An endophytic bacterium R3 of Actinidia arguta and its application

Abstract

The present invention discloses an endophytic bacterium R3 of Actinidia arguta and its application in promoting plant growth, which relates to the fields of bioinformatics, agronomy, and microbiology. Specifically, by analyzing the environmental factors of the sampling sites of Actinidia arguta and the community structure of root endophytic bacteria, and combining with the classical microbial isolation and culture methods, an endophytic bacterium R3 of Actinidia arguta that can promote plant growth is screened out. The endophytic bacterium R3 of Actinidia arguta significantly increases the total chlorophyll content, glutamine synthetase activity, and auxin content of maize, and at the same time significantly increases the plant height, root length, and biomass of maize.

Description

Technical Field

[0001] The present invention relates to the fields of bioinformatics, agronomy, and microbiology, and specifically relates to screening an endophyte that can promote plant growth by sampling environmental factors and root endophyte community structure of Actinidia arguta, combined with classical microbial isolation and culture methods. Background Art

[0002] Actinidia arguta has strong growth potential and large growth amount. The longest vine can reach 30 m, and the diameter of the branch can reach 15 cm. After years of collection and evaluation of Actinidia arguta resources, we found that it has strong adaptability, can grow under 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 that Actinidia arguta recruits a large number of growth-promoting functional bacteria from the environment during the process of adapting to the environment.

[0003] Endophytic bacteria are microorganisms that colonize the internal tissues of living plants without causing any direct and obvious disease symptoms. Most endophytic bacteria are beneficial to plants, can survive in plants and establish a symbiotic relationship with them. Some endophytic bacteria play a role in promoting plant growth, especially in enhancing the adaptability of plants to adverse environments, thereby increasing their growth amount. However, so far, there has been no research report on endophytic bacteria of Actinidia arguta.

[0004] Plant growth-promoting bacteria (PGPB) refer to microorganisms with promoting activity for plant growth. These bacteria have obvious direct promoting effects on seed germination, root growth, flowering, etc. during the plant growth process. Plant growth-promoting bacteria mainly involve multiple genera such as Rhodococcus, Burkholderia, Methylobacterium, Sphingomonas, Bacteroides, Enterobacter, Pseudomonas, and Massilia. 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 to limit diseases caused by plant pathogens and improve the disease resistance and stress resistance of plants; secretion of growth hormones such as auxin, gibberellin, and cytokinin; having 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. Therefore, it is very necessary to provide an endophytic bacteria strain of Actinidia arguta to promote plant growth. Summary of the Invention

[0006] The object of the present invention is to provide an endophytic bacteria R3 of Actinidia arguta and its application in promoting plant growth.

[0007] The endophytic bacterium (Enterobacter amnigenus) R3 of Actinidia arguta of the present invention is preserved in the China General Microbiological Culture Collection Center (CGMCC). The preservation date is September 23, 2024, and the preservation number is CGMCC NO. 32045. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0009] By using bioinformatics means, through analyzing the environmental factors of the sampling sites of Actinidia arguta and the root endophytic bacterial community structure, and combining with the classical microbial isolation and culture methods, the endophytic bacterium R3 of Actinidia arguta that can promote plant growth is screened out from Actinidia arguta. Molecular identification shows that it belongs to the genus Enterobacter, and it is named as the endophytic bacterium (Enterobacter amnigenus) R3 of Actinidia arguta. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the preservation number is CGMCC NO. 32045.

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

[0011] First, after performing 16s rDNA amplicon sequencing on the microbial communities of the roots, stems, leaves, fruits, and seeds of Actinidia arguta collected in Zuojia Town, Jilin City, Jilin Province in different seasons, the endophytic bacterial community structure is analyzed, and an association analysis is carried out with environmental factors to find out the endophytic bacteria that are significantly correlated with environmental factors and sample tissues. Then, using a specific culture medium, the target genus of bacteria is isolated from Actinidia arguta samples, and a growth-promoting bacterial culture strategy that can effectively improve plant growth is established. It is determined that 6 out of the top 10 genera with the highest abundance of endophytic bacteria in Actinidia arguta have growth-promoting effects, including the genus Methylobacterium, Sphingomonas, Enterobacter, Bacteroides, Pseudomonas, and Massilia.

[0012] Then, 12 screening culture media are designed for the above 6 genera, and then an endophytic bacterium of 1 target genus is enriched by using the above solid culture medium. After amplification, sequencing and qualitative analysis of the 16s rDNA conserved sequence and physiological and biochemical identification, it is named as the endophytic bacterium R3 of Actinidia arguta, 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 (Enterobacter amnigenus) R3 of Actinidia arguta described in the invention to the potted plants of corn seedlings, and sets a blank control group and a group applying the bacterial agent. After 45 days of inoculation, the growth parameters of corn are measured. The results show that compared with the control, the total chlorophyll content, glutamine synthetase activity and auxin content are all significantly increased after the inoculation treatment, and the increase ratio of glutamine synthetase activity is as high as 763.96%; after inoculation, the plant height, root length and biomass are significantly increased, the root system increases by 142.73%, and the fresh weight of the above-ground part increases by 96.07%. Promoting the growth of plants is beneficial to its application in agricultural production.

[0015] The beneficial effects of the present invention:

[0016] Compared with the existing technology, the present invention provides an endophytic bacterium derived from Actinidia arguta, namely the endophytic bacterium R3 of Actinidia arguta, which significantly increases the total chlorophyll content, glutamine synthetase activity and auxin content of corn, and at the same time significantly increases the plant height, root length and biomass of corn. Specific embodiments

[0017] The following further describes the present invention in detail with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0018] Example 1

[0019] 1. Sampling of Actinidia arguta samples

[0020] From January to December 2021, roots, stems, leaves and fruits of Actinidia arguta were collected from the Actinidia arguta nursery in Zuojia Town, Jilin City, Jilin Province in each season, placed in an ice box and immediately disinfected after being taken back to the laboratory for subsequent experiments.

[0021] 2. DNA extraction and PCR amplification

[0022] Use E.Z.N.A. The soil DNA kit was used to extract the DNA of endophytic bacteria from the tested samples of Actinidia arguta. After the extraction was completed, 1% agarose gel electrophoresis was used to detect the quality of the extracted genomic DNA, and NanoDrop2000 was used to measure the DNA concentration and purity. Using the above-extracted DNA as a template, primers carrying Barcode sequences were used to perform PCR amplification on the V3-V4 variable region of the 16S rRNA gene, with 3 replicates for each sample. Primer sequences: 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 in Actinidia arguta

[0027] Mix the three replicated PCR products, and then recover the PCR products using a 2% agarose gel. The AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.A) was used to purify the recovered products. Then, the quality of the recovered DNA was detected by 2% agarose gel electrophoresis, and the recovered products were quantitatively detected using a QuantusTM Fluorometer (Promega, Madison, WI, USA). The NEXTflexTM Rapid DNA Seq Kit (BiooScientific, Austin, TX, U.S.A) was used to construct the library: (1) adapter ligation; (2) use magnetic beads to screen and remove self-ligated adapter fragments; (3) enrich the library template by PCR amplification; (4) recover the PCR products with magnetic beads to obtain the final library. Sequencing was performed using the Illumina Miseq PE300 / NovaSeq PE250 platform (Shanghai Majorbio Bio-pharm Technology Co., Ltd.). The Usearch software (version 11) was used to divide the operational taxonomic units (OTUs) at a sequence similarity level of 97%. The RDP classifier (version 2.11) was used to perform OTU species taxonomic annotation by aligning with the Silva 16S rRNA gene database (v138), and the Silva 16S rRNA database (version 138) was aligned with a set alignment threshold of 70%. The raw data after amplicon sequencing has been uploaded to the National Microbiology Science Data Center, and the data registration number is PP549865.1. Using the BLAST search program of the National Center for Biotechnology Information (NCBI), the partial nucleotide sequence of the 16S rDNA gene of the strain was compared with the similar sequences retrieved from the DNA database, and it was found that the similarity with Enterobacter amnigenus with GenBank accession number JQ864378.1 was 99.86%, and R3 was determined to be Enterobacter amnigenus.

[0028] 4. Bioinformatics analysis

[0029] The Alpha diversity index was calculated using Mothur software (version 1.30.2), and the inter-group T-test difference analysis of the Alpha diversity index, community bar chart, and community pie chart analysis were performed using R software (version 3.3.1). The inter-group difference analysis of Beta diversity was carried out using QIIME software; the environmental factor correlation analysis used the RDA model (R language version 2.4.3). The multi-group comparison of species differences was performed using the stats package of R (version 3.3.1) and the scipy package of Python (v1.0.0); the FAPROTAX (1.2.1) database was used for the functional prediction analysis of endophytic bacteria, and the COG family information corresponding to the endophytic bacteria OTU was obtained by comparison. Through data analysis, the top 10 genera of endophytic bacteria with the highest 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 was richer in winter than in other seasons, and the types of bacteria in the roots were richer than those in other organs, thus clarifying the sampling season and sampling site of the samples and increasing the probability of screening out the target strains.

[0030] 5. Isolation and Identification of Growth-Promoting Endophytic Bacteria

[0031] Refer to the list of preservation media for strains in the German National Collection of Microorganisms DSMZ (https: / / www.dsmz.de / ). According to the growth characteristics of strains of the genera Methylobacterium, Sphingomonas, Enterobacter, Bacteroides, Pseudomonas, and Massilia, the optimal growth medium for the target genus of microorganisms was obtained, and freeze-dried powder of Actinidia arguta roots was added to the medium to make the endophytic bacteria more adaptable to the growth characteristics of the medium, enabling them to grow faster and making it easier to isolate rare strains from Actinidia arguta roots. The specific operations are as follows: Take fresh Actinidia arguta roots, place them in a foam box with an ice pack, and immediately bring them back to the laboratory. Rinse them under running water for 30 min, then cut the roots and stems into stem segments of 2-3 cm on a sterile laminar flow bench. First, disinfect them with 75% ethanol for 90 s, wash them 3 times with sterile water, then continuously shake and disinfect them with 2.5% NaClO for 6-8 min, and finally wash them 3 times with sterile water and blot off the excess water with sterile filter paper for use; Take 100 μL of the sterile water used to rinse the roots for the last time and spread it on the isolation medium, and culture it at 28 °C for 2 d. If no colonies grow on the isolation medium plate, it is considered that the surface disinfection is thorough, otherwise it is not. For samples with incomplete disinfection, resample and disinfect and repeat the above steps. Use a blender with a sterile inner surface to crush 2 g of the surface-disinfected fresh Actinidia arguta roots, and then add 3 mL of sterile water. Pipette 200 μL of the sample resuspension and spread it on the optimal growth medium for the target genus for the screening of endophytic bacteria. There are 168 samples, and 3 replicates are made for each sample. Lightly burn the periphery of the plate with the outer flame of an alcohol lamp and then seal it with a sealing film. Place the above plates in a constant temperature incubator at 30 °C for 1-2 days, transfer the grown colonies to the corresponding liquid medium, and culture them with shaking at 30 °C and 200 rpm for 12 h for strain preservation and DNA extraction. Amplify and sequence the bacterial conserved region 16S rDNA of the 93 isolated endophytic bacterial strains for species identification. The primers used are (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACTT-3′). The PCR reaction system is 2.5 μL of 10×PCR Buffer, 1 μL of dNTP (each 10 mM), 1.5 μL of Taq Plus DNA Polymerase (5 U·μL -1 ) 1, 8 μL of 50 mM MgSO4, 1 μL of the upstream primer, 1 μL of the downstream primer, 1 μL of Template (genomic DNA 20-50 ng·μL -1 ) 1, and 9.5 μL of ddH2O.

[0032] The amplified gene fragments were subjected to 1% agarose gel electrophoresis to detect the quality and concentration of the amplified gene fragments. Qualified amplified fragments were recovered using the SanPrep Column DNA Gel Extraction Kit (SK8131) from Sangon Biotech, and then sent to Sangon Biotech for sequencing. After obtaining the 16S rDNA sequence results of the sequencing, that is, the gene sequence shown in SEQ ID NO:1, log in to the NCBI website, and compare the 16S rDNA sequence obtained by sequencing in the Nucleotide database through BLAST to find sequences with a similarity greater than 97%, so as to determine which family and genus the strain belongs to.

[0033] 6. Growth-promoting pot experiment

[0034] After screening the endophytic bacterium (Enterobacter amnigenus) R3 of Actinidia arguta, a pot experiment was carried out to verify the growth-promoting effect of the strain. A single-factor completely randomized block experiment was adopted. Maize seedlings with consistent growth vigor were selected. There were a total of 2 treatment groups, namely the blank control group (CK group) and the Actinidia arguta endophytic bacterium R3 inoculant group (R3 group). Each treatment group had 3 replicates, and each replicate had 7 pots of seedlings. After activating the Actinidia arguta endophytic bacterium R3 strain, it was inoculated into a triangular flask containing 100 mL of sterile LB liquid medium at an inoculation amount of 1%, and placed in a shaker for constant temperature culture (30 °C, 190 r·min -1 ) for 12 h, and then the bacterial liquid was centrifuged in a centrifuge at 4 °C and 6000 r·min -1 for 10 min. After removing the supernatant, the Actinidia arguta endophytic bacterium R3 cells were obtained. The Actinidia arguta endophytic bacterium R3 cells were suspended with a part of 0.85% sterile (NH4)2SO4 solution, and then the suspension was diluted. The OD 600 of the bacterial suspension was measured with a spectrophotometer, and the OD 600 value was adjusted to 0.3 - 0.4 (about 10 6 -10 7 CFU·mL -1 ) with sterile (NH4)2SO4 solution for standby. Seven days after the emergence of the seedlings, the Actinidia arguta endophytic bacterium R3 bacterial liquid was inoculated. The potted seedlings were watered thoroughly the evening before inoculation to ensure that they did not need to be watered for more than 3 days. The Actinidia arguta endophytic bacterium R3 bacterial liquid was slowly injected along the stem root of the maize seedlings with a sterile syringe (the needle removed) for 10 mL. The Actinidia arguta endophytic bacterium R3 bacterial liquid was inoculated for the second time 10 mL at an interval of 7 days, and a total of 2 inoculations were carried out. The blank control group used sterile water instead of the Actinidia arguta endophytic bacterium R3 bacterial liquid, and the rest of the operations were the same as those of the Actinidia arguta endophytic bacterium R3 inoculant group. During the test period, water was regularly watered and weeds were removed to ensure the normal growth conditions of the test seedlings.

[0035] 7. Results of the pot experiment

[0036] After 45 days of pot experiments, the effects on promoting plant growth are shown in Table 2-4: Compared with the control group under the same cultivation conditions, inoculation with the endophytic bacterium R3 of Actinidia arguta can significantly increase the total chlorophyll content, glutamine synthetase activity, and indole-3-acetic acid (IAA) content in leaves. Among them, the increase ratio of glutamine synthetase activity is as high as 763.96%; after inoculation, the plant height, root length, and biomass increase significantly. The root system increases by 142.73%, and the fresh weight of the above-ground part increases by 96.07%.

[0037] Table 2 Photosynthetic pigment content and nitrogen assimilation-related enzyme activities of maize.

[0038]

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

[0040]

[0041] Table 4 Growth parameters of maize.

[0042]

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

[0044]

[0045] Example 2

[0046] To achieve the optimal growth medium of the present invention, the following technical solutions are adopted for preparation: Weigh 10 g of peptone, 5 g of sodium chloride, 3 g of beef extract, 15 g of agar powder and 10 g of Actinidia arguta freeze-dried powder; dissolve in ionized water and make up the volume to 1000 mL; after heating and boiling, dispense into Erlenmeyer flasks and sterilize at 121 °C for 22 minutes to obtain the optimal growth medium.

[0047] Example 3

[0048] The formulations of 12 kinds of screening media are as follows:

[0049] (1) Tryptic Soy Agar Medium (TSA): 15.0 g of tryptone, 5.0 g of soya peptone, 5.0 g of sodium chloride, 13.0 g of agar and distilled water are made up to 1.0 L, pH 7.3 ± 0.21.

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

[0051] (3) Nutrient Broth Solid Medium (LB solid): 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, distilled water is made up to 1.0 L, final pH 7.2 ± 0.2.

[0052] (4) KB Agar Medium: 20.0 g / L of proteose peptone, 1.5 of dipotassium hydrogen phosphate, 1.5 g / L of magnesium sulfate (MgSO4·7H2O), 15.0 g / L of agar, distilled water is made up to 1.0 L, final pH 7.2 ± 0.2.

[0053] (5) Tryptic Soy Broth (TSB): Tryptone: 17.0 g / L, Soya 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 is made up to 1.0 L, pH 7.3 ± 0.2.

[0054] (6) Columbia Blood Agar Plate (CA - B): 10.0 g / L of casein tryptic digest, 3.0 g / L of heart infusion tryptic digest, 1.0 g / L of corn starch, 5.0 g / L of meat peptone, 5.0 g / L of yeast extract, 5.0 g / L of sodium chloride, 20.0 g / L of agar, distilled water is made up to 1.0 L, pH 7.3 ± 0.21.

[0055] (7) AC Agar Medium: It shows 20 g / L of peptone, 3 g / L of beef extract, 3 g / L of yeast extract, 3 g / L of malt extract, 5 g / L of glucose, 0.2 g / L of vitamin C, 13 g / L of agar, made up to 1.0 L with distilled water, pH 7.1 - 7.3.

[0056] (8) Modified Nutrient Agar Medium: 10 g / L of peptone, 5 g / L of sodium chloride, 3 of beef extract, 15 g / L of agar powder, 10 g / L of Actinidia arguta freeze-dried powder, made up to 1.0 L with distilled water, pH 7.2 ± 0.2.

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

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

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

[0060] (12) 0.50 g / L of yeast powder, 0.50 g / L of casein peptone, 0.50 g / L of casein amino acid, 0.50 g / L of glucose, 0.50 g / L of soluble starch, 0.30 g / L of sodium pyruvate, 0.30 g / L of K2HPO4, 0.05 g / L of MgSO4(7H2O), 15.00 g / L of agar, made up to 1.0 with distilled water, adjusted to the final pH 7.2 with K2HPO4 or KH2PO4.

[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 essence of the present invention.

Claims

1. An application of endophytic bacteria (Enterobacter amnigenus) R3 of Actinidia arguta in promoting chlorophyll synthesis in plant leaves, characterized in that: The plant is corn, and 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. An application of endophytic bacteria (Enterobacter amnigenus) R3 of Actinidia arguta in promoting the activity of glutamine synthetase in plant leaves, characterized in that: The plant is corn, and 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.

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