBG-PGPR-19 bacterial cells were short rod-shaped, with blunt ends, a length of 0.5~1.0 μm, and a width of 0.2~0.5 μm ( Figure 2 ).

[0043] 3. Molecular biological identification of strains

[0044] A single colony was picked and placed in NB medium, cultured overnight at 30 °C and 200 r / min, and then DNA was extracted using a bacterial genomic DNA extraction kit. Using DNA as a template, the 16S rDNA gene sequence was amplified using the upstream primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and the downstream primer 1492R (5'-GGYTACCTTGTTACGACTT-3').

[0045] The 50 μL PCR amplification reaction system was: 25 μL Taq enzyme Mix, 1 μL upstream primer 27F, 1 μL downstream primer 1492R, 1 μL DNA template, and 22 μL sterile water.

[0046] The PCR amplification reaction conditions were as follows: pre-denaturation at 95 °C for 7 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, cycle number: 95 °C for 30 s, 30 cycles; final extension at 72 °C for 10 min; and hold at 12 °C for 10 min.

[0047] After the amplified product was detected by 1% agarose gel electrophoresis, the purified sample was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of strain CNBG-PGPR-19 is shown in SEQ ID NO.1. The measured gene sequence was searched for homologous sequences in the GenBank database (NCBI), and the strains with higher similarity were determined by BLAST analysis. At the same time, a phylogenetic tree was constructed using MEGA11 software to clarify the species relationship of the strains. The results showed that strain CNBG-PGPR-19 had the highest similarity with Aeromonas media, and clustered with Aeromonas media. Therefore, strain CNBG-PGPR-19 can be determined to be Aeromonas media ( Figure 3 ).

[0048] The strain CNBG-PGPR-19 is classified and named Aeromonas media CNBG-PGPR-19, and has been deposited in the China General Microbiological Culture Collection with the deposit number CGMCC No. 23675. The deposit date is October 28, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0049] Example 2

[0050] 1. Growth curve and selenium tolerance of strain CNBG-PGPR-19

[0051] A single colony of CNBG-PGPR-19 was picked up and placed in NB liquid medium. After activation for 2 generations at a 5% inoculum, it was inoculated into NB liquid medium containing sodium selenite at concentrations of 0 (control), 20 mM, 60 mM, and 100 mM at a 2% inoculum, and cultured at 30°C and 180 rpm. After 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h, the OD value was measured at 600 nm to obtain the growth curve and observe the color change.

[0052] The results are as follows Figure 4 As shown, after the strain CNBG-PGPR-19 was cultured in a medium with a selenium concentration of 20 mM for 12 h, a light red color began to appear, indicating that the strain had reduced part of the inorganic selenium into red elemental nano-selenium; as the culture time increased, the red color of the culture medium gradually deepened, while the color of the NB culture medium without inoculation of the strain did not change significantly at different times.

[0053] The results are as follows Figure 5 As shown, after 96 h of cultivation, the three selenium-containing culture media all turned red, and the color of the culture medium with a selenium concentration of 100 mM was slightly lighter than that of the other two culture media, indicating that the reduction of inorganic selenium by the strain in the culture medium with high selenium concentration was limited to a certain extent.

[0054] The results are as follows Figure 6 As shown in the figure, the results of the growth curve showed that the strain CNBG-PGPR-19 could grow normally in NB medium with sodium selenite concentrations of 20 mM, 60 mM, and 100 mM, and its growth characteristics in the three mediums were similar. However, compared with the control group, the growth and reproduction rate of the strain CNBG-PGPR-19 in the selenium-containing NB medium was inhibited, and the concentration of the final growth plateau was about half of that of the control.

[0055] 2. Selenium reduction ability of strain CNBG-PGPR-19

[0056] A single colony of CNBG-PGPR-19 was picked up and placed in NB liquid medium. After activation for 2 generations at a 5% inoculum, it was inoculated into NB liquid medium with a sodium selenite concentration of 20 mM at a 2% inoculum and cultured at 30 °C and 180 rpm. 10 mL of sample was taken at 0, 24 h, 48 h, and 72 h, respectively. The supernatant was taken after centrifugation at 10,000 rpm for 20 min, filtered with a 0.22 μm sterile filter, and the selenium content in the supernatant was determined by ICP-OES. The selenium reduction amount of the strain was obtained based on the calculation of the selenium reduction amount in the supernatant.

[0057] The results are as follows Figure 7 As shown in the figure, the selenium content in the supernatant of the culture medium of strain CNBG-PGPR-19 showed a significant decreasing trend with time, especially at 48 h, the selenium content decreased sharply, and after 72 h of culture, the selenium concentration decreased to 17.16 mM. The selenium reduction amount of the strain showed an opposite trend to the selenium concentration. With the increase of time, the selenium reduction amount gradually increased. At 48 h, the selenium content increased sharply. After 72 h of culture, the maximum selenium reduction amount reached 214.35 mg / L.

[0058] In summary, strain CNBG-PGPR-19 plays a very prominent role in the reduction and transformation of inorganic selenium.

[0059] 3. Effects of strain CNBG-PGPR-19 on green vegetable growth and selenium content

[0060] ① Preparation of CNBG-PGPR-19 bacterial culture

[0061] A single colony of CNBG-PGPR-19 was picked up and placed in NB liquid medium. After activation for 2 generations at a 5% inoculum, it was inoculated into NB liquid medium at a 2% inoculum and cultured at 30 °C and 180 rpm for 24 h. The colony was removed from the shaker and set aside (the bacterial solution concentration was 3.2 × 10 9 CFU / mL).

[0062] ②Effects of strain CNBG-PGPR-19 on growth and selenium content of Brassica chinensis

[0063] The soil from the nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province was used as the cultivation soil, and 200 g of soil (passed through a 2 mm sieve) was placed in each container. Dwarf yellow cabbage was sown in the seedling medium, and the cabbage seedlings were transplanted after germination. There were two treatments in the experiment, namely sodium selenite solution + liquid NB medium (CK) and sodium selenite solution + CNBG-PGPR-19 bacterial solution. Three pots were set up in each treatment, and one cabbage was transplanted in each pot. After the cabbage seedlings were slow to grow, 50 mL of bacterial solution containing sodium selenite (30 μL 16 mg / L sodium selenite solution and 10 mL bacterial solution were mixed and diluted to 50 mL with water) and liquid medium containing sodium selenite (30 μL 16 mg / L sodium selenite solution and 10 mL liquid NB medium were diluted to 50 mL with water) were poured into each pot. After the cabbage was harvested, the roots, aboveground parts and total fresh weight of the plants were measured. After the plants were dried, the total selenium and inorganic selenium in the plants were determined by hydride atomic fluorescence spectrometry. The content of organic selenium was the difference between the total selenium and inorganic selenium contents.

[0064] The results are as follows Figure 8 As shown in the figure, after applying CNBG-PGPR-19 bacterial solution to the soil, the roots, aboveground parts and total fresh weight of the cabbage plants were significantly higher than those in the control group, which were increased by 24.48%, 19.24% and 20.56% respectively.

[0065] The results are as follows Fig. 9 As shown in the results, the application of CNBG-PGPR-19 bacterial solution promoted the increase of total selenium and organic selenium content in green vegetables. Compared with the control group, the total selenium and organic selenium content of green vegetables in the treatment group increased by 30 μg / kg and 440 μg / kg, respectively, while the content of inorganic selenium, which is toxic to the human body, decreased by 190 μg / kg.

[0066] In summary, strain CNBG-PGPR-19 has a significant promoting effect on the growth of green vegetables and the improvement of selenium content (especially organic selenium).

Claims

1. A strain of Aeromonas intermedia CNBG-PGPR-19, which is classified as Aeromonas intermedia ( Aeromonas media ), has been deposited in the China General Microbiological Culture Collection Center, the deposit number is: CGMCC No. 23675, the deposit date is: October 28, 2021, and the deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. Use of Aeromonas intermedius CNBG-PGPR-19 according to claim 1 in reducing and converting inorganic selenium.

3. Use of Aeromonas intermedius CNBG-PGPR-19 according to claim 1 in promoting the growth of green vegetables.

4. The use according to claim 3, characterized in that: The method of promoting the growth of green vegetables includes promoting the increase of the total fresh weight of green vegetables, promoting the growth of the root system of green vegetables, and promoting the growth of the above-ground part of green vegetables.

5. Use of the Aeromonas intermedius CNBG-PGPR-19 described in claim 1 in promoting an increase in the total selenium content in green vegetables and / or promoting an increase in the organic selenium content in green vegetables and / or promoting a decrease in the inorganic selenium content in green vegetables.

6. Use of the bacterial liquid of Aeromonas intermedius CNBG-PGPR-19 according to claim 1 in promoting the growth of green vegetables.

7. Use of the bacterial liquid of Aeromonas intermedius CNBG-PGPR-19 according to claim 1 in promoting an increase in the total selenium content in green vegetables and / or promoting an increase in the organic selenium content in green vegetables and / or promoting a decrease in the inorganic selenium content in green vegetables.

8. Use of Aeromonas intermedia CNBG-PGPR-19 according to claim 1 in preparing a selenium-enriched bacterial agent.

9. Use of Aeromonas intermedius CNBG-PGPR-19 according to claim 1 in the production of elemental nano-selenium.

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