Methylobacterium separated from myrica rubra root nodules and application thereof

Abstract

The invention discloses methylobacterium separated from myrica rubra root nodules and application thereof, and belongs to the technical field of microorganisms. The methylobacterium is separated from myrica rubra root nodule tissue, is named as Methylobacterium sp.creature, is preserved in the China Center for Type Culture Collection on March 14, 2024, and has the preservation number of CCTCC NO: M 2024480. When the strain acts on the root system of a plant seedling, the growth of lateral roots can be promoted, especially the lateral roots with binary branches are generated, and the symbiotic bacteria can be promoted to colonize on a nodulation plant to form nodules.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a methyl bacterium separated from a bayberry root nodule and application thereof in promoting the growth of plant lateral roots. Background Art

[0002] Symbiosis in nature is mainly divided into two categories, the interaction between legumes and rhizobia and the interaction between actinomycete rhizobium and Frankia. Both types of symbiosis will form a special symbiotic structure - nodules. Symbiotic bacteria use nitrogen in the atmosphere to convert it into ammonia that can be used by plants, thereby supplementing the nitrogen elements needed by plants. Bayberry, as a typical actinomycete rhizobium plant, interacts with Frankia and other auxiliary strains to establish an effective symbiotic system to help plants grow healthily.

[0003] Through the isolation of endophytes in bayberry nodules, people found that in addition to Frankia, there are some other nitrogen-fixing bacteria and auxiliary bacteria colonizing the inside of the nodules. One type of nitrogen-fixing bacteria is Methylobacterium, which can use methanol as a carbon source for growth. Methylotrophy is a type of microorganism that can use non-CC bond low-carbon compounds (such as methane, methanol, formaldehyde, etc.) and is widely present in nature. Because most of these bacteria are rod-shaped, they are also called Methylotrophic bacteria.

[0004] Most of the bacteria of the genus Methylobacterium are pink in color and are usually called PPFM bacteria (pink-pigmented facultatively methylotrophic bacteria). PPFM bacteria usually parasitize on the roots, stems, and leaves of plants, and are most abundant on the leaves. PPFM uses one-carbon compounds (such as methanol) secreted by plants as a carbon source and produces plant hormones such as auxin (IAA) and cytokinin (CK), and has a positive effect on plant nitrogen metabolism, seed germination, photosynthesis, plant nutrition, etc.

[0005] For example, patent document CN107841479A discloses that a strain of Methylobacterium sp. MN28958 was isolated and screened from peanut leaves, and it was made into a microbial seed coating agent and applied to corn seeds, which can significantly promote the growth of corn seedlings. Wang Gaiping et al. isolated a strain of Methylobacterium sp. W-1 from saline soil samples in the Urho area in the northern Xinjiang Uygur Autonomous Region. The strain produces auxin, which has a growth-promoting effect on the growth of Arabidopsis seedlings (Isolation and Growth-Promoting Potential of a Salt-Tolerant Methylobacterium sp. W-1. Microbiology Bulletin, 2021, 48(11): 4134-4144.)

[0006] At present, there has been no functional research report on Methylobacterium isolated from bayberry root nodule tissue. The special living environment gives microorganisms special biological activities. In-depth research on this type of microorganisms is of great value to the development of agriculture. Summary of the invention

[0007] The purpose of the present invention is to provide a new methyl bacterium, which is used to interact with plant root systems to promote plant lateral root development, thereby enhancing plant growth.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention isolates a new methylobacterium from the root nodule tissue of bayberry, and its morphological characteristics are: forming light red circular colonies on methanol culture medium, and the individual morphology of the strain is rod-shaped; Gram staining is negative; its 16s rDNA sequence is shown in SEQ ID No. 1, and the strain is identified as belonging to Methylbacterium goingesingense in molecular phylogenetic taxonomy, and it is named Methylobacterium sp. cucegen. It was deposited in China Center for Type Culture Collection on March 14, 2024, with a deposit number of CCTCC NO: M 2024480, and the deposit address is Wuhan University, Wuhan, China.

[0010] Furthermore, the culture medium of the Methylobacterium is a DSMZ culture medium containing methanol.

[0011] Furthermore, the culture condition of the Methylobacterium is 28-30°C.

[0012] The present invention has found that inoculating Methylobacterium sp. cucegen into the roots of plant seedlings can promote the growth of lateral roots, thereby increasing the biomass of the plants.

[0013] Therefore, the present invention provides the use of the methylbacterium in promoting plant growth.

[0014] Furthermore, the promoting plant growth includes promoting the growth of plant lateral roots.

[0015] Furthermore, the methyl bacterium promotes the production of dichotomous branched lateral roots in plants. Studies have shown that the methyl bacterium can cause the plant root system to produce dichotomous branched lateral roots.

[0016] Furthermore, the plant is a leguminous plant or an actinomycete nodulation plant that is symbiotic with rhizobia, and the methylbacterium promotes plant nodulation. Studies have shown that the increase in the dichotomous branching lateral roots of plant roots helps promote the colonization of symbiotic bacteria in the plant roots to form nodule structures.

[0017] Furthermore, the plant is a cash crop, which includes any one of the following types: Leguminosae, Solanaceae and Cruciferae.

[0018] Furthermore, the legume plant is alfalfa, the Solanaceae plant is tomato, and the cruciferous plant is rapeseed.

[0019] The results showed that Methylobacterium promoted the increase of dichotomous lateral roots in alfalfa, and mixed rhizobium inoculation could promote plant nodulation. Methylobacterium could promote the increase of dichotomous lateral roots in tomato and rapeseed.

[0020] Further, the application includes: preparing the Methylobacterium grown to the logarithmic growth phase into a bacterial suspension (OD 600 =1.0), inoculated into the root tips of young plants and cultured.

[0021] The invention also provides a microbial preparation for promoting the growth of plant lateral roots, wherein the active ingredient of the microbial preparation comprises the methylobacterium.

[0022] The present invention has the following beneficial effects:

[0023] The invention provides a new methylobacterium sp. cucegen, which can act on the root system of plant seedlings to promote the development of lateral roots, especially the generation of dichotomous branched lateral roots, and is beneficial to promoting the colonization of symbiotic bacteria on nodule plants to form nodules. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The growth of Methylobacterium on DSMZ medium.

[0025] Figure 2 This is a Gram stain microscopic examination of Methylobacterium.

[0026] Figure 3 Constructing a molecular evolutionary tree for Methylobacterium.

[0027] Figure 4 The Methylobacterium suspension promotes the growth of dichotomous lateral roots of various plants; A is a lateral root type pattern diagram; B is the morphology of lateral roots of different types of bayberry roots; C is the morphology of dichotomous lateral roots in alfalfa, tomato and rapeseed after inoculation with Methylobacterium.

[0028] Figure 5The growth phenotype of alfalfa promoted by Methylobacterium suspension at different concentrations; A is a model diagram, B is the growth phenotype of alfalfa seedlings, C is the statistics of the total fresh weight of alfalfa plants affected by different concentrations of Methylobacterium, D is the statistics of the increase in the length of underground roots of alfalfa caused by different concentrations of Methylobacterium, E is the statistics of the number of lateral roots of alfalfa caused by different concentrations of Methylobacterium, and F is the statistics of the production of dichotomous lateral roots of alfalfa caused by different concentrations of Methylobacterium.

[0029] Figure 6 The nodulation phenotype of alfalfa promoted by Methylobacterium suspension, where A is the phenotype of alfalfa and B is the statistics of the number of nodules. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] DSMZ solid culture medium, components: KNO3 1g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.02g, Na2HPO4 0.23g, NaH2PO4 0.07g, FeSO4·7H2O 1mg, CuSO4·5H2O 5μg, H3BO3 10μg, MnSO4·5H2O 10μg, ZnSO4·7H2O 70μg, MoO3 10μg, Agar 12g, deionized water 1L and 5mL methanol, PH 6.8.

[0033] Fahraeus slant medium, components: 5mM NH4Cl, 1mM CaCl2, 0.5mM MgSO4, 0.6mM K2HPO4, 0.8mM Na2HPO4, 20μM Fe citrate, with 0.1mg MnSO4, 0.1mg CuSO4, 0.1mg ZnSO4, 0.1mg H3BO3, and 0.1mg Na2MoO4, deionized water 1L, PH=6.7, Agar 15g.

[0034] Example 1: Isolation and identification of Methylobacterium cucegen strains

[0035] 1. Isolation and purification of Methylobacterium cucegen strain

[0036] Step 1, sample collection, collecting bayberry plants in Lanxi area, washing the roots with deionized water, and collecting a large number of bayberry root nodules;

[0037] Step 2, disinfection of the nodule surface: wash the nodule with deionized water for 3 times, clean the surface with 75% ethanol for 60 seconds, wash it with deionized water for 3 times, then clean the surface with 10% sodium hypochlorite for 10 minutes, and wash it with deionized water for 5 times;

[0038] Step 3, strain isolation and purification, cut the washed nodules into several small portions with a clean blade, place them on DSMZ plates, and culture them upside down in a 28°C incubator. Keep observing until obvious colonies grow on the plate, pick out single colonies and further purify them using the dilution coating method to obtain purified single colonies, which are then preserved for the next step of identification.

[0039] 2. Strain identification

[0040] 2.1 Morphological identification

[0041] Activate Methylobacterium from -80℃ and streak culture on DSMZ methanol solid medium. Incubate in dark at 28℃ for 3-5 days. Light red round colonies can be seen. Figure 1 As shown. The individual morphology of Methylobacterium is rod-shaped and Gram staining is negative, such as Figure 2 shown.

[0042] 2.2 Molecular identification

[0043] The 16S rDNA amplification and sequence identification of Methylobacterium cucegen strains and the construction of phylogenetic evolutionary tree were carried out as follows:

[0044] Take Methylobacterium grown to the logarithmic growth phase, centrifuge at 6,000g for 10 min, collect the cells, wash twice with sterile water, and then wash once with TE (Tris 50mmol / L, EDTA 20mmo1 / L) buffer at pH8.0, collect the cells, add 2×CTAB extraction buffer (2% CTAB, 100mmo1 / L Tris-HCl, 20mmol / L EDTA, 1.4mo1 / L NaCl, pH8.0) and grind thoroughly in a sterile mortar: transfer to a centrifuge tube, bathe at 65°C for 1h, centrifuge at 6000g for 5min twice, collect the supernatant, add an equal volume of chloroform:isoalcohol (24:1) solution, mix thoroughly, centrifuge at 13000g for 20min, and collect the aqueous phase.

[0045] Add 1 / 5 volume of 5% CTAB buffer (5% CTAB, 0.35 mol / L NaCl) and mix thoroughly, incubate in a 65°C water bath for 10 min, add an equal volume of chloroform:isoamyl alcohol (24:1) solution to extract once, centrifuge at 13000 g for 20 min, and collect the aqueous phase.

[0046] The DNA was precipitated by adding 1 / 10 volume of 3 mol / L NaAc and 2 volumes of ethanol, rinsed with 70% ethanol, dried naturally, dissolved in TE buffer (pH 7.5) containing RNase, and the quality of the DNA was detected by 0.8% agarose gel electrophoresis.

[0047] After obtaining genomic DNA, 16s rDNA fragment was amplified using 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT) as universal primers.

[0048] The PCR reaction system was as follows: 0.5 μL template DNA, 10 μL 2× Green Taq Mix, 0.5 μL each of upstream and downstream primers, and ddH2O was added to 20 μL.

[0049] The PCR cycle conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, 29-30 cycles in steps 2-4, extension at 72°C for 10 min, and low temperature storage at 10°C for 30 min.

[0050] The PCR product was subjected to electrophoresis using 1% agarose gel and observed using a gel imager to analyze whether the target band of the correct size was amplified. If so, gel recovery was performed.

[0051] The recovered products were sent to the company for sequencing, and the sequences were compared with the BLAST sequence in the NCBI database to construct a phylogenetic tree, such as Figure 3 The results showed that the sequence of strain cucegen was 99% similar to that of Methylobacterium, which determined that strain cucegen belonged to Methylobacterium goesingense in molecular phylogenetic taxonomy.

[0052] The above morphological and molecular identification results showed that the newly isolated strain belonged to Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Methylobacteriaceae, and Methylobacterium. It was named Methylobacterium sp. cucegen. It was deposited in China Center for Type Culture Collection on March 14, 2024, with the deposit address: Wuhan, China, Wuhan University, with the deposit number CCTCC NO: M 2024480, and was identified as alive on March 21, 2024.

[0053] Example 2: Cultivation of Methylobacterium

[0054] 1. Prepare DSMZ solid culture medium: KNO3 1 g, MgSO4·7H2O 0.2 g, CaCl2·2H2O0.02 g, Na2HPO4 0.23 g, NaH2PO4 0.07 g, FeSO4·7H2O 1 mg, CuSO4·5H2O 5 μg, H3BO3 10 μg, MnSO4·5H2O 10 μg, ZnSO4·7H2O 70 μg, MoO3 10 μg, Agar 12 g, deionized water 1 L and methanol 5 mL, pH 6.8.

[0055] 2. Take out the bacteria preservation tube from the -80℃ refrigerator, streak the activated Methylobacterium on the DSMZ plate, invert and culture it in a 28℃ incubator for 3-5 days. The one that grows several small red colonies is Methylobacterium cucegen. Pick a single clone colony, shake it in 4mL DSMZ liquid culture medium, and culture it in a 28℃ shaker at 220rpm for 3-5 days until the logarithmic growth phase, which can be used for subsequent experiments.

[0056] Example 3: Effect of Methylobacterium on the Growth of Bifurcated Lateral Roots of Different Plants

[0057] Step 1, seed germination, washing alfalfa, tomato, and rapeseed seeds with deionized water, disinfecting with sodium hypochlorite, and then planting on a water agar plate for germination;

[0058] Step 2, seed transplanting, transferring the germinated plant seedlings to Fahraeus slant medium;

[0059] Step 3, inoculate Methylobacterium, streak on DSMZ plate to activate Methylobacterium, then pick a single clone colony and shake it in DSMZ liquid medium at 28℃220rpm until the logarithmic growth phase, centrifuge at 4℃ to obtain fresh bacteria, resuspend in sterile water to OD 600=1, pipette 10 μL of Methylobacterium suspension and drop it on the root tip of the seedling, add an equal amount of sterile water to the control, and place the seedling in a 25℃ light incubator and culture it vertically for 1 week.

[0060] Step 4: Statistics of plant growth phenotypes: Use a Nikon camera to photograph the growth of seedlings, and count the number of lateral roots of plants in the experimental group and the control group and the occurrence of dichotomous lateral roots.

[0061] The results are as follows Figure 4 As shown, the number of lateral roots in the roots of alfalfa, tomato and rapeseed seedlings in the control group was small, and the distance between adjacent lateral roots was far; the number of lateral roots in the roots of alfalfa, tomato and rapeseed seedlings in the experimental group was large, and the distance between adjacent lateral roots was very close, and even two lateral roots were formed at the position of the same lateral root primordium. We describe this phenomenon as "binary branching lateral roots".

[0062] Example 4: Growth promotion experiment of Methylobacterium (taking alfalfa as an example)

[0063] 1. Prepare fresh alfalfa seeds (purchased from Yongdeng, Lanzhou, Gansu), disinfect with 95% concentrated sulfuric acid for 5-7 minutes, discard the waste liquid, place the centrifuge tube on ice, wash with ddH2O for 3 times, change to 10% NaClO for disinfection for 5-7 minutes, discard the waste liquid, change to ddH2O for washing 5-7 times, keep at 4°C overnight, and plant the seeds on 0.8% water agar plates the next day. After the seeds germinate, remove the seed coat and transfer to Fahraeus slant culture medium.

[0064] 2. Prepare Methylobacterium grown to the logarithmic growth phase, centrifuge at 4200 rpm for 10 min at 4°C, and resuspend in ddH2O to OD 600 =1.5. Further gradient dilution to OD 600 =0.6, 0.3 and 0.15.

[0065] 3. After the alfalfa seedlings were restored for 2 days, 10 μL of Methylobacterium suspension of different concentrations was dripped onto the root tips of the seedlings. An equal volume of ddH2O was dripped onto the control group. After absorption, the seedlings were placed in a 25°C light incubator for vertical culture for one week. The physiological phenotypes of the alfalfa plants were counted, including the fresh weight of the plants, the length of the main root, the number of lateral roots, the branching phenotype of the lateral roots, etc.

[0066] like Figure 5 As shown in the figure, compared with the control group, the biomass of alfalfa seedlings inoculated with Methylobacterium increased, and the higher the inoculation concentration, the more biomass accumulated. Specifically, the total fresh weight of the plants and the number of lateral roots increased with the increase of inoculation concentration, and the proportion of dichotomously branched lateral roots also increased with the increase of inoculation concentration.

[0067] Example 5: Experiment on promoting nodulation of alfalfa by increasing dichotomous branched lateral roots

[0068] As in Example 4, 10 μL of Methylobacterium suspension (OD 600 =1.5), and the control group was dripped with an equal volume of ddH2O at the same position. After absorption, the alfalfa seedlings were placed in a 25°C light incubator for vertical culture for 1 week; then the alfalfa in the experimental group and the control group were transferred into the sterilized matrix (vermiculite: perlite = 3:1), and the roots were inoculated with alfalfa Sinorhizobium Sm2011 the next day. Each small pot was inoculated with dilution to OD 600 =0.05 volume of 10mL bacterial suspension, supplemented with nitrogen-free BNM culture medium (formula: CaSO4·2H2O 344mg per L, MES 390mg per L, Nod Majors 5mL per L, Nod Minors I 5mL per L, Nod Minors II 5mL per L, Fe-EDTA 5mL per L, pH=6.5), and the physiological phenotypes of alfalfa plants and the number of nodules of the plants were counted 4 weeks after inoculation.

[0069] The results are as follows Figure 6 As shown, compared with the control group, the biomass of alfalfa seedlings inoculated with Methylobacterium increased and the average number of nodules per plant increased significantly after inoculation with Rhizobium.

[0070] Although the embodiments of the present invention have been described, it will be appreciated by those skilled in the art that some changes and modifications may be made to the embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A methylbacterium, characterized in that The methylobacterium was isolated from the root nodule tissue of bayberry and named Methylobacterium sp. cucegen. It was deposited in the China Center for Type Culture Collection on March 14, 2024, with the deposit number CCTCC NO: M 2024480.

2. The methylbacterium according to claim 1, characterized in that The culture medium of the methylbacterium is a DSMZ culture medium containing methanol.

3. The methylbacterium according to claim 1 or 2, characterized in that The culture condition of the Methylobacterium is 25-30°C.

4. Use of the methylbacterium according to any one of claims 1 to 3 in promoting plant growth.

5. The use according to claim 4, characterized in that The promoting plant growth includes promoting the growth of plant lateral roots.

6. The use according to claim 5, characterized in that The Methylobacterium promotes the production of dichotomously branched lateral roots in plants.

7. The use according to any one of claims 4 to 6, characterized in that: The plant is a leguminous plant or an actinomycete nodulation plant that is symbiotic with rhizobia, and the methylbacterium promotes plant nodulation.

8. The use according to any one of claims 4 to 6, characterized in that: The plants are alfalfa, tomato and rapeseed.

9. The use according to any one of claims 4 to 6, characterized in that: The application comprises: inoculating the methylbacterium suspension into the root tips of plant seedlings and culturing.

10. A microbial preparation for promoting the growth of plant lateral roots, characterized in that: The active ingredient thereof comprises the methyl bacterium as described in any one of claims 1 to 3.

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