CN111763650A

CN111763650A - Nitrogen-fixing methylobacterium strain and application thereof

Info

Publication number: CN111763650A
Application number: CN202010784714.1A
Authority: CN
Inventors: 杨婷; 史发超; 安玉兴; 孙东磊; 卢颖林
Original assignee: Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
Current assignee: Guangdong Institute of Bioengineering Guangzhou Cane Sugar Industry Research Institute
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2020-10-13

Abstract

The invention relates to a nitrogen-fixing methylobacterium strain and application thereof. The methylobacterium has a collection number of GDMCC No. 61052. The methylobacterium can fix nitrogen biologically, and has wide development and utilization values in agricultural production.

Description

Nitrogen-fixing methylobacterium strain and application thereof

Technical Field

The invention relates to the field of microbiology, in particular to a nitrogen-fixing methylobacterium strain and application thereof.

Background

During the growth process, the plant interacts with various microorganisms, mainly including plant rhizosphere microorganisms, phyllospheric microorganisms and endophytes. These microorganisms can promote plant growth by direct or indirect means such as nitrogen fixation, phosphate solubilization, auxin secretion, increased plant resistance, and the like. In agricultural production, the beneficial microorganisms inoculated by external sources can promote the growth of plants and increase the crop yield, and meanwhile, the environmental friendliness and the sustainability of the action effect are advocated in modern agriculture with more and more importance on the ecological environment.

Methylobacterium (Methylobacterium) is a type of facultative aerobic bacteria that can utilize monocarbons including methanol, methylamine, etc. as carbon sources. Bacteria of the genus Methylobacterium are pink due to their ability to synthesize carotenoids, and are also called Pink-segmented Facultivative methylotrophics (PPFM). Methylobacterium is widely present in soil, air, water areas and other environments, and in plant phyllosphere and rhizosphere, has the functions of generating plant hormone, antagonizing plant pathogenic microorganisms, relieving abiotic stress and the like, and has great development and utilization values in agricultural production.

Disclosure of Invention

Based on this, the object of the present invention is to provide a novel methylobacterium YT2019B002, which can fix nitrogen effectively.

The specific technical scheme is as follows:

a methylobacterium YT2019B002, wherein the deposited number of the methylobacterium YT2019B002 is GDMCC No. 61052.

The invention also aims to provide an application of the methylobacterium YT2019B002 in biological nitrogen fixation.

The invention also aims to provide an application of the methylobacterium YT2019B002 in improving the nitrogen content of soil.

The invention also aims to provide an application of the methylobacterium YT2019B002 in preparing a soil conditioner.

Another object of the present invention is to provide a biological agent, the active ingredient of which comprises the above methylobacterium YT2019B 002.

Another objective of the present invention is to provide a method for culturing the methylobacterium YT2019B002, comprising the following steps: the methylobacterium YT2019B002 is inoculated in a culture medium and cultured at the temperature of 20-30 ℃. Further, culturing at 22-28 ℃; further, the culture is carried out at 24 to 26 ℃.

In some of these embodiments, the medium is AMS medium.

The Methylobacterium YT2019B002(Methylobacterium sp.) is preserved in the Guangdong province microbial strain preservation center (GDMCC, address: No. 59 building 5 of Michelia furiosu 100 of Guangzhou, Guangzhou province) specified by the national intellectual property office in 6.9.2020, and the preservation date is as follows: year 2020, 6/9, accession number: GDMCCNo: 61052.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers a new microorganism methylobacterium YT2019B002 belonging to the methylobacterium for the first time, which can carry out biological nitrogen fixation and has wide development and utilization values in agricultural production.

Drawings

FIG. 1 shows the shape and gram stain profile of the strain YT2019B002 on the medium (A: shape of the culture on AMS medium; B: gram stain);

FIG. 2 is a gel electrophoresis chart of the PCR amplification result of the specific primer (mxaF gene), (M: DL2000 Marker; 1, 2: YT2019B002 strain; 3, 4: negative control);

FIG. 3 is a phylogenetic tree constructed by the Neighbor-Joining (NJ) method based on the 16S rRNA sequence; the strain Escherichia coli 562(J01859) is an outlier,^T: a model strain;

FIG. 4 is a nitrogen fixation capacity plate assay of strain YT2019B002 (A: CK Normal AMS Medium; B: NH-free)₄Cl nitrogen deficient AMS medium).

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The present invention will be described in further detail with reference to specific examples.

The material and the method are as follows:

separating the material source: the separation material, namely, the sugarcane seedlings (Saccharum officinarum 'Badila') is collected from the east Yong town of south sand area of Guangzhou, Guangdong province, and after collection, the ice box is preserved at low temperature and brought back to a laboratory, and the ice box is preserved at low temperature of-20 ℃ for functional microorganism separation.

Solid AMS medium: according to the Methylobacillus bacteria obligate culture Medium in Whittenbury et al 1970, the main component comprises K₂HPO₄0.7g，KH₂PO₄0.54g，MgSO₄·7H₂O 1.0g，CaCI₂·2H₂O 0.2g，FeSO₄·7H₂O 4mg，NH₄Cl 0.5g，ZnSO₄·7H₂O 100.0mcg，MnCl₂·4H₂O 30.0mcg，H₃BO₃300.0mcg，CoCl₂·6H₂O 200.0mcg，CuCl₂·2H₂O 10.0mcg，NiCl₂·6H₂O 20.0mcg，Na₂MoO₄·2H₂O60.0 mcg, Agar 15.0g, distilled water 1L, pH 6.8, sterilizing at 121 deg.C for 30min, cooling to 50-55 deg.C, adding 1mL of filter sterilized methanol per 100mL of culture medium, mixing, and pouring onto a flat plate.

The separation method comprises the following steps: the sugarcane rhizosphere soil is prepared into 10 by a gradient dilution method^-6-10^-8g/mL soil suspension, uniformly coating the suspension on a solid AMS culture medium, carrying out inversion culture at a constant temperature of 25 ℃, observing, separating and purifying a strain producing pink pigment, and naming the strain as YT2019B002, inoculating the strain into a nutrient broth liquid culture medium, shaking at a speed of 160rpm of 25 ℃ for 48 hours, adding 30% glycerol aqueous solution (v/v) according to a volume ratio of 1:1, and storing the strain at a temperature of-80 ℃ for later use.

Identification of the strain YT2019B 002:

1. determination of physicochemical Properties of Strain YT2019B002

Streak-culturing strain YT2019B002 on soybean casein agar culture medium TSA (OXOID) solid culture medium at 25 deg.C for 5 days, picking single colony, gram-staining, and culturing

2Systems gram negative bacteria identification card (GN card) for carbon source utilization, enzyme activity and drug resistance detection.

2. Molecular biological identification of strain YT2019B002

YT2019B002 single colony DNA was extracted by a thermal cracking method, and the type of the strain was confirmed by mxaF gene amplification using the specific primers mxa f1003(5-GCGGCACCAACTGGGGCTGGT-3 ', SEQ ID NO:2) and mxa r1561 (5-GGGCAGCATGAAGGGCTCCC-3', SEQ ID NO: 3). Meanwhile, the 16S rRNA fragment is amplified by 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO:4) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3', SEQ ID NO:5), the PCR product is sent to Shanghai biological engineering (Shanghai) GmbH for sequencing, and the sequence is used for identifying the accurate type of YT2019B002 by constructing a phylogenetic tree with the sequence of a similar species.

Determination of enzyme activity of strain YT2019B002

And (3) measuring the nitrogen fixation activity of the strain: inoculating the strain in the absence of a nitrogen source(NH₄Cl) on AMS solid medium to add NH₄Using a normal AMS culture medium of Cl as a control, inoculating bacteria by a streak method, carrying out inverted culture at a constant temperature of 28 ℃ for 7 days, observing the growth condition of the strain, and repeating 3 treatments each time.

Determination of dextranase Activity: the strain was streaked on KM medium and contained 0.4% (NH) as a main component₄)₂SO₄，0.01％NaCI，0.01％MgSO₄，0.01％CaCI₂0.05% yeast extract, 33mg Fe (III) -EDTA, 0.05M potassium phosphate buffer (pH 7.0), 0.04% sodium carboxymethylcellulose (CMC), Agar 15.0g, distilled water 1L, sterilized at 121 ℃ for 30 min. After 3 days of inverted culture at constant temperature of 30 ℃, 0.1% Congo red solution is poured into the flat plate to dye for 30min, 1M NaCI solution fades for 10min, and whether a transparent ring is generated or not is observed.

Assay of Hemophilus Activity reference is made to the method of Shin et al (2001.) A ① 60.5.5 mg Chromel S in 50ml deionised water ② 10ml ferric iron solution (1mM FeCl)₃·6H₂O, 10mM hydrochloric acid as a solvent), ③ 72.9.9 mg of CTAB is dissolved in 40ml of deionized water, the three solutions are mixed and metered to 100ml, the pH is adjusted to be neutral, sterilization is carried out at 121 ℃ for 20min, 30.24g of pegs are added into 900ml of water agar culture medium, the pH is 6.8, sterilization is carried out at 121 ℃ for 20min, A and B solutions are mixed and poured into a flat plate, inoculation is carried out at 30 ℃ for 3d of constant temperature inversion culture, and then the result is observed and recorded.

ACC activity assay: referring to the Penrose and Glick (2003) method, the strain was inoculated into a 50mL Erlenmeyer flask containing 10mL AMS liquid medium, shaken at 28 ℃ for 4-5 days at 120rpm, centrifuged at 3000g for 5min to collect the cells, washed twice with filter-sterilized 0.1M Tris-HCI (pH 7.5), resuspended in 1mL 0.1M Tris-HCI (pH 7.5), 200. mu.L of the supernatant was spread on DF microelement medium containing 3mM ACC as the only nitrogen source to add (NH)₄)₂SO₄The DF culture medium (0.2% w/v) is used as a positive control, and the growth condition of the strain is observed after the coated culture dish is placed at the constant temperature of 28 ℃ for 3d of inverted culture.

As a result:

1. identification

The strain YT2019B002 is gram-stained red (as shown in figure 1) and is a gram-negative bacterium. Colony morphology: the colony on the AMS solid medium is opaque and light pink, has the diameter of 1-3mm, protrudes upwards, has neat edges and is dry.

Physical and chemical Property characterization As shown in Table 1, using

2System and

2 gram negative bacteria identification card (GN), through the determination of 47 kinds of carbon source utilization, enzyme activity and drug resistance and a negative control reaction in total, the primary identification YT2019B002 is Methylobacterium sp bacteria.

TABLE 1 physicochemical Properties of Strain YT2019B002

Note: 95% to 100% positive; v-6% to 94% positive; positive-0% to 5%.

Using the DNA of the strain YT2019B002 as a template and amplification with a specific primer of Methylobacterium, a specific band with a fragment size of 560bp was obtained (fig. 2), and it was confirmed that YT2019B002 was a bacterium of the genus Methylobacterium. The 16S rRNA sequence (shown as SEQ ID NO: 1) of the strain YT2019B002 is compared with the sequence in the NCBI database, and the similarity of the sequence and the M.populi model strain BJ001(Access No.: AY251818) is the highest and reaches 99.49 percent. A phylogenetic tree is constructed by using 16S rRNA sequences of other species of Methylobacterium in NCBI as reference and Escherichia coli (Access No.: J01859) as outer population and adopting a Neighbor-Joining method (NJ), and strains YT2019B002 and M.populi (AY251818) in the obtained optimal tree form an independent branch (as shown in figure 3) with high support rate (96).

The 16S rRNA gene sequence of the methylobacterium YT2019B002 is shown as SEQ ID NO: 1, and the following components:

SEQ ID NO:1：

CAGCTTACACATGCAGTCGAACGGGCTTCTTCGGAAGTCAGTGGCAGACGGGTGAGTAACACGTGGGAACGTGCCCTTCGGTTCGGAATAACTCAGGGAAACTTGAGCTAATACCGGATACGCCCTTATGGGGAAAGGTTTACTGCCGAAGGATCGGCCCGCGTCTGATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGgCCtTAGGGTTGTAAAGCTCTTTTGTCCGGGACGATAaTGACGGTACCGGAAGAATAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGCCGATTAAGTCGGGGGTGAAAGCCTGTGGCTCAACCACAGAATTGCCTTCGATACTGGTTGGCTTGAGACCGGAAGAGGACAGCGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCCGGTTCTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGCCGTTGGCCTGCTTGCAGGTCAGTGGCGCCGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCARAAaCCTWACCATcCCCTTGACATGGCaTGTTACCTCGAGAGATCGGGGATCCTCTTCGRAGGCGTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCACGTCCTTAGTTGCCATCATTCAGTTGGGCACTCTAGGGAGACTGCCGGTGATAAGCCGCGAGGAAGGTGTGGATGACGTCAAGTCCTCATGGCCCTTACGGGATGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGACGCGAAACCGCGAGGTTGAGCAAATCCCCAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGGGTGCATGAAGGCGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTCTTACCCGACGGCGCTGCGCCAACCGCAAGGGGGCAGGCGACCACGGTAGTCGCAC。

2. measurement of Nitrogen fixation Capacity

The results of the main activity assay related to growth promotion show that the strain YT2019B002 can grow on AMS medium without nitrogen source (FIG. 4), and can react with NH₄The colony growth on the normal AMS medium with Cl as the nitrogen source has no obvious difference, so that the strain YT2019B002 has the capability of fixing nitrogen.

YT2019B002 was unable to grow on DF medium with ACC as the sole nitrogen source and also had no glucanase and siderophin activity as determined by plate.

In conclusion, the strain YT2019B002 can carry out biological nitrogen fixation.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Guangdong province bioengineering research institute (Guangzhou sugar industry institute)

<120> nitrogen-fixing methylobacterium strain and application thereof

<130>2020-08-05

<160>5

<170>PatentIn version 3.3

<210>1

<211>1380

<212>DNA

<213>Artificial Sequence

<400>1

cagcttacac atgcagtcga acgggcttct tcggaagtca gtggcagacg ggtgagtaac 60

acgtgggaac gtgcccttcg gttcggaata actcagggaa acttgagcta ataccggata 120

cgcccttatg gggaaaggtt tactgccgaa ggatcggccc gcgtctgatt agcttgttgg 180

tggggtaacg gcctaccaag gcgacgatca gtagctggtc tgagaggatg atcagccaca 240

ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat attggacaat 300

gggcgcaagc ctgatccagc catgccgcgt gagtgatgaa ggccttaggg ttgtaaagct 360

cttttgtccg ggacgataat gacggtaccg gaagaataag ccccggctaa cttcgtgcca 420

gcagccgcgg taatacgaag ggggctagcg ttgctcggaa tcactgggcg taaagggcgc 480

gtaggcggcc gattaagtcg ggggtgaaag cctgtggctc aaccacagaa ttgccttcga 540

tactggttgg cttgagaccg gaagaggaca gcggaactgc gagtgtagag gtgaaattcg 600

tagatattcg caagaacacc agtggcgaag gcggctgtct ggtccggttc tgacgctgag 660

gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat 720

gaatgccagc cgttggcctg cttgcaggtc agtggcgccg ctaacgcatt aagcattccg 780

cctggggagt acggtcgcaa gattaaaact caaaggaatt gacgggggcc cgcacaagcg 840

gtggagcatg tggtttaatt cgaagcaacg cgcaraaacc twaccatccc cttgacatgg 900

catgttacct cgagagatcg gggatcctct tcgraggcgt gcacacaggt gctgcatggc 960

tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccacgtc 1020

cttagttgcc atcattcagt tgggcactct agggagactg ccggtgataa gccgcgagga 1080

aggtgtggat gacgtcaagt cctcatggcc cttacgggat gggctacaca cgtgctacaa 1140

tggcggtgac agtgggacgc gaaaccgcga ggttgagcaa atccccaaaa gccgtctcag 1200

ttcggattgc actctgcaac tcgggtgcat gaaggcggaa tcgctagtaa tcgtggatca 1260

gcacgccacg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1320

tggtcttacc cgacggcgct gcgccaaccg caagggggca ggcgaccacg gtagtcgcac 1380

<210>2

<211>21

<212>DNA

<213>Artificial Sequence

<400>2

gcggcaccaa ctggggctgg t 21

<210>3

<211>20

<212>DNA

<213>Artificial Sequence

<400>3

gggcagcatg aagggctccc 20

<210>4

<211>20

<212>DNA

<213>Artificial Sequence

<400>4

agagtttgat cmtggctcag 20

<210>5

<211>22

<212>DNA

<213>Artificial Sequence

<400>5

tacggytacc ttgttacgac tt 22

Claims

1. The methylobacterium YT2019B002 is characterized in that the deposited number of the methylobacterium YT2019B002 is GDMCC No. 61052.

2. The use of methylobacterium YT2019B002 in claim 1 for biological nitrogen fixation.

3. The use of methylobacterium YT2019B002 in the improvement of soil nitrogen content as claimed in claim 1.

4. The use of methylobacterium YT2019B002 in claim 1 for preparing soil conditioner.

5. A biological agent characterized in that its active ingredient comprises Methylobacterium YT2019B002 as claimed in claim 1.

6. The method for culturing methylobacterium YT2019B002, which comprises the steps of: the methylobacterium YT2019B002 is inoculated in a culture medium and cultured at the temperature of 20-30 ℃.

7. The culture method according to claim 6, wherein the medium is AMS medium.