Composition for reducing cadmium absorption of rice and application

Abstract

The invention provides a composition for reducing cadmium absorption of rice and application, and relates to the technical field of biology. In the composition, the ratio of the viable count of the bacillus subtilis N16 to the viable count of the Burkholderia sp. Y1 in the composition is (3 to 5): 1, and the ratio of the viable count of the Burkholderia sp. Y1 to the viable count of the Burkholderia sp. Y1 in the composition is (3 to 5): 1. The composition can reduce cadmium absorption of rice, and after the composition is applied to rice planting, the composition can significantly increase the content of root iron plaque and reduce the cadmium content of rice roots, spike stalks and grains.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a composition for reducing cadmium absorption in rice and an application thereof. Background Art

[0002] With the acceleration of industrialization and urbanization, soil cadmium pollution has become increasingly serious and has become a major hidden danger threatening agricultural sustainable development and food safety worldwide. Cadmium is a highly toxic heavy metal element that has high stability and mobility in the soil and is easily absorbed and accumulated by crops.

[0003] On the one hand, cadmium in the soil comes from a wide range of sources, including human activities such as mining, metal smelting, sewage irrigation, irrational use of fertilizers and pesticides, as well as natural factors such as soil parent materials. Once cadmium enters the soil environment, it can enter crops through root absorption and other pathways, which not only inhibits the growth and development of crops, causing plants to be short, leaves to turn yellow, and roots to be damaged, resulting in reduced or even total crop yields; on the other hand, more seriously, cadmium will be transmitted through the food chain and accumulated in the human body, causing kidney damage, bone lesions and other chronic diseases, endangering human health.

[0004] Traditional soil cadmium pollution remediation methods, such as the soil import method and soil replacement method in physical remediation, can quickly reduce the soil cadmium content in the short term, but the cost is high, the amount of work is large, and it is easy to cause soil structure damage and fertility loss; chemical remediation uses chemical modifiers, which has certain results, but may introduce secondary pollution and the long-term effect is unstable. As an environmentally friendly technology, bioremediation has gradually attracted attention, among which the use of microorganisms to reduce rice cadmium absorption has shown unique advantages. Microorganisms can affect the bioavailability of cadmium through various mechanisms. For example, some microorganisms can secrete metabolites such as organic acids and biosurfactants, which react with cadmium to form complexes and precipitation, reduce the activity of cadmium in soil solution, and make it difficult for crop roots to absorb it; some microorganisms can also change the redox potential of the soil and promote the transformation of cadmium to insoluble forms. However, a single strain of bacteria often has limited functions in a complex soil ecosystem, and it is difficult to comprehensively and efficiently deal with the problem of cadmium pollution. Therefore, it is urgent to develop a combination of strains to work synergistically to effectively reduce rice cadmium absorption, ensure the quality and safety of agricultural products, and achieve sustainable utilization of cadmium-contaminated farmland. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention aims to provide a composition for reducing cadmium absorption by rice and its application.

[0006] One of the objects of the present invention is to provide a composition for reducing cadmium absorption in rice, comprising Bacillus subtilis ( Bacillus subtilis ) N16 and Burkholderia ( Burkholderia sp. )Y1; The Bacillus subtilis ( Bacillus subtilis )N16 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31392; The Burkholderia spp. Burkholderia sp. )Y1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 17, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30686.

[0007] Preferably, Bacillus subtilis N16 and Burkholderia ( Burkholderia sp. )The ratio of the number of live bacteria in Y1 is 3~5:1.

[0008] The second object of the present invention is to provide a composition for reducing cadmium absorption by rice for use in remediation of heavy metal contaminated soil and reduction of heavy metal transport in crops.

[0009] Preferably, the composition for reducing cadmium absorption of rice is a solid bacterial agent or a liquid bacterial agent.

[0010] Preferably, the composition for reducing cadmium absorption of rice is further prepared into a microbial fertilizer.

[0011] Beneficial effects of the present invention: The Bacillus subtilis provided by the present invention ( Bacillus subtilis ) N16 and Burkholderia ( Burkholderia sp. ) The composition of Y1 can reduce the cadmium absorption of rice. After being applied to rice planting, the composition can significantly increase the root iron film content and reduce the cadmium content in rice roots, panicles and grains. DETAILED DESCRIPTION

[0012] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0013] Microorganisms involved in the embodiments: A strain of Bacillus subtilis ( Bacillus subtilis ) N16, deposited on July 22, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31392; A Burkholderia strain ( Burkholderia sp.)Y1, deposited on May 17, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30686.

[0014] Strain isolation and screening: The strain of the invention is derived from fresh cadmium-rich soil in Bailang area of ​​Tibet.

[0015] Take 1g of fresh cadmium-rich soil from Bailang, Tibet, put it into a clean conical bottle with glass beads and 100ml of sterile water, and put it in a shaker at 120rpm for 1h. The concentration of the soil solution is 10 -2 The purpose of adding glass beads is to allow the soil to be fully ground so that the microorganisms in it can be better released into the solution.

[0016] Dilution, take 6 sterile test tubes, add 9ml sterile water to each, mark 1, 2, 3, 4, 5, 6 respectively, take 1ml soil solution from the conical flask, add to test tube No. 1, and mix well. At this time, the concentration of test tube No. 1 is 10 -4 Then take 1 ml of liquid from test tube No. 1 and add it to test tube No. 2, and so on, so that the concentration of the soil solution is 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 .

[0017] For plate preparation, add 0.3 g of ammonium chloride (NH4Cl), 2 g of manganese carbonate (MnCO3·H2O), 1 g of yeast extract, and 20 g of agar to 1000 ml of deionized water, sterilize in an autoclave at 121°C for 20 minutes, cool to 50°C, add 1 ml each of mixed vitamin solution and trace element solution, and 2 ml of ferric citrate solution.

[0018] The formula of trace element solution in solid improved fibroblast culture medium (amount added per liter) is: MgSO4·7H20, 3g; NaC1, 1g; EDTA, 0.5g; MnSO4·H20, 0.5g; FeSO4·7H2O, 0.1g; Co(NO3)2·6H2O, 0.1 g; CaCl2, 0.1g; ZnSO4·7H2O, 0.1g; NiCl2·6H2O, 0.02g; CuS04·5H2O, 0.01g; KAl(SO4)2, 0.01 g; H3BO3, 0.01g; Na2MoO4·2H2O, 0.01g; Na2WO4·2H2O, 0.01 g; Na2SeO3, 0.001g.

[0019] Vitamin solution formula (amount added per liter): Folic acid, 2.000 mg; Pyridoxine hydrochloride / B6, 10.000 mg; Riboflavin / B2, 5.000 mg; Biotin, 2.000 mg; Thiamine / B1, 5.000 mg; Nicotinic acid / B3, 5.000 mg; Calcium pantothenate / B5, 5.000 mg; Vitamin B12, 0.100 mg; p-Aminobenzoic acid / Bx, 5.000 mg; Thioctic acid / Vn, 5.000 mg; Monopotassium phosphate, 900 mg. Ferric citrate solution: Dissolve 15g of trisodium citrate and 14.15g of ferrous sulfate heptahydrate in a small amount of water and add to a volumetric flask. Then add water to make up to 100ml. Divide into 2ml clean centrifuge tubes and store in a -20℃ refrigerator. Thaw when needed.

[0020] S5: Spreading: Take 0.1 ml of the solution from each test tube with different soil solution concentrations and spread it on the modified Trichoderma solid culture medium. After sealing the medium with a sealing film, place it in a cell culture incubator at 25°C in the dark for 3 days.

[0021] S6: Isolation and purification: Use a disposable sterile inoculation loop to pick colonies with the required characteristics from a plate containing multiple colonies, and streak them on a new plate until there are no foreign bacteria and a single colony is formed, thus obtaining two different single colonies N16 and Y1.

[0022] S7: Expansion culture: The single colony with characteristics screened out is inoculated into liquid LB culture medium by aseptic operation and cultured in a shaker at 25°C and 120 rpm for 24 hours to form a bacterial suspension.

[0023] S8: Preservation of bacterial strains: The bacterial suspension prepared in S7 and glycerol are added into sterile freezing tubes at a ratio of 3:7 to make glycerol tubes and stored at -20°C.

[0024] S9: Strain identification: The strain preserved in the glycerol tube was inoculated into 50 ml LB medium and placed in a shaker at 25°C and 120 rpm for incubation for 24 hours. The medium was then poured into a 50 ml sterile centrifuge tube and centrifuged for 10 minutes. The supernatant was discarded and the precipitate was divided into 1.5 ml sterile centrifuge tubes using aseptic operation. The 16S rDNA was sent for strain identification. The strain N16 was identified as Bacillus subtilis and the strain Y1 was identified as Burkholderia sp.

[0025] Pot experiment: First, prepare the bacterial suspension, prepare LB liquid culture medium, and dispense it into 200 ml conical flasks, 50 ml per flask. Pipette 100 μL of the strain preserved in the glycerol tube into the conical flask, and place it in a shaker at 120 rpm for 24 hours to prepare a bacterial suspension. Use sterile water to adjust the OD600 value of the bacterial suspension to 0.5. At this time, the number of live cells in 1 ml of N16 and Y1 bacterial suspension is about 5.0×10 9 , Centrifugation: Pour the bacterial solution into a sterile centrifuge tube according to the application ratio, centrifuge at 4000r / min for 10min, discard the supernatant, add water to the precipitate and mix well to obtain the centrifuged bacterial solution, which is used to apply to the potting soil.

[0026] Soak X24 rice seeds in a 1:1 hydrogen peroxide solution for 30 minutes for disinfection, then rinse with clean water and place in a vermiculite tray for cultivation. Move the plants to pots when they grow to about 20 cm, and apply centrifuged bacterial solution when they bloom.

[0027] The experiment was divided into 5 groups, namely control group, experimental group 1, experimental group 2, experimental group 3 and experimental group 4.

[0028] Control group: No bacteria were added to the soil during the X24 rice flowering period. After the rice matured, the roots, cobs and grains were collected. The cadmium content of each part of the rice after the iron oxidizing bacteria solution was applied was counted.

[0029] The number of living strains in the experimental group 1 was N16:Y1 = 3:0. During the experiment, a pot experiment was used. During the flowering period of rice, 1.5×10 11 cfu / g of Bacillus subtilis N16 was added to the soil without adding Burkholderia. After the rice matured, the roots, cobs and grains were collected. The cadmium content of each part of the rice after the iron oxidizing bacterial solution was calculated.

[0030] The number ratio of living organisms in the two experimental groups was N16:Y1 = 3:1. During the experiment, a pot experiment was used. During the flowering period of rice, 1.5×10 11 cfu / g of Bacillus subtilis N16 and 5×10 10The centrifuged bacterial solution of Burkholderia Y1 with a concentration of 100 cfu / g was added to the soil, and the roots, cobs and grains were collected after the rice matured. The cadmium content of each part of the rice after the iron-oxidizing bacterial solution was calculated.

[0031] The number ratio of living organisms in the three experimental groups was N16:Y1 = 4:1. During the experiment, a pot experiment was used. During the flowering period of rice, 2×10 11 cfu / g of Bacillus subtilis N16 and 5×10 10 The centrifuged bacterial solution of Burkholderia Y1 with a concentration of 100 cfu / g was added to the soil, and the roots, cobs and grains were collected after the rice matured. The cadmium content of each part of the rice after the iron-oxidizing bacterial solution was calculated.

[0032] The number ratio of living organisms in the four experimental groups was N16:Y1 = 5:1. During the experiment, a pot experiment was used. During the flowering period of rice, 2.5×10 11 cfu / g of Bacillus subtilis N16 and 5×10 10 The centrifuged bacterial solution of Burkholderia Y1 with a concentration of 100 cfu / g was added to the soil, and the roots, cobs and grains were collected after the rice matured. The cadmium content of each part of the rice after the iron-oxidizing bacterial solution was calculated.

[0033] The DCB method was used to measure the iron film content of rice roots. A DCB solution was prepared according to the ratio of 0.3 mol / L sodium citrate + 1 mol / L sodium bicarbonate + 3g sodium dithionite. The washed fresh rice roots were placed in the DCB solution and soaked for 30 minutes. The roots were taken out and the volume was adjusted to 50. The solution was filtered and the iron content in the solution was detected by ICP-MS. The test results are shown in Table 1.

[0034] Nitric acid digestion: weigh 0.25g of plant sample into a plant digestion tube, add 8ml of 70% nitric acid overnight, put into the digestion furnace, heat at 80℃ for 1.5h, heat to 120℃ for 1.5h, heat to 150℃ for 3h, then open the cover to volatilize the nitric acid, wait until the liquid in the digestion tube is about 1ml, digestion is complete, and use a dispenser to make the volume 50ml. Note: This process needs to be carried out in a fume hood.

[0035] ICP-MS detection: the liquid sample of the rice root iron film content measured by the DCB method and the liquid sample after nitric acid digestion were filtered into a test tube with filter paper and the cadmium content was detected by ICP-MS instrument. The test results are shown in Table 1.

[0036] Table 1 Iron film content and cadmium content in various parts of rice roots

[0037] The above data show that the experimental group 1 only applied 1.5×10 11After the centrifuged bacterial solution of Bacillus subtilis N16 with a concentration of cfu / g was added to the soil, the iron film content of rice roots increased by 6.3%, the cadmium content of rice roots decreased by 3.1%, the cadmium content of rice panicles decreased by 11.2%, and the cadmium content of rice grains decreased by 27.9%.

[0038] In the second experiment, 1.5×1011 cfu / g of Bacillus subtilis N16 and 5×10 10 cfu / g of Burkholderia Y1 centrifuged bacterial liquid was added to the soil, N16:Y1=3:1, the iron film content of rice roots increased by 102.6%, the cadmium content of rice roots decreased by 23.5%, the cadmium content of rice panicles decreased by 11.7%, and the cadmium content of rice grains decreased by 32.3%.

[0039] In the third experiment, 2×10 11 cfu / g of Bacillus subtilis N16 and 5×10 10 cfu / g of Burkholderia Y1 centrifuged bacterial liquid was added to the soil, N16:Y1=4:1, the iron film content of rice roots increased by 99.3%, the cadmium content of rice roots decreased by 40.7%, the cadmium content of rice panicles decreased by 33.6%, and the cadmium content of rice grains decreased by 23.8%.

[0040] In the fourth experiment, 2.5 × 10 11 cfu / g of Bacillus subtilis N16 and 5×10 10 cfu / g of Burkholderia Y1 centrifuged bacterial liquid was added to the soil, N16:Y1=5:1, the iron film content of rice roots increased by 93.9%, the cadmium content of rice roots decreased by 36.5%, the cadmium content of rice panicles decreased by 35.4%, and the cadmium content of rice grains decreased by 43.8%.

[0041] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with the technical features with similar functions disclosed in this application (but not limited to) by each other.

Claims

1. A composition for reducing cadmium absorption in rice, characterized in that: Including Bacillus subtilis ( Bacillus subtilis ) N16 and Burkholderia ( Burkholderia sp. )Y1; The Bacillus subtilis ( Bacillus subtilis )N16 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31392; The Burkholderia spp. Burkholderia sp. )Y1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 17, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30686.

2. The composition for reducing cadmium absorption in rice according to claim 1, characterized in that: The Bacillus subtilis N16 and Burkholderia Burkholderia sp. )The ratio of the number of live bacteria in Y1 is 3~5:

1.

3. Use of the composition for reducing cadmium absorption by rice as claimed in any one of claims 1 to 2 in the remediation of heavy metal contaminated soil and the reduction of heavy metal transport in crops.

4. The use of the composition for reducing cadmium absorption by rice as claimed in claim 3 in the remediation of heavy metal contaminated soil and the reduction of heavy metal transport in crops, characterized in that: The composition for reducing cadmium absorption of rice is a solid bacterial agent or a liquid bacterial agent.

5. Use of the composition for reducing cadmium absorption of rice as claimed in claim 3 in the remediation of heavy metal contaminated soil and the reduction of heavy metal transport in crops, characterized in that: The composition for reducing cadmium absorption of rice is further prepared into a microbial fertilizer.

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