Acinetobacter calcoaceticus and application thereof
Through the selenium oxidation and reduction function of Acinetobacter calcium acetate ES28, the problem of selenium enrichment in plants is solved, safe and efficient selenium absorption of plants is achieved, the risk of selenium poisoning is reduced, and the plant's disease resistance and growth performance is improved.
Patent Information
- Application Number
- CN202411646646.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to effectively utilize microorganisms to promote plant selenium enrichment, and there is also the problem of excessive or low selenium, which leads to selenium poisoning or lack.
Using Acinetobacter calcium acetate ES28, this strain has both selenium oxidation and reduction functions, which can oxidize SeMet to selenite and reduce Se(IV) to red nanoselenium to promote plant absorption of selenium.
Through the application of Acinetobacter calcium acetate ES28, the plant is enriched in selenium, reducing the risk of selenium poisoning, and improving the plant's disease resistance and growth performance.
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Figure CN119614416A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of agricultural microbial technology, and specifically to a calcoaceticus Acinetobacter and its application. The present invention also relates to the application of the bacterium as a growth-promoting bacterium in the creation of microbial organic fertilizer products. Background Art
[0002] Selenium (Se) is an essential trace element for life, with multiple functions such as anti-oxidation, anti-cancer, anti-virus, and immunity enhancement. Selenium has therapeutic and preventive effects on many diseases such as Keshan disease, cancer, and Alzheimer's disease.
[0003] Selenium exists in soil in various forms, including organic selenium, elemental selenium, water-soluble selenium, adsorbed selenium and mineral selenium. There are four valence states of selenium, including Se(-II), Se(0), Se(IV) and Se(VI), of which Se(-II) is mainly found in organic selenium in organisms, such as selenomethionine and selenocysteine. Elemental Se(0) is very rare in nature and is mostly found in selenium ores. Se(IV) and Se(VI) are mainly found in inorganic selenium in the form of selenite and selenate, and are the main forms of selenium that plants can absorb and utilize. Generally speaking, elemental selenium is basically non-toxic, while selenite is the most toxic of the four valence states.
[0004] Microbial metabolism of selenium mainly includes selenium reduction, oxidation, assimilation and methylation. At present, microorganisms (including bacteria, fungi, etc.) have been studied in detail in terms of selenium reduction and assimilation mechanisms. Some important key genes and enzymes have also been found in the methylation and oxidation of selenium, but the research on related mechanisms is still lacking. Under natural conditions, the oxidation of selenium is a very slow process. Only in recent years have scientists discovered microorganisms with obvious selenium oxidation functions such as T3F4, which accelerates the oxidation process of inorganic selenium and is also beneficial for plants to absorb inorganic selenium and convert it into non-toxic and harmless organic selenium, thereby achieving safe and efficient selenium supplementation for the human body.
[0005] However, the higher the selenium content in the soil, the better. Excessive selenium can cause symptoms of selenium poisoning in humans, animals and plants, such as Keshan disease and carcinogenesis. Studies have found that microorganisms reduce the highly toxic selenite or selenate in the soil to insoluble and less bioavailable elemental selenium (Se0), resulting in its removal from the solution. Too low or lacking selenium can cause reduced immunity of the body. Through microbial enhancement of selenium, plants can effectively absorb selenium and use it as a food source to alleviate the lack of selenium intake in humans and animals.
[0006] Currently, the synthesis and extraction of organic selenium are mainly based on chemical methods, but the technology is difficult and the cost is high. Utilizing the selenium-reducing properties of microorganisms is a green and efficient way. Plant selenium enrichment mainly includes applying selenium fertilizers in the soil and leaves and mixing seeds, but it is limited by the absorption efficiency of plants, and selenium pollution, selenium poisoning and other problems are more prominent. Using microorganisms to oxidize selenium is safer, more reliable and less costly.
[0007] The strain Acinetobacter calcoaceticus ES28 provided by the present invention belongs to the strain of the genus Acinetobacter of the family Moraxellaceae. It is currently reported that the strain has the ability to tolerate salt and alkali, can secrete siderophores, indole-3-acetic acid (IAA), and the ability to dissolve potassium, can promote plant growth, improve plant tolerance to salt and alkali, and has good ecological benefits, economic benefits, and social benefits.
[0008] So far, no strains of the genus Acinetobacter in the family Moraxellaceae have been reported to have selenium oxidation and reduction capabilities. Summary of the invention
[0009] The purpose of the present invention is to provide a calcoacetic acid bacterium and its application, wherein the calcoacetic acid bacterium ES28 also has the ability to produce auxin, secrete siderophore, dissolve phosphorus and potassium, and take into account the plant growth promotion and disease resistance.
[0010] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:
[0011] A calcoaceticus acinetobacter, the strain is named as: calcoaceticus acinetobacter ES28 (Acinetobactercalcoaceticus ES28), and the deposit number is GDMCC65292.
[0012] As an improvement, it is used in the reduction of selenium.
[0013] As an improvement, it is used in selenium oxidation.
[0014] As an improvement, it is used in the preparation of plant selenium-enriched growth-promoting microbial agents.
[0015] The advantages of the present invention compared with the prior art are:
[0016] The calcoaceticus Acinetobacter ES28 of the present invention, which has both selenium oxidation and reduction functions, has the ability to oxidize SeMet into selenite that can be directly absorbed and utilized by plants, thereby promoting selenium enrichment in plants; and has the ability to reduce Se(IV) into red nano-selenium (SeNPs); the strain is an effective supplement to the resources of selenium oxidation and selenium reduction strains.
[0017] The calcoacetic acid Acinetobacter ES28 of the present invention also has the ability to produce auxin, secrete siderophores, dissolve phosphorus and potassium, and other abilities to promote plant growth and resist diseases, providing a new strategy for using soil microorganisms to promote plant selenium enrichment, growth promotion, and disease resistance, thereby achieving plant quality improvement and yield increase, and providing a green, environmentally friendly, and safe method for microbial restoration in selenium-poisoned areas and plant selenium enrichment in selenium-deficient areas, which is of great significance to promoting human health development. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a phylogenetic analysis diagram between the calcoaceticus Acinetobacter ES28 having both selenium oxidation and reduction capabilities of the present invention and other members of the genus Acinetobacter (based on 16S rDNA sequence analysis);
[0019] Figure 2 This is a schematic diagram of the oxidation of elemental selenium by the strain ES28 of the present invention;
[0020] Figure 3 This is a schematic diagram of the conversion rate of the strain ES28 of the present invention under conditions of different Se(Ⅳ) concentrations;
[0021] Figure 4 It is the growth-promoting ability of the strain ES28 of the present invention. DETAILED DESCRIPTION
[0022] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings.
[0023] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.
[0024] Example 1: Isolation and identification of strain ES28
[0025] Sample collection: In May 2023, the applicant collected the selenium-rich soil sample used in the present invention from the surface soil of a selenium-rich farmland in Lichuan City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province.
[0026] Bacteria isolation and purification: Weigh 10g of soil sample and add it to a triangular flask containing 90mL of sterile water to make a soil suspension. Take 1mL of soil suspension and add it to 50mL of meat extract peptone medium. Shake and culture it in a shaker at 25℃ and 100rpm for 24h. Transfer the bacterial suspension to a meat extract peptone liquid medium containing 1mM sodium selenite at a 5% inoculum amount and shake and culture it at 25℃ and 100rpm for 24h. Repeat this three times. Take 1mL of fermentation broth and add it to 9mL of sterile water to gradually dilute to 10-4, 10-5 and 10-6. Take 100μL respectively and spread it on 200mg / L, 400mg / L, 600mg / L, 800mg / L, 1000mg / LSe(IV) meat extract peptone solid culture medium. Spread 3 plates for each gradient and culture at 25℃ for 48h. Select single colonies for streak purification and send them to sequencing company for detection after colony PCR. After confirmation of purification, store them in culture storage tubes according to 50% glycerol: bacterial solution, mark them and store them at -80℃.
[0027] Screening of selenium oxidizing bacteria: Add LB medium with a final concentration of 0.10 g L-1 elemental selenium powder to the strain at a 1% inoculum, and culture for 120 hours at 28°C and 150 rpm on a shaker. Centrifuge the supernatant at 12,000 rpm for 10 minutes, and use high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) to detect the content of Se(Ⅳ). If the Se(Ⅳ) content increases significantly, it means that the bacteria has oxidative properties for elemental selenium.
[0028] Screening of selenium-reducing bacteria and evaluation of their reducing ability: The strain was inoculated with 1% of the R2A liquid medium with a final concentration of 5mM, 10mM, 20mM, 50mM, and 100mM Se(Ⅳ), and cultured on a shaking table at 25°C and 120rpm for 72h. Every 24h, 1mL of the supernatant was sampled, centrifuged at 12000rpm for 10min, 400μL of the supernatant was aspirated, and 1mL of 1M Na2S solution was added to dissolve the red elemental Se(0) in the solution. After standing at room temperature for 1h to form a stable and homogeneous solution, the absorbance was detected (A500nm), and its concentration was calculated based on the Se(0) standard curve. If the Se(Ⅳ) content is significantly reduced and red nanoparticles are produced, it means that the bacteria has the ability to reduce and transform Se(Ⅳ).
[0029] After comparative analysis of the selenium oxidation and reduction abilities of the screened strains, a new strain with both selenium oxidation and reduction abilities was finally obtained ( Figure 1 ).
[0030] Example 2: Oxidation curve of selenomethionine by strain ES28
[0031] The strain ES28 was added to the LB medium with a final concentration of 0.1 μg L-1 sterile elemental selenium powder at a 1% inoculum, with 50 mL of liquid medium per bottle, 3 replicates, and a blank control without bacteria was set up. The culture was placed at 28°C and shaken at 150 rpm for 120 hours, and samples were taken every 24 hours, 2 mL each time, and added to a clean centrifuge tube for testing.
[0032] The sample to be tested was centrifuged at 12000 rpm for 10 min, the supernatant was taken and filtered through a 0.22 μm filter membrane, and stored in a refrigerator at -20°C. The Se(IV) content was detected by HPLC-HG-AFS.
[0033] The measured Se(Ⅳ) concentration (μg L-1) was used as the ordinate and time as the abscissa to construct the single substance selenium oxidation curve ( Figure 2 ).
[0034] Example 3: Reduction ability of strain ES28 for Se(IV)
[0035] Drawing of the standard curve of single-substance selenium: add 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 μL 100 mM Na2SeO3, add 100 μL 1M hydroxylamine hydrochloride, and fill up to 400 μL with sterile water, repeat 3 times for each gradient. After shaking at room temperature for 15 minutes, add 1 mL 1 mol / L Na2S solution to each tube to dissolve Se(0) to form a stable homogeneous solution, let stand at room temperature for 1 hour, measure the absorbance at 500 nm, and calculate the Se(0) concentration based on this.
[0036] The strain was inoculated with 1% of R2A liquid medium with final concentrations of 5mM, 10mM, 20mM, 50mM, and 100mM Se(Ⅳ), 50mL of liquid medium per bottle, and repeated 3 times. A blank control without bacteria was also set up. The culture was placed at 25°C and shaken at 120rpm for 72h. 1mL of the sample was taken and centrifuged at 12000rpm for 10min. 400μL of the supernatant was aspirated and 1mL of 1M Na2S solution was added to dissolve the red elemental Se(0) in the solution. The solution was allowed to stand at room temperature for 1h to form a stable and homogeneous solution, and then the absorbance was detected (A500nm).
[0037] The Se(IV) assimilation and utilization curve of strain ES28 was constructed with the measured Se(0) concentration (μM) as the ordinate and the initial Se(IV) concentration as the abscissa ( Figure 3 ). The strain ES28 has a significant reduction ability for Se(IV), and the conversion rate is different at different concentrations, with the maximum conversion rate being 92%.
[0038] Example 4: Detection of growth-promoting indexes of strain ES28
[0039] Determination of phytase activity in fermentation broth: strain ES28 was inoculated with 1% inoculum in 100mL liquid ASW medium, cultured at 37℃ 200rpm for 120h, and 200μL fermentation broth was taken every 24h, centrifuged at 4℃ to remove the supernatant, and stored in a refrigerator at 4℃ for later use. Dry an appropriate amount of potassium dihydrogen phosphate at 105℃ for 2h, cool in a dryer, accurately weigh 1.36g, dissolve in sterile water and dilute to 100mL. The concentration of the stock solution is 100mM, and the stock solution is diluted to 0, 10μM, 30μM, 50μM, and 70μM in sequence. Take 100μL of phosphorus standard solution of each concentration and ES28 fermentation broth and add them to 900μL of the prepared 2mM sodium phytate solution. The fermentation broth is first added with the stop solution and then the sodium phytate solution as a control. The reaction was allowed to stand in a 50℃ water bath for 30 minutes, 750μL stop solution was added to terminate the enzyme reaction, 1.5mL colorimetric solution was added to the mixture, and the mixture was allowed to stand at room temperature for 15 minutes. The absorbance at a wavelength of 700nm was detected by an enzyme reader. The enzyme activity unit (U) is defined as the amount of enzyme that releases 1μmol of phosphate per minute. The highest phytase activity was measured at 96h, which was 0.26U mL-1.
[0040] ELISA quantitative determination of indoleacetic acid production: strain ES28 was inoculated in an LB test tube at 1% inoculation, cultured at 37°C and 170rpm until OD600 = 1.0, then washed 3 times with sterile deionized water, and the seed solution concentration was adjusted to OD600 = 1.0. The ELISA kit was used to continuously determine the IAA content produced by strain ES28 in tryptophan Landy fermentation broth, sampling every 24h for 120h, with 3 replicates at each time point. The ELISA kit determined the IAA concentration range of 2ngmL-1-200ng mL-1. The ES28 fermentation broth of different periods was appropriately diluted, and the IAA content in the fermentation broth was determined and calculated in combination with the standard curve. The highest indoleacetic acid content was measured at 72h, which was 12mg L-1.
[0041] ELISA quantitative determination of nitrogenase activity: strain ES28 was inoculated in an LB test tube at 1% inoculation, cultured at 37°C and 170rpm until OD600 = 1.0, then washed 3 times with sterile deionized water, and the seed solution concentration was adjusted to OD600 = 1.0. The nitrogenase activity of strain ES28 and nitrogen-fixing Pseudomonas stutzeri A1501 (positive control) was detected using a microbial nitrogenase ELISA kit (product number: RJ23242). The nitrogenase activity of strain ES28 was finally measured to be 18.5U L-1, while that of control A1501 was 17.5U L-1.
[0042] Iron carrier production capacity determination: strain ES28 was inoculated in an LB test tube at 1% inoculation, cultured at 37°C and 170rpm to OD600 = 1.0, then washed 3 times with sterile deionized water, and the seed solution concentration was adjusted to OD600 = 1.0. The seed solution was inoculated into the MKB medium at 1% inoculation and cultured for 2 days at 30°C and 170rpm. The fermentation broth was centrifuged at 10000rpm for 5min to obtain the supernatant, and the supernatant and CAS detection solution were mixed in equal proportions. After sufficient reaction at room temperature, the absorbance value As at a wavelength of 630nm was measured on a multifunctional microplate reader; the sterile uninoculated MKB medium was used as a control, and the absorbance value was measured as Ar. The relative iron carrier production of the strain was calculated using the formula 1-As / Ar. The larger the calculated value, the stronger the ability of the strain to produce iron carriers. The experiment was repeated three times. The relative iron carrier production of strain ES28 was finally measured to be 0.45.
[0043] Determination of phosphate-dissolving and potassium-dissolving ability: strain ES28 was inoculated in an LB test tube at a 1% inoculation rate, cultured at 37°C and 170rpm until OD600 = 1, then washed 3 times with sterile deionized water, and the seed solution concentration was adjusted to OD600 = 1. Take 1mL of seed solution and inoculate it in 100mL of liquid LB culture medium, cultured at 37°C and 170rpm until OD600 = 1.0, wash the bacteria with sterile water 3 times, and resuspend the bacteria with an equal volume of sterile water. Take 200μL of the bacterial resuspension and inoculate it on solid calcium phytate culture medium, inorganic phosphorus solid culture medium and potassium-dissolving culture medium, respectively, culture at 30°C for 3d, and observe the degradation zone ( Figure 4 ).
[0044] Deposit information of the biological material of the present invention:
[0045] Name: Acinetobactercalcoaceticus ES28 with both selenium oxidation and reduction capabilities.
[0046] Proposed taxonomic name: Acinetobacter sp.
[0047] Deposit number: GDMCC No:65292.
[0048] Preservation unit: Guangdong Microbiological Culture Collection Center.
[0049] Address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
[0050] Preservation time: October 18, 2024.
[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.
Claims
1. A calcoaceticus Acinetobacter, characterized in that: The strain is named as Acinetobacter calcoaceticus ES28, and its deposit number is GDMCC65292.
2. The Acinetobacter calcoaceticus ES28 according to claim 1 is characterized in that: Application in selenium reduction.
3. The Acinetobacter calcoaceticus ES28 according to claim 1 is characterized in that: Application in selenium oxidation.
4. The Acinetobacter calcoaceticus ES28 according to claim 1 is characterized in that: Application in the preparation of plant selenium-enriched growth-promoting microbial agents.