Copper-resistant plant growth-promoting endophyte, screening method and application thereof

Abstract

The present invention belongs to the field of heavy metal pollution remediation technology, and specifically discloses a copper-resistant plant growth-promoting endophyte and a screening method and application. The endophyte is Bacillus Bacillus sp The screening method is to cut the sterilized Commelina communis leaves and place them in NA medium at 28°C for 72 hours; the culture medium is transferred to a medium containing 100 mg / L Cu 2+ The culture medium was purified to single colonies by plate streaking method and then stored; the genomic DNA of endophytes was extracted, the gene fragments were amplified with universal primers, and the taxonomic status of the strains was determined by NCBI BLAST comparison after sequencing; the IAA production capacity and ACC deaminase activity of endophytes were determined, and the plant growth promotion potential of endophytes was quantitatively determined; the endophytes were inoculated in a gradient of Cu 2+ Rice culture system to determine the effects on plant growth and alleviation of heavy metal stress.

Description

Technical Field

[0001] The present invention relates to the technical field of heavy metal pollution remediation, and in particular to a copper-resistant plant growth-promoting endophyte, a screening method and an application thereof. Background Art

[0002] With the development of industrialization and agricultural intensification, heavy metal contamination of soils has become a serious problem in agricultural ecosystems worldwide, especially copper (Cu) contamination, whose main sources include metal mining, industrial waste, and excessive use of copper-containing fertilizers and fungicides. Although copper is an essential trace element for plant growth, excessive copper levels in soil can significantly inhibit plant growth, manifesting as reduced biomass, increased oxidative stress, and heavy metal accumulation, ultimately leading to reduced crop yields.

[0003] As one of the most important food crops, rice's high and stable yields are crucial to food security. Heavy metal pollution not only affects the rice's root absorption and transport systems, leading to root deformation and growth retardation, seriously impacting rice yield and quality; it also interferes with rice's photosynthesis and respiration, reducing its disease resistance and increasing the incidence of diseases and pests, leading to significant yield declines. Furthermore, heavy metal pollution can lead to the accumulation of heavy metals in rice grains, posing a serious threat to human health.

[0004] Currently, the main measures to control heavy metal pollution in rice include soil remediation technology and the cultivation of rice varieties with low heavy metal accumulation. Soil remediation technologies such as soil removal and replacement, chemical cleaning or phytoremediation are effective, but they are costly and time-consuming. Therefore, researchers are committed to cultivating rice varieties with low cadmium accumulation through gene editing technology. For example, gene mutation rice cultivated using the CRISPR / Cas9 system significantly reduced the cadmium content in the grain without affecting the yield. In addition, the team of Hu Peisong, an academician of the Chinese Academy of Engineering, successfully created a two-line hybrid rice with ultra-low accumulation of cadmium and arsenic in the grain, providing a solution for complex pollution areas.

[0005] Traditional soil remediation techniques (such as physical and chemical methods) suffer from high costs and destructiveness, while cultivating rice varieties with low heavy metal accumulation is time-consuming, costly, and risky. Therefore, plant-microbe co-remediation has attracted widespread attention as an economical, efficient, and environmentally friendly remediation technology. Plant growth-promoting bacteria (PGPRs) have significant potential for alleviating heavy metal stress and increasing crop yields due to their ability to fix nitrogen, solubilize phosphate, secrete plant hormones (such as indoleacetic acid, IAA), and enhance stress-resistant enzymes (such as ACC deaminase). However, existing research on endophytes that regulate heavy metal toxicity in rice is limited in its understanding of the regulatory mechanisms and has primarily focused on cadmium and chromium pollution, with little research on PGPRs targeting copper (Cu) pollution. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides a copper-resistant plant growth-promoting endophyte, a method for screening the copper-resistant plant growth-promoting endophyte, and an application of the copper-resistant plant growth-promoting endophyte.

[0007] The copper-resistant plant growth-promoting endophyte of the present invention is achieved as follows: the endophyte is Bacillus sp. Bacillus sp .D2, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on December 18, 2024, with the deposit number CGMCC No.33107.

[0008] The screening method of the present invention for copper-resistant plant growth-promoting endophytes is achieved by comprising the steps of host treatment, gradient screening, molecular identification, growth-promoting property analysis, and anti-Cu function verification, specifically as follows:

[0009] A. Host treatment: Cut the sterilized Commelina leaves into segments, take 2-3 segments and place them in a 50 mg / L Cu 2+ NA medium, cultured at 28 °C for 72 h;

[0010] B. Gradient screening: The endophyte culture solution obtained from the above constant temperature culture was transferred to a medium containing 100 mg / L Cu 2+ The culture medium was purified to single colonies by plate streaking method and then stored at 4°C;

[0011] C. Molecular identification: Genomic DNA of endophytes was extracted, and the 16S rRNA gene fragment was amplified using universal primers 27F / 1492R. After sequencing, the taxonomic status of the species was determined by NCBI BLAST comparison.

[0012] D. Growth-promoting properties analysis: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of the endophytes to quantitatively determine the plant growth-promoting potential of the endophytes;

[0013] E. Anti-Cu function verification: Endophytes were inoculated in a gradient of Cu 2+ A rice culture system was developed to determine its effects on plant growth and alleviating heavy metal stress.

[0014] Furthermore, in step C, genomic DNA of the endophyte was extracted using a Bacteria Genomic DNA Kit, and the DNA purity and integrity were verified using a Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. Subsequently, the genomic DNA of the endophyte that passed the DNA purity and integrity verification was amplified with the universal primers 27F / 1492R to amplify the 16S rRNA gene fragment. The amplified product was then sequenced, and finally, based on the sequencing results, a phylogenetic tree was constructed using the NCBI platform to determine the taxonomic status of the endophyte.

[0015] Furthermore, the IAA capacity determination process in step D is as follows:

[0016] D10, prepare 0~20μg / mL gradient IAA standard solution, then add Salkowski colorimetric reagent to measure OD 530 To establish the IAA standard curve;

[0017] D20: Inoculate the activated endophytes after molecular identification into LB liquid medium containing 200 mg / L L-tryptophan and culture at 28°C and 180 rpm for 48 h. Take 1 mL of the bacterial solution and mix it evenly with an equal volume of Salkowski color developer. Let it stand in the dark at room temperature for 30 min. If the color turns pink, it is a positive endophyte capable of producing IAA. Measure its OD 530 ;

[0018] D30, 1mL sterile water and an equal volume of Salkowski colorimetric reagent were used as blank controls, and quantitative experiments were performed according to the D20 steps. The samples were placed in a dark environment at room temperature for 30 minutes, and the OD values were measured. 530 The IAA content was calculated based on the IAA standard curve.

[0019] Furthermore, the ACC deaminase activity determination process in step D is as follows:

[0020] D60: The endophytes identified by molecular identification were activated and washed with Tris-HCl buffer at pH 7.6, then suspended in Tris-HCl containing 0.3% toluene at pH 8.5, and then 20 mM ACC substrate was added and reacted at 30°C for 15 min;

[0021] D70, after the reaction is completed, add 0.56M HCl to terminate the reaction, centrifuge the supernatant and add 2,4-dinitrophenylhydrazine, then react in a 30℃ water bath for 30min, then add 2mol / L NaOH to cause color reaction, and measure OD 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

[0022] Furthermore, the anti-Cu function verification process in step E is as follows:

[0023] E10. Germinate the sterilized rice seeds on a culture dish. After germination for 3 days in a 25°C incubator, select rice seeds at the same growth stage and transfer them to a black culture box filled with sterilized Hoagland nutrient solution.

[0024] E20, 7 days after transplanting, blank control group, single bacteria inoculation group, gradient Cu 2+ stress group (200-500 μmol / L) and bacteria-copper compound treatment group;

[0025] E30, the rice of the above groups were grown in an artificial climate incubator, and the phenotypic parameters, biomass, chlorophyll content and antioxidant enzyme activity of the rice of different groups were measured, and the measured parameters were compared to verify the endophytic bacteria. Bacillus sp. Alleviating effects of D2 on rice growth and heavy metal stress.

[0026] Furthermore, the chlorophyll content is determined by first extracting the leaf pigment using 80% acetone, then measuring the absorbance of the acetone-extracted supernatant at 663 nm and 645 nm, and then calculating the chlorophyll a and chlorophyll b contents according to the Arnon formula.

[0027] Furthermore, the step E also includes the determination of Cu content in rice tissues: first, the rice tissue sample is digested, and then the Cu content in each tissue is determined by ICP-OES. 2+ The transport coefficient TF was then calculated, i.e., TF = Cu content in the shoots / Cu content in the roots.

[0028] Furthermore, step E further includes root scanning electron microscopy analysis: after the rice tissue sample is fixed with glutaraldehyde, dehydrated with ethanol gradient and freeze-dried, the root surface morphology and bacterial colonization are observed by scanning electron microscopy.

[0029] The application of the copper-resistant plant growth-promoting endophyte of the present invention is achieved as follows: the copper-resistant plant growth-promoting endophyte is used to alleviate copper toxicity of rice.

[0030] Beneficial effects of the present invention:

[0031] 1. The present invention aims to solve the problem of crop toxicity stress caused by copper-contaminated soil, and uses the copper-enriched plant Commelina communis ( Commelina communis ) was used as the research object, and an endophyte of Commelina communis with copper resistance and plant growth-promoting function was screened for the first time. Bacillus sp. D2 (CGMCC No. 33107), clarify its physiological and biochemical characteristics, and explore its role and mechanism in alleviating copper toxicity in rice, providing new ideas and technical support for the remediation of heavy metal pollution.

[0032] 2. Endophytes of the present invention Bacillus sp. D2 by gradient Cu 2+ The strains were obtained through stress screening and 16S rRNA gene sequencing, and verified by anti-Cu function, which not only ensured accurate classification, but also revealed that the endophytes had both indoleacetic acid (IAA) production and ACC deaminase activity, thus ensuring high copper tolerance, significantly increasing rice biomass, chlorophyll content and antioxidant enzyme activity, and reducing the Cu transport coefficient.

[0033] 3. Endophytes of the present invention Bacillus sp. D2 directly promotes the development of rice roots by producing indoleacetic acid (IAA), thereby alleviating the growth inhibition caused by copper stress; and by decomposing ethylene precursors (ACC), it can reduce the adverse ethylene level of rice, thereby reducing oxidative stress damage; and after the endophyte colonizes the rice root system, it can reduce the root's sensitivity to Cu 2+ The absorption of heavy metals was enhanced and the transport to the aboveground parts was reduced (the transport coefficient TF decreased significantly), thereby reducing the risk of heavy metal accumulation in grains.

[0034] 4. Endophytes of the present invention Bacillus After inoculation of rice with sp. D2, the Cu resistance function was verified, and the aboveground biomass of rice seedlings increased by 47.61% and the length increased by 34.42%, while the Cu resistance of stem and leaf tissues increased by 1.3%. 2+ The contents decreased by 63.42% and 37.88% respectively, and Cu 2+ Endophyte inoculation Bacillus sp. D2, there was no rupture or collapse of the root surface and a large number of bacteria were present on the surface, indicating that the rice was inoculated with endophytes. Bacillus sp. D2 can alleviate Cu 2+ Adverse effects on growth under stress.

[0035] 5. This invention uses scanning electron microscopy to observe endophyte colonization and root morphology in rice roots, combined with gene sequencing data, to provide a basis for subsequent mining of endophyte functional genes (such as copper transporter regulatory genes). Furthermore, through endophyte screening methods (such as treatment with the host Commelina communis), this method can be extended to other heavy metal pollution phytoremediation systems, showing potential for cross-species application.

[0036] 6. The application of the copper-resistant plant growth-promoting endophytes of the present invention in alleviating copper toxicity of rice not only has no secondary pollution risk compared to chemical chelating agents or soil replacement, but also has low cost; Bacillus sp. D2 can be inoculated by soaking seeds or watering, which is easy to operate and suitable for different planting patterns.

[0037] In summary, compared with traditional physical and chemical remediation technologies for treating heavy metal pollution in rice, the present invention has the characteristics of being environmentally friendly, low-cost, and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram showing the principle of endophyte separation and its effect on alleviating Cu toxicity in rice according to the present invention;

[0039] In the figure: (a) morphology of the plant from which the endophytes were isolated, (b) scanning electron micrograph of the endophytes, (c) phylogenetic tree of the endophytes, and (d) schematic diagram of the control mechanism of endophyte-mediated alleviation of Cu toxicity in rice.

[0040] Figure 2 for Figure 1 Enlarged view of Figure (c);

[0041] Figure 3 Comparison of IAA and ACC production by the endophytes of the present invention;

[0042] Figure: 13 strains of Cu-resistant endophytic bacteria were isolated and purified from Commelina communis leaves in four regions, including two strains from Changsha, named C-1 and C-2; four strains from Liuyang, named L-1, L-4, L-6, and L-8; three strains from Zhuzhou, named Z-1, Z-2, and Z-3; and four strains from Daye, named D-2, D-3, D-4, and D-6.

[0043] Figure 4 Comparison of the length and biomass of rice under different treatment conditions in the examples of the present invention;

[0044] In the figure: (a) is a comparison of rice length and morphology, (b) is a comparison of rice length (where a, b...e are significant differences, the same below), (c) is a comparison of rice biomass;

[0045] Figure 5 Comparison of copper distribution and transport coefficient in rice tissues under different treatments in the examples of the present invention;

[0046] In the figure: (a) shows the comparison of copper content in different tissues of rice, (b) shows the copper transfer coefficient of rice;

[0047] Figure 6 Comparison of antioxidant enzyme activities in rice leaves and roots under different treatments in the examples of the present invention;

[0048] In the figure, (a), (c), and (e) are comparisons of leaf activity, and (b), (d), and (f) are comparisons of root activity;

[0049] Figure 7 These are scanning electron micrographs of rice roots under different treatments in the examples of the present invention;

[0050] In the figure: (a) shows the surface morphology of rice roots under normal conditions, (b) shows the surface morphology of rice roots after adding D2 treatment, and (c) shows the surface morphology of rice roots after adding 400 µM Cu 2+ Surface morphology of rice roots after treatment. (d) Figure 4 shows the addition of 400 µM Cu 2+ Surface morphology of rice roots after +D2 treatment. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0052] like Figures 1 to 7 As shown, the copper-resistant plant growth-promoting endophyte of the present invention is Bacillus Bacillus sp . D2, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on January 3, 2025, with the deposit number CGMCC No.33107.

[0053] The screening method for copper-resistant plant growth-promoting endophytes of the present invention includes host treatment, gradient screening, molecular identification, growth-promoting property analysis, and anti-Cu function verification steps, and specifically includes the following steps:

[0054] A. Host treatment: Cut the sterilized Commelina leaves into segments, take 2-3 segments and place them in a 50 mg / L Cu 2+ NA medium, cultured at 28 °C for 72 h;

[0055] B. Gradient screening: The endophyte culture solution obtained from the above constant temperature culture was transferred to a medium containing 100 mg / L Cu 2+ The culture medium was purified to single colonies by plate streaking method and then stored at 4°C;

[0056] C. Molecular identification: Genomic DNA of endophytes was extracted, and the 16S rRNA gene fragment was amplified using universal primers 27F / 1492R. After sequencing, the taxonomic status of the species was determined by NCBI BLAST comparison.

[0057] D. Growth-promoting properties analysis: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of the endophytes to quantitatively determine the plant growth-promoting potential of the endophytes;

[0058] E. Anti-Cu function verification: Endophytes were inoculated in a gradient of Cu 2+ A rice culture system was developed to determine its effects on plant growth and alleviating heavy metal stress.

[0059] In step C, endophyte genomic DNA was extracted using a Bacteria Genomic DNA Kit. The DNA purity and integrity were then verified using a Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. The endophyte genomic DNA, which passed the DNA purity and integrity verification, was then amplified using universal primers 27F / 1492R for the 16S rRNA gene fragment. The amplified product was then sequenced. Finally, based on the sequencing results, a phylogenetic tree was constructed using the NCBI platform to determine the taxonomic status of the endophyte.

[0060] The IAA capacity determination process in step D is as follows:

[0061] D10, prepare 0~20μg / mL gradient IAA standard solution, then add Salkowski colorimetric reagent to measure OD 530 To establish the IAA standard curve;

[0062] D20: Inoculate the activated endophytes after molecular identification into LB liquid medium containing 200 mg / L L-tryptophan and culture at 28°C and 180 rpm for 48 h. Take 1 mL of the bacterial solution and mix it evenly with an equal volume of Salkowski color developer. Let it stand in the dark at room temperature for 30 min. If the color turns pink, it is a positive endophyte capable of producing IAA. Measure its OD 530 ;

[0063] D30, 1mL sterile water and an equal volume of Salkowski colorimetric reagent were used as blank controls, and quantitative experiments were performed according to the D20 steps. The samples were placed in a dark environment at room temperature for 30 minutes, and the OD values were measured. 530 The IAA content was calculated based on the IAA standard curve.

[0064] The ACC deaminase activity determination process in step D is as follows:

[0065] D60: The endophytes identified by molecular identification were activated and washed with Tris-HCl buffer at pH 7.6, then suspended in Tris-HCl containing 0.3% toluene at pH 8.5, and then 20 mM ACC substrate was added and reacted at 30°C for 15 min;

[0066] D70, after the reaction is completed, add 0.56M HCl to terminate the reaction, centrifuge the supernatant and add 2,4-dinitrophenylhydrazine, then react in a 30℃ water bath for 30min, then add 2mol / L NaOH to cause color reaction, and measure OD 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

[0067] The anti-Cu function verification process in step E is as follows:

[0068] E10. Germinate the sterilized rice seeds on a culture dish. After germination for 3 days in a 25°C incubator, select rice seeds at the same growth stage and transfer them to a black culture box filled with sterilized Hoagland nutrient solution.

[0069] E20, 7 days after transplanting, blank control group, single bacteria inoculation group, gradient Cu 2+ stress group (200-500 μmol / L) and bacteria-copper compound treatment group;

[0070] E30, the rice of the above groups were grown in an artificial climate incubator, and the phenotypic parameters, biomass, chlorophyll content and antioxidant enzyme activity of the rice of different groups were measured, and the measured parameters were compared to verify the endophytic bacteria. Bacillus sp. Alleviating effects of D2 on rice growth and heavy metal stress.

[0071] The chlorophyll content was determined by first extracting the leaf pigment using 80% acetone, then measuring the absorbance of the supernatant extracted with acetone at 663 nm and 645 nm, and then calculating the chlorophyll a and chlorophyll b contents according to the Arnon formula.

[0072] The step E also includes the determination of Cu content in rice tissues: first, the rice tissue samples are digested, and then the Cu content in each tissue is determined by ICP-OES. 2+ The transport coefficient TF was then calculated, i.e., TF = Cu content in the shoots / Cu content in the roots.

[0073] The step E also includes root scanning electron microscopy analysis: after the rice tissue sample is fixed with glutaraldehyde, dehydrated with ethanol gradient and freeze-dried, the root surface morphology and bacterial colonization are observed by scanning electron microscopy.

[0074] The invention provides an application of the copper-resistant plant growth-promoting endophyte in alleviating copper toxicity in rice.

[0075] Example 1

[0076] S100: Place the Commelina communis ( Commelina communis ) Cut the leaves into pieces of appropriate size (such as 1cm×1cm pieces), and then take 2~3 pieces and place them on the Cu 2+ The cells were cultured in a 50 mg / L NA medium at 28°C for 72 h.

[0077] S200: The endophyte culture solution obtained by the above constant temperature culture is transferred to the Cu 2+The culture medium was placed upside down in a 28°C incubator for 48 hours, with 3 replicates for each treatment. The colonies were picked according to their size, morphology, color, and transparency, and the plate streak method was used to culture the colonies on a plate containing Cu. 2+ The culture medium was streaked and purified continuously to obtain single colonies, which were stored in a 4°C refrigerator for future use.

[0078] S300: Genomic DNA from endophytic and rhizospheric bacteria of Commelina communis was extracted using the Bacteria Genomic DNA Kit according to the manufacturer's instructions. DNA concentration and integrity were determined using a Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. DNA that had passed DNA purity and integrity verification was then used as a template for PCR amplification of the 16S rRNA gene fragment using universal primers 27F and 1492R, synthesized by Beijing Qingke Biotechnology Co., Ltd. The amplified products were verified by 1% agarose gel electrophoresis and then sent to Qingke Biotechnology Co., Ltd. (Beijing) for sequencing. The resulting sequence was then compared with the endophytes by BLAST analysis on the NCBI (https: / / www.ncbi.nlm.nih.gov) platform to identify endophytes with high similarity. Figure 2 For bacteria Bacillus sp. D2 phylogenetic tree, through the bacterial Bacillus sp. D2 were sequenced and phylogenetic tree analyzed, and it was found that Bacillus huizhouensis strain The homology of CA98 reached 99.93%, which can be confirmed to be Bacillus; Bacillus The GenBank accession number of sp. D2 is ON000250.

[0079] S400: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of endophytes, analyze the growth-promoting properties of the different endophytes obtained above for anti-Cu bacteria, select the endophytes with the best growth-promoting properties, and quantitatively determine the plant growth-promoting potential of endophytes.

[0080] The process of determining the ability of endophytes to produce indoleacetic acid (IAA) is as follows:

[0081] S410. Prepare indoleacetic acid (IAA) standard solutions at concentrations of 0, 1, 2, 5, 10, and 20 µg / mL. Then add Salkowski colorimetric reagent (50 mL of H2SO4: 1 mL of 0.5 M FeCl3) and measure the OD. 530 To establish a standard curve of indoleacetic acid.

[0082] S420. The activated endophytes after molecular identification were inoculated into LB liquid medium containing 200 mg / L L-tryptophan and cultured at 28°C and 180 rpm for 48 h. 1 mL of the bacterial solution was mixed evenly with 1 mL of Salkowski color developer and allowed to stand at room temperature in the dark for 30 min. If the color turned pink, it was a positive endophyte capable of producing IAA. The OD value was measured. 530 .

[0083] S430, use 1 mL of sterile water and 1 mL of Salkowski colorimetric reagent as blank controls, and perform quantitative experiments according to the steps in S420. Let the mixture stand in a dark environment at room temperature for 30 minutes, and measure the OD 530 The IAA content was calculated based on the indoleacetic acid standard curve.

[0084] The determination process of ACC deaminase activity produced by endophytes is as follows:

[0085] S460. After activation of the molecularly identified endophytes, culture them in DFa medium at 28°C for 48 h, then centrifuge at 8000 rpm for 10 min at 4°C, remove the supernatant, and add 5 mL of 0.1 mol / L Tris-HCl buffer (pH 7.6). Centrifuge and remove the supernatant. Resuspend the cells in 600 µL of 0.1 mol / L Tris-HCl (pH 8.5), then add 30 µL of 0.3% toluene, rapidly shake for 30 s, and mix thoroughly to obtain a mixture. Add 200 µL of the above mixture and 20 µL of 0.5 mol / L ACC to a 1.5 mL centrifuge tube and incubate at 30°C for 15 min.

[0086] S470. After the reaction is complete, add 1 mL of 0.56 M HCl and mix well to terminate the reaction. Then, centrifuge at 16,000 rpm for 5 minutes at room temperature. Take 0.5 mL of the supernatant obtained by centrifugation and mix it evenly with 400 µL of 0.56 mol / L HCl. Then, add 150 µL of 2,4-dinitrophenylhydrazine and react in a 30°C water bath for 30 minutes. Then, add 1 mL of 2 mol / L NaOH to cause a color reaction and measure the OD. 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

[0087] The results are as follows Figure 3 As shown, in the absence of Cu 2+ Under stress, all bacteria have the ability to produce IAA, and the range of bacterial IAA production is 1.67~9.72µg / mL. Among them, the highest IAA production is from bacteria D-2 (i.e., endophytic bacteria Bacillus sp. D2); at 100 mg / L Cu 2+Under these conditions, the IAA production of most bacteria increased, among which D-2 increased rapidly, which was 3.5 times that of normal conditions; 200 mg / L Cu 2+ Under stress, the IAA production of bacteria decreased.

[0088] ACC deaminase has been shown to be present in endophytes that promote plant growth. It can convert ACC into ammonia and α-ketobutyrate, reducing the ethylene content in plants. Plants generally produce only a small amount of ethylene, but when plants are under stress, the level of endogenous ethylene tends to increase significantly. 2+ Under stress, the ACC deaminase content increased significantly. Figure 3 It can be seen that there is no Cu 2+ Under stress, the ACC deaminase activity of bacteria L-1 and L-4 was higher, and 100 mg / L Cu 2+ Under these conditions, ACC deaminase activity increased, and the increase of bacteria D-2 was the largest.

[0089] S500: Inoculate endophytes into a gradient of Cu 2+ The rice culture system with a concentration of 200-500 μmol / L was used to determine its effect on plant growth and the alleviation of heavy metal stress. The specific determination process is as follows:

[0090] S510. Germinate the sterilized rice seeds on a culture dish. After germination for 3 days in a 25°C incubator, select rice seeds at the same growth stage and transfer them to a black culture box (12 cm long × 8 cm wide × 13 cm high) filled with sterilized Hoagland nutrient solution (pH 5.8).

[0091] S520, 7 days after transplanting, different treatments were carried out: (1) blank group (CK); (2) inoculation of endophytes Bacillus sp D2; (3) 200µmol / L Cu 2+ ; (4) Inoculation Bacillus sp D2 + 200 µmol / L Cu 2+ ; (5) 300µmol / L Cu 2+ ; (6) Inoculation Bacillus sp D2+300µmol / L Cu 2+ ; (7) 400µmol / L Cu 2+ ; (8) Inoculation Bacillus sp D2+400µmol / L Cu 2+ ; (9) 500µmol / L Cu 2+ ; (10) Inoculation Bacillus sp D2+500µmol / L Cu 2+ .

[0092] Rice in S530 and the aforementioned groups were grown in an artificial climate incubator (parameters set at 25°C, 70% relative humidity, and 22,000 Lux / 16h and 0 Lux / 8h) for 14 days. Rice seedling tissues were collected and the phenotypic parameters (root length, plant height), biomass (fresh weight, dry weight), chlorophyll content, and antioxidant enzyme (POD, APX, CAT) activities of the rice in different groups were measured. The measured parameters were compared to verify the endophytic bacteria. Bacillus sp. Alleviating effects of D2 on rice growth and heavy metal stress.

[0093] The specific process of measuring the phenotypic parameters, biomass and chlorophyll of different groups of rice is as follows:

[0094] S531. Root and shoot lengths of rice seedlings were measured by photographing and using ImageJ online software, which can be accessed at https: / / rsb.info.nih.gov / ij / .

[0095] S532. The seedling tissues were then divided into roots and stems, and the fresh weights of the two components were recorded; they were then dried and weighed to determine the dry weight, which was used to represent the biomass.

[0096] To assess the chlorophyll content of aboveground parts, 0.1 g of fresh tissue homogenate was added to 10 mL of 80% acetone to extract leaf pigments. The absorbance of the acetone-extracted supernatant was measured at 663 nm and 645 nm, respectively, using a UV-visible spectrophotometer (LabTech, UV 8100D, China). The chlorophyll a and b contents in the supernatant were then calculated according to the Arnon formula.

[0097] The results of the test are as follows Figure 4 As shown, different concentrations of Cu 2+ Add to inoculum Bacillus sp. D2 (abbreviated as D2 in the figure, the same below) in the culture medium of rice seedlings to find the optimal Cu 2+ With the concentration of heavy metal Cu 2+ The higher the concentration, the more obvious the inhibitory effect on rice seedling growth. 2+ When the concentration is 200µmol / L, 300µmol / L, 400µmol / L and 500µmol / L, it has a certain inhibitory effect on the growth of rice, among which 400µmol / L and 500µmol / L have the greatest inhibitory effect. Bacillus sp. D2, in 400µmol / L Cu 2+ The best mitigation effect is achieved under the following conditions. 2+At a concentration of 400µmol / L, the growth of rice seedlings was significantly inhibited, with the aboveground plant height decreasing by 32.2%, and the aboveground and underground biomass decreasing by 47.49% and 19.89%, respectively (e.g. Figure 4 c), growth inhibition and biomass reduction are the most obvious toxic reactions of plants when facing heavy metal stress. Bacillus sp. D2, the most significant effect on alleviating the toxicity of heavy metal Cu was observed. The aboveground biomass increased by 47.61% and the length increased by 34.42%. This preliminarily indicated that the inoculated bacteria Bacillus sp. D2 can effectively alleviate the damage caused by Cu stress on rice seedlings, so 400µmol / L Cu 2+ Conduct follow-up experiments.

[0098] The specific process of determining the activities of antioxidant enzymes (POD, APX, CAT) in different groups of rice is as follows:

[0099] S535. Weigh 0.1 g of fresh rice leaves, add 1 mL of phosphate buffer (pH 7.8), grind on ice, transfer to a test tube, centrifuge at 8000 g for 10 min at 4°C, and collect the supernatant for analysis.

[0100] S536. Mix 20 mL of the working solution in the CAT kit and keep it warm in a 25°C water bath for 10 minutes.

[0101] S537. Take 1 mL of CAT working solution and add 35 µL of sample supernatant. Measure the absorbance at a wavelength of 240 nm for 5 seconds and 1 minute, respectively. Determine the CAT content based on the absorbance.

[0102] The activities of S538, APX and POD were determined using corresponding kits.

[0103] To combat the negative effects of reactive oxygen species (ROS), plants activate their own antioxidant defense system to eliminate ROS. This is an important part of a plant's defense against heavy metal poisoning, primarily including APX, POD, and CAT. Superoxide dismutase catalyzes the conversion of superoxide radicals into less toxic H2O2, serving as the first line of defense against oxidation. Ascorbic acid peroxidase catalyzes the oxidation of ascorbic acid by H2O2, serving as a major consumer of ascorbic acid in plants. CAT and POD convert H2O2 into H2O and O2. Test results are as follows: Figure 6 As shown, toxic Cu 2+ The antioxidant enzyme activities of rice seedlings decreased under copper stress, with APX and CAT activities in leaves decreasing by 69.18% and 44.54%, respectively, and APX activity in roots decreasing by about 50%. BacillusAfter inoculation with sp. D2, the POD activity in rice leaves and roots increased by 70.3% and 12.3%, respectively, and the APX and CAT activities also showed the same trend. Bacillus sp D2 significantly increased the activities of antioxidant enzymes, indicating that inoculation can effectively enhance the defense ability of rice seedlings against Cu toxicity-induced oxidative stress and reduce oxidative damage in plant cells.

[0104] S600: Rice tissue samples were digested and then the Cu content in each tissue was determined by ICP-OES. 2+ The concentration is then used to calculate the transport coefficient TF, which is TF = Cu content in the aboveground part / Cu content in the root. The specific measurement process is as follows:

[0105] S610. Take rice plants and separate them into roots, stems, and leaves. Wash them three times with deionized water, then soak them in 20 mM ethylenediaminetetraacetic acid (EDTA) solution for 20 minutes to remove heavy metal ions on the surface. Repeat this three times, and then dry them in a drying oven at 105°C.

[0106] S620. Take 0.2 g of dried plant roots, stems and leaves, cut them into pieces, add 8 mL of HNO3 and 2 mL of HClO4 respectively into a tetrafluoroethylene tube, and use a fully automatic digestion instrument to thoroughly digest them.

[0107] S630, then filter the digestion solution with a 0.22µm filter membrane and use ICP-OES to determine the Cu content in each tissue. 2+ Concentration, and finally calculate the transport coefficient (TF) = Cu content in above-ground tissues / Cu content in underground tissues.

[0108] After preliminary experiments, the following treatment groups were finally determined for the Cu content determination experiment: (1) blank control group (CK); (2) inoculated endophyte Bacillus sp D2 (D2); (3) Add 400µmol / L Cu 2+ Coercion (Cu 2+ ); (4) 400µmol / L Cu 2+ Inoculum under stress Bacillus sp . D2(D2+Cu 2+ ). The result is as follows Figure 5 As shown, Cu 2+ Under the treatment, the Cu content in the root was 1904.36 mg / kg±37.4 mg / kg, Cu 2+ Inoculum under stress Bacillus sp The Cu content after D2 was 3826.26 mg / kg±119.9 mg / kg, which increased by about 1 times. The Cu content of the stem and leaf tissues after inoculation was 3826.26 mg / kg±119.9 mg / kg, which increased by about 1 times. 2+The contents of Cu and Cr decreased by 63.42% and 37.88% respectively. 2+ The transport coefficient of the inoculated bacteria was smaller, and less heavy metals were transferred to the stems and leaves of rice seedlings. Bacillus sp . D2 leads to more Cu absorption by roots 2+ , but less Cu 2+ transported to aboveground tissues.

[0109] S700: After the rice tissue samples were fixed with glutaraldehyde, dehydrated with ethanol gradients, and freeze-dried, the root surface morphology and bacterial colonization were observed using a scanning electron microscope. The specific operation of the scanning electron microscope is as follows:

[0110] S710, bacteria collection Bacillus sp Rice root tissues at D2 and under different treatments were fixed with 2.5% glutaraldehyde at 4°C overnight and then washed three times with phosphate buffered saline (PBS) (0.1 mol / L).

[0111] S720, then add 30%, 50%, 70%, 90%, and 100% ethanol for step-by-step dehydration, each dehydration for 30 minutes. Then, pre-freeze the dehydrated tissue at -80°C overnight, and then dry it in a vacuum freeze dryer. Finally, place the dried rice root tissue on a metal slide, spray it with gold, and observe.

[0112] Observation results such as Figure 7 The surface morphology of rice roots under different treatments is shown. Under normal treatment, the root cell surface is smooth, without rupture, and very few microorganisms exist ( Figure 7 a); vaccination Bacillus The surface of the rice roots of sp. D2 remained smooth and intact, with no root cell rupture and the presence of certain microorganisms ( Figure 7 b) Add 400µmol / L Cu 2+ After treatment, it was found that the root surface became rough, and the root epidermis and cortex tissue were partially broken, indicating that the root system was poisoned by heavy metals to a certain extent ( Figure 7 c);Cu 2+ Inoculum Bacillus sp. D2, there was no rupture or collapse of the root surface, and a large number of bacteria were present on the surface ( Figure 7 d), indicating bacteria Bacillus sp. D2 can colonize in the rice root system and effectively alleviate the damage of heavy metals to root tissues.

[0113] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A copper-resistant plant growth-promoting endophyte, wherein the endophyte is Bacillus Bacillus sp. D2, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on January 3, 2025, with the deposit number CGMCC No. 33107.

2. Use of the copper-resistant plant growth-promoting endophyte according to claim 1 in alleviating copper toxicity in rice.

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