CN113322214B

CN113322214B - Atractylodes macrocephala endophytic bacterium and application thereof

Info

Publication number: CN113322214B
Application number: CN202110784739.6A
Authority: CN
Inventors: 朱波; 秦路平; 吴威; 刘世禹; 何玟叶; 张轲杰
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2022-03-11
Anticipated expiration: 2041-07-12
Also published as: CN113322214A

Abstract

The invention discloses an atractylis ovata endophytic bacterium and application thereof, and belongs to the technical field of endophytic bacterium resource utilization and plant disease control. The endophytic bacteria is obtained by separating living body of Atractylodes macrocephala Koidz of Compositae by endophytic bacteria separation and purification technology, and is classified and named as Rhodococcus sp AM201 of Actinomycetes, and the strain preservation number is CGMCC No. 22155. The invention also discloses the application of the endophytic bacteria: can inhibit the growth of pathogenic bacteria of the root rot of the bighead atractylodes rhizome, induce the bighead atractylodes rhizome to generate systemic disease resistance, reduce the gene expression of the BXL2, At1g65750 and BZIP43 of the bighead atractylodes rhizome, and reduce the disease index of the root rot of the bighead atractylodes rhizome by 35.71 percent. The endophytic bacteria have obvious effect of preventing and treating the bighead atractylodes rhizome root rot and bring wide application prospect to the field of biological prevention and treatment of bighead atractylodes rhizome fungal diseases.

Description

Atractylodes macrocephala endophytic bacterium and application thereof

Technical Field

The invention relates to the technical field of plant endophyte resource utilization and plant disease prevention and control, in particular to an atractylis ovata endophyte and application thereof in prevention and control of atractylis ovata root rot.

Background

The traditional Chinese medicine rhizoma atractylodis macrocephalae is dried rhizome of Atractylodes macrocephala koidz of the family compositae and the genus atractylodis, is one of famous medicinal materials 'eight Zhejiang province', is warm in nature, bitter and sweet in taste, enters spleen and stomach channels, and has the effects of strengthening spleen and replenishing qi, eliminating dampness and promoting diuresis, stopping sweating, preventing miscarriage and the like. The large head atractylodes rhizome is widely used clinically, is known as the first important herb for strengthening spleen and tonifying qi, and is called as 'ten prescriptions and nine prescriptions' and 'southern surgery and north ginseng'.

Root rot is a main disease in bighead atractylodes rhizome cultivation, can cause serious harm to bighead atractylodes rhizome in the whole growing period, has the perennial incidence rate of about 30 percent, can achieve 60 to 70 percent of stifle death rate in seriously ill fields, even is completely harvested, and is one of the main obstacles for restricting the production and development of bighead atractylodes rhizome. The bighead atractylodes rhizome root rot is a vascular bundle systemic disease and is caused by complex infection of various pathogenic bacteria, and main pathogenic bacteria are Fusarium sp. After the fusarium infects a plant host, the fusarium can degrade the plant cell wall, change the cell membrane permeability, prevent ATP synthesis and inhibit the growth of plant roots. After the plant cell wall is degraded, pectin blocks the duct of the host plant, which prevents the host plant from absorbing water, resulting in plant wilting death. Fusarium overwintering in soil mainly by using mycelium, chlamydospore and other host disease residues, the chlamydospore can survive for at least more than 5-10 years in the soil, and root rot is very difficult to control. At present, the prevention and treatment of the root rot of the bighead atractylodes rhizome mainly focuses on crop rotation, stubble replacement and pesticide spraying, but has the problems of limited land resources, excessive pesticide residues, environmental pollution, drug resistance of pathogenic bacteria and the like.

Endophytic bacteria (Endophytic bacteria) refer to a class of bacteria that live in plant cells or within plant tissues at a certain period of their life history without causing significant disease to the plant tissues. In a broad sense, they include saprophytic bacteria that grow on the vegetative surface at a certain stage in life history, latent pathogenic bacteria and mycorrhizal bacteria that are not temporarily harmful to the host. The endophytic bacteria of plants are a special biological group, are wide in distribution and multiple in variety, and have extremely rich biological diversity from various aspects such as host plant species, self variety, heredity, ecological environment and the like. The relationship between the endophytic bacteria and the host plant is very complex and is generally considered as a mutualistic symbiotic relationship, on one hand, the host provides sites and nutrients required for growth of the endophytic bacteria, and on the other hand, the endophytic bacteria play an important role in the growth and development and systemic evolution processes of the host. Researches show that the existence of endophytic bacteria can enhance the disease resistance of plants to certain pathogenic microorganisms, and the endophytic bacteria are important microorganism resources in the technical field of plant prevention and control.

However, no report is currently found about the application of the atractylis ovata endophytic bacteria in preventing and treating the root rot of atractylis ovata.

Disclosure of Invention

The invention aims to provide a bighead atractylodes rhizome endophytic bacterium and application thereof in preventing and treating bighead atractylodes rhizome root rot, aiming at the defects of the prior art.

In a first aspect of the present invention, there is provided a medicinal plant endophytic bacterium, designated Rhodococcus sp 201. The endophytic bacteria is obtained by separating and purifying endophytic bacteria from living Atractylodes macrocephala Koidz of Compositae, and is identified as Rhodococcus sp 201 of Actinomycetes by microorganism classification. The strain is preserved, the preservation number of the strain is CGMCC No.22155, the preservation date is 2021, 4 and 9 days, the preservation unit is China general microbiological culture Collection center (CGMCC), the address is No. 3 of Xilu No.1 of Beijing, Chaoyang, and the postal code is 100101.

The solid culture of the endophytic bacteria is characterized in that:

culturing in beef extract culture medium (NA) at 26 deg.C in dark for rapid growth, wherein the colony is yellow-white, and the edge is in the shape of dish-shaped gear, as shown in FIG. 1.

The 16S rRNA amplification electrophoresis pattern of the endophytic bacteria is shown in figure 2, and the base sequence is shown in SEQ ID NO. 1.

The sequences were aligned at NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi) and found to have 100.00% homology to Rhodococcus japonicum (MN826591) and Rhodococcus erythropolis (MT549100) of Rhodococcus erythropolis, Chongshengi, Chongqing. Multiple sequence alignments were performed using MEGA7.0, and 1 sequence was randomly picked and aligned 1000 times by Neighbor-joining (NJ) analysis. A phylogenetic tree of the endogenous bacteria of the invention is shown in FIG. 3.

In a second aspect of the invention, the application of the endophytic bacteria in preventing and treating the root rot of the bighead atractylodes rhizome is provided.

According to the application, the AM201 bacterial suspension can inhibit the growth of pathogenic bacteria of the root rot of the bighead atractylodes rhizome by a plate confronting method, the condition of the root rot of the bighead atractylodes rhizome can be effectively relieved by applying the AM201 bacterial suspension, the bacterial suspension is prepared by adopting the endophytic bacteria through an NB culture medium, and the formula of the NB culture medium is as follows: 10g of peptone, 3g of beef powder, 5g of sodium chloride, 1000mL of distilled water, pH 7.2. + -. 0.2.

The application is mainly realized by the following technical scheme: (1) taking the endophytic bacteria strain, picking a small amount of thallus by using an inoculating needle under the aseptic condition, inoculating the thallus into a sterilized NB liquid culture medium, and carrying out dark culture for 48h under the conditions of 26 ℃, 65% humidity and 180 rpm; (2) sucking the bacterial suspension under aseptic condition, and adjusting the bacterial suspension to OD₆₀₀Obtaining the bacterial suspension as shown in figure 4, wherein the bacterial suspension is 0.6-1.0. (3) By adopting a filter paper plate confronting method, the AM201 bacterial suspension can effectively inhibit the growth of the pathogenic bacteria of the root rot of the bighead atractylodes rhizome, namely the fusarium oxysporum f.oxysporum, the fusarium solani and the fusarium semitectum f.incarnatum, and the highest inhibition rate reaches 74.47 +/-0.03% (figure 5). (4) The AM201 bacterial suspension is applied to the diseased plant of the white atractylodes rhizome in a root irrigation mode, the application amount is 5 mL/time/3 d and lasts for 21d, the condition of the root rot of the white atractylodes rhizome can be effectively relieved, and the expressions of BXL2, At1g65750 and BZIP43 genes are reduced (fig. 6 and 7).

The invention has the advantages that:

the endophytic bacterium Rhodococcus erythropolis AM201 disclosed by the invention can obviously inhibit the growth of pathogenic bacteria of the root rot of the bighead atractylodes rhizome by virtue of a living bacterium suspension prepared by a liquid culture medium, can down-regulate the expression of genes BXL2, At1g65750 and BZIP43, and can effectively relieve the condition of the root rot of the bighead atractylodes rhizome by 35.71%. The biological control effect of rhodococcus AM201 on the root rot of bighead atractylodes rhizome has great value in popularization and application in the agricultural field.

Drawings

The invention is further explained below with reference to the figures and examples;

FIG. 1: colony morphology of Rhodococcus AM201(Rhodococcus sp.) on NA solid medium;

FIG. 2: 16S rRNA sequence gel electrophoresis pattern based on AM201(Rhodococcus sp.) amplified by 16S rRNA primer;

FIG. 3: 16S rRNA-based Rhodococcus AM201(Rhodococcus sp.) phylogenetic tree (NJ method);

FIG. 4: a live bacterial suspension prepared from Rhodococcus AM201(Rhodococcus sp.);

FIG. 5: inhibiting effect of Rhodococcus AM201(Rhodococcus sp.) on hypha growth of pathogenic bacteria of root rot of white atractylodes, wherein a is radius of fungus colony; b, AM201 inhibits the growth of 3 kinds of rhizoma atractylodis macrocephalae pathogenic bacteria, CK is Fusarium oxysporum, Fusarium solani and Fusarium incarnatum respectively, and the center is the form of the pathogenic bacteria after the AM201 confronts;

FIG. 6: rhodococcus AM201(Rhodococcus sp.) suspension is effective in relieving the root rot disease of Atractylodis rhizoma; a, rhizoma Atractylodis Macrocephalae as control group; b, root rot largehead atractylodes rhizome; c, a root rot largehead atractylodes rhizome and AM201 bacterial liquid;

FIG. 7: rhodococcus AM201(Rhodococcus sp.) suspension can reduce the expression of genes BXL2, At1g65750 and BZIP43 of root rot bighead atractylodes rhizome,^**P<0.01。

Detailed Description

The endophytic bacteria of the present invention are strains isolated from living bodies of potentially healthy Atractylodes macrocephala in Zhejiang.

Example 1:

the endophytic bacteria are obtained by separation according to the following steps: taking the rhizome of fresh healthy rhizoma Atractylodis Macrocephalae from Zhejiang river, washing with clear water to remove surface impurities. Cutting appropriate size rhizome, wrapping gauze, and performing three-step surface disinfection treatment with 75% ethanol solution for 1min, 1% sodium hypochlorite solution for 3min, and 75% ethanol solution for 30 s. And (3) sterilizing, taking out, adding quartz sand and 20mL of PBS buffer solution, fully grinding to slurry, taking 100 mu L of supernatant, coating the supernatant on the surface of the NA culture medium, and placing the NA culture medium in a 28 ℃ mould incubator for dark culture for 2-3 d. The method comprises the steps of purifying the atractylis ovata endophytic bacteria by a line drawing method, and regularly observing until a single colony is separated, so as to finally obtain the endophytic bacteria strain AM201, wherein the colony is yellow white as a whole and has a disc-shaped gear-shaped edge, and is stored at a low temperature of 4 ℃ as shown in figure 1.

Taking a freezing storage tube of the AM201 strain, picking a little thallus by using an inoculating needle under the aseptic condition, placing the thallus in a newly-prepared NB culture medium for activation, carrying out shading culture at 26 ℃ until the thallus is mature, extracting the DNA of the strain according to an improved CTAB method, and carrying out PCR amplification on the 16S rRNA gene by using 27F and 1492R primers. PCR reaction cycle parameter amplification step: initial denaturation at 1.95 ℃ for 3 min; denaturation at 2.94 ℃ for 40 s; annealing at 3.52 ℃ for 50 s; stretching at 4.72 deg.C for 1 min; 5.2-4 repeating the cycle 35 times; 6.72 ℃, 10min, expand. And (3) sending the PCR product detected by agarose gel electrophoresis to Shanghai's chemical company for sequence determination. The sequence determined from the PCR product was used as a target sequence, the GenBank database in NCBI was searched for homologous sequences, a reference sequence most similar to the morphotype sequence was downloaded, and the phylogenetic status of the strain to be identified was determined using the Neighbor-joining (NJ) method for phylogenetic analysis, as shown in FIGS. 2 and 3. The endophytic bacterial strain is classified and named as Rhodococcus sp 201 with the preservation number of CGMCC No. 22155.

Example 2:

taking a freezing storage tube of the AM201 strain, selecting a proper amount of thallus to an NB culture medium under the aseptic condition, culturing for 48h under the conditions of 26 ℃, darkness and 180rpm until the OD of the bacterial liquid is reached₆₀₀0.6-1.0; making three pathogenic fungi of Atractylodes macrocephala root rot into fungus blocks with a sterile puncher with a diameter of 5mm, respectively inoculating into the center of freshly prepared PDA culture medium, placing 3 equidistant sterile filter paper sheets (with a diameter of 6mm) at 2cm of the center of the culture medium, and sucking 5 μ L of bacterial suspension with a pipette to soak the filter paper sheets. mu.L of NB medium served as negative control and each treatment was repeated 3 times. Placing into a mould incubator, culturing at 26 + -2 deg.C in dark environment for 5-7d, and measuring relative antibacterial rate. The result shows that AM201 endophytic bacteria can effectively inhibit the growth of the pathogenic bacteria of the bighead atractylodes rhizome root rot, namely fusarium oxysporum f.oxysporum, fusarium solani f.incarnatum, and the highest inhibition rate reaches 74.47 +/-0.03%, as shown in figure 5.

Example 3:

taking a freezing storage tube of the AM201 strain, picking a small amount of thalli by using an inoculating needle under the aseptic condition, inoculating the thalli into a sterilized NB liquid culture medium, and carrying out dark culture for 48h under the conditions of 26 ℃, 65% humidity and 180 rpm; further, the bacterial suspension is sucked under aseptic conditions and adjusted to OD₆₀₀And (3) 0.6-1.0, applying the white atractylodes rhizome diseased plant in a root irrigation mode, wherein the application amount is 5 mL/time/3 d, the duration is 21d, the same volume of distilled water is used as a control group, and n is 30. Investigating and counting the number of diseased plants, incidence, injury area and death rate of diseased plants of Atractylodis rhizoma, and performing blood cell countingDetecting the number of pathogenic bacteria, and calculating Disease Index (DI) [ Sigma (disease grade plant number multiplied by representative value)/total plant number multiplied by highest grade representative value]X 100. The bacterial suspension was found to alleviate the root rot disease of atractylodes macrocephala koidz, wherein the Disease Index (DI) of the root rot disease of atractylodes macrocephala koidz is 54.44 ± 8.01, which is respectively reduced by 35.71% compared with the root rot disease model group, as shown in fig. 6. Taking an Atractylodes macrocephala Koidz plant treated by AM201 bacterial suspension, extracting Atractylodes macrocephala Koidz Total RNA by using an SV Total RNA Isolation System kit (Z3105, Promega), purifying the Atractylodes macrocephala Koidz Total RNA by using magnetic beads with poly-T oligonucleotides, separating mRNA with poly-A from the Total RNA, fragmenting the purified mRNA for 8min at 94 ℃, carrying out reverse transcription on the collected mRNA fragments into double-strand cDNA by using Super Script II reverse transcriptase, reacting the purified cDNA for 0.5h under the action of an End Repair Mix enzyme System, purifying by using AMPure XPbeads, immediately carrying out Adapter ligation reaction after the reaction is finished, adding Adapters at two ends of the double-strand DNA fragment, carrying out PCR reaction on the reaction product of the step, and selectively amplifying DNA fragments of which two ends contain Adapters. The specific PCR reaction condition is that firstly, the reaction is carried out for 30s at 98 ℃; then reacting at 98 ℃ for 10s, at 60 ℃ for 30s and at 72 ℃ for 30s, and circulating for 15 times; finally, the reaction is carried out for 5min at 72 ℃. After the reaction is finished, AMPure XPbeads are used for purifying the enriched and amplified DNA fragments, supernatant is absorbed and stored at the temperature of 20 ℃ below zero, the prepared transcriptome library is subjected to quantitative analysis by using the Qubit dsDNA Assay Kit, and the size of the library fragments is analyzed by using the Bioanalyzer 2100DNA-1000 Kit. Quality-qualified transcriptome libraries were sequenced on the Solexa HiSeq 2000 system. The results show that AM201 bacterial suspension intervention can significantly reduce the expression of root rot and bighead atractylodes rhizome genes BXL2, At1g65750 and BZIP43, as shown in FIG. 7.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Sequence listing

<110> Zhejiang university of traditional Chinese medicine

<120> Atractylodes macrocephala endophytic bacterium and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 1377

<212> DNA

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gtcgagcggt aaggcctttc ggggtacacg agcggcgaac gggtgagtaa cacgtgggtg 60

atctgccctg cacttcggga taagcctggg aaactgggtc taataccgga tatgacctcc 120

tatcgcatgg tgggtggtgg aaagatttat cggtgcagga tgggcccgcg gcctatcagc 180

ttgttggtgg ggtaatggcc taccaaggcg acgacgggta gccgacctga gagggtgacc 240

ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 300

gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag ggatgacggc cttcgggttg 360

taaacctctt tcagcaggga cgaagcgcaa gtgacggtac ctgcagaaga agcaccggct 420

aactacgtgc cagcagccgc ggtaatacgt agggtgcaag cgttgtccgg aattactggg 480

cgtaaagagt tcgtaggcgg tttgtcgcgt cgtttgtgaa aaccagcagc tcaactgctg 540

gcttgcaggc gatacgggca gacttgagta ctgcagggga gactggaatt cctggtgtag 600

cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcgggtct ctgggcagta 660

actgacgctg aggaacgaaa gcgtgggtag cgaacaggat tagataccct ggtagtccac 720

gccgtaaacg gtgggcgcta ggtgtgggtt ccttccacgg aatccgtgcc gtagctaacg 780

cattaagcgc cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg 840

ggcccgcaca agcggcggag catgtggatt aattcgatgc aacgcgaaga accttacctg 900

ggtttgacat ataccggaaa gctgcagaga tgtggccccc cttgtggtcg gtatacaggt 960

ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1020

aacccctatc ttatgttgcc agcacgtcat ggtggggact cgtaagagac tgccggggtc 1080

aactcggagg aaggtgggga cgacgtcaag tcatcatgcc ccttatgtcc agggcttcac 1140

acatgctaca atggccagta cagagggctg cgagaccgtg aggtggagcg aatcccttaa 1200

agctggtctc agttcggatc ggggtctgca actcgacccc gtgaagtcgg agtcgctagt 1260

aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1320

acgtcatgaa agtcggtaac acccgaagcc ggtggcttaa ccccttgtgg gagggag 1377

Claims

1. An Atractylodes macrocephala endophytic bacterium, which is obtained by separating Atractylodes macrocephala of Atractylodes of Compositae, and is classified and named as Rhodococcus (R) of ActinomycetesRhodococcussp.) AM201 with the preservation number of CGMCC No. 22155.

2. An application of the atractylis ovata endophytic bacterium of claim 1 in preventing and treating atractylis ovata root rot is characterized in that the application is as follows: the bacterial suspension prepared by endophytic bacterium AM201 is applied to the bighead atractylodes rhizome root rot disease strain in a root irrigation manner, so that the resistance of the bighead atractylodes rhizome to the root rot is enhanced.

3. The use according to claim 2, wherein endophytic bacterium AM201 inhibits the pathogenic bacterium Fusarium oxysporum (F.oxysporum) of the root rot disease of Atractylodes macrocephalaFusarium oxysporum) Fusarium solani (F.sp.), (Fusarium solani) With Fusarium semitectum (F.), (Fusarium incarnatum) The growth of (2).

4. The use according to claim 3, wherein the suspension of AM201 is prepared by the following steps: (1) selecting the endophytic bacteria strain, and selecting a small amount of bacteria by using an inoculating needle under the aseptic conditionInoculating the body into sterilized NB liquid culture medium, and dark culturing at 26 deg.C and 65% humidity under 180rpm for 48 h; (2) sucking the bacterial suspension under aseptic condition, and adjusting the concentration of the bacterial suspension to OD₆₀₀And (5) obtaining the product, wherein the = 0.6-1.0.

5. The use of claim 4, wherein the formulation of NB liquid medium is: 10g of peptone, 3g of beef powder, 5g of sodium chloride, 1000mL of distilled water, pH 7.2. + -. 0.2.

6. The use according to claim 4, characterized in that the amount applied is 5 mL/dose/3 d for 21 d.

7. The use of claim 2, wherein the endophytic bacteria inhibit the growth of pathogenic bacteria hyphae of the white atractylodes rhizome by a plate confronting method, and the inhibition rate is calculated according to the formula: the bacteriostasis rate = (the growth radius of pathogenic bacteria of a control group-the growth radius of pathogenic bacteria of a confronting group)/the growth radius of pathogenic bacteria of the control group multiplied by 100 percent.

8. The use as claimed in claim 2, wherein the enhanced resistance of Atractylodis rhizoma is manifested by a decrease in the phenotypic disease index of root rot and a down-regulation of the expression of the genes BXL2, At1g65750 and BZIP 43.

9. The use according to claim 8, wherein the Disease Index (DI) is calculated according to the formula DI = [ Σ (number of diseased plants x number of representative) per total number of plants and x number of highest degree of disease ] × 100.