Actinidia arguta endophytic bacterium R3 and application thereof

Abstract

The invention discloses an actinidia arguta endophytic bacterium R3 and application thereof in promoting plant growth, relates to the fields of bioinformatics, agronomy and microbiology, and in particular relates to an actinidia arguta endophytic bacterium R3 and application thereof in promoting plant growth by combining a classical microorganism isolation culture method for sampling field environmental factors and root endophytic bacterium community structures of actinidia arguta. The actinidia arguta endophytic bacterium R3 capable of promoting plant growth is screened out. The actinidia arguta endophytic bacteria R3 can obviously increase the total chlorophyll content, the glutamine synthetase enzyme activity and the auxin content of the corn, and can obviously increase the plant height, the root length and the biomass of the corn.

Description

Technical Field

[0001] The invention relates to the fields of bioinformatics, agronomy and microbiology, and in particular to screening out an endophyte capable of promoting plant growth by combining environmental factors of a sampling site of Actinidia arguta and the structure of endophytic bacteria community in the roots with a classical microbial isolation and culture method. Background Art

[0002] Actinidia arguta has strong growth potential and large growth volume. The vine can reach up to 30 meters in length and the branch diameter can reach 15 cm. After years of collecting and evaluating Actinidia arguta resources, we found that it has strong adaptability, can grow in relatively harsh conditions, and shows good environmental adaptability. These specificities of Actinidia arguta, in addition to being determined by the plant's own genes, may be due to the fact that Actinidia arguta recruits a large number of growth-promoting functional bacteria into the environment during the process of adapting to the environment.

[0003] Endophytic bacteria are microorganisms that live in the internal tissues of living plants without causing any direct, obvious disease symptoms. Most endophytic bacteria are beneficial to plants and can survive in plants and establish a symbiotic relationship with them. Some endophytic bacteria promote plant growth, especially in improving the ability of plants to adapt to adverse environments, thereby increasing their growth. However, so far, no research has been reported on endophytic bacteria in Actinidia arguta.

[0004] Plant growth-promoting bacteria (PGPB) refers to microorganisms that have the activity of promoting plant growth. This type of bacteria has a significant direct promoting effect on seed germination, root growth, flowering, etc. during plant growth. Plant growth-promoting bacteria mainly involve multiple genera such as Ralstonia, Burkholderia, Methylobacterium, Sphingomonas, Bacteroides, Enterobacter, Pseudomonas and Marseilles. They promote plant growth through various mechanisms, such as: biological nitrogen fixation, phosphorus solubilization, potassium solubilization ability; production of siderophores, antibiotics, hydrolases and antibacterial volatile organic compounds and other substances, limit diseases caused by plant pathogens, improve plant disease resistance and stress resistance; secrete growth hormones such as auxin, gibberellins and cytokinins; have functions such as ACC deaminase activity.

[0005] Since endophytic bacteria have the characteristics of low culture cost, rapid growth, short fermentation cycle, and good synergistic effect with plants, it is necessary to provide an endophytic bacterial strain of Actinidia arguta to promote plant growth. Summary of the invention

[0006] The invention aims to provide an endophytic bacterium R3 of Actinidia arguta and application thereof in promoting plant growth.

[0007] The endophytic bacteria (Enterobacter amnigenus) R3 of Actinidia arguta of the present invention is deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration, with a deposit date of September 23, 2024, a deposit number of CGMCCNO.32045, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] In order to achieve the above purpose, the technical solution provided by the present invention is:

[0009] By using bioinformatics methods, the environmental factors of the sampling site of kiwifruit and the structure of the endophytic bacterial community in the roots were analyzed, combined with the classic microbial isolation and culture methods, and the endophytic bacteria R3 of kiwifruit that can promote plant growth were screened from kiwifruit. Molecular identification showed that it belongs to the genus Enterobacter, and it was named as endophytic bacteria of kiwifruit (Enterobacter amnigenus) R3. It was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection on September 23, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC NO.32045.

[0010] The endophytic bacteria R3 of Actinidia arguta of the present invention is obtained by the following method:

[0011] First, the microbial communities of the roots, stems, leaves, fruits and seeds of Actinidia arguta collected from Zuojia Town, Jilin City, Jilin Province in different seasons were sequenced by 16s rDNA amplicon to analyze the structure of the endophytic bacterial community, and the association analysis with environmental factors was performed to find out the endophytic bacteria that were significantly associated with environmental factors and sample tissues. Then, the target genera were isolated from the Actinidia arguta samples using a specific culture medium, and a growth-promoting bacterial culture strategy that can effectively improve plant growth was established. It was determined that 6 of the top 10 genera of endophytic bacteria in Actinidia arguta had a growth-promoting effect, including Methylobacterium, Sphingomonas, Enterobacter, Bacteroides, Pseudomonas, and Massilia.

[0012] Then, 12 screening culture media were designed for the above-mentioned 6 bacterial genera, and then the endophytic bacteria of one target genus were enriched using the above-mentioned solid culture medium. After 16s rDNA conservative sequence amplification, sequencing qualitative analysis and physiological and biochemical identification, it was named as endophytic bacteria of Actinidia arguta R3, and its strain preservation number is CGMCC NO.32045.

[0013] Another object of the present invention is to provide the use of the endophytic bacteria of Actinidia arguta.

[0014] The present invention designs a plant cultivation strategy, applies the endophytic bacteria (Enterobacteramnigenus) R3 of Actinidia arguta described in the invention to a corn seedling potted plant, and sets a blank control group and a bacterial agent application group. After 45 days of inoculation, the corn growth parameters are measured. The results show that the total chlorophyll content, glutamine synthetase activity and auxin content of the inoculated bacteria treatment are significantly increased compared with the control, among which the glutamine synthetase activity is increased by as much as 763.96%; the plant height, root length and biomass increase significantly after inoculation, the root system increases by 142.73%, and the fresh weight of the aboveground part increases by 96.07%. Promoting the growth of plants is beneficial to its application in agricultural production.

[0015] Beneficial effects of the present invention:

[0016] Compared with the prior art, the present invention provides an endophytic bacterium derived from Actinidia arguta, namely, Actinidia arguta endophytic bacterium R3. Actinidia arguta endophytic bacterium R3 can significantly increase the total chlorophyll content, glutamine synthetase activity and auxin content of corn, and at the same time significantly increase the plant height, root length and biomass of corn. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with specific examples, and the following examples will help those skilled in the art to further understand the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0018] Example 1

[0019] 1. Sampling of Actinidia arguta

[0020] From January to December 2021, every season we went to the kiwifruit orchard in Zuojia Town, Jilin City, Jilin Province to collect the roots, stems, leaves and fruits of kiwifruit. We placed them in ice boxes and brought them back to the laboratory for disinfection immediately for subsequent experiments.

[0021] 2. DNA Extraction and PCR Amplification

[0022] Using EZNA The soil DNA kit was used to extract the DNA of endophytic bacteria in the test samples of Actinidia arguta. After the extraction, the quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined by NanoDrop2000. The above-extracted DNA was used as a template, and the primers carrying the Barcode sequence were used to PCR amplify the variable region of the 16S rRNA gene V3-V4, and each sample was repeated 3 times. Primer sequence: Forward799F (5'-AACMGGATTAGATACCCKG-3'), Reverse 1193R (5'-ACGTCATCCCCACCTTCC-3'). The PCR amplification system and amplification conditions are shown in Table 1.

[0023] Table 1 PCR amplification system and amplification conditions.

[0024]

[0025]

[0026] 3. Purification and sequencing of 16s rDNA amplicons of endophytic bacteria from Actinidia arguta

[0027] The PCR products of three replicates were mixed, and then the PCR products were recovered using 2% agarose gel. The recovered products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). Then, the quality of the recovered DNA was tested by 2% agarose gel electrophoresis, and the recovered products were quantitatively tested using QuantusTM Fluorometer (Promega, Madison, WI, USA). The library was constructed using NEXTflexTM Rapid DNA Seq Kit (BiooScientific, Austin, TX, USA): (1) adapter connection; (2) using magnetic beads to screen and remove adapter self-ligated fragments; (3) using PCR amplification to enrich the library template; (4) using magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using Illumina's Miseq PE300 / NovaSeq PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). Usearch software (version 11) was used to divide the taxonomic units (OTU) at a sequence similarity level of 97%. The OTU species taxonomic annotation was performed using RDP classifier (version 2.11) to compare with the Silva 16S rRNA gene database (v138), and the comparison threshold was set to 70% for comparison with the Silva 16S rRNA database (version 138). The raw data after amplicon sequencing has been uploaded to the National Center for Microbiological Sciences Data Center, and the data accession number is PP549865.1. The partial nucleotide sequence of the 16S rDNA gene of the strain was compared with the similar sequence retrieved from the DNA database using the BLAST search program of the National Center for Biotechnology Information (NCBI), and it was found that the similarity with the Enterobacter amnigenus (Enterobacter amnigenus) with GenBank accession number JQ864378.1 was 99.86%, confirming that R3 was Enterobacter amnigenus.

[0028] 4. Bioinformatics Analysis

[0029] The Alpha diversity index was calculated using Mothur software (version 1.30.2), and the Alpha diversity index was analyzed by T test between groups, community bar graphs and community pie charts, and Beta diversity was analyzed between groups using R software (version 3.3.1). PERMANOVA analysis was performed using QIIME software; the RDA model (R language vsession 2.4.3) was used for environmental factor association analysis. The stats package of R (version 3.3.1) and the scipy package of Python (v1.0.0) were used for multi-group comparison of species difference analysis; the FAPROTAX (1.2.1) database was used to perform functional prediction analysis of endophytic bacteria, and the COG family information corresponding to the endophytic bacterial OTU was obtained by comparison. Through data analysis, the top 10 genera of endophytic bacteria abundance in Actinidia arguta were obtained, and 6 genera with growth-promoting functions were identified, specifically: Methylobacterium, Sphingomonas, Enterobacter, Bacteroides, Pseudomonas, and Massilia, which determined the scope of the screening method for the subsequent isolation of growth-promoting endophytic bacteria. And through data analysis, it was found that the diversity of endophytic bacteria in winter is richer than in other seasons, and the types of bacteria in the roots are richer than in other organs, thus clarifying the collection season and collection site of the samples, and increasing the probability of screening the target strains.

[0030] 5. Isolation and identification of growth-promoting endophytes

[0031] With reference to the list of culture media for strain preservation of the German National Collection of Microorganisms DSMZ (https: / / www.dsmz.de / ), the optimal growth medium for the target species of microorganisms was obtained according to the growth characteristics of strains of the genera Methylobacterium, Sphingomonas, Enterobacter, Bacteroides, Pseudomonas and Marseilles, and freeze-dried powder of Actinidia arguta roots was added to the culture medium to make endophytic bacteria better adapt to the growth characteristics of the culture medium, accelerate their growth, and make it easier to isolate rare strains from the roots of Actinidia arguta. The specific operation is as follows: take fresh kiwifruit roots, place them in a foam box with ice packs, take them back to the laboratory immediately, rinse them under running water for 30 minutes, then cut the roots and stems into 2-3cm stem segments on a sterile clean bench, first disinfect them with 75% ethanol for 90 seconds, wash them with sterile water three times, then use 2.5% NaClO to shake and disinfect them for 6-8 minutes, and finally wash them with sterile water three times, and use sterile filter paper to absorb excess water for standby use; take 100μL of the sterile water used to rinse the roots for the last time and apply it to the separation medium, culture it at 28℃ for 2 days, if there is no colony growth on the separation medium plate, it is considered that the surface is thoroughly disinfected, otherwise it is not thorough, and the samples that are not thoroughly disinfected are re-sampled and disinfected to repeat the above steps. Use a blender with a sterile inner surface to crush 2g of fresh kiwifruit roots that have been surface disinfected, and then add 3mL of sterile water. 200 μL of the sample resuspension was applied to the optimal growth medium of the target species for the screening of endophytic bacteria. 168 samples were prepared in triplicate for each sample. The outer flame of an alcohol lamp was used to lightly burn the outer periphery of the plate and then sealed with a sealing film. The plate was placed in a 30°C constant temperature incubator for 1 to 2 days, and the grown colonies were transferred to the corresponding liquid culture medium and cultured at 30°C and 200 rpm for 12 hours for strain preservation and DNA extraction. The 16S rDNA of the conserved region of the bacteria was amplified and sequenced for species identification of the 93 isolated endophytic bacterial strains. The primers used were (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′). The PCR reaction system was 10×PCR Buffer 2.5 μL, dNTP (each 10 mM) 1 μL, Taq Plus DNA Polymerase (5 U·μL -1 )1. 50 mM MgSO 4 8μL, upstream primer 1μL, downstream primer 1μL, Template (genomic DNA 20-50ng·μL -1 )1 μL, ddHO 2 2. O 9.5 μL.

[0032] The amplified gene fragments were subjected to 1% agarose gel electrophoresis to detect the quality and concentration of the amplified gene fragments. The qualified amplified fragments were recovered using the SanPrep column DNA gel recovery kit (SK8131) of Sangon Biotech Co., Ltd. and then sent to Sangon Biotech Co., Ltd. for sequencing. After obtaining the sequencing 16S rDNA sequence results, i.e., the gene sequence shown in SEQ ID NO: 1, log in to the NCBI website, compare the sequenced 16S rDNA sequence in the Nucleotide database through BLAST, find out the sequence with a similarity greater than 97%, and thus determine which family and genus the strain belongs to.

[0033] 6. Growth promotion pot experiment

[0034] After screening the endophytic bacteria (Enterobacter amnigenus) R3 of Actinidia arguta, a pot experiment was conducted to verify the growth-promoting effect of the strain. A single-factor completely randomized block experiment was used to select corn seedlings with consistent plant growth. A total of two treatment groups were set up, including a blank control group (CK group) and an endophytic bacteria R3 agent group (R3 group) of Actinidia arguta. Each treatment group was repeated 3 times, with 7 pots of seedlings each time. After activating the endophytic bacteria R3 strain of Actinidia arguta, it was inoculated into a triangular flask containing 100 mL of sterile LB liquid culture medium at a rate of 1%, and placed in a shaking incubator for constant temperature culture (30°C, 190 r·min -1 ) for 12 h, and then the bacterial solution was heated at 4 °C and 6000 r / min -1 Centrifuge for 10 min, remove the supernatant to obtain the endophytic bacteria R3 cells of Actinidia arguta, and use part of 0.85% sterile (NH4) 2 SO 4 The endophytic bacteria R3 of Actinidia arguta were suspended in the solution, and then the suspension was diluted and the OD was measured by spectrophotometer. 600 The concentration of bacterial suspension was determined by sterile (NH4) 2 SO 4 Solution adjustment OD 600 Value to 0.3-0.4 (about 10 6 -10 7 CFU·mL -1) and set aside. Start inoculating the endophytic bacteria R3 solution of Actinidia arguta 7 days after emergence. Water the potted seedlings thoroughly in the evening of the day before inoculation to ensure that no watering is required for more than 3 days. Use a sterile syringe (without the needle) to slowly inject 10mL of the endophytic bacteria R3 solution of Actinidia arguta along the stems and roots of the corn seedlings. Inoculate 10mL of the endophytic bacteria R3 solution of Actinidia arguta a second time after an interval of 7 days. Inoculate twice in total. The blank control group uses sterile water instead of the endophytic bacteria R3 solution of Actinidia arguta. The rest of the operations are the same as those of the endophytic bacteria R3 agent group of Actinidia arguta. Water and weed regularly during the test period to ensure normal growth conditions for the test seedlings.

[0035] 7. Potted Plant Experiment Results

[0036] After 45 days of potted experiments, the effects on promoting plant growth are shown in Table 2-4: Compared with the control group under the same culture conditions, inoculation with endophytic bacteria R3 of Actinidia arguta can significantly increase the total chlorophyll content, glutamine synthetase activity and auxin (IAA) content of the leaves, among which the increase in glutamine synthetase activity is as high as 763.96%; after inoculation, plant height, root length and biomass increased significantly, the root system increased by 142.73%, and the fresh weight of the aboveground part increased by 96.07%.

[0037] Table 2 Contents of photosynthetic pigments and activities of nitrogen assimilation-related enzymes in corn.

[0038]

[0039] Table 3 Hormone content parameters of corn leaves.

[0040]

[0041] Table 4. Corn growth parameters.

[0042]

[0043] The gene sequence of endophytic bacteria R3 in Actinidia arguta is shown in SEQ ID NO: 1:

[0044]

[0045] Example 2

[0046] In order to achieve the optimal growth culture medium of the present invention, the following technical scheme is adopted: weigh 10g of peptone, 5g of sodium chloride, 3g of beef extract, 15g of agar powder and 10g of kiwifruit freeze-dried powder; dissolve them in deionized water and make the volume to 1000mL; heat and boil, then divide them into Erlenmeyer flasks and sterilize them at 121°C for 22 minutes to obtain the optimal growth culture medium.

[0047] Example 3

[0048] The 12 screening culture medium formulas are as follows:

[0049] (1) TSA medium: 15.0 g tryptone, 5.0 g soytone, 5.0 g sodium chloride, 13.0 g agar and distilled water to make up to 1.0 L, pH 7.3 ± 0.21.

[0050] (2) Nutrient agar medium (NA): peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, distilled water to 1.0 L, final pH 7.3 ± 0.2.

[0051] (3) Nutrient broth solid medium (LB solid): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, distilled water to 1.0 L, final pH 7.2±0.2.

[0052] (4) KB agar medium: prion peptone 20.0 g / L, dipotassium hydrogen phosphate 1.5, magnesium sulfate (MgSO 4 7H 2 O) 1.5 g / L, agar 15.0 g / L, distilled water to 1.0 L, final pH 7.2 ± 0.2.

[0053] (5) Tryptone soy broth (TSB): Tryptone: 17.0 g / L, soy peptone: 3.0 g / L, sodium chloride: 5.0 g / L, glucose: 2.5 g / L, anhydrous potassium dihydrogen phosphate: 2.5 g / L, distilled water to 1.0 L, pH 7.3±0.2.

[0054] (6) Columbia blood agar (CA-B): 10.0 g / L casein pancreatic digest, 3.0 g / L heart pancreatic digest, 1.0 g / L corn starch, 5.0 g / L meat peptic digest, 5.0 g / L yeast extract, 5.0 g / L sodium chloride, 20.0 g / L agar, distilled water to 1.0 L, pH 7.3 ± 0.21.

[0055] (7) AC agar medium: 20 g / L peptone, 3 g / L beef extract, 3 g / L yeast extract, 3 g / L malt extract, 5 g / L glucose, 0.2 g / L vitamin C, 13 g / L agar, distilled water to 1.0 L, pH 7.1-7.3.

[0056] (8) Improved nutrient agar medium: peptone 10 g / L, sodium chloride 5 g / L, beef extract 3, agar powder 15 g / L, Actinidia arguta freeze-dried powder 10 g / L, distilled water to 1.0 L, pH 7.2±0.2.

[0057] (9) 5 g / L peptone, 30 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, distilled water to 1.0 L, pH to 7.0-7.2.

[0058] (10) Meat broth 10 g / L, peptone 5 g / L, yeast powder 3 g / L, D(+)-glucose 5 g / L, starch 1 g / L, sodium chloride 5 g / L, sodium acetate 3 g / L, L-cysteine ​​hydrochloride 0.5 g / L, distilled water to 1.0 L, agar 15 g / L, pH 6.8±0.22.

[0059] (11) Gelatin peptone 16.0 g / L, tryptone 10.0 g / L, anhydrous potassium sulfate 10.0 g / L, anhydrous magnesium chloride 1.4 g / L, cetyltrimethylammonium bromide 0.2 g / L, nalidixic acid 0.015 g / L, agar 15.0 g / L, pH 7.1±0.2.

[0060] (12) Yeast powder 0.50 g / L, protein 0.50 g / L, casein amino acid 0.50 g / L, glucose 0.50 g / L, soluble starch 0.50 g / L, sodium pyruvate 0.30 g / L, K 2 HPO 4 0.30 g / L, MgSO 4 (7H 2 O) 0.05 g / L, agar 15.00 g / L, distilled water to 1.0, K 2 HPO 4 or KH2PO4 to a final pH of 7.2.

[0061] The above content describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific implementation methods, and those skilled in the art can make modifications within the scope of the claims without affecting the essential content of the present invention.

Claims

1. An endophytic bacterium of Actinidia arguta (Enterobacter amnigenus) R3, characterized in that: The endophytic bacteria R3 of Actinidia arguta is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.32045 and the deposit date: September 23, 2024.

2. The use of the endophytic bacteria R3 of Actinidia arguta in promoting plant growth as claimed in claim 1, characterized in that: The plant is corn.

3. The use of the endophytic bacteria R3 of Actinidia arguta fruit as claimed in claim 1 in promoting chlorophyll synthesis in plant leaves, characterized in that: The plant is corn.

4. The use of the endophytic bacteria R3 of Actinidia arguta in promoting the activity of glutamine synthetase in plant leaves as claimed in claim 1, characterized in that: The plant is corn.

5. The use of the endophytic bacteria R3 of Actinidia arguta fruit as claimed in claim 1 in promoting the increase of auxin content in plant leaves, characterized in that: The plant is corn.

6. The use according to claim 2, characterized in that The promoting plant growth specifically includes: promoting plant height growth, increasing root length, and increasing biomass.

7. Use of the endophytic bacteria R3 of Actinidia arguta as claimed in claim 1 in corn planting.

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