Fungal virus mutant as well as construction method and application thereof

Abstract

The invention discloses a fungal virus mutant as well as a construction method and application thereof, and belongs to the technical field of fungal virus modification. The fungal virus mutant is obtained by point mutation of an initiation codon ATG for coding a protein p18 in an infectious clone pSK-ABC into ACG, a coding gene of the protein p18 is located in an open reading frame of a component C at the 3533rd-5463rd sites of a complete sequence of the infectious clone pSK-ABC, and a nucleotide sequence of the infectious clone of the mutant is shown as SEQ ID NO: 1. The phenotype of the mutant virus infected strain is observed to find that the Mp18 mutant virus can reduce the growth speed of host fungi, and causes the characteristics of abnormal host colonial morphology, conidium yield and pathogenicity reduction and the like; besides, when the attenuated strain induced by the Mp18 mutant and the virulent strain are co-inoculated, scab extension on wheat leaves can be effectively inhibited, so that the effect of preventing and controlling the fusarium graminearum virulent strain is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of fungal virus modification, and in particular to a fungal virus mutant and a construction method and application thereof. Background Art

[0002] Fungal viruses are viruses that replicate and proliferate in fungi and are widely present in fungi. 70% to 80% of plant diseases in agricultural production are caused by pathogenic fungi. Fungal viruses can reduce the pathogenicity of plant pathogenic fungi and convert them into endophytic fungi to promote plant growth and disease resistance. Therefore, fungal viruses are an important type of biocontrol resource. Fungal viruses are widely present in major fungal groups. According to their genome composition and replication mode, fungal viruses can be divided into five types: double-stranded RNA (dsRNA) viruses, positive single-stranded RNA (+ssRNA) viruses, negative single-stranded RNA (-ssRNA) viruses, reverse transcriptase single-stranded RNA (RT ssRNA) viruses, and single-stranded DNA (ssDNA) viruses.

[0003] Fusarium graminearum gemytripvirus 1 (FgGMTV1) is a multi-component single-stranded circular DNA virus, containing three single-stranded circular DNA fragments: DNA-A, DNA-B and DNA-C, with molecular sizes of 1316nt, 1320nt and 1309nt, respectively, encoding replication initiation protein (Rep), coat protein (CP) and unknown functional protein. The inventor team has successfully constructed the infectious clone of FgGMTV1 and clarified the relationship between the various components: DNA-A and DNA-B are necessary for virus replication and infection, and the joint infection of the two can significantly inhibit the growth, spore production and pathogenicity of the host fungus, but the virus is unstable and will not be vertically transmitted through conidia; the replication and proliferation of DNA-C depends on DNA-A and DNA-B. The virus containing three components is stable and can be transmitted through conidia, but it does not affect the phenotype and pathogenicity of the host fungus. In addition, by deletion modification of the DNA-C component, the VIGS system p26-D4 based on the fungal virus FgGMTV1 was successfully constructed. By inserting gene fragments related to pathogenicity or toxin synthesis of Fusarium graminearum, the system can obtain stable attenuated strains and effectively prevent and control wheat fusarium wilt.

[0004] Fungal diseases account for more than 70% to 80% of plant diseases, seriously reducing the yield and quality of crops. In the past 50 years, the prevention and control of fungal diseases of crops has mainly relied on chemical pesticides, and the excessive use of chemical pesticides threatens food safety, leading to environmental pollution and increased resistance of pathogens. Wheat head blight (Fusarium head blight, FHB) is a global disease caused mainly by various Fusarium species such as Fusarium graminearum. At present, the prevention and control of wheat head blight mainly relies on chemical control and agricultural management. Fungal viruses can inhibit the spread of pathogens, do not cause harm to plants, and have no pesticide residues. They have the advantages of being green, safe, and sustainable. The use of fungal viruses for biological control of plant fungal diseases is considered to be a highly promising prevention and control strategy. At present, the application of fungal viruses is mainly limited by the insufficient resource mining of fungal viruses. The vast majority of identified fungal viruses are asymptomatic infections, that is, they have no effect on the phenotype and pathogenicity of host pathogenic fungi. For example, when the three components of Fusarium graminearum virus FgGMTV1 co-infect, it shows asymptomatic infection, which does not affect the phenotype and pathogenicity of the host Fusarium graminearum. Therefore, through the molecular modification of the start codon of the fungal virus encoding protein, fungal virus mutants that weaken the pathogenicity of the host fungus can be mined, the application of existing fungal viruses can be expanded, and more available fungal virus resources can be provided for the prevention and control of fungal diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a fungal virus mutant and a construction method and application thereof to solve the problems existing in the above-mentioned prior art. The mutant virus infection can effectively reduce the growth rate of the host fungal strain, cause abnormal host colony morphology, reduce conidia production and pathogenicity, etc., and can induce the host's weak toxic characteristics, thereby achieving the purpose of effectively preventing and controlling the highly toxic strains of wheat fusarium.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a fungal virus mutant, which is obtained by point mutation of the start codon ATG encoding protein p18 in an infectious clone pSK-ABC to ACG, wherein the encoding gene of the protein p18 is located in the open reading frame of component C at positions 3533-5463 of the full sequence of the infectious clone pSK-ABC.

[0008] Preferably, the nucleotide sequence of the fungal virus mutant infectious clone obtained after point mutation is as shown in SEQ ID NO: 1, wherein the point mutation method is to mutate the 3792th position of the infectious clone pSK-ABC full sequence from A to G, and the 5101st position from A to G.

[0009] The present invention also provides a method for constructing a fungal virus mutant, comprising the step of point mutating the start codon ATG encoding protein p18 in the infectious clone pSK-ABC to ACG; the encoding gene of the protein p18 is located in the open reading frame of component C at positions 3533-5463 of the full sequence of the infectious clone pSK-ABC.

[0010] Preferably, the construction method comprises the following steps: based on the complete sequence of the infectious clone pSK-ABC, mutating position 3792 of the complete sequence of the infectious clone pSK-ABC from A to G and mutating position 5101 from A to G.

[0011] The invention also provides an application of the fungal virus mutant in preventing and controlling wheat fusarium head blight.

[0012] Preferably, the fungal virus mutant achieves the purpose of preventing and controlling wheat fusarium head blight by reducing the pathogenicity of the strong strain that causes wheat fusarium head blight.

[0013] Preferably, the virulent strain causing wheat fusarium rust includes Fusarium graminearum.

[0014] The present invention also provides an application of the fungal virus mutant in any of the following items:

[0015] (1) Reduce the growth rate of host strains;

[0016] (2) reduce the conidia production of the host strain;

[0017] (3) reduce the pathogenicity of host strains;

[0018] (4) Promoting abnormal morphology of host strains;

[0019] (5) Promoting the delay of the extension of lesions on wheat leaves;

[0020] The host strains include Fusarium graminearum, which causes wheat fusarium rust.

[0021] The present invention also provides a method for preventing and controlling wheat fusarium wilt, comprising the steps of using the fungal virus mutant to transfect wild-type Fusarium graminearum to construct a weak strain, and then treating wheat with the weak strain.

[0022] Preferably, the fungal virus mutants in the weak strains are horizontally transmitted to the strong strains that cause wheat fusarium rust, thereby reducing the pathogenicity of the strong strains to achieve the purpose of preventing and controlling wheat fusarium rust; the strong strains that cause wheat fusarium rust include Fusarium graminearum.

[0023] The present invention discloses the following technical effects:

[0024] The present invention transforms component C in the infectious clone pSK-ABC, mutates the 3792nt of component C from A to G, and mutates the 5101nt from A to G (see SEQ ID NO: 1 for details), thereby obtaining a mutant Mp18. Experiments have shown that the Mp18 mutant virus can reduce the growth rate of the host fungus, cause abnormal host colony morphology, reduce conidia yield and pathogenicity, and other characteristics. Further, when the attenuated strain induced by the Mp18 mutant is co-inoculated with the virulent strain PH-1 in wheat, it is found that the attenuated strain induced by the Mp18 mutant effectively reduces the extension of the lesions, thereby achieving the effect of preventing and controlling the virulent strain of wheat fusarium. The present invention expands the types of existing fungal viruses and provides more available fungal virus resources for the prevention and control of fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 Identification of proteins encoded by DNA-C component: A: Genomic structure and encoding ORF of DNA-C component, predicted ORFs are indicated by arrows; B: RT-PCR analysis of transcription of p9, p18 and p26 in strains infected with pSK-ABC, where M: DNA marker; RNA: negative control (direct PCR amplification using completely digested total RNA as template); cDNA: cDNA / 18S: positive control (PCR amplification of EF-1α fragment after reverse transcription using random primers); cDNA / p9, p18, p26: PCR amplification of corresponding fragments after reverse transcription using specific primers for p9, p18 and p26, respectively; C: Promoter activity analysis of p18 and p26, fluorescence observation after culturing strains p18(500)-GFP and p26(500)-GFP for 2 days, PH-1 (EV, control) as negative control, RP27-GFP as positive control; Scale bar: 250μm;

[0027] Figure 2 Schematic diagram of p9, p18 and p26 point mutants;

[0028] Figure 3 Southern blot hybridization results of DNA extracted from strains infected with virus mutants Mp9, Mp18, Mp26, Mp9.18, Mp9.26, Mp18.26 and Mp9.18.26;

[0029] Figure 4 The phenotypic identification results of virus mutants Mp9, Mp18, Mp26, Mp9.18, Mp9.26, Mp18.26 and Mp9.18.26 infecting strains; A: Comparison of colony morphology of mutant infecting strains; B: Comparison of growth rates of mutant infecting strains; C: Comparison of colony diameters of mutant infecting strains; D: Conidia yield of mutant infecting strains; E: Pathogenicity detection of mutant infecting strains; F: Statistical results of the number of diseased spikelets per wheat ear;

[0030] Figure 5 Results of biological control analysis of the attenuated strain induced by mutant Mp18: A: Schematic diagram of the experiment on controlling the virulent strain of wheat Gibberella with the attenuated strain induced by mutant Mp18; B: Statistical results of the disease incidence and lesion size of wheat leaves; C: morphological observation of strains isolated from diseased wheat leaves; D: Detection results of the toxicity of the isolated strains. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The single-stranded DNA virus FgGMTV1 of Fusarium graminearum used in the embodiment of the present invention was isolated and identified by this laboratory, see LIP. (2020). A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious as cloned DNA and purified virions. Science Advances 6, eaay9634. The infectious clone pSK-ABC used was optimized and transformed by this laboratory, see Zhang LH. (2023). A Mycovirus VIGS Vector Confers Hypovirulence to a Plant Pathogenic Fungus to Control Wheat FHB. Advanced Science. The wild-type Fusarium graminearum strain PH-1 used was preserved by this laboratory.

[0037] The present invention first analyzes the possibility of the protein encoded by the DNA-C component of the Fusarium graminearum single-stranded DNA virus FgGMTV1. The specific strategy is: predicting the ORF encoded by the DNA-C component, and confirming that the DNA-C component encodes p9, p18 and p26 proteins by analyzing the promoter activity and transcription activity of the predicted ORF. Figure 1 Then, by making point mutations in the start codons of the proteins p9, p18 and p26 encoded by the DNA-C component (see Figure 2 ) to verify the replication of mutant viruses (see Figure 3 ). Furthermore, by observing and measuring the phenotype of the strain infected by the mutant virus, it was found that the p18 mutant virus can reduce the growth rate of the host fungus, cause abnormal host colony morphology, reduce conidia production and pathogenicity (see Figure 4 ). Furthermore, the attenuated strain induced by the Mp18 virus mutant was used as a biological control factor, and its control effect on the virulent strain of wheat fusarium was investigated by co-inoculation. The results showed that when the attenuated strain induced by Mp18 was co-inoculated with the virulent strain, the extension of the lesions could be effectively reduced, thereby achieving the effect of controlling the virulent strain of wheat fusarium (see Figure 5 ). The above specific strategies are further described below with specific embodiments.

[0038] Example 1 Identification of the protein encoded by DNA-C component

[0039] 1. Prediction of ORF encoding the viral FgGMTV1 DNA-C component

[0040] Through ORFfinder( https: / / www.ncbi.nlm.nih.gov / orffinder / ), SoftBerry( http: / / www.softberry.com / berry.phtml ), Augustus (http: / / bioinf.uni-greifswald.de / augustus / submission) and other websites to predict DNA-C encoding ORF.

[0041] 2. Construction of promoter activity detection vector

[0042] The infectious cloning plasmid pSK-ABC was used as a template to amplify the 500 bp genomic sequence upstream of the start codon (ATG) of p18 and p26 as the promoter sequence. The above fragments were inserted into the pGTN vector treated with restriction endonucleases KpnⅠ and BamHI using homologous recombinase to obtain recombinant plasmids p18(500)-GFP and p26(500)-GFP.

[0043] 3. Preparation of protoplasts of Fusarium graminearum

[0044] The Gibberella inoculated on the PDA plate was transferred to a 250mL Erlenmeyer flask containing 100mL CMC liquid culture medium (1g NaNO3 solid, 0.25g magnesium sulfate MgSO4·7H2O, 0.5g potassium dihydrogen phosphate KH2PO3, 0.5g yeast powder, and the volume was adjusted to 500mL after dissolution. 0.75g sodium carboxymethyl cellulose (CMC) was weighed in 100mL Erlenmeyer flasks respectively, and 50mL of dissolved CMC liquid was added to each flask, which was sealed and sterilized by high pressure for standby use), and cultured at 25℃ and 180rpm for 3-5 days (4 days in this experiment); the culture medium was filtered with sterilized three-layer lens paper, centrifuged at 4000rpm for 5min at room temperature, and the supernatant was discarded; sterilized water was added, the conidia precipitate was resuspended and diluted to 10 7 spores / mL; 3×10 7Transfer the spores to a 250mL Erlenmeyer flask containing 100mL YEPD liquid culture medium and shake at 180rpm for 12-14h (12h in this experiment) at 25℃; filter the culture medium cultured overnight with three layers of sterile lens paper, rinse with sterile water 2-3 times (twice in this experiment), then rinse with 1.2M KCl 1-2 times (twice in this experiment), drain with sterile filter paper, and collect the mycelium; transfer the mycelium to 20mL protoplast enzymatic solution with sterile tweezers, shake gently at 90rpm, 28℃ for 2-3h (2h in this experiment), and examine the plasmid and quantity of the protoplasts under a microscope every 0.5h. Filter the enzymatic hydrolysis mixture with two layers of sterile lens paper into a sterile 50 mL centrifuge tube and rinse twice with 1.2 M KCl solution to wash off as many protoplasts as possible; centrifuge the protoplasts at 2600 rpm and 4 °C for 1 min, discard the supernatant, and resuspend with ice-cold STC buffer (weigh 2.22 g CaCl2, 40 g sucrose, then add 20 mL 1 mol / L Tris-HCl (pH = 8) solution, make up to 200 mL, and sterilize for later use), and wash the protoplasts three times under the above centrifugation conditions; finally, resuspend the protoplasts with STC buffer and adjust its final concentration to 2–5 × 10 7 / mL (3×10 7 / mL); dispense into 200 μL / tube, add dimethyl sulfoxide (DMSO) with a final concentration of 7%, mix well, and store at -80°C.

[0045] 4. PEG-mediated transformation

[0046] The protoplasts were taken out from -80℃ and melted on ice. 200μL PH-1 protoplasts and 30μg recombinant plasmid p18(500)-GFP or p26(500)-GFP were added to a 2mL centrifuge tube, gently mixed, and placed on ice for 20min. 1.25mL PTC solution (4g of PEG 4000 was weighed and dissolved in 10mL STC solution, heated in a microwave oven for 3s until completely melted, sterilized with a sterilized 20μm filter, and prepared for use) was added dropwise to the 2mL centrifuge tube, mixed with a pipette tip, and then allowed to stand at room temperature for 20min. The protoplasts in the 2mL centrifuge tube were transferred to a 15mL centrifuge tube and filtered with 5mL TB3 solution (containing 50 μg / mL ampicillin; TB3 solution: weigh 0.75 g of peptone, 0.75 g of yeast powder, 50 g of sucrose, dissolve in distilled water and make up to 250 mL, dispense and sterilize) was used to repeatedly rinse the 2 mL centrifuge tube, and then added to the above 15 mL centrifuge tube, and gently shaken overnight (16 h) at 25°C and 90 rpm; the protoplast regeneration was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, 600 μL of STC was added to resuspend the precipitate, 20 mL of TB3 medium (TB3 solution with 1% agar and 200 mg / mL neomycin) was added, mixed and poured into a 15 cm diameter plate as the lower culture medium; after solidification, 20 mL TB3 medium (containing 1% agar, 400 mg / mL neomycin) was used as the lower medium; cultured in the dark at 25°C for 3-5 days (4 days in this experiment), single colonies were picked and placed on PDA plates containing 400 mg / mL neomycin, and molecular identification and further observation were performed after three subcultures.

[0047] Take 1 μg of high-quality RNA and use DNase I to fully digest the genomic DNA. The digestion system is as follows:

[0048] Table 1 Digestion system

[0049]

[0050] The reaction was carried out at 37°C for 1 hour, and the reaction was terminated at 65°C for 5 minutes, and the reaction was immediately placed on ice. A reverse transcription reaction was then carried out using a reverse transcription kit (Yeasen, 11121ES60) according to the method provided by the kit. After obtaining cDNA, PCR reaction was immediately carried out or stored at -20°C. The detection primers used are shown in Table 2.

[0051] Table 2 Primers for detecting p9, p18 and p26 transcripts

[0052]

[0053]

[0054] 5. Fluorescence observation

[0055] Take fresh activated p18(500)-GFP and p26(500)-GFP strains, place them in the center of the PDA plate, and place a coverslip 2-3 cm away from the bacterial block. Culture at 25°C in the dark until the hyphae extend to 1 / 3 of the coverslip, then take the coverslip for fluorescence observation. Use strain PH-1(EV) as a negative control and strain RP27-GFP as a positive control. Fluorescence observation was performed using a Zeiss upright fluorescence microscope.

[0056] 6. Results

[0057] The DNA-C component was predicted to encode three relatively large ORFs, which are located in the DNA-C genome. Figure 1 A. The largest ORF (including the stop codon) is located at 892-206 in the DNA-C genome, encoding a protein with a size of 228 amino acids (aa) and a molecular weight of 25.8 kDa, so it is named p26; the smallest ORF is located at 1214-978 in the DNA-C genome, encoding a protein with a size of 78 amino acids (aa) and a molecular weight of 8.6 kDa, so it is named p9; another ORF has introns, which are located at 1127-919 in the DNA-C genome, and the exons (including the stop codon) are located at 1153-1128 and 918-447, encoding a protein with a size of 165 amino acids (aa) and a molecular weight of 18.9 kDa, so it is named p18 (see Figure 1 Protoplast transformation and fluorescence observation showed that the 500 bp genomic sequence upstream of the translation start site (ATG) of p18 and p26 proteins both had promoter activity, and the promoter activity of p18 upstream was stronger (see Figure 1 Since the promoter sequences of p9 and p18 in the first 500 bp are highly overlapped, it is believed that the p9 promoter also has promoter activity. RT-PCR experiments confirmed that transcripts of p9, p18, and p26 could be detected in the strain infected with pSK-ABC, and that p18 did contain intron sequences (see Figure 1 (middle B).

[0058] Example 2 Construction and infection of p9, p18 and p26 start codon point mutant viruses based on the infectious clone pSK-ABC

[0059] The present invention is based on the infectious clone pSK-ABC, and the start codons of p9, p18 and p26 are mutated. Figure 2As shown, firstly, the start codons of p9, p18 and p26 were subjected to single base mutation. The mutation principle is to mutate one base without changing the translation of other upstream or downstream protein amino acids. The start codon ATG of p9 was mutated to ATC and named Mp9; the start codon ATG of p18 was mutated to ACG and named Mp18; the start codon ATG of p26 was mutated to ACG and named Mp26. In order to detect whether there is synergy between different proteins, double mutations of p9 and p18, p9 and p26, p18 and p26, and triple mutations of p9, p18 and p26 were designed at the same time, named Mp9:p18, Mp9:p26, Mp18:p26, Mp9:p18:p26, respectively. The design of mutant primers is shown in Table 3.

[0060] Table 3 Primers for constructing p9, p18 and p26 point mutants

[0061]

[0062] The above 7 mutants were transfected into protoplasts of wild-type Fusarium graminearum strain PH-1 to obtain 7 transfected strains.

[0063] The methods of Fusarium graminearum protoplast preparation, PEG-mediated transfection (same method as above), DNA extraction, Southern hybridization, etc. refer to the standard operating procedures in "Molecular Cloning".

[0064] Specific construction strategies such as Figure 2 The results showed that according to the infectivity experiment, the above 7 mutants successfully infected the PH-1 strain and replicated successfully (see Figure 3 ). Since DNA-C is not necessary for the virus FgGMTV1 to infect strains, and the hybridization results showed that the DNA-C component can be detected in strains infected with all mutants, it is proved that the termination of expression of p9, p18 and p26 does not affect the replication of the DNA-C component.

[0065] Example 3 Phenotypic and Pathogenicity Analysis of Virus-Infected Strains with p9, p18 and p26 Start Codon Mutants

[0066] 1. Determination of colony morphology and growth rate of mutant virus-infected strains

[0067] Use a 5mm puncher to punch out a bacterial cake of the transfectant strain and place it in the center of the PDA culture medium plate to activate the strain; use a 5mm puncher to punch out a bacterial cake at the edge of the colony of the activated strain and transfer the bacterial cake to the center of the PDA culture medium plate, repeating five times respectively; after culturing at 25℃ in the dark for 4 days, observe the colony morphology and measure the colony diameter and conduct statistical analysis.

[0068] 2. Determination of conidia yield

[0069] (1) Take five cakes of the same size from the center half of the colony of fresh strains; for low-toxic strains, take cakes from the part where the mycelium growth is obviously abnormal;

[0070] (2) Place the bacterial cake in CMC liquid medium (equal amount of liquid in each bottle), with 3 replicates for each strain;

[0071] (3) After culturing for 5 days at 25°C and 160 rpm in the dark, spores were collected and the spore suspension was mixed and the conidia concentration (spore production) was measured using a blood count plate.

[0072] 3. Determination of pathogenicity of strains

[0073] Wheat variety Yangmai 158 was planted and used for wheat ear inoculation when the wheat grew to the flowering stage. Conidia of each transfectant strain were prepared according to the above method, and the spore concentration was adjusted to 3×10 5 / mL; select wheat ears with basically the same growth, and when you find the fifth spikelet from bottom to top, use your fingers to separate the inner and outer glume of the spikelet. Pipette 10μL of the prepared spore liquid or fungus block and place it at the root between the inner and outer glume, and gently close it to maintain its natural state. Mark the inoculated spikelets with a marker and hang a label on the wheat stalk to record the inoculation strain, inoculation method, inoculation date, inoculator and other information. Inoculate at least 15 wheat ears with each strain; use a fresh-keeping bag to keep moist and culture for 48 hours; remove the fresh-keeping bag and continue to culture for 14 days, observe the disease condition of the wheat ears, cut the wheat ears, take pictures and count the number of diseased spikelets per ear.

[0074] The results showed that, through the detection of colony diameter, growth rate, spore production and pathogenicity of mutant virus-infected strains, it was found that when p18 was not expressed, that is, infection with the Mp18 mutant virus could significantly reduce the growth rate of the host strain and cause serious morphological abnormalities (see Figure 4 AC), while p9 and p26 cannot be expressed, that is, infection with Mp9 and Mp26 mutant viruses has no significant effect on the growth of host strains; at the same time, when infected with Mp18 mutant viruses, the conidia production and pathogenicity of the host strains are significantly reduced (see Figure 4 medium DF), which has potential as a biological control agent.

[0075] The sequence shown in SEQ ID NO: 1 below is the complete sequence of the Mp18 mutant (without vector). Point mutations are made based on the complete sequence of the infectious clone pSK-ABC (without vector) (patent authorization number: ZL202310441503.1). The complete sequence of the infectious clone pSK-ABC (without vector) is described in the patent (authorization number: ZL202310441503.1), the shaded background is the restriction site (1-18nt, 1760-1771nt, 3509-3532nt, 5464-5501nt), the dotted line is the sequence of component A (19-1759nt), the straight underline is the sequence of component B (1772-3508nt), and the wavy line is the sequence of component C (3533-5463nt). Double restriction enzyme digestion with HindⅢ (1-6nt) and NotⅠ (5494-5501nt) was used to ligate the sequence into the pBluescript SK+ cloning vector.

[0076] Based on the complete sequence of the infectious clone pSK-ABC (without the vector), the Mp18 mutant mutated the start codon ATG of the encoding protein p18 to ACG, specifically, in the component C sequence, the 3792nt position was mutated from A to G (marked with lowercase g), and the 5101nt position was mutated from A to G (marked with lowercase g).

[0077]

[0078]

[0079]

[0080]

[0081] Example 4 Control effect of the weakly toxic strain induced by mutant Mp18 on the highly toxic strain of wheat fusarium

[0082] Based on the results of Example 3, the control effect of the attenuated strain induced by the mutant virus Mp18 infection on the virulent strain of wheat fusarium was further tested. In this example, the co-infection method was selected to verify the control effect of the attenuated strain induced by the mutant virus Mp18.

[0083] 1. Co-infection method

[0084] 10 μL of mycelial suspension (1×10 4 cfu / mL), and 10 μL of the virulent strain PH-1 of Fusarium graminearum marked with the neomycin resistance gene neo (VF) mycelial suspension (1×10 4cfu / mL) were inoculated into the same wheat leaf. After 6 days, the disease condition of the wheat leaf was observed (see Figure 5 (A).

[0085] 2. Re-isolation and culture of strains and detection of viral infection

[0086] Pick the diseased part of wheat leaves, disinfect with 75% ethanol for 3 minutes, rinse with sterile water to remove residual ethanol, put it into PDA medium, culture at 25°C for 2-3 days, and observe the growth and morphology of hyphae. The newly isolated strains were transferred to new PDA medium with cellophane for DNA extraction and detection of recombinant viruses, and PDA medium containing 400 mg / mL neomycin (neo) for strain genotype screening. The DNA extraction of strains and Southern hybridization of viruses refer to the standard operating procedures in "Molecular Cloning".

[0087] Results: In the co-infection assay, compared with the control group, the extension of leaf lesions on wheat leaves infected with the attenuated strain of Mp18 was significantly delayed, and the protection efficiency on one-week-old and three-week-old leaves reached 82.2% and 89.5%, respectively (see Figure 5 The strain re-isolation experiment showed that the mutant virus Mp18 could be horizontally transmitted from the attenuated strain to the highly virulent strain PH-1. neo , thereby reducing the pathogenicity of the strain (see Figure 5 The comprehensive results prove that the mutant virus Mp18 based on the DNA virus FgGMTV1 can induce the host's attenuated toxicity and can effectively control the virulent strains of wheat fusarium under experimental conditions.

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A fungal virus mutant, characterized in that: The fungal virus mutant is obtained by point mutation of the start codon ATG encoding protein p18 in the infectious clone pSK-ABC to ACG, and the encoding gene of the protein p18 is located in the open reading frame of component C at positions 3533-5463 of the full sequence of the infectious clone pSK-ABC.

2. The fungal virus mutant according to claim 1, characterized in that The nucleotide sequence of the fungal virus mutant infectious clone obtained after point mutation is shown in SEQ ID NO: 1, wherein the point mutation method is to mutate the 3792th position of the infectious clone pSK-ABC full sequence from A to G, and the 5101st position from A to G.

3. A method for constructing a fungal virus mutant, characterized in that: The method comprises the step of point mutation of the start codon ATG of the protein p18 encoded in the infectious clone pSK-ABC to ACG; the gene encoding the protein p18 is located in the open reading frame of component C at positions 3533-5463 of the full sequence of the infectious clone pSK-ABC.

4. The construction method according to claim 3, characterized in that: The construction method comprises the following steps: based on the complete sequence of the infectious clone pSK-ABC, the 3792nd position of the complete sequence of the infectious clone pSK-ABC is mutated from A to G, and the 5101st position of A is mutated from A to G.

5. Use of the fungal virus mutant according to claim 1 or 2 in preventing and controlling wheat fusarium head blight.

6. The use according to claim 5, characterized in that The fungal virus mutant achieves the purpose of preventing and controlling wheat fusarium head blight by reducing the pathogenicity of the strong strain causing wheat fusarium head blight.

7. The use according to claim 6, characterized in that The virulent strains that cause wheat fusarium rust include Fusarium graminearum.

8. Use of the fungal virus mutant according to claim 1 or 2 in any of the following: (1) Reduce the growth rate of host strains; (2) reduce the conidia production of the host strain; (3) reduce the pathogenicity of host strains; (4) Promoting abnormal morphology of host strains; (5) Promoting the delay of the extension of lesions on wheat leaves; The host strains include Fusarium graminearum, which causes wheat fusarium rust.

9. A method for preventing and controlling wheat scab, characterized in that: The method comprises the steps of transfecting a wild-type Fusarium graminearum with the fungal virus mutant according to claim 1 or 2 to construct a weak strain, and then treating wheat with the weak strain.

10. The method according to claim 9, characterized in that The fungal virus mutants in the weak strains are horizontally transmitted to the strong strains that cause wheat fusarium rust, and the purpose of preventing and controlling wheat fusarium rust is achieved by reducing the pathogenicity of the strong strains; the strong strains that cause wheat fusarium rust include Fusarium graminearum.

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