CN118047848A - Pear anthracnose ribotoxin CfRibo1, and coding gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and provides a pear anthracnose pathogen ribotoxin CfRibo1, a coding gene and application thereof, wherein ribotoxin CfRibo1 is protein with an amino acid sequence shown in SEQ ID NO. 1; the inoculation mode simulating the diversity of microorganisms in the nature proves that the ribotoxin CfRibo1 provided by the invention is of great importance to the pathogenicity of the pear anthracnose, and the knockout mutant obtained by utilizing PEG-mediated genetic transformation almost loses the pathogenicity to the host when being inoculated with the microorganisms separated from the periphery of the host leaves, meanwhile, cfRibo1 is a key factor for infection and pathogenicity of the pear anthracnose in the natural environment, can be used as a molecular target for preventing and treating the pear anthracnose, and has important application value.

Description

Pear anthracnose ribotoxin CfRibo1, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a pear anthracnose pathogen ribotoxin CfRibo1, and a coding gene and application thereof.

Background

China is the world-wide largest pear production and consumption country, and the pear industry has become the pillar industry for local economic development and income increasing and enrichment of peasants in China. However, pear anthracnose caused by pear anthracnose pathogen is one of the major diseases on pears, and causes major loss of pear yield in China every year, and has become a main factor for restricting the healthy development of pear industry in China. After 2007, pear anthracnose rapidly spreads in pear planting areas of yellow river channels in China, and damages fruit diseases to cause large-area rot of pears, so that huge economic losses are caused for a plurality of pear varieties such as Dangshan pear, huangguan pear, pear and the like. After 2009, pear anthracnose gradually bursts in the pear producing area in south China, and leaves can be damaged to cause abnormal breakfast of leaves, so that the pear yield in Zhejiang, anhui, hubei, jiangsu, sichuan provinces is reduced sharply;

The pathogenic factors of the pear anthracnose are unknown, and the pathogenic mechanism is unclear, so that the pear anthracnose is a main reason that the pear anthracnose is difficult to effectively control. Numerous studies have shown that effectors (effectors) secreted by pathogenic bacteria play a critical role in pathogenic processes of pathogens. The genome sequence of the colletotrichum pyriform is published in 2012 at the earliest, the predicted codes of the genome sequence are more than 700 effectors, and a plurality of effector genes are highly expressed in the interaction process of the germ and a host, but only few effector genes are reported so far, and the reports of pathogenic key effector genes are less. Therefore, the germ effector is systematically excavated, a key pathogenic factor is found, a scientific basis can be provided for a pathogenic mechanism of the germ effector, and the germ effector has important guiding significance for the scientific prevention and control of pear anthracnose in the future.

Therefore, a pear anthracnose pathogen ribotoxin CfRibo1, and a coding gene and application thereof are provided by the person skilled in the art to solve the problems provided by the background technology.

Disclosure of Invention

In order to solve the technical problems, the invention provides a pear anthracnose pathogen ribotoxin CfRibo1, and a coding gene and application thereof, so as to solve the problems in the prior art.

A pear anthracnose pathogen ribotoxin CfRibo1, wherein ribotoxin CfRibo1 is a protein with an amino acid sequence shown in SEQ ID NO.1, and the protein can be (A1), (A2), (A3) or (A4); (A1) is a protein shown as SEQ ID NO. 1; (A2) A fusion protein obtained by ligating a tag to the N-terminus or C-terminus of the protein (A1); (A3) A protein derived from the protein shown in (A1) or (A2) which is obtained by substituting one or more amino acid residues, is derived from the colletotrichum pyriformis and is related to the protein activity shown in SEQ ID NO. 1; (A4) A sequence which has 80% identity or more with the amino acid sequence of the protein shown in SEQ ID NO.1 and has a similar function to the amino acid sequence shown in SEQ ID NO. 1;

Specifically, the protein of (A3) wherein the substitution of one or more amino acid residues is a substitution of not more than 4 amino acid residues; the protein in the (A3) can be synthesized artificially or can be obtained by synthesizing the coding gene and then performing biological expression; the coding gene of the protein in (A3) can be obtained by replacing one or more codons of amino acid residues with the DNA sequence shown in SEQ ID NO.2 and connecting the tag coding sequence shown in the table 1 at the 5 'end or the 3' end.

Preferably, the SEQ ID NO.1 consists of 176 amino acid residues, and in order to facilitate purification of the protein of (A1), the tag shown in Table 1 above may be attached at the amino-or carboxy-terminus of the protein shown in SEQ ID NO. 1:

TABLE 1 sequence of tags

A coding gene of pear anthracnose germ ribotoxin CfRibo1, which codes for pear anthracnose germ ribotoxin CfRibo1 according to claims 1-2.

Preferably, the coding of the pear anthracnose pathogen ribotoxin CfRibo is shown as SEQ ID NO. 2.

Preferably, the SEQ ID NO.2 consists of 531 nucleotides, and SEQ ID NO.2 encodes a protein (CfRibo 1) shown in SEQ ID NO. 2;

Preferably, the nucleic acid molecule encoding the protein CfRibo1 may be a DNA molecule shown as (B1) or (B2) or (B3) or (B4): (B1) a DNA molecule with a coding region shown in SEQ ID NO. 2; (B1) a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 2; (B3) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) or (B1) and which codes for CfRibo protein; (B4) A DNA molecule derived from Pyricularia pyrifolia and having 75% or more identity with the DNA molecule defined in (B1) or (B2) and encoding CfRibo.sup.1 protein;

Wherein the DNA molecule may be DNA such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the protein CfRibo of the present invention may be easily mutated by one of ordinary skill in the art using known methods, such as site-directed mutagenesis; those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein CfRibo1 isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the protein CfRibo1 is encoded.

The term "identity" as used herein refers to sequence similarity of natural nucleotide sequences. "identity" includes a nucleotide sequence having 75% or more of the nucleotide sequence of the protein (CfRibo 1) consisting of the amino acid sequence shown in SEQ ID NO.1 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

Preferably, providing said nucleotide molecule-related biological material, including recombinant vectors and recombinant bacteria; the recombinant vector is a recombinant vector pColdTF-CfRibo; the recombinant strain can be obtained by introducing a recombinant vector of the nucleic acid molecule into a starting microorganism.

Preferably, the recombinant vector containing any of the above nucleic acid molecules may be a recombinant plasmid obtained by inserting a DNA molecule shown in SEQ ID No.2 into a multiple cloning site of an expression vector; the recombinant vector pCold TF-CfRibo can be a recombinant plasmid obtained by replacing a small DNA fragment between restriction enzymes NdeI and BamHI of the vector pCold TF with a DNA molecule shown in SEQ ID NO. 2; the starting microorganism is bacteria or fungi; the bacteria are specifically escherichia coli BL21 (DE 3);

Preferably, the recombinant microorganism containing any one of the above nucleic acid molecules may specifically be BL21 (DE 3)/pCold TF-CfRibo1; the BL21 (DE 3)/pCold TF-CfRibo1 may be a recombinant E.coli obtained by introducing the recombinant plasmid pCold TF-CfRibo1 into E.coli BL21 (DE 3).

Preferably, the application of the pear anthracnose ribotoxin CfRibo1 or the coding gene thereof is any one or more of the following:

D1 Use in modulating ribonuclease activity;

D2 Use in modulating antimicrobial activity;

D3 Application in regulating and controlling the pathogenicity of pear anthracnose germs;

d4 To inhibit and/or kill colletotrichum pyritinoides.

Preferably, said use comprises the use of D1) to D4) by inhibiting transcription or inactivating the coding gene of SEQ ID NO.1, or inhibiting translation of said RNA molecule, or inhibiting and/or inactivating the activity of the CfRibo protein of SEQ ID NO. 1.

Preferably, in the application, the pathogenicity of the pear anthracnose pathogen is regulated by inhibiting the transcription of the coding gene or the translation of the RNA molecule or inhibiting and/or inactivating the activity of CfRibo protein, so that the pear anthracnose pathogen can be inhibited and/or killed.

A method for reducing pathogenicity of colletotrichum pyritinoides, comprising the steps of: inhibiting transcription of the coding gene as described above or inhibiting translation of the RNA molecule as described above, or inhibiting and/or inactivating the activity of the CfRibo protein as described above.

Preferably, the reduction of the pathogenicity of the colletotrichum pyricola is to reduce the infectivity of the colletotrichum pyricola to a host and/or the pathogenicity of the colletotrichum pyricola to the host, the inactivation of the protein is realized by the inhibition or the reduction of the expression of a coding gene of the protein to be inhibited or inactivated, the expression of the coding gene is gene knockout, and the gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination; gene knockout is the inactivation of a particular target gene by a change in DNA sequence;

The method for knocking out the gene is polyethylene glycol (PEG) -mediated genetic transformation, specifically, a target gene knockout box is transferred into pear anthracnose bacteria to screen and obtain knocking out inactivated recombinant bacteria of the target gene, and the target gene knockout box is a recombinant nucleic acid fragment containing a sequence 600-800bp upstream of the target gene to be knocked out, a resistance screening fragment (which can be neomycin phosphotransferase gene (neo) or hygromycin B phosphotransferase gene (hph)) and a sequence 600-800bp downstream of the target gene to be knocked out which are connected in sequence;

the application of the substances for inhibiting CfRibo protein expression and/or activity in preparing the pear anthracnose bactericide/bacteriostat also belongs to the protection scope of the invention.

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

The CfRibo A provided by the invention has antibacterial activity, is a resolution factor of germ occupying microbial ecological niches, plays a key role in the pathogenic process of pear anthracnose infection, almost loses pathogenicity to a host when being inoculated with microorganisms separated from the host girth by utilizing PEG-mediated genetic transformation, and CfRibo A is a key factor of pear anthracnose infection and pathogenicity in natural environment, can be used as a molecular target for pear anthracnose prevention and control, and has important application value.

Drawings

FIG. 1 is a schematic structural diagram of the CfRibo protein sequence of the present invention;

FIG. 2 is a diagram showing the relative expression levels of CfRibo gene of the present invention at different infection time points of Pyricularia pyrifolia;

FIG. 3 is a schematic diagram showing the detection of CfRibo protein and prokaryotic expression according to the present invention;

FIG. 4 is a schematic diagram showing the detection of ribonuclease activity of CfRibo protein of the invention;

FIG. 5 is a schematic diagram of the vegetative growth phenotype of the CfRibo gene knockout mutant of the invention on PDA medium;

FIG. 6 is a schematic diagram showing the pathogenicity analysis of CfRibo A gene knockout mutant according to the present invention.

Detailed Description

Embodiments of the present invention are described in further detail below with reference to the accompanying drawings and examples. The following examples are illustrative of the present invention, but are not intended to limit the scope of the present invention, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Embodiment one: cloning of pear anthracnose ribotoxin CfRibo1 coding gene

(1) The protein CfRibo and the coding gene (or cDNA) thereof of the pear anthracnose germ in the embodiment can be obtained by taking cDNA of a pear anthracnose germ strain DSCF-02 stored in the laboratory as a template and amplifying a primer consisting of 5'-ATCGAAGGTAGGCATATGAACCCCATCGCCCTCGAAAAG-3' (SEQ ID NO. 3) and 5'-AAGCTTGAATTCGGATCCTTAAGCGGTAAGGGCAAGCAG-3' (SEQ ID NO. 4) to obtain a PCR amplification product of about 531 bp;

(2) Sequencing the PCR amplification product; sequencing results show that the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 2; the structure of CfRibo protein sequence corresponding to SEQ ID NO.2 nucleotide is shown in figure 1.

Embodiment two: analysis of expression pattern of CfRibo coding genes in infection of hosts

(1) Inoculating pear fruit with pear anthracnose germ conidium, sampling after 0h,12h,24h,36h and 48h respectively, extracting total RNA of the sample with Vazyme company plant total RNA extraction kit, and detecting RNA content and quality with a spectroradiometer;

(2) Taking 1 mug RNA as a template, carrying out cDNA synthesis according to the usage instructions of Vazyme company HISCRIPT II reverse transcription kit, and taking a proper amount of reverse transcription product for subsequent real-time quantitative PCR reaction;

(3) Primers for detecting CfRibo gene are 5'-CTATGCCAACAACCAGCAG-3' (SEQ ID NO. 5) and 5'-AGACGAGATGATGGGGAAC-3' (SEQ ID NO. 6), and primers for detecting CfActin gene are 5'-ATCAACCCCAAGTCCAACAG-3' (SEQ ID NO. 7) and 5'-CGATTTCACGCTCGGCAGT-3' (SEQ ID NO. 8); the detection result is shown in figure 2; the results show that CfRibo gene up-regulates expression during infection of the host.

Embodiment III: cfRibo1 recombinant protein expression and purification

(1) The pCold TF vector was digested with NdeI and BamHI endonucleases to obtain a linearized pCold TF vector, and the PCR amplified product of example 1 was ligated to the linearized pCold TF vector using ClonExpress one-step cloning kit from Vazyme to obtain a recombinant plasmid pCold TF-CfRibo 1;

(2) Transferring pCold TF-CfRibo1 recombinant plasmid into escherichia coli BL21 (DE 3) to be competent through a heat shock method, screening by using ampicillin, selecting a monoclonal to an LB culture medium for culturing, and performing expansion culture in 100mL of LB according to the proportion (1:100) until OD 600=0.6; then IPTG was added at a final concentration of 0.3mM for induction expression (16 ℃ C., 200rpm for 24 hours); suspending the cultured strain in lysis buffer (20mM Na2HPO4, 300mM NaCl,pH7.4,1mg/mL lysozyme,1mM PMSF,1.98mM mercaptoethanol), ultrasonic crushing for 15min, centrifuging to obtain supernatant which is CfRibo recombinant protein crude protein;

(3) Filling the chromatographic column with a proper amount of Ni-NTA (Thermo Scientific) resin; according to the instructions, cfRibo1 recombinant protein crude protein was slowly added to the column, cfRibo1 recombinant protein was eluted with twice the resin bed volume of elution buffer (250 mM imidazole in PBS, pH 7.4) and dialyzed with PBS (20 mM sodium phosphate, 300mM sodium chloride; pH 7.4) buffer; the CfRibo recombinant protein obtained is shown in FIG. 3.

Embodiment four: cfRibo1 analysis of recombinant protein ribonuclease Activity

(1) Extracting and obtaining total RNA of pear fruits and total RNA of pear anthracnose germs respectively by the method described in the embodiment 2;

(2) Mixing appropriate amount of total RNA of fructus Pyri fruit and total RNA of fructus Pyri anthracnose pathogen with 1 μm CfRibo recombinant protein, incubating at 37deg.C for 30min, and detecting by gel electrophoresis; the detection result is shown in fig. 4; the result shows that CfRibo1 recombinant protein can effectively degrade total RNA of pear fruits and total RNA of pear anthracnose bacteria, and CfRibo is proved to have ribonuclease activity;

Fifth embodiment: growth phenotype of CfRibo A1 knockout mutant on PDA Medium

(1) Extracting and obtaining genome DNA of the strain DSCF-02 by using FastPure Cell/Tissue Total RNA Isolation Kit kit of Vazyme company according to operation instruction; amplifying a primer consisting of 5'-GTGAGCTCGGTACCGGATCCTGTGGCTTTGATTTGCTGA-3' (SEQ ID NO. 9) and 5'-ACTAGCTCCAGCCAAGCCGTTGCTGTAACGGATTTGAGA-3' (SEQ ID NO. 10) to obtain a fragment of 663bp at the upstream of the CfRibo gene, amplifying a primer consisting of 5 'AGATGCCGACCGGGTTAGGGAGTAATAATGGTGGC-3' (SEQ ID NO. 11) and 5'-TGAGTAAGGTTACCGAATTCCGTTGATGTCAAGCGTTG-3' (SEQ ID NO. 12) to obtain a fragment of 673bp at the downstream of the CfRibo gene, and fusing the two fragments with the hph gene fragment to obtain a CfRibo gene knockout box;

(2) Transferring CfRibo gene knockout boxes into protoplasts of colletotrichum pyricum through PEG mediated genetic transformation, and obtaining CfRibo gene knockout transformants through hygromycin B as resistance screening;

(3) Culturing wild strains DSCF-02 and CfRibo gene knockout mutants of the pear anthracnose pathogen in a PDA culture medium (28 ℃), observing the shape of a colony after 3d, counting the radius of the colony and photographing; the results of vegetative growth are shown in FIG. 5; the results showed that CfRibo gene knockout mutant showed no significant differences in growth phenotype compared to wild type DSCF-02.

Example six: pathogenicity analysis of CfRibo gene knockout mutant on pear fruit

(1) Hyphae of wild-type strains DSCF-02 and CfRibo gene knockout mutants of colletotrichum pyriform are placed in a PDB culture medium to be cultured for 4 days (28 ℃ C., 180 rpm), and a large amount of conidia are formed in the culture solution through microscopic observation; filtering the culture solution by using Miracloth filter cloth, and centrifuging the filtrate to obtain conidium of germ;

(2) Sampling pear leaves from an Anhui Dangshan county orchard pear, and separating and purifying the pear leaves to obtain bacteria around the leaves; is a common separation method for plant pathology research;

(3) Conidia were diluted with sterile water to the appropriate concentration (1 x 106 conidia/mL), and periphyllal bacteria concentration was diluted with sterile water to od600=0.2; the diluted conidia of the DSCF-02 and CfRibo gene knockout mutants are respectively mixed with diluted periphyllum bacteria and inoculated on pear fruits; the pear fruit variety is Dangshan pear; the pathogenicity analysis results are shown in FIG. 6; the results showed that CfRibo gene knockout mutant had almost lost pathogenicity compared to wild-type DSCF-02; this suggests CfRibo that it is a key causative agent of the species B.pyriformis.

From the above, the pear anthracnose ribose toxin CfRibo1 provided by the invention has antibacterial activity, is important for the pear anthracnose to occupy the microbial ecological niche and infect a host, can be used as a molecular target in pear anthracnose prevention and control, and has important application prospects in the development of novel bactericide/bacteriostat.

The sequence is described below

Amino acid sequence of CfRibo protein: SEQ ID NO.1

MVSLKSTLAVALCAVLAAANPIALEKRQGTTGLGSIRCGDAKYSRKQVDEAVAEGCRLYANNQQVGTSEYPHRFNNREGLTFDTSGPYQEFPIISSGNYTGRAPGPDRVVFDPDYRGSCVFVGAMTHTGAVQRNGFVSCNESSSSSGSGSSAASSITTSGSFGVLLPVLSLLALTA;

Nucleotide sequence of CfRibo gene: SEQ ID NO.2

ATGGTCAGCCTCAAATCCACCCTCGCCGTGGCCCTCTGCGCCGTCCTCGCCGCCGCCAACCCCATCGCCCTCGAAAAGCGCCAGGGCACCACCGGCCTCGGCTCCATCCGCTGCGGCGACGCAAAGTACTCCCGCAAGCAGGTCGACGAGGCCGTCGCCGAGGGCTGCCGCCTCTATGCCAACAACCAGCAGGTCGGCACCAGCGAGTACCCCCACCGCTTCAACAACCGCGAGGGGCTCACCTTCGACACCTCCGGCCCCTACCAAGAGTTCCCCATCATCTCGTCTGGAAACTACACTGGCAGGGCACCCGGCCCCGACCGTGTGGTCTTCGACCCCGACTACCGGGGCAGCTGCGTCTTTGTCGGCGCAATGACCCACACCGGCGCCGTCCAGCGCAACGGCTTTGTCTCGTGCAATGAGAGCTCTTCAAGCAGCGGCAGTGGTAGCTCGGCGGCCTCGAGTATCACTACCTCAGGCTCATTTGGGGTCCTACTGCCTGTGCTGTCTCTGCTTGCCCTTACCGCTTAA;

PCR primer amplification sequences in example 1:

SEQ ID NO.3

ATCGAAGGTAGGCATATGAACCCCATCGCCCTCGAAAAG；

SEQ ID NO.4

AAGCTTGAATTCGGATCCTTAAGCGGTAAGGGCAAGCAG；

PCR primer amplification sequences in example 2:

SEQ ID NO.5

CTATGCCAACAACCAGCAG；

SEQ ID NO.6

AGACGAGATGATGGGGAAC；

SEQ ID NO.7

ATCAACCCCAAGTCCAACAG；

SEQ ID NO.8

CGATTTCACGCTCGGCAGT；

PCR primer amplification sequences in example 5:

SEQ ID NO.9

GTGAGCTCGGTACCGGATCCTGTGGCTTTGATTTGCTGA；

SEQ ID NO.10

ACTAGCTCCAGCCAAGCCGTTGCTGTAACGGATTTGAGA；

SEQ ID NO.11

AGATGCCGACCGCGGGTTAGGGAGTAATAATGGTGGC；

SEQ ID NO.12

TGAGTAAGGTTACCGAATTCCGTTGATGTCAAGCGTTG。

Reference is made to:

Methods for genetic transformation of anthrax bacteria are disclosed in literature "Liu W,Liang X,Gleason M L,et al.Transcription factor CfSte12 of Colletotrichum fructicola is a key regulator of early apple Glomerella leaf spot pathogenesis.Applied andenvironmental microbiology,2020,87(1):e02212-20.".

The method of inoculating pathogens is disclosed in document "Shang S,Wang B,Zhang S,et al.A novel effector CfEC92 of Colletotrichum fructicola contributes to glomerella leaf spot virulence by suppressing plant defences at the early infection phase.Molecular plant pathology,2020,21(7):936-950.".

While embodiments of the present invention have been shown and described above for purposes of illustration and description, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. A pear anthracnose germ ribotoxin CfRibo1, characterized in that: the ribotoxin CfRibo is protein with an amino acid sequence shown as SEQ ID NO. 1.

2. A pear anthracnose pathogen ribotoxin CfRibo1 according to claim 1, wherein: the SEQ ID NO.1 consists of 176 amino acid residues.

3. The coding gene of the pear anthracnose pathogen ribotoxin CfRibo1 is characterized in that: the coding gene codes for the pear anthracnose germ ribotoxin CfRibo1 according to claims 1-2.

4. The coding gene of claim 3, wherein: the coding of the pear anthracnose pathogen ribotoxin CfRibo is shown as SEQ ID NO. 2.

5. The coding gene of claim 4, wherein: the SEQ ID NO.2 consists of 531 nucleotides, and the SEQ ID NO.2 codes for a protein shown as SEQ ID NO. 2.

6. The coding gene of claim 5, wherein: providing said nucleotide molecule related biological material, including recombinant vectors and recombinant bacteria; the recombinant vector is a recombinant vector pColdTF-CfRibo; the recombinant strain can be obtained by introducing a recombinant vector of the nucleic acid molecule into a starting microorganism.

7. An application of pear anthracnose germ ribotoxin CfRibo1 or a coding gene thereof is characterized in that: the application is any one or more of the following:

D1 Use in modulating ribonuclease activity;

D2 Use in modulating antimicrobial activity;

D3 Application in regulating and controlling the pathogenicity of pear anthracnose germs;

d4 To inhibit and/or kill colletotrichum pyritinoides.

8. The use according to claim 7, wherein: the application comprises the application of D1) -D4) by inhibiting transcription or inactivating the coding gene of SEQ ID NO.1, or inhibiting translation of the RNA molecule, or inhibiting and/or inactivating the activity of CfRibo protein of SEQ ID NO. 1.

9. A method of reducing the pathogenicity of colletotrichum pyritinoides as defined in any one of claims 7-8, wherein: comprising the following steps: inhibiting transcription of the coding gene or inhibiting translation of the RNA molecule, or inhibiting and/or inactivating the activity of the CfRibo protein.

10. A method of reducing the virulence of pear anthracnose pathogen as in claim 9, wherein: the reduction of the pathogenicity of the colletotrichum pyritinoides is to reduce the infectivity and/or pathogenicity of the colletotrichum pyritinoides on a host, the inactivation of the protein is realized by the inhibition or reduction of the activity to be inhibited or the expression of a coding gene of the protein to be inactivated, and the expression of the coding gene is gene knockout.