Rice endophyte for promoting rice growth and reducing cadmium as well as preparation method and application of rice endophyte

Abstract

The invention provides a rice endophyte for promoting rice growth and reducing cadmium as well as a preparation method and application of the rice endophyte, and relates to the field of environmental microbiology and agricultural microorganisms. The rice endophyte is stenotrophomonas, is named as Stentrophomonas sp.R5, is preserved in the China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, the postcode is 430072, the preservation number is CCTCC NO: M 20231758, and the preservation date is September 20, 2023. The rice endophyte not only has the capabilities of producing IAA, dissolving phosphorus, producing siderophores and the like for promoting the growth and development of rice, but also has an efficient inhibition effect on absorption and transportation of cadmium in the rice, particularly, the cadmium reduction amplitude of mature rice grains reaches up to 57.69%, and the rice endophyte has little influence on microbial communities in a soil environment, and has a good application prospect. Excessive interference is not caused, and conversion of available nutrients in the soil is promoted.

Description

Technical Field

[0001] The present invention relates to the fields of environmental microbiology and agricultural microbiology, and particularly relates to an endophytic bacterium of rice for promoting rice growth and reducing cadmium, and a preparation method and application thereof. Background Art

[0002] Cadmium (Cd) is a heavy metal element that is highly toxic and non-essential to the human body. Compared with other heavy metal pollutants, Cd has fluidity and is easily absorbed by plant roots and transferred to the above-ground parts, and then enters the human body through the food chain, posing a hazard to human health and the entire ecosystem. In recent years, some scholars have found through research that about 10% of the rice sold in the markets across the country has exceeded the cadmium standard. Long-term consumption of rice with excessive cadmium content can cause various diseases. Therefore, restricting the accumulation of cadmium in rice and reducing the harm caused by cadmium to human health through the food chain has become an urgent task.

[0003] Regarding the cadmium pollution problem in rice, the remediation methods include physical remediation (such as soil replacement, deep plowing, and electro-remediation to reduce or eliminate the cadmium content in the soil), which has a significant cadmium reduction effect. However, the soil replacement method has the following defects: high input cost, difficult implementation, and difficult to be widely applied on a large scale. Chemical remediation (adding chemical reagents to adsorb and chelate cadmium ions) has the advantages of simple operation and good cadmium reduction effect. The disadvantages are that the chemical reagents may react with the nutrients required by the plants, resulting in the plants being unable to obtain sufficient nutrients and causing secondary pollution. For biological remediation (using the life activities of animals, plants, and microorganisms such as cell metabolism (bioprecipitation and biotransformation), biosorption, bioaccumulation, extracellular precipitation, and efflux to reduce or change the form of cadmium to a less dangerous form), there are still some deficiencies: when coexisting with local microorganisms under environmental pressure and natural conditions, the microbial functions and survival rates will decrease. These influencing factors include chemical and physical properties such as substrates, nutrients, pH, temperature, and inoculation density. For example, some microorganisms are aerobic microorganisms, and adding them may compete with rice for oxygen and inhibit the growth of rice. The addition of some bacteria may also cause significant changes in the composition and structure of the soil bacterial community, thereby affecting the stability of the soil microecology.

[0004] Generally speaking, compared with traditional physical and chemical remediation, green and safe bioremediation has the advantages of low cost, easy operation, sustainability, and no secondary pollution, and is attracting more and more attention. In particular, in recent years, the use of plant endophytes to adsorb heavy metals to enhance the heavy metal remediation ability of plants themselves has gradually become a research hotspot. During the long-term symbiosis between endophytes and host plants, a relationship of co-evolution and mutual benefit has been established. Currently, reports on rice endophytes mainly focus on their growth promotion effects on rice, antibacterial activities, and resistance to rice blast, etc., and are mainly applied in fields such as the extraction of natural active substances, the production of biological fertilizers or biological pesticides. There are few reports on screening and isolating cadmium-tolerant endophytes in rice to help rice reduce cadmium stress. Summary of the Invention

[0005] The present invention provides a rice endophyte for promoting rice growth and reducing cadmium, and its preparation method and application, aiming to solve the above problems existing in the background technology.

[0006] To achieve the above object, an embodiment of the present invention provides a rice endophyte for promoting rice growth and reducing cadmium. The rice endophyte belongs to the genus Stenotrophomonas, named Stenotrophomonas sp. R5, and is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postcode 430072, deposit number CCTCC NO: M20231758, and the deposit date is September 20, 2023.

[0007] Based on the general concept of an invention, an embodiment of the present invention provides a preparation method of the above-mentioned rice endophyte for promoting rice growth and reducing cadmium, including the following steps:

[0008] S1: Select plump and full rice seeds, and perform surface disinfection to ensure thorough sterilization;

[0009] S2: Place the surface-disinfected rice seeds in a sterilized mortar, add liquid nitrogen and grind them thoroughly into a powder. Weigh and inoculate them into 250 mL of 1 / 2LB, TSA, R2A, NB, and rice soup liquid media respectively with a sterilized weighing spoon, and place them in a shaker for cultivation;

[0010] S3: Respectively suck 1 mL of the bacterial liquid and dilute it by different multiples, then respectively suck 100 μL of the diluted bacterial suspension and spread it on the corresponding solid medium, place it in an incubator for inverted cultivation, regularly observe the growth of the strains on the plate, and after growing for a period of time, pick out with an inoculation loop and perform streak cultivation on the corresponding solid medium to obtain the purified rice endophyte.

[0011] Preferably, the specific process of surface disinfection in step S1 is as follows: after soaking with absolute ethanol, pour out the absolute ethanol, wash with sterilized water 5 times, then soak with NaClO, and wash with sterilized water 5 times.

[0012] More preferably, the method for inspecting thorough sterilization of surface disinfection is as follows: aspirate 100 μL of the sterilized water used to rinse the rice seeds finally and spread it on a 1 / 2 LB solid medium, place it in a constant temperature incubator for 2 - 5 days, and observe whether there are strains growing on the medium to determine whether the surface disinfection and sterilization of the rice seeds are thorough.

[0013] Preferably, the shaker speed in step S2 is 180 rpm / min, the temperature is 28 °C, and the incubation time is 36 h.

[0014] Preferably, the different dilution multiples in step S3 are 1×10 4 、1×10 5 and 1×10 6 times.

[0015] Preferably, the temperature of the constant temperature incubator in step S3 is 28 °C.

[0016] Preferably, the strain growth time in step S3 is 2 - 5 days.

[0017] Preferably, the prepared rice endophyte bacteria are mixed evenly according to the ratio of the bacterial liquid to sterile glycerol of 1:1, aspirate 1.5 mL and dispense it into 2 - mL centrifuge tubes, and store it in an environment of -80 °C for standby.

[0018] The embodiment of the present invention also provides the application of the above - mentioned rice endophyte bacteria for promoting rice growth and reducing cadmium or the rice endophyte bacteria prepared by the above - mentioned preparation method in rice.

[0019] The above - mentioned scheme of the present invention has the following beneficial effects:

[0020] (1) A kind of rice endophyte bacteria for promoting rice growth and reducing cadmium of the present invention belongs to the genus Stenotrophomonas, named Stenotrophomonas sp.R5, deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postcode 430072, deposit number CCTCC NO: M 20231758, deposit date September 20, 2023.

[0021] (2) Compared with some aerobic bacteria that compete with rice for oxygen in the soil and inhibit the growth and development of rice, the rice endophyte bacteria of the present invention not only have the ability to promote the growth and development of rice such as producing IAA, dissolving phosphorus, and producing siderophores, but also play an efficient inhibitory role in the absorption and transport of cadmium in rice.

[0022] (3) When certain microorganisms coexist with local microorganisms under environmental stress and natural conditions, their survival rate and function decrease, especially the effect of remediating cadmium-polluted soil is greatly reduced. However, the endophytic bacteria of rice in the present invention can significantly reduce the cadmium content in rice plants, and the cadmium reduction rate in mature rice grains is as high as 57.69%.

[0023] (4) Compared with the fact that certain microorganisms will significantly change the composition and structure of the original microbial community after being added to the soil environment and have a negative impact on the original ecological environment, the endophytic bacteria of rice in the present invention have little impact on the microbial community in the soil environment. They will not cause too much interference and can promote the transformation of available nutrients in the soil. Detailed implementation manners

[0024] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments.

[0025] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0026] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0027] The present invention provides an endophytic bacteria of rice for promoting rice growth and reducing cadmium, and its preparation method and application, aiming at the existing problems.

[0028] Example 1 Isolation, screening and identification of endophytic bacteria of rice

[0029] 1. Isolation, screening and culture of endophytic bacteria in rice seeds

[0030] Select plump and full Huanghuazhan rice seeds. First, soak them in 75% anhydrous ethanol for 10 minutes, then pour out the anhydrous ethanol, wash them 5 times with sterile water, and then soak them in 5% NaClO for 10 minutes and wash them 5 times with sterile water. Pipette 100 μL of the sterile water for the last rinse of the rice seeds and spread it on a 1 / 2LB solid medium, and place it in a constant temperature incubator for 2 - 5 days to observe whether there are strains growing on the medium to ensure that the surface of the rice seeds is thoroughly disinfected.

[0031] After surface-sterilizing rice seeds, place them in a sterilized mortar, add a small amount of liquid nitrogen, and grind them thoroughly until they become powdery. Then, use a sterilized weighing spoon to inoculate the rice seed powder into 250 mL of 1 / 2LB, TSA, R2A, NB, and rice soup liquid media respectively. After culturing in a shaker (28 °C, 180 rpm / min) for 36 hours, respectively aspirate 1 mL of the bacterial liquid and dilute it by 1×10 4 、1×10 5 and 1×10 6After dilution by a certain multiple, 100 μL of the bacterial suspension was respectively pipetted and spread on the corresponding solid medium, and then placed in an inverted position in a constant temperature incubator at 28 °C for incubation. The growth of the strains on the plate was observed regularly. After the strains grew for 2 - 5 days, strains with different morphological characteristics on the plate were selected with an inoculation loop and streaked on the corresponding solid medium to obtain purified single bacteria. After activating the single bacteria, they were mixed evenly according to the ratio of bacterial liquid:sterile glycerol = 1:1, and 1.5 mL was pipetted and dispensed into 2 mL centrifuge tubes, then stored in a -80 °C refrigerator for later use. In this study, 5 liquid media with different nutrient levels (1 / 2LB, TSA, R2A, NB, and rice soup liquid medium) were used to isolate endophytic bacteria from rice seeds, with the expectation of obtaining more and more complete endophytic bacteria from rice seeds. Among them, the preparation method of NB liquid medium is as follows: Weigh 10.0 g of glucose, 3.0 g of beef extract, and 5.0 g of peptone, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, dispense into Erlenmeyer flasks, seal them, and sterilize them at 121 °C for 30 minutes using the autoclave method, and use them after cooling. The preparation method of TSA liquid medium is as follows: Weigh 5.0 g of sodium chloride, 15.0 g of tryptone, and 5.0 g of soybean papain hydrolyzate, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, dispense into Erlenmeyer flasks, seal them, and sterilize them at 121 °C for 30 minutes using the autoclave method, and use them after cooling. The preparation method of R2A liquid medium is as follows: Weigh 0.5 g of peptone, 0.5 g of yeast extract powder, 0.3 g of potassium dihydrogen phosphate, 0.5 g of casein hydrolyzate, 0.5 g of glucose, 0.5 g of soluble starch, 0.024 g of anhydrous magnesium sulfate, and 0.3 g of sodium pyruvate, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, dispense into Erlenmeyer flasks, seal them, and sterilize them at 121 °C for 30 minutes using the autoclave method, and use them after cooling. The preparation method of NB liquid medium is as follows: Weigh 10.0 g of glucose, 3.0 g of beef extract, and 5.0 g of peptone, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, dispense into Erlenmeyer flasks, seal them, and sterilize them at 121 °C for 30 minutes using the autoclave method, and use them after cooling. Rice soup liquid medium: Weigh 200.0 g of rice, boil it with 1000 mL of water for 1 h, filter it with double-layer gauze, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, dispense into Erlenmeyer flasks, seal them, and sterilize them at 121 °C for 30 minutes using the autoclave method, and use them after cooling.

[0032] 2. Identification of 16S rDNA of endophytic bacteria

[0033] The isolated and screened endophytic bacteria were inoculated into 1 / 2LB liquid medium for activation. After centrifugation using a high-speed refrigerated centrifuge, the bacterial cells were collected, and the genomic DNA of the strains was extracted using a bacterial DNA extraction kit according to the instructions. The amplified products of the strain DNA were sent to Hunan SaiSiwei Biotechnology Co., Ltd. for sequencing and identification. After obtaining the gene sequences of the endophytic bacteria, they were aligned online with the NCBI database to identify the genus and species of the endophytic bacteria. The results showed that the rice endophytic bacterial strain belongs to the genus Stenotrophomonas, and it was named Stenotrophomonas sp. R5.

[0034] Example 2 Analysis of the growth-promoting effect of rice endophytic bacteria

[0035] (1) Qualitative analysis of IAA production function

[0036] The isolated and screened rice endophytic bacteria were inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured in a shaker (28 °C, 180 rpm / min) for 36 hours. 50 μL of the cultured bacterial suspension and 50 μL of Salkowski's colorimetric solution were pipetted into the round holes of a white ceramic plate. The positive control was 50 μL of IAA (50 mg / L) + 50 μL of Salkowski's colorimetric solution. After standing for 30 minutes in a dark and temperature-constant environment, the color change was observed.

[0037] (2) Qualitative analysis of phosphorus-solubilizing function

[0038] The isolated and screened rice endophytic bacteria were inoculated onto a solid medium for detecting the phosphorus-solubilizing function of the strains. Whether a phosphorus-solubilizing circle appeared on the plate was observed within 2 - 7 days, and the size (diameter, mm) of the strain and the phosphorus-solubilizing circle was recorded.

[0039] (3) Qualitative analysis of nitrogen-fixing function

[0040] The isolated and screened rice endophytic bacteria were inoculated onto a solid medium for detecting the nitrogen-fixing function of the strains, and their growth was observed within 2 - 7 days.

[0041] (4) Analysis of Cd tolerance of endophytic bacteria

[0042] The isolated and screened rice endophytic bacteria were inoculated into 1 / 2LB liquid medium and cultured in a shaker (28 °C, 180 rpm / min) for 36 hours. 100 μL of the bacterial suspension was respectively spread onto 1 / 2LB solid medium containing different Cd concentrations (1 mmol / L and 4 mmol / L), and then placed in an incubator (28 °C) for inverted culture. Their growth was observed within 2 - 7 days.

[0043] (5) Analysis of siderophore production function of endophytic bacteria

[0044] Inoculate the isolated and screened rice endophytes onto CAS plates, place them in an incubator at a constant temperature (28 °C) for inverted culture, observe whether there is an orange-yellow halo around the strains inoculated on the plates within 2 - 7 days, and record the sizes (diameter, mm) of the strains and the halos.

[0045] The results of the above functional analyses are shown in the following table:

[0046]

[0047] (The ability of endophytic bacteria to produce siderophores is reflected by the formula HD / CD, where CD: diameter of the strain, HD: diameter of the yellow halo.)

[0048] (Tolerance of endophytic bacteria strains to heavy metal cadmium: "+": colonies present, "++": more colonies, "-": no colonies.)

[0049] (Analysis of the growth-promoting ability of strains: "+": has this function, "-": does not have this function.)

[0050] The results show that rice endophytes have certain growth-promoting and Cd-resistant abilities.

[0051] Example 3 Application of Rice Endophytes in Rice

[0052] Research on the bacteria themselves found that rice endophytes have certain growth-promoting and Cd-resistant abilities. When applying this rice endophyte to field production, the expected effects can be achieved.

[0053] Compared with CK (blank group), the contents of ammonium nitrogen, nitrate nitrogen, available phosphorus, and available potassium in the soil at the maturity stage of rice inoculated with rice endophytes all increased, and the contents of ammonium nitrogen, nitrate nitrogen, and available potassium increased significantly. Among them, the increase rates of rice inoculated with rice endophytes reached 15.21%, 24.77%, and 24.69% respectively. This shows that rice endophytes can increase the nutrient content in the soil and can significantly increase the dry matter weights of the roots, stem sheaths, leaves, and brown rice of rice plants. Among them, the increase rates of rice inoculated with rice endophytes reached 11.09%, 7.33%, 15.46%, and 9.38% respectively. This shows that rice endophytes can promote the growth and development of crops.

[0054] Inoculating rice with rice endophytes can also significantly reduce the Cd content in rice grains (P < 0.05). At the maturity stage of rice, the Cd content in rice grains without adding strains was 0.26 mg·kg -1 , and the Cd content in rice grains inoculated with rice endophytes was 0.15 mg·kg -1 , and the Cd reduction rate for rice grains at the maturity stage was 42.31%. This shows that rice endophytes can significantly reduce the Cd content in rice grains.

[0055] The rhizosphere microbial community is one of the important factors determining plant growth and metal bioavailability. The microbial community is generally described by microbial diversity indices, evenness indices, richness indices, as well as microbial structure and species composition. However, inoculation with rice endophytes has no significant effect on the diversity, structure, and relative abundance of the bacterial community composition in the rhizosphere soil of rice, indicating that inoculation with rice endophytes does not affect the original microbial environment of the soil.

[0056] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A rice endophyte that promotes rice growth and reduces cadmium, characterized in that, The rice endophyte is of the genus Stenotrophomonas, named Stenotrophomonas sp. R5, preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postcode 430072, preservation number CCTCC NO: M 20231758, and the preservation date is September 20, 2023.

2. The preparation method of a rice endophyte for promoting rice growth and reducing cadmium according to claim 1, characterized in that It includes the following steps: S1: Select plump and full rice seeds, conduct surface disinfection to ensure thorough sterilization; S2: Place the surface-disinfected rice seeds in a sterilized mortar, add liquid nitrogen and grind thoroughly until powdery, weigh with a sterilized weighing spoon and inoculate them into 250 mL of 1 / 2LB, TSA, R2A, NB and rice soup liquid media respectively, and place them in a shaker for cultivation; S3: Respectively suck 1 mL of the bacterial liquid and dilute it by different multiples, then respectively suck 100 μL of the diluted bacterial suspension and spread it on the corresponding solid medium, place it in an incubator for inverted cultivation, regularly observe the growth of the strains on the plate, after growing for a period of time, pick out with an inoculation loop and streak-culture on the corresponding solid medium to obtain purified rice endophytes.

3. The preparation method of a rice endophyte for promoting rice growth and reducing cadmium according to claim 2, characterized in that, The specific process of surface disinfection in step S1 is: soak with absolute ethanol, pour out the absolute ethanol, wash 5 times with sterilized water, then soak with NaClO, and wash 5 times with sterilized water.

4. The preparation method of an endophytic bacterium of rice for promoting rice growth and reducing cadmium according to claim 3, characterized in that, The method for inspecting thorough sterilization of surface disinfection: suck 100 μL of the sterilized water for the last rinsing of rice seeds and spread it on the 1 / 2LB solid medium, place it in an incubator for 2 - 5 days, observe whether there are strains growing on the medium, so as to determine whether the surface disinfection and sterilization of rice seeds are thorough.

5. The preparation method of an endophytic bacterium of rice for promoting rice growth and reducing cadmium according to claim 2, characterized in that, In step S2, the shaker speed is 180 rpm / min, the temperature is 28 °C; the cultivation time is 36 h.

6. The preparation method of an endophytic bacterium of rice for promoting rice growth and reducing cadmium according to claim 2, characterized in that, In step S3, the different dilution multiples are 1×10 4 , 1×10 5 and 1×10 6 times.

7. The preparation method of a rice endophyte for promoting rice growth and reducing cadmium according to claim 2, characterized in that, In step S3, the temperature of the incubator is 28 °C.

8. The preparation method of an endophytic bacterium of rice for promoting rice growth and reducing cadmium according to claim 2, characterized in that, In step S3, the growth time of the strains is 2 - 5 days.

9. The preparation method of a rice endophyte for promoting rice growth and reducing cadmium according to claim 2, characterized in that, The prepared rice endophytes are mixed evenly according to the ratio of bacterial liquid to sterile glycerol of 1:1, suck 1.5 mL and dispense it into 2 mL centrifuge tubes, and store it in an environment of -80 °C for standby.

10. The application of the rice endophyte for promoting rice growth and reducing cadmium as described in claim 1 or the rice endophyte prepared by the preparation method described in any one of claims 2 - 9 in rice.

Images