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

Abstract

The invention belongs to the technical field of heavy metal pollution remediation, and particularly discloses a copper-resistant plant growth-promoting endophyte as well as a screening method and application thereof. The endophyte is bacillus sp. D2, and the preservation number of the endophyte is CGMCC (China General Microbiological Culture Collection Center) No.33107. The screening method comprises the following steps: cutting sterilized dayflower leaves, and culturing in an NA culture medium at a constant temperature of 28 DEG C for 72 hours; transferring the culture solution to a culture medium containing 100mg / L of Cu < 2 + >, purifying the culture solution to a single colony through a plate streaking method, and storing the single colony The method comprises the following steps: extracting endophyte genome DNA (Deoxyribonucleic Acid), amplifying gene segments by using a universal primer, sequencing, and comparing and determining strain classification status through NCBI BLAST; determining the IAA production capacity and ACC deaminase activity of the endophyte, and quantitatively determining the plant growth promoting potential of the endophyte; the endophyte is inoculated into a gradient Cu < 2 + >-containing rice culture system, and the relieving effect on plant growth and heavy metal stress is measured.

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 and a screening method and application thereof. Background Art

[0002] With the development of industrialization and agricultural intensification, heavy metal pollution in soil has become a serious problem in global agricultural ecosystems, especially copper (Cu) pollution, the main sources of which include metal mining, industrial waste and excessive use of copper-containing fertilizers and fungicides. Although copper is an essential trace element for plant growth, when the copper content in the soil is too high, it will significantly inhibit plant growth, manifested as reduced biomass, increased oxidative stress and heavy metal enrichment, ultimately leading to reduced crop yields.

[0003] As one of the most important food crops, rice's high and stable yields are related to food security. Heavy metal pollution not only affects the root absorption and transportation system of rice, leading to root deformation, slow growth and other problems, seriously affecting rice yield and quality; but also interferes with rice's photosynthesis and respiration, reduces its disease resistance, increases the incidence of diseases and pests, and leads to a significant drop in yield; and heavy metal pollution leads to the enrichment and accumulation of heavy metal elements in rice grains, thus posing a serious threat to human health.

[0004] At present, the main measures to control heavy metal pollution in rice include soil remediation technology and cultivating rice varieties with low heavy metal accumulation. Soil remediation technologies such as soil removal and replacement, chemical cleaning or phytoremediation are effective, but costly and time-consuming. Therefore, researchers are committed to cultivating low cadmium accumulation rice varieties through gene editing technology. For example, genetically mutated rice cultivated by the CRISPR / Cas9 system significantly reduced the cadmium content in the grains 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 grains, providing a solution for complex polluted areas.

[0005] Traditional soil remediation technologies (such as physical and chemical methods) have disadvantages such as high cost and strong destructiveness, while the cultivation of low heavy metal accumulation rice varieties has a long cycle, high cost and high risk. Therefore, plant-microorganism combined remediation has attracted widespread attention as an economical, efficient and environmentally friendly remediation technology. Among them, plant growth-promoting bacteria (PGPR) have the ability to fix nitrogen, dissolve phosphorus, secrete plant hormones (such as indoleacetic acid, IAA) and stress-resistant enzymes (such as ACC deaminase), thereby enhancing the tolerance of plants to heavy metals. Therefore, they have important potential in alleviating heavy metal stress and increasing crop yields. However, the existing research on endophytes for heavy metal poisoning in rice not only has a limited understanding of the regulatory mechanism, but also mainly focuses on the research of cadmium and chromium pollution, and there is little research on plant growth-promoting bacteria for 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 endophytes of the present invention are achieved as follows: the endophytes are Bacillus sp. Bacillus sp .D2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on December 18, 2024, with the deposit number CGMCC No.33107.

[0008] The screening method of the copper-resistant plant growth-promoting endophytes of the present invention is achieved by comprising the steps of host treatment, gradient screening, molecular identification, growth-promoting property analysis, and anti-Cu function verification, and the specific contents are as follows: A. Host treatment: Cut the sterilized Commelina leaf into segments, take 2-3 segments and place them in a 50 mg / L Cu 2+ NA medium was cultured at 28°C for 72 h; 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; C. Molecular identification: The genomic DNA of endophytes was extracted, and the 16S rRNA gene fragment was amplified with universal primers 27F / 1492R. After sequencing, the taxonomic status of the species was determined by NCBI BLAST comparison; D. Analysis of growth-promoting properties: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of endophytes and quantitatively determine the plant growth-promoting potential of endophytes; E. Anti-Cu function verification: Endophytes were inoculated in a gradient Cu 2+ A rice culture system was developed to determine its effects on plant growth and alleviating heavy metal stress.

[0009] Furthermore, in the step C, the genomic DNA of the endophyte is extracted using a Bacteria Genomic DNA Kit, and then the DNA purity and integrity are verified by a Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. Subsequently, the genomic DNA of the endophyte that has passed the DNA purity and integrity verification is amplified with the universal primers 27F / 1492R for the 16S rRNA gene fragment, and then the amplified product is sequenced. Finally, based on the sequencing results, a phylogenetic tree is constructed through the NCBI platform to determine the taxonomic status of the endophyte.

[0010] Furthermore, the IAA capability determination process in step D is as follows: D10, prepare 0~20μg / mL gradient IAA standard solution, then add Salkowski colorimetric reagent to measure OD 530 To establish the IAA standard curve; D20, inoculate the activated endophytes after molecular identification into LB liquid medium containing 200 mg / L L-tryptophan, culture at 28°C and 180 rpm for 48 h, take 1 mL of 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 is pink, it is a positive endophyte that can produce IAA, and measure its OD 530 ; D30, use 1mL sterile water and an equal volume of Salkowski colorimetric reagent as blank control, and perform quantitative experiments according to the D20 steps. Let it stand in the dark at room temperature for 30 minutes and measure the OD 530 , the IAA content was calculated according to the IAA standard curve.

[0011] Furthermore, the ACC deaminase activity determination process in step D is as follows: D60, the endophytes identified by molecular identification were activated and washed with Tris-HCl buffer at pH 7.6, then suspended with Tris-HCl containing 0.3% toluene and pH 8.5, and then 20 mM ACC substrate was added to react at 30°C for 15 min; D70, after the reaction is completed, add 0.56M HCl to terminate the reaction, centrifuge to obtain the supernatant and add 2,4-dinitrophenylhydrazine, then react in a 30℃ water bath for 30min, then add 2mol / L NaOH to produce a color reaction, and measure OD 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

[0012] Furthermore, the anti-Cu function verification process in step E is as follows: E10, germinating the sterilized rice seeds on a culture dish, and after germination for 3 days in a 25° C. incubator, selecting rice seeds of the same growth period and transferring them to a black culture box filled with sterilized Hoagland nutrient solution; 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; E30, the rice in the above groups were grown in an artificial climate incubator, and the phenotypic parameters, biomass, chlorophyll content and antioxidant enzyme activity of rice in different groups were measured, and the measured parameters were compared to verify the endophytic bacteria Bacillus sp.Effects of D2 on rice growth and alleviating heavy metal stress.

[0013] Furthermore, the chlorophyll content is determined by first using 80% acetone to extract leaf pigments, then measuring the absorbance of the supernatant extracted with acetone at 663nm and 645nm, and then calculating the content of chlorophyll a and chlorophyll b according to the Arnon formula.

[0014] Furthermore, the step E also includes the determination of the Cu content of rice tissues: firstly, the rice tissue samples are digested, and then the Cu in each tissue is determined by ICP-OES. 2+ The transport coefficient TF was then calculated, i.e., TF = Cu content in the aboveground part / Cu content in the root part.

[0015] Furthermore, 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.

[0016] The application of the copper-resistant plant growth-promoting endophytes of the present invention is achieved as follows: the aforementioned copper-resistant plant growth-promoting endophytes are used to alleviate copper toxicity of rice.

[0017] Beneficial effects of the present invention: 1. The present invention aims to solve the problem of crop toxicity stress caused by copper-contaminated soil by using the copper-enriching 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.

[0018] 2. Endophytes of the present invention Bacillus sp. D2 by gradient Cu 2+ The stress screening and 16S rRNA gene sequencing were obtained, and the anti-Cu function was verified, which not only ensured the accuracy of classification, but also the endophyte had both indoleacetic acid (IAA) production and ACC deaminase activity, thus ensuring high copper tolerance, significantly improving the biomass, chlorophyll content and antioxidant enzyme activity of rice, and reducing the Cu transport coefficient.

[0019] 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 endophytes colonize the rice roots, they can reduce the root's sensitivity to Cu2+ The absorption of heavy metals in grains can be enhanced and the transport to the aboveground parts can be reduced (the transport coefficient TF decreases significantly), thereby reducing the risk of heavy metal accumulation in grains.

[0020] 4. Endophytes of the present invention Bacillus After sp. D2 was inoculated into rice, 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 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 root cells on the root surface, and a large number of bacteria were present on the surface, indicating that rice inoculated with endophytes Bacillus sp. D2 can alleviate Cu 2+ Adverse effects on growth under stress.

[0021] 5. The present invention uses scanning electron microscopy to observe the colonization of endophytes and the root morphology of rice roots, combined with gene sequencing data, to provide a basis for subsequent endophyte functional gene mining (such as copper transporter regulatory genes); and through endophyte screening methods (such as host Commelina treatment), it can be extended to other heavy metal pollution plant remediation systems, and has cross-species application potential.

[0022] 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; and the endophytes Bacillus sp. D2 can be inoculated by soaking seeds or watering, which is easy to operate and suitable for different planting patterns.

[0023] 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

[0024] Figure 1 This is a schematic diagram of the separation of endophytes and their mitigation of Cu toxicity to rice in the present invention; In the figure: (a) shows the morphology of the plant from which the endophytes were isolated, (b) shows the scanning electron micrograph of the endophytes, (c) shows the phylogenetic tree of the endophytes, and (d) shows the control principle diagram of the mitigation of Cu toxicity in rice by endophytes; Figure 2 for Figure 1 Enlarged view of Figure (c); Figure 3 Comparison of IAA and ACC production of endophytes of the present invention; In the figure: 13 strains of Cu-resistant endophytic bacteria were isolated and purified from Commelina communis leaves in four regions, including 2 strains from Changsha, named C-1 and C-2; 4 strains from Liuyang, named L-1, L-4, L-6 and L-8; 3 strains from Zhuzhou, named Z-1, Z-2 and Z-3; 4 strains from Daye, named D-2, D-3, D-4 and D-6; Figure 4 The length biomass comparison of rice under different treatment conditions in the embodiments of the present invention; 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; Figure 5 Comparison of copper distribution and transport coefficient in rice tissues under different treatments in the embodiments of the present invention; In the figure: (a) shows the comparison of copper content in different tissues of rice, (b) shows the copper transfer coefficient of rice; Figure 6 Comparison of antioxidant enzyme activities in rice leaves and roots under different treatments in the embodiments of the present invention; In the figure, (a), (c) and (e) are comparisons of leaf activity, and (b), (d) and (f) are comparisons of root activity; Figure 7 The scanning electron microscope images of rice roots under different treatments in the embodiments of the present invention; 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

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is 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 used to limit the present invention.

[0026] 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 Microorganism Culture Collection Administration (CGMCC) on January 3, 2025, with the deposit number CGMCC No.33107.

[0027] The screening method of the copper-resistant plant growth-promoting endophyte of the present invention comprises the steps of host treatment, gradient screening, molecular identification, growth-promoting property analysis, and anti-Cu function verification, and the specific contents are as follows: A. Host treatment: Cut the sterilized Commelina leaf into segments, take 2-3 segments and place them in a 50 mg / L Cu 2+ NA medium was cultured at 28°C for 72 h; 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; C. Molecular identification: The genomic DNA of endophytes was extracted, and the 16SrRNA gene fragment was amplified with universal primers 27F / 1492R. After sequencing, the taxonomic status of the species was determined by NCBI BLAST comparison; D. Analysis of growth-promoting properties: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of endophytes and quantitatively determine the plant growth-promoting potential of endophytes; E. Anti-Cu function verification: Endophytes were inoculated in a gradient Cu 2+ A rice culture system was developed to determine its effects on plant growth and alleviating heavy metal stress.

[0028] In the step C, the genomic DNA of the endophyte is extracted using a Bacteria Genomic DNA Kit, and then the DNA purity and integrity are verified by a Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. Subsequently, the genomic DNA of the endophyte that has passed the DNA purity and integrity verification is amplified with the universal primers 27F / 1492R for the 16SrRNA gene fragment, and then the amplified product is sequenced. Finally, according to the sequencing results, a phylogenetic tree is constructed through the NCBI platform to determine the taxonomic status of the endophyte.

[0029] The IAA capability determination process in step D is as follows: D10, prepare 0~20μg / mL gradient IAA standard solution, then add Salkowski colorimetric reagent to measure OD 530 To establish the IAA standard curve; D20, inoculate the activated endophytes after molecular identification into LB liquid medium containing 200 mg / L L-tryptophan, culture at 28°C and 180 rpm for 48 h, take 1 mL of 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 is pink, it is a positive endophyte that can produce IAA, and measure its OD 530 ; D30, use 1mL sterile water and an equal volume of Salkowski colorimetric reagent as blank control, and perform quantitative experiments according to the D20 steps. Let it stand in the dark at room temperature for 30 minutes and measure the OD 530 , the IAA content was calculated according to the IAA standard curve.

[0030] The ACC deaminase activity determination process in step D is as follows: D60, the endophytes identified by molecular identification were activated and washed with Tris-HCl buffer at pH 7.6, then suspended with Tris-HCl containing 0.3% toluene and pH 8.5, and then 20 mM ACC substrate was added to react at 30°C for 15 min; D70, after the reaction is completed, add 0.56M HCl to terminate the reaction, centrifuge to obtain the supernatant and add 2,4-dinitrophenylhydrazine, then react in a 30℃ water bath for 30min, then add 2mol / L NaOH to produce a color reaction, and measure OD 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

[0031] The anti-Cu function verification process in step E is as follows: E10, germinating the sterilized rice seeds on a culture dish, and after germination for 3 days in a 25° C. incubator, selecting rice seeds of the same growth period and transferring them to a black culture box filled with sterilized Hoagland nutrient solution; 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; E30, the rice in the above groups were grown in an artificial climate incubator, and the phenotypic parameters, biomass, chlorophyll content and antioxidant enzyme activity of rice in different groups were measured, and the measured parameters were compared to verify the endophytic bacteria Bacillus sp. Effects of D2 on rice growth and alleviating heavy metal stress.

[0032] The chlorophyll content is determined by first using 80% acetone to extract leaf pigments, then measuring the absorbance of the supernatant extracted with acetone at 663nm and 645nm, and then calculating the content of chlorophyll a and chlorophyll b according to the Arnon formula.

[0033] The step E also includes the determination of the Cu content in rice tissues: firstly, the rice tissue samples are digested, and then the Cu in each tissue is determined by ICP-OES. 2+ The transport coefficient TF was then calculated, i.e., TF = Cu content in the aboveground part / Cu content in the root part.

[0034] 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.

[0035] The invention discloses an application of the aforementioned copper-resistant plant growth-promoting endophyte in alleviating copper toxicity of rice.

[0036] Example 1

[0037] S100: Place the Commelina communis ( Commelina communis ) The leaves were cut into pieces of appropriate size (e.g. 1 cm × 1 cm pieces), and then 2 to 3 pieces were placed on the Cu 2+ The cells were cultured in a 50 mg / L NA medium at 28°C for 72 h.

[0038] S200: The endophyte culture solution obtained from the above constant temperature culture is transferred to Cu 2+ The culture medium was placed upside down in a 28°C incubator for 48 hours. Three replicates were set for each treatment. Colonies were selected according to their size, shape, color, and transparency. 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.

[0039] S300: The genomic DNA of endophytic and rhizosphere bacteria of Commelina communis was extracted using the Bacteria Genomic DNA Kit. The experimental operation was carried out according to the instructions. The concentration and integrity of the DNA were then determined by Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. The DNA with qualified DNA purity and integrity was then used as a template to perform PCR amplification of the 16S rRNA gene fragment using universal primers 27F and 1492R synthesized by Beijing Qingke Biotechnology Co., Ltd. The amplified product was verified by 1% agarose gel electrophoresis and sent to Qingke Biotechnology Co., Ltd. (Beijing) for sequencing. The obtained sequencing sequences were subjected to BLAST comparison analysis on the NCBI (https: / / www.ncbi.nlm.nih.gov) platform to obtain 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 determined to be Bacillus; endophyte Bacillus The GenBank accession number of sp. D2 is ON000250.

[0040] S400: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of endophytes, analyze the growth-promoting properties of anti-Cu bacteria of the different endophytes obtained above, select the endophytes with the best growth-promoting properties, and quantitatively determine the plant growth-promoting potential of endophytes.

[0041] The process of determining the ability of endophytes to produce indoleacetic acid (IAA) is as follows: S410. Prepare standard solutions of indoleacetic acid (IAA) at concentrations of 0, 1, 2, 5, 10, and 20 µg / mL, and then add Salkowski colorimetric reagent (50 mL of H 2 SO 4 :1mL0.5M FeCl 3 ), determine OD 530 To establish a standard curve of indoleacetic acid.

[0042] S420, inoculate the activated endophytes after molecular identification into LB liquid medium containing 200 mg / L L-tryptophan, culture at 28°C and 180 rpm for 48 h, take 1 mL of bacterial solution and mix it evenly with 1 mL of Salkowski color developer, let it stand in a dark environment at room temperature for 30 min, if the color is pink, it is a positive endophyte that can produce IAA, and measure its OD 530 .

[0043] 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 of S420. Let it stand in a dark environment at room temperature for 30 minutes, and measure its OD 530 The IAA content was calculated based on the indoleacetic acid standard curve.

[0044] The determination process of ACC deaminase activity produced by endophytes is as follows: S460, after the molecular identification, the endophytes were activated and cultured in DFa medium at 28°C for 48h, then centrifuged at 8000r / min for 10min at 4°C, the supernatant was removed, and then 5mL of 0.1mol / L Tris-HCl buffer at pH 7.6 was added, and the supernatant was removed after centrifugation; then 600µL of 0.1mol / L Tris-HCl at pH 8.5 was used for suspension, and then 30µL of 0.3% toluene was added, and the mixture was shaken rapidly for 30s to be fully mixed to obtain a mixed solution; 200µL of the above mixed solution and 20µL of 0.5mol / L ACC were added to a 1.5mL centrifuge tube, and the mixture was reacted at 30°C for 15min; S470. After the above reaction is completed, add 1 mL of 0.56 M HCl and mix well to terminate the reaction. Then centrifuge at 16000 r / min at room temperature for 5 min. 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 min. Then add 1 mL of 2 mol / L NaOH to react and measure the OD. 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

[0045] 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, 3.5 times that of normal conditions; 200 mg / L Cu 2+ Under stress, the IAA production of bacteria decreased.

[0046] 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, so Cu 2+ Under stress, the content of ACC deaminase 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 in bacterial D-2 was the largest.

[0047] S500: Inoculate endophytes in a gradient of Cu 2+ (200~500μmol / L) rice culture system to determine its effect on plant growth and alleviation of heavy metal stress. The specific determination process is as follows: 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).

[0048] S520, 7 days after transplanting, different treatments were carried out: (1) blank control (CK); (2) inoculation of endophytes Bacillus sp . D2; (3) 200 µmol / L Cu 2+ ;(4)Inoculation Bacillus sp . D2+200 µmol / L Cu 2+ ; (5) 300 µmol / L Cu 2+ ;(6)Inoculation Bacillus sp . D2+300µmol / L Cu 2+ ; (7) 400 µmol / L Cu 2+ ; (8) Inoculation Bacillus sp . D2+400µmol / L Cu 2+ ; (9) 500 µmol / L Cu 2+ ;(10)Inoculation Bacillus sp . D2+500µmol / L Cu 2+ .

[0049] The rice in the above groups was grown in an artificial climate incubator (the parameters were set at 25°C, 70% relative humidity, and 22,000 Lux / 16h and 0 Lux / 8h) for 14 days. The rice seedling tissues were collected, and then 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. Effects of D2 on rice growth and alleviating heavy metal stress.

[0050] The specific process of determining the phenotypic parameters, biomass and chlorophyll of different groups of rice is as follows: S531. Root and shoot lengths of rice seedlings were measured by taking photos and using ImageJ online software, which can be accessed at https: / / rsb.info.nih.gov / ij / .

[0051] 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 represented the biomass.

[0052] S533. To evaluate the chlorophyll content of the aboveground part, 0.1 g of fresh tissue homogenate was added to 10 mL of 80% acetone to extract leaf pigments; then, a UV-visible spectrophotometer (LabTech, UV 8100D, China) was used to measure the absorbance of the acetone-extracted supernatant at 663 nm and 645 nm, respectively, and then the contents of chlorophyll a and b in the supernatant were calculated according to the Arnon formula.

[0053] 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 heavy metal Cu 2+ The higher the concentration, the more obvious the inhibitory effect on the growth of rice seedlings. 2+ When the concentration was 200µmol / L, 300µmol / L, 400µmol / L and 500µmol / L, the growth of rice was inhibited to a certain extent, among which 400µmol / L and 500µmol / L had the greatest inhibitory effect. Bacillus sp. D2, at 400µmol / L Cu 2+ The mitigation effect is best under the condition of 2+ At a concentration of 400 µmol / L, the growth of rice seedlings was significantly inhibited, with the height of the aboveground part 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, with the aboveground biomass increasing by 47.61% and the length increasing by 34.42%, which preliminarily indicated that the inoculated bacteria Bacillus sp. D2 can effectively alleviate the damage caused by Cu stress to rice seedlings, so 400µmol / L Cu 2+ Conduct follow-up experiments.

[0054] The specific process of determining the antioxidant enzyme (POD, APX, CAT) activities of different groups of rice is as follows: S535. Weigh 0.1 g of fresh rice leaves, add 1 mL of phosphate buffer (pH 7.8), grind on ice and transfer to a test tube. Centrifuge at 4°C and 8000 g for 10 min and take the supernatant for testing.

[0055] S536. Mix 20 mL of the working solution in the CAT kit and keep it warm in a 25°C water bath for 10 min.

[0056] S537. Take 1 mL of CAT working solution and add it to 35 µL of sample supernatant. Measure the absorbance at a wavelength of 240 nm for 5 s and 1 min, respectively. Determine the CAT content based on the absorbance value.

[0057] The activities of S538, APX and POD were determined using corresponding kits.

[0058] In order to combat the negative effects of reactive oxygen species (ROS), plants activate their own antioxidant defense system to remove ROS, which is an important part of plants’ own defense against heavy metal toxicity. It mainly includes APX, POD and CAT. Superoxide dismutase catalyzes the conversion of superoxide radicals into less toxic H 2 O 2 It is the first line of defense against oxidation. Ascorbate peroxidase catalyzes H 2 O 2 Oxidizing ascorbic acid is an important consumer of ascorbic acid in plants. The role of CAT and POD is to convert H 2 O 2 Converted to H 2 O and O 2 The test results are as follows: Figure 6 As shown, toxic Cu 2+ The antioxidant enzyme activities of rice seedlings decreased under the treatment, among which the APX and CAT activities in leaves decreased by 69.18% and 44.54%, respectively, and the APX activity in roots decreased by about 50%. Bacillus sp. D2, the POD activity in rice leaves and roots increased by 70.3% and 12.3%, respectively, and the APX and CAT activities showed the same trend. Bacillus sp D2 significantly increased the activities of antioxidant enzymes, indicating that inoculation can effectively enhance the defense of rice seedlings against Cu toxicity-induced oxidative stress and reduce oxidative damage in plant cells.

[0059] S600: Rice tissue samples were digested and then the Cu content in each tissue was determined by ICP-OES. 2+ Concentration, then calculate the transport coefficient TF, that is, TF = aboveground Cu content / root Cu content. The specific determination process is as follows: S610. Take rice plants and separate them into roots, stems and leaves. Wash them three times with deionized water, and then soak them in 20 mM ethylenediaminetetraacetic acid (EDTA) solution for 20 minutes to remove heavy metal ions on the surface. Repeat three times, and then place them in a drying oven at 105°C to dry.

[0060] S620, take 0.2g of dried plant roots, stems and leaves, cut them into pieces, add 8mL of HNO 3 and 2 mL of HClO 4 Thoroughly digest the samples in a polytetrafluoroethylene tube using a fully automatic digester.

[0061] S630, then filter the digestion solution with a 0.22µm filter membrane, and use ICP-OES to determine the Cu in each tissue2+ Concentration, and finally calculate the transport coefficient (TF) = Cu content in above-ground tissues / Cu content in underground tissues.

[0062] 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 400 µmol / L Cu 2+ Coercion (Cu 2+ ); (4) 400 µmol / L Cu 2+ Inoculated bacteria under stress Bacillus sp . D2(D2+Cu 2+ ). The results are 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+ Inoculated bacteria under stress Bacillus sp . The Cu content after D2 was 3826.26 mg / kg ± 119.9 mg / kg, which increased by about 1 times. 2+ The contents of Cu and Cu 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 causes more Cu absorption by the roots 2+ , but less Cu 2+ Transported to aboveground tissues.

[0063] S700: After the rice tissue samples were fixed with glutaraldehyde, dehydrated with ethanol gradient and freeze-dried, the root surface morphology and bacterial colonization were observed by scanning electron microscopy. The specific operation of scanning electron microscopy is as follows: S710, bacteria collection Bacillus sp The root tissues of rice under D2 and 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).

[0064] S720, and then add 30%, 50%, 70%, 90%, and 100% ethanol respectively for step-by-step dehydration, each dehydration for 30 minutes. Then, the dehydrated tissue was pre-frozen at -80°C overnight, and then dried in a vacuum freeze dryer. Finally, the dried rice root tissue was placed on a metal slide, sprayed with gold, and observed.

[0065] Observation results such as Figure 7The 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 sp. D2, the surface of the rice root remained smooth and intact, with no root cell rupture and the presence of certain microorganisms ( Figure 7 b) Add 400 µmol / 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 root system of rice and effectively alleviate the damage of heavy metals to root tissues.

[0066] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope 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 Microorganism Culture Collection Administration (CGMCC) on January 3, 2025, with the deposit number CGMCCNo.33107.

2. A method for screening the copper-resistant plant growth-promoting endophyte according to claim 1, characterized in that: It includes host treatment, gradient screening, molecular identification, growth-promoting characteristics analysis, and anti-Cu function verification steps. The specific contents are as follows: A. Host treatment: Cut the sterilized Commelina leaf into segments, take 2-3 segments and place them in a 50 mg / L Cu 2+ NA medium was cultured at 28°C for 72 h; 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; C. Molecular identification: The genomic DNA of endophytes was extracted, and the 16S rRNA gene fragment was amplified with universal primers 27F / 1492R. After sequencing, the taxonomic status of the species was determined by NCBI BLAST comparison; D. Analysis of growth-promoting properties: Determine the indoleacetic acid (IAA) production capacity and ACC deaminase activity of endophytes and quantitatively determine the plant growth-promoting potential of endophytes; E. Anti-Cu function verification: Endophytes were inoculated in a gradient Cu 2+ A rice culture system was developed to determine its effects on plant growth and alleviating heavy metal stress.

3. The method for screening copper-resistant plant growth-promoting endophytes according to claim 2, characterized in that: In step C, the genomic DNA of the endophyte is extracted using a Bacteria Genomic DNA Kit, and then the DNA purity and integrity are verified by Nanodrop ND-1000 spectrophotometer and 1% agarose gel electrophoresis. Subsequently, the genomic DNA of the endophyte that has passed the DNA purity and integrity verification is amplified with universal primers 27F / 1492R for 16S rRNA gene fragments, and then the amplified products are sequenced. Finally, according to the sequencing results, a phylogenetic tree is constructed through the NCBI platform to determine the taxonomic status of the endophyte.

4. The method for screening copper-resistant plant growth-promoting endophytes according to claim 2, characterized in that: The IAA capability determination process in step D is as follows: D10, prepare 0~20μg / mL gradient IAA standard solution, then add Salkowski colorimetric reagent to measure OD 530 To establish the IAA standard curve; D20, inoculate the activated endophytes after molecular identification into LB liquid medium containing 200 mg / L L-tryptophan, culture at 28°C and 180 rpm for 48 h, take 1 mL of 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 is pink, it is a positive endophyte that can produce IAA, and measure its OD 530 ; D30, use 1mL sterile water and an equal volume of Salkowski colorimetric reagent as blank control, and perform quantitative experiments according to the D20 steps. Let it stand in the dark at room temperature for 30 minutes and measure the OD 530 , the IAA content was calculated according to the IAA standard curve.

5. The method for screening copper-resistant plant growth-promoting endophytes according to claim 2, characterized in that: The ACC deaminase activity determination process in step D is as follows: D60, the endophytes identified by molecular identification were activated and washed with Tris-HCl buffer at pH 7.6, then suspended with Tris-HCl containing 0.3% toluene and pH 8.5, and then 20 mM ACC substrate was added to react at 30°C for 15 min; D70, after the reaction is completed, add 0.56M HCl to terminate the reaction, centrifuge to obtain the supernatant and add 2,4-dinitrophenylhydrazine, then react in a 30℃ water bath for 30min, then add 2mol / L NaOH to produce a color reaction, and measure OD 540 The ACC deaminase activity was calculated based on the α-ketobutyrate standard curve.

6. The method for screening copper-resistant plant growth-promoting endophytes according to claim 2, characterized in that: The anti-Cu function verification process in step E is as follows: E10, germinating the sterilized rice seeds on a culture dish, and after germination for 3 days in a 25° C. incubator, selecting rice seeds of the same growth period and transferring them to a black culture box filled with sterilized Hoagland nutrient solution; 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; E30, the rice in the above groups were grown in an artificial climate incubator, and the phenotypic parameters, biomass, chlorophyll content and antioxidant enzyme activity of rice in different groups were measured, and the measured parameters were compared to verify the endophytic bacteria Bacillus sp. Effects of D2 on rice growth and alleviating heavy metal stress.

7. The method for screening copper-resistant plant growth-promoting endophytes according to claim 6, characterized in that: The chlorophyll content is determined by first using 80% acetone to extract leaf pigments, then measuring the absorbance of the supernatant extracted with acetone at 663nm and 645nm, and then calculating the content of chlorophyll a and chlorophyll b according to the Arnon formula.

8. The method for screening copper-resistant plant growth-promoting endophytes according to claim 2, characterized in that: The step E also includes the determination of the Cu content in rice tissues: firstly, the rice tissue samples are digested, and then the Cu in each tissue is determined by ICP-OES. 2+ The transport coefficient TF was then calculated, i.e., TF = Cu content in the aboveground part / Cu content in the root part.

9. The method for screening copper-resistant plant growth-promoting endophytes according to claim 8, characterized in that: 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.

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

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