Application of the FgFd1 gene in regulating the growth, reproduction, pathogenicity, and stress resistance of Fusarium graminearum

Abstract

This invention provides the application of the FgFd1 gene in regulating the growth, reproduction, pathogenicity, and stress resistance of *Fusarium graminearum*, belonging to the field of genetic engineering technology. The nucleotide sequence of the FgFd1 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the FgFd1 gene is shown in SEQ ID NO.1. The *Fusarium graminearum* FgFd1 gene knockout mutant (ΔFgFd1) obtained by this invention exhibits slower growth rate, reduced conidia number, decreased pathogenicity, and reduced stress resistance compared to the wild-type (WT) strain. This invention provides a technical basis for exploring the molecular mechanisms of sporulation and pathogenicity of *Fusarium graminearum*, and provides potential molecular targets for developing disease control measures targeting sporulation inhibition, showing broad application prospects in plant fungal disease research.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of the FgFd1 gene in regulating the growth, reproduction, pathogenicity, and stress resistance of Fusarium graminearum. Background Technology

[0002] Wheat is a key crop for ensuring my country's food security, and its safe production is directly related to sustainable agricultural development and people's livelihood needs. Fusarium graminearum, a fungal blight disease of wheat, is a devastating global epidemic. After infection, water-soaked brown spots appear on wheat ears and spread rapidly. A pinkish mold layer develops on the diseased areas, eventually forming withered white ears, leading to shriveled and shrunken grains with a significant decrease in starch and protein content. In general epidemic years, it can cause 10%-30% yield loss, while in epidemic years, losses can reach 80%-90%, or even total crop failure. In particular, during infection, Fusarium graminearum produces mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) on the diseased areas. Contamination of wheat grains not only renders the wheat inedible but can also cause acute poisoning in humans and animals, including fever, vomiting, and diarrhea. Long-term ingestion poses carcinogenic and teratogenic risks, seriously threatening food safety and human health.

[0003] The spread and infection of *Fusarium graminearum* are highly dependent on sporulation. The fungus overwinters as mycelium in diseased plant debris, soil, or seeds. The following spring, when temperature and humidity are suitable (15–25℃, relative humidity >80%), it rapidly produces a large number of conidia and ascospores, which can be dispersed by air currents to the wheat ears. During the flowering to grain-filling stage, it invades the flowers and tissues, completing primary and secondary infections, directly determining the extent and intensity of the disease. Therefore, sporulation is a crucial step in *Fusarium graminearum* completing its life cycle and establishing a disease cycle, and is also an important target for controlling Fusarium head blight.

[0004] Identifying and analyzing key genes regulating sporulation in Fusarium graminearum and clarifying their biological functions is crucial for understanding the pathogenic mechanism of this fungus and can also provide potential molecular targets for developing new disease control strategies that target sporulation inhibition. Currently, research on genes related to sporulation regulation in Fusarium graminearum is still limited, especially regarding the function of the FgFd1 gene in regulating the growth, reproduction, pathogenicity, and stress resistance of Fusarium graminearum. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of the FgFd1 gene in regulating the growth, reproduction, pathogenicity, and stress resistance of Fusarium graminearum, offering new clues for the control of plant pathogenic fungi.

[0006] In view of this, one of the objectives of the present invention is to provide the application of FgFd1 in regulating the pathogenicity and / or growth rate and / or asexual reproduction rate and / or stress resistance of Fusarium graminearum, wherein FgFd1 is a protein with an amino acid sequence as shown in SEQ ID NO.1 or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a protein with an amino acid sequence as shown in SEQ ID NO.1.

[0007] The asexual reproduction rate is the sporulation rate, and the stress resistance includes the ability to resist oxidative stress and the integrity of the cell wall.

[0008] A second objective of this invention is to provide the application of the aforementioned FgFd1-related biological materials in regulating the pathogenicity and / or growth rate and / or asexual reproduction rate and / or stress resistance of Fusarium graminearum, wherein the biological materials include nucleic acid molecules encoding FgFd1 or expression cassettes, recombinant vectors or recombinant microorganisms containing said nucleic acid molecules.

[0009] The asexual reproduction rate is the sporulation rate, and the stress resistance includes the ability to resist oxidative stress and the integrity of the cell wall.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2.

[0011] A third objective of this invention is to provide the application of the aforementioned FgFd1 as a target in the design and screening of antifungal drugs.

[0012] The fourth objective of this invention is to provide the application of the aforementioned FgFd1 or the aforementioned biological material in the cultivation of FgFd1 gene-deficient Fusarium graminearum with reduced pathogenicity and / or reduced growth rate and / or reduced asexual reproduction rate and / or reduced stress resistance.

[0013] The reduced asexual reproduction rate refers to a reduced sporulation rate, and the reduced stress resistance includes a reduced ability to resist oxidative stress and a reduced cell wall integrity.

[0014] The fifth objective of this invention is to provide a method for cultivating a Fusarium graminearum strain with reduced pathogenicity and / or reduced growth rate and / or reduced asexual reproduction rate and / or reduced stress resistance, comprising the step of reducing the expression level and / or activity of the aforementioned FgFd1 in Fusarium graminearum to obtain a Fusarium graminearum strain with a reduced FgFd1 gene function.

[0015] The reduced asexual reproduction rate refers to a reduced sporulation rate, and the stress resistance includes a reduced ability to resist oxidative stress and a reduced cell wall integrity.

[0016] Furthermore, the method for reducing the expression level and / or activity of FgFd1 in Fusarium graminearum is achieved by knocking out, inhibiting, or silencing the expression of the gene encoded by FgFd1 in Fusarium graminearum.

[0017] Furthermore, the knockout method is a homologous recombination method.

[0018] Furthermore, the method of homologous recombination involves introducing the homologous recombination fragment for homologous recombination into the protoplast of Fusarium graminearum.

[0019] The sixth objective of this invention is to provide the application of any of the methods described above in the prevention and control of diseases caused by Fusarium graminearum.

[0020] The beneficial effects of the gene FgFd1, which regulates sporulation of Fusarium graminearum, and its application are as follows:

[0021] This invention aims to elucidate the sporulation mechanism of Fusarium graminearum. Based on previous transcriptome analysis by our research group, a gene with functional domain annotations was identified. However, annotation using CDD and pfam online software revealed that this gene lacked protein domain annotations, classifying it as a novel gene, which we named FgFd1.

[0022] The *Fusarium graminearum* FgFd1 gene knockout mutant (ΔFgFd1) obtained in this invention exhibits a slower growth rate compared to the wild-type (WT) strain and the complement (Com-FgFd1). The spore morphology of the knockout mutant shows no significant change compared to the wild-type and complement. However, its stress resistance is reduced compared to the wild-type and complement. The mycelial inhibition rate of the knockout mutant increases under hydrogen peroxide and Congo red treatment, indicating reduced oxidative stress resistance and cell wall integrity. Sporulation is significantly reduced, suggesting that the FgFd1 gene may regulate *Fusarium graminearum* spore production.

[0023] The FgFd1 gene of this invention plays a very important role in the pathogenicity of Fusarium graminearum. Wild-type and complement strains are highly pathogenic, causing obvious brown necrosis in wheat leaves, while the FgFd1 knockout mutant only shows browning symptoms at the inoculation site of wheat leaves. Attached Figure Description

[0024] Figure 1 This is a diagram of the Fusarium graminearum FgFd1 gene knockout strategy of the present invention (schematic diagram of Split-PCR gene knockout method).

[0025] Figure 2 Agarose gel electrophoresis image of the FgFd1 knockout transformant for verification of the present invention;

[0026] Figure 3 A schematic diagram illustrating the gene complementation strategy using the pKNTG vector;

[0027] Figure 4 The inhibition rate statistics of the wild type, knockout ΔFgFd1 and complement (Com-FgFd1) of this invention under different concentrations of H2O2 stress are shown in the figure.

[0028] Figure 5 The wild-type, knockout ΔFgFd1 and complement (Com-FgFd1) of this invention are shown in the statistical graph of inhibition rates under different concentrations of Congo red stress.

[0029] Figure 6 The statistical graph shows the mycelial growth rate of the wild type, knockout ΔFgFd1 and complement (Com-FgFd1) of this invention after 72 h of growth on PDA medium.

[0030] Figure 7 The diagram shows the conidial yield statistics of the wild type of this invention, including the knockout body ΔFgFd1 and the complement body (Com-FgFd1).

[0031] Figure 8 Spore morphology observation diagrams of the wild type, knockout body ΔFgFd1 and complement body (Com-FgFd1) of this invention;

[0032] Figure 9 The diagram shows the pathogenicity determination of the wild type, knockout ΔFgFd1 and complement (Com-FgFd1) of this invention on detached wheat leaves. Detailed Implementation

[0033] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are all conventional commercially available products.

[0034] The plant materials, strains, and related vectors used in the following embodiments of the present invention are described below:

[0035] The main strains include Fusarium graminearum strain YJ-1.

[0036] Main vector: pKOV21 plasmid containing the hygromycin resistance gene sequence (Baosai Biotechnology, product number pK072). Complementation vector pKNTG (Amp, G-418 resistance) was preserved in the inventor's laboratory.

[0037] Main reagents: Trizol, purchased from Beijing Cooler Master Technology Co., Ltd.; 2×Taq PCR StarMix (Dye) and StarScript II cDNA first-strand synthesis premix (including genomic removal), purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.; Agarose gel DNA recovery kit and plasmid miniprep kit, purchased from Tiangen Biotech (Beijing) Co., Ltd.; DNA extraction kit, purchased from Omega Bio-Tek; BioGold™ Seamless EFF Cloning Kit, purchased from Zhejiang Boerjin Technology Co., Ltd.; Ampicillin (Amp) and genimycin sulfate (G418), purchased from Beijing Solarbio Technology Co., Ltd.; Hygromycin B, purchased from Roche.

[0038] The main culture media are: PDA medium (potato glucose agar medium): Weigh 200 g of potato, 20 g of glucose, and 17 g of agar, add deionized water to make up to 1 L, and autoclave at 121℃ for 20 min.

[0039] PDB medium (potato glucose broth): Weigh 200 g of potatoes and 20 g of glucose, add deionized water to a final volume of 1 L, and autoclave at 121℃ for 20 min.

[0040] CMC medium (sodium carboxymethyl cellulose medium): 15 g sodium carboxymethyl cellulose, 1 g yeast extract, 1 g potassium dihydrogen phosphate (KH2PO2), 1 g ammonium nitrate (NH4NO3), 0.5 g magnesium sulfate heptahydrate (MgSO4·7H2O), and deionized water to a final volume of 1 L.

[0041] YEPD medium: 20 g glucose, 10 g peptone, 3 g yeast extract, and deionized water to a final volume of 1 L.

[0042] TB3 medium: 200 g sucrose, 3 g casamino acid, 3 g yeast extract, and deionized water to a final volume of 1 L.

[0043] Bottom agar medium: 200 g sucrose, 3 g casaminoacid, 3 g yeast extract, 10 g agar, and deionized water to a final volume of 1 L.

[0044] Top agar medium: 200 g sucrose, 3 g casamino acid, 3 g yeast extract, 15 g agar, and deionized water to a final volume of 1 L.

[0045] All the above-mentioned culture medium raw materials were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0046] Example 1: Cloning, knockout mutants and complement mutants of the Fusarium graminearum FgFd1 gene.

[0047] 1.1 Extraction of genomic DNA from Fusarium graminearum.

[0048] Add 100 mg of wild-type bacterial strain sample to a sterile 2 mL centrifuge tube, freeze in liquid nitrogen, and disrupt using a cell disruptor. Add 600 μL of FG1 Buffer and vortex to ensure all tissue clumps are evenly dispersed in the solution. Incubate at 65°C for 10 min, shaking 2-3 times during this period. Add 140 μL of FG2 Buffer and shake to mix, place on ice for 5 min, and centrifuge at 10000 g for 10-15 min. Transfer the supernatant to a new 1.5 mL centrifuge tube, then add 0.5 times the volume of isopropanol and vortex to mix. Immediately centrifuge at 10000 g for 2 min, carefully discarding the supernatant without touching the precipitate. Invert the centrifuge tube on absorbent paper for 1 min to drain the liquid completely. Add 300 μL of sterile water heated to 65°C, shake to dissolve the precipitate, add 4 μL of RNase A, and vortex to mix. Add 150 μL of FG3 Buffer and 300 μL of isopropanol. Mix 1 μL of anhydrous ethanol and shake gently. Place the HiBind® DNA column into a 2 mL collection tube, pass the solution through the column, and centrifuge at 10,000 g for 1 min at room temperature. Discard the supernatant. Place the HiBind® DNA column back into a new 2 mL collection tube, add 750 μL of DNA Wash Buffer (dilute with anhydrous ethanol according to the instructions before use), centrifuge at 10,000 g for 1-2 min at room temperature, discard the filtrate, and repeat this step once. Place the HiBind® DNA column back into the 2 mL collection tube, centrifuge at 1,000 g for 2 min at room temperature, discard the supernatant, invert and air dry. Place the HiBind® DNA column into a new 1.5 mL centrifuge tube, add 50-100 μL of preheated Elution buffer (65℃), incubate at room temperature for 5 min, centrifuge at 10,000 g for 1 min, and elute the DNA. The DNA can be stored for a short period at -20℃.

[0049] 1.2 Gene FgFd1 knockout.

[0050] Preparation of protoplasts for wild-type strains: Wild-type Fusarium graminearum strains were activated on PDA medium and cultured at 25℃ for 2-3 days. They were then transferred to fresh PDA medium. Once fresh mycelia emerged, appropriately sized mycelial blocks were transferred to 50 mL of CMC medium and cultured at 25℃ and 175 rpm for 3-5 days. The CMC medium from the previous shaking culture was filtered through sterile filter paper to obtain a spore suspension. The suspension was centrifuged at 3500 rpm for 10 min at room temperature. The supernatant was discarded, and 75 mL of sterile YEPD medium was added. After mixing, the suspension was transferred to a 250 mL Erlenmeyer flask and cultured at 25℃ and 175 rpm for approximately 12 h using a shaker. The YEPD medium from the previous shaking culture was filtered through sterile three-layer lens paper and rinsed with sterile water. 0.5 g of the filtered mycelia were weighed into a 50 mL centrifuge tube and 10 g of spores were added. Mix mL of freshly prepared enzyme hydrolysate, and incubate at 30℃ with shaking at 90 rpm for 1-2 h, observing the protoplast lysis during this period. Once the hyphae have successfully lysed, filter the enzyme hydrolysate obtained in the previous step through one layer of sterilized filter paper and three layers of lens paper (three layers of lens paper on the outside, one layer of filter paper on the inside). Centrifuge the filtrate at 3500 rpm for 8 min at room temperature. Discard the supernatant, add 10 mL of STC medium to resuspend the protoplasts, centrifuge at 3500 rpm for 8 min at room temperature, and repeat this step once. Discard the supernatant, add a certain amount of STC medium to adjust the protoplast concentration to 5 × 10⁻⁶. 7 The number of protoplasts is 1 per mL, indicating that the preparation of protoplasts is complete.

[0051] Gene knockout fragments were constructed using the Split-PCR method. Figure 1 Using DNA as a template, a reverse complementary sequence of primer HYG-F was added to the 5' end of primer FgFd1-2R, and a reverse complementary sequence of primer HYG-R was added to the 5' end of primer FgFd1-3F. Then, approximately 1 kb homologous arm sequences upstream (A) and downstream (B) of the FgFd1 gene were amplified using primers FgFd1-1F / FgFd1-2R and FgFd1-3F / FgFd1-4R, respectively. Using pKOV21 as a template, the first half (H1) and the second half (H2) of the hygromycin resistance gene (HPH) were amplified using primers HYG-F / HY-R and HYG-R / YG-F, respectively. The sequences are shown in Table 1 below.

[0052] Table 1 Primer sequences used for FgFd1 gene knockout

[0053]

[0054] A and H1, B and H2 were linked via homologous recombination and added together to the protoplasts of YJ-1. After gentle mixing, the mixture was incubated at room temperature for 20 min. 1 mL of filtered, sterile 40% PTC solution was added, and the mixture was gently mixed and incubated at room temperature for 20 min. 5 mL of TB3 medium was added, and the mixture was incubated overnight at 90 rpm in a 25°C shaker. 100 mL of melted Bottom agar medium was added to the TB3 solution from the previous step, and a certain amount of hygromycin was added to achieve a final concentration of 300 μg / mL. After mixing, the mixture was poured into sterile culture dishes, and after the medium solidified, it was inverted and incubated overnight at 25°C. A certain amount of hygromycin was added to melted Top agar medium to achieve a final concentration of 400 μg / mL, and this mixture was poured onto the top layer of the previous medium. After the medium solidified, it was inverted and incubated for 3 days in a 25°C shaker. Approximately 3 days later, after single colonies have grown in Topagar medium, they are transferred to PDA medium and incubated at 25°C for subsequent DNA extraction and PCR detection. The verification results are as follows: Figure 2 As shown, FgFd1-1F and HYG-R, and HYG-F and FgFd1-4R showed the expected bands; FgFd1-5F and FgFd1-6R, FgFd1-1F and FgFd1-6R, and FgFd1-5F and FgFd1-4R showed no bands, proving that the knockout strain ΔFgFd1 was successfully constructed.

[0055] 1.3 FgFd1 gene complementation.

[0056] Constructing the complementation vector pKNTG ( Figure 3 Using DNA as a template, the FgFd1 target gene and its first 1.5 kb fragment were amplified using primers Com-FgFd1-F and Com-FgFd1-R. This fragment was then ligated into the pKNTG vector using homologous recombination. The vector was then mixed with protoplasts of ΔFgFd1 (preparation and transformation were the same as the FgFd1 knockout steps described above; sequence information is shown in Table 2). Verification was performed using primers FgFd1-F and pKNTG-R, and FgFd1-5F and FgFd1-6R. The expected bands appeared, confirming the successful construction of the complement strain Com-FgFd1.

[0057] Table 2 Primer sequences used for FgFd1 gene complementation

[0058]

[0059] Example 2: The effect of the FgFd1 gene on the response to abiotic stress.

[0060] Wild-type strains YJ-1, ΔFgFd1, and Com-FgFd1 were activated, and mycelial cakes were collected from the edge of the colonies using a 5 mm punch and inoculated into the center of PDA plates containing different stress reagents. The stress reagents and their final concentrations were H2O2 (0.05%, 0.1%) and Congo red (300 mg / L, 600 mg / L), respectively. PDA plates without stress reagents served as controls. After incubation at 25°C for 3 days, the colony diameter was measured using the cross-crossing method, and the inhibition rate was calculated. Each strain was tested in triplicate.

[0061]

[0062] Compared with the wild-type strain, the knockout mutant ΔFgFd1 showed a significantly increased inhibition rate (p<0.05) on plates containing H2O2, indicating a weakened tolerance to these stress factors. Figure 4 Under 0.05% H2O2 conditions, the phenotype of the complement strain Com-FgFd1 recovered to a level similar to that of the wild-type strain, but was still significantly lower than the inhibition rate of the knockout mutant ΔFgFd1, indicating that the antioxidant capacity of the knockout mutant ΔFgFd1 was reduced.

[0063] Compared with the wild-type strain, the knockout mutant ΔFgFd1 showed a significantly increased inhibition rate on plates containing Congo red (p<0.05), indicating that its tolerance to these stress factors was weakened. Figure 5 Under conditions of 300 mg / L or 600 mg / L Congo red, the phenotype of the complemented strain Com-FgFd1 was restored to a level similar to that of the wild-type strain, and was significantly lower than the inhibition rate of the knockout mutant ΔFgFd1, indicating that the cell wall integrity of the knockout mutant ΔFgFd1 was reduced.

[0064] Therefore, knocking out the FgFd1 gene significantly reduced the stress resistance of Fusarium graminearum.

[0065] Example 3: Effects of the FgFd1 gene on mycelial growth, sporulation and spore germination of Fusarium graminearum.

[0066] Growth rate determination: Mycelial discs of the three strains were inoculated in the center of PDA plates and incubated at 25°C for 3 days. The colony diameter was measured. Results showed that the mycelial growth rate of the reintroduced strain Com-FgFd1 recovered to the wild-type level. The colony diameter of ΔFgFd1 was significantly smaller than that of the wild-type and Com-FgFd1, indicating that knocking out FgFd1 led to a decrease in mycelial growth rate. Figure 6 ).

[0067] Sporulation yield determination: Three mycelial discs were inoculated into 50 mL of CMC liquid medium and incubated at 25℃ and 180 rpm for 3 days. The number of conidia was counted using a hemocytometer. The results showed that the sporulation yield of ΔFgFd1 was significantly lower than that of the wild-type and Com-FgFd1 strains. Figure 7 The reintroduced strain recovered to wild-type levels, indicating that the FgFd1 gene is related to the sporulation capacity of Fusarium graminearum, and knocking out FgFd1 leads to a decrease in sporulation of Fusarium graminearum.

[0068] Spore and hyphal morphology observation: Conidia produced in CMC medium were collected and their morphology was observed under a microscope. Results showed no significant differences in spore morphology (length, number of septa) among the three strains. Figure 8 The strains were inoculated onto PDA medium with slides inserted. After culturing for 1 day, the slides were removed to observe the hyphal morphology. The results showed that there was no significant difference in hyphal morphology among the three strains.

[0069] Example 4: Effect of FgFd1 gene on pathogenicity of Fusarium graminearum.

[0070] In vitro inoculation of wheat leaves: After surface disinfection and germination of wheat seeds (Bainong 207, provided by Professor Ou Xingqi of Henan University of Science and Technology), the seeds were transferred to a fruit and vegetable basket with a filter screen, spread evenly on the filter screen, placed on a tray, and water was added to the bottom of the filter screen. After the wheat reached the two-leaf stage, leaves with uniform growth were removed and placed in a petri dish for inoculation with 5 mm mycelium cakes. The results were observed and statistically analyzed after 3 days of incubation at 25℃.

[0071] The experimental results showed that the wild-type and reinjected strains produced longer lesions on wheat leaves, while the knockout strains only showed smaller lesions near the inoculation site on wheat leaves. Figure 9 The results indicate that the FgFd1 gene has a significant impact on the pathogenicity of Fusarium graminearum on wheat leaves, and knocking out FgFd1 leads to a decrease in the pathogenicity of Fusarium graminearum.

[0072] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Use of FgFd1 in modulating the pathogenicity and / or growth rate and / or asexual reproduction rate and / or stress resistance of Fusarium graminearum, characterized in that, The FgFd1 amino acid sequence is as shown in SEQ ID NO.1, or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein as shown in SEQ ID NO.

1.

2. Use of the FgFd1 -related biomaterial as defined in claim 1 for modulating the pathogenicity and / or growth rate and / or rate of asexual reproduction and / or stress resistance of Fusarium graminearum, characterized in that, The biological material includes a nucleic acid molecule encoding FgFd1 or an expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule.

3. Use according to claim 2, wherein the compound is ###0002### The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. The use of FgFd1 as a target in the design and screening of antifungal drugs as described in claim 1.

5. The use of the FgFd1 as described in claim 1 or the biological material as described in claim 2 or 3 in the cultivation of transgenic Fusarium graminearum with reduced pathogenicity and / or reduced growth rate and / or reduced asexual reproduction rate and / or reduced stress resistance.

6. A method for cultivating *Fusarium graminearum* strains with reduced pathogenicity and / or reduced growth rate and / or reduced asexual reproduction rate and / or reduced stress resistance, characterized in that... The method includes the step of reducing the expression level and / or activity of FgFd1 as described in claim 1 in Fusarium graminearum to obtain a Fusarium graminearum with FgFd1 gene function deficiency.

7. The method as described in claim 6, characterized in that, The method for reducing the expression level and / or activity of FgFd1 as described in claim 1 in Fusarium graminearum is achieved by knocking out, inhibiting, or silencing the expression of the gene encoding FgFd1 in the recipient Fusarium graminearum.

8. The method as described in claim 7, characterized in that, The knockout method is homologous recombination.

9. The method as described in claim 8, characterized in that, The method of homologous recombination involves introducing a homologous recombination fragment into the protoplast of Fusarium graminearum.

10. The application of the method according to any one of claims 6-9 in the prevention and control of diseases caused by Fusarium graminearum.

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