Application of FgCON7 and FgNDK1 gene combination in preparation of drug for preventing and treating wheat scab

Abstract

The application belongs to the field of plant disease prevention and control and molecular biological technology, and specifically discloses application of FgCON7 and FgNDK1 genes in preparation of a wheat scab prevention and treatment agent, wherein by spraying dsRNA of the FgCON7 and FgNDK1 genes, the genes are successfully knocked out in fusarium graminearum, the obtained fusarium graminearum knockout mutant has a slow colony growth and development rate, a reduced number of conidia, a decreased ATP synthesis amount, a reduced mycotoxin synthesis amount, and can effectively reduce the infection ability of fusarium graminearum on a wheat plant. Pathogenicity experiments show that the double-target joint use can serve as an effective RNAi mode, and the inhibition effect is better than that of single-target inhibition, and the antifungal efficiency is higher.

Description

Technical Field

[0001] This invention relates to the fields of plant disease control and molecular biotechnology, particularly the application of the combined FgCON7 and FgNDK1 genes in the preparation of agents for controlling wheat scab. Background Technology

[0002] Wheat scab is caused by Fusarium graminearum (a fungus), and mainly manifests as seedling rot, ear rot, and stem rot. It leads to reduced wheat yield and quality, and produces fungal toxins such as DON, threatening human, animal, and ecological safety.

[0003] In its early stages, *Fusarium graminearum* parasitizes the host in a living manner, cleverly suppressing the host's innate immune response. It then rapidly transitions to a necrotic vegetative stage, concentrating on secreting various pathogenic factors to destroy host cells. The pathogen primarily overwinters as dormant hyphae and reproductive structures in crop residues and diseased plant parts in the field. The following spring, when temperature and humidity are suitable, spores produced in the sexual stage serve as the primary source of primary infection. These spores are dispersed over long distances by air currents and rain splash, precisely reaching the susceptible flowers or young tissues of the host. After successful infection and disease development, numerous asexually reproducing spores are produced on the surface of the diseased parts. These spores repeatedly reinfect throughout the growing season using the same wind and rain vectors, causing the disease to spread rapidly in the field. Meanwhile, infected seeds are transported long distances across regions through human intervention.

[0004] Currently, the main methods for treating plant diseases and pests include chemical pesticides, physical control, and traditional agricultural practices. Chemical pesticides are widely used due to their rapid effectiveness and low cost, but long-term reliance leads to increased pesticide resistance in pests, a reduction in natural enemies, soil and water pollution, and excessive residues in agricultural products, and can easily disrupt the ecological balance. Physical control methods, such as trapping and isolation, are environmentally friendly, but are cumbersome, costly, and inefficient, making it difficult to cover large areas of farmland. Traditional agricultural practices, such as crop rotation, intercropping, and breeding disease-resistant varieties, have some effect, but they are time-consuming, resistance is prone to degradation, and their ability to cope with sudden outbreaks of diseases and pests is limited. The limitations of these methods have prompted people to turn to more sustainable solutions, and biopesticides have emerged as a result.

[0005] In recent years, RNA interference (RNAi) technology, as an emerging gene silencing tool, has provided a novel approach to plant disease control. Spray-induced gene silencing (SIGS) technology specifically silences target genes of pathogens by exogenously spraying double-stranded RNA (dsRNA) of key pathogen genes, thereby achieving disease control. This technology has significant advantages such as strong targeting, environmental friendliness, and no harmful residues. However, the control effect of SIGS technology is highly dependent on the selection of target genes. The genome of Fusarium graminearum is large and complex, with the Unigene sequence predicted to contain 12,833 genes, and the genome contains a large number of single nucleotide polymorphisms (SNPs), most of which are distributed in discontinuous regions near chromosome telomeres. Finding and validating effective pathogen-related genes from such a large genome, especially gene combinations that can achieve efficient disease control through synergistic regulation, is extremely challenging.

[0006] More importantly, the pathogenicity of *Fusarium graminearum* is regulated by a multi-gene network. Silencing a single target gene often results in limited control efficacy due to gene functional redundancy or compensatory effects of the pathogen, and single targets are prone to escape due to gene mutations. Therefore, it is urgent to discover multi-gene target combinations that can synergistically regulate the pathogenicity of *Fusarium graminearum*, thereby breaking gene functional redundancy by simultaneously interfering with multiple pathogenicity-related pathways, improving the effectiveness of RNAi control, and reducing the risk of pathogen resistance. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the application of the combined FgCON7 and FgNDK1 genes in the preparation of agents for controlling wheat scab.

[0008] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is the application of the combined FgCON7 and FgNDK1 genes in the preparation of agents for controlling wheat scab. This involves preparing dsRNA formulations targeting the FgCON7 and FgNDK1 genes respectively, and simultaneously exogenously spraying these dsRNA formulations onto wheat plants. The gDNA sequence of the FgCON7 gene is shown in SEQ ID NO.1; the cDNA sequence of the FgCON7 gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the FgCON7 gene is shown in SEQ ID NO.3; the gDNA sequence of the FgNDK1 gene is shown in SEQ ID NO.4; the cDNA sequence of the FgNDK1 gene is shown in SEQ ID NO.5; and the amino acid sequence of the protein encoded by the FgNDK1 gene is shown in SEQ ID NO.6.

[0009] The second technical solution provided by the present invention is a fungicide for controlling wheat scab, which is composed of dsRNA preparations of the FgCON7 gene and the FgNDK1 gene.

[0010] Furthermore, the concentration of the dsRNA preparations for both the FgCON7 and FgNDK1 genes is 20 ng / μL.

[0011] The third technical solution provided by this invention is a method for preventing and controlling wheat scab, comprising the following steps: During the wheat growth process, the agent for controlling wheat scab as described in claim 2 or claim 3 is applied externally.

[0012] Compared with existing technologies, this invention successfully knocked out *Fusarium graminearum* by exogenously spraying dsRNA of the FgCON7 and FgNDK1 genes. The resulting *Fusarium graminearum* knockout mutants exhibited slower colony growth and development, reduced conidia number, decreased ATP synthesis, and lower mycotoxin synthesis, effectively reducing the infectivity of *Fusarium graminearum* on wheat plants. Pathogenicity experiments showed that the combined use of dual targets is an effective RNAi method, with better inhibitory effects and higher antifungal efficiency than single-target inhibition. Attached Figure Description

[0013] Figure 1 Agarose gel electrophoresis images of PCR amplification products and dsRNA synthesis products of FgCON7 and FgNDK1 genes: A is the electrophoresis image of PCR amplification products; B is the electrophoresis image of dsRNA transcription products; C is the electrophoresis image of dsRNA purified by magnetic beads; M is DL2000 DNA Marker; 1 is the purified dsCON7; 2 is the purified dsNDK1.

[0014] Figure 2 This image shows a comparison of colony diameters of Fusarium graminearum wild-type, single-target treated, and dual-target treated on PDA medium.

[0015] Figure 3 A comparative diagram showing the changes in hyphal branching of Fusarium graminearum wild-type, single-target treatment, and dual-target treatment.

[0016] Figure 4 This is a statistical chart showing the ATP synthesis of wild-type Fusarium graminearum, single-target therapy, and dual-target therapy.

[0017] Figure 5 A comparative diagram showing the inhibition of conidial internalization in wild-type Fusarium graminearum, single-target therapy, and dual-target therapy.

[0018] Figure 6Laser confocal microscopy images showing the morphology and quantity of ATP within Fusarium hyphae of wild-type Fusarium graminearum after single-target therapy and dual-target therapy.

[0019] Figure 7 The control and inhibition effects of wild-type Fusarium graminearum, single-target therapy, and dual-target therapy on Fusarium graminearum on wheat leaves are shown in the figures: A represents the control effect; B represents the inhibition effect.

[0020] Figure 8 The images show the control and storage protection effects of Fusarium graminearum wild-type, single-target therapy, and dual-target therapy on Fusarium graminearum on wheat ears.

[0021] Figure 9 This is a statistical chart showing the synthesis of the fungal toxin DON under wild-type Fusarium graminearum, single-target therapy, and dual-target therapy. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0023] All experimental materials used in the examples are conventional materials in the art and can be purchased commercially. Experimental methods not specified with detailed conditions were performed according to standard experimental methods or the supplier's recommended operating instructions. The *Fusarium graminearum* ZKCC number was ZKCC3088, purchased from Zhongke Quality Inspection; the tested wheat was "Shentai No. 2". The composition and preparation method of the culture media used in the examples are as follows: PDA medium (g / L): Potato extract powder 12, glucose 20, agar 14. Weigh 46g, heat to dissolve in 1000ml purified water, and autoclave at 121℃ for 20min.

[0024] PDB medium (g / L): 300g potato extract powder, 20g glucose. Weigh 24g, heat to dissolve in 1000ml purified water, and autoclave at 121℃ for 20min.

[0025] YEPD medium (g / L): yeast extract 10g, peptone 20g, glucose 20g. Weigh 50g, heat to dissolve in 1000ml purified water, and autoclave at 115℃ for 15min.

[0026] TBI medium (g / L): sucrose 30, ammonium sulfate 2, potassium dihydrogen phosphate 1, magnesium sulfate heptahydrate 0.5, potassium chloride 0.2, ferrous sulfate heptahydrate 0.01. Weigh 34.01 g, heat to dissolve in 1000 ml of purified water, and autoclave at 121℃ for 15 min. After cooling, aseptically add 200 μl of trace element solution.

[0027] Trace element solution (g / 50mL): citric acid 2.5, zinc sulfate heptahydrate 2.5, copper sulfate pentahydrate 0.125, manganese sulfate monohydrate 0.125, boric acid 0.025, sodium molybdate dihydrate 0.025. Weigh 5.2g, heat to dissolve in 50ml of purified water, filter sterilize through a 0.22μm bacterial filter, and store at 4℃ protected from light.

[0028] Example 1: Screening of Fusarium graminearum genes FgCON7 and FgNDK1 This embodiment first performed transcriptome analysis on Fusarium graminearum to obtain two Fusarium graminearum genes related to pathogenicity: FgCON7 and FgNDK1. The full-length gDNA sequences of the Fusarium graminearum FgCON7 and FgNDK1 genes are shown in SEQ ID NO.1 and SEQ ID NO.4; the cDNA sequences of the Fusarium graminearum FgCON7 and FgNDK1 genes are shown in SEQ ID NO.2 and SEQ ID NO.5; and the encoded amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.6.

[0029] Example 2: Synthesis of dsRNA from the FgNDK1 and FgCON7 genes of Fusarium graminearum Based on sequence characteristics, secondary structure, and number of target sites, select the dsRNA synthesis region and design primers: CON7-dsF taatacgactcactatagggACCGTGCATGGTGAAAATCT CON7-dsR taatacgactcactatagggTGGCAGACCAACACGAGGT NDK1-dsF taatacgactcactatagggGCGTTCATTGCTCGGTATTGA NDK1-dsR taatacgactcactatagggGCCATGCGCGAATAGGACTA The T7 promoter sequence was added to the 5′ end of both the forward and reverse primers. Primer information used in the experiment is shown in the table above. Using recombinant plasmids containing the full-length sequences of the FgCON7 and FgNDK1 genes as templates, PCR amplification was performed using the aforementioned primers. PCR products were purified using a MagBeads PCR Cleanup Kit to remove impurities such as primers, enzymes, and mononucleotides. The purified PCR products were then analyzed by 1% agarose gel electrophoresis, and the results are shown below. Figure 1 As shown in A, the target band is clearly visible.

[0030] dsRNA was synthesized via in vitro transcription using the T7 RNA Transcription Kit Plus, following the kit instructions. The transcription products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 1As shown in Figure B, the synthesized dsRNA band is visible. The qualified dsRNA samples were purified using the MagBeads dsRNA Purification Kit to remove impurities such as free nucleotides and proteins. The electrophoresis results of the purified dsRNA are shown below. Figure 1 As shown in C. From Figure 1 As can be seen, this embodiment obtained high-purity dsRNA products: dsCON7 and dsNDK1.

[0031] Example 3: Effects of FgCON7 and FgNDK1 gene silencing on Fusarium graminearum colony growth Prepare the culture medium according to the PDA medium instructions, pour it into petri dishes and let it solidify. Prepare 20 ng / μL solutions of dsEGFP, dsCON7, and dsNDK1. Pour 0.5 ml of the prepared dsRNA solution into the medium, spread it evenly with a spreader, and inoculate Fusarium graminearum in the center of the medium. Incubate the dishes upside down at 27°C for 7 days. Observe and record the status of Fusarium graminearum on days 0, 1, 3, 5, and 7. Results are shown below. Figure 2 The results showed that single targets could inhibit the growth of Fusarium graminearum, but combined target treatments showed the most severe inhibition, demonstrating stronger inhibitory potential.

[0032] Example 4: Effects of FgCON7 and FgNDK1 gene silencing on hyphal branching of Fusarium graminearum Prepare the liquid culture medium according to the PDB instructions, dispense it into 250ml Erlenmeyer flasks, and after cooling, add the prepared 20 ng / μL dsEGFP, dsCON7, and dsNDK1 solutions, mix well, and place a small amount of Fusarium graminearum sample into the prepared liquid culture medium. Incubate in a shaker at 27℃ for 5 days. Take a small amount of hyphae and place it in a 1.5ml EP tube, gently grind it with the provided grinding rod to disperse the hyphae, add 1ml of water and mix well by blowing. Take 0.1ml of the bacterial solution and drop it onto a glass slide, gently cover it with a coverslip, and observe the hyphal branching state under an optical microscope. The results are shown below. Figure 3 dsNDK1 has little effect on hyphal branching, and the ability of the dual-target combined treatment to inhibit hyphal branching may be entirely derived from dsCON7.

[0033] Example 5: Effects of FgCON7 and FgNDK1 gene silencing on ATP synthesis in Fusarium graminearum Prepare liquid culture medium according to the PDB instructions, dispense into 250ml Erlenmeyer flasks, cool, and then add 20 ng / μL of the prepared dsEGFP, dsCON7, and dsNDK1 solutions. Mix well, and place a small amount of *Fusarium graminearum* sample into the prepared liquid culture medium. Incubate at 27°C for 1, 3, 5, and 7 days. Place a small amount of mycelium into a 1.5ml EP tube and perform ATP content detection according to the ATP content assay kit (BC0305, Solarbio). Plot a bar graph with the number of days on the x-axis and ATP content on the y-axis. Results are shown below. Figure 4 Compared with the negative control group, the ATP content in the single-target group and the double-target group was significantly inhibited (the inhibition of ATP content in the CON7 group may be related to its inhibition of mycelial and spore growth).

[0034] Example 6: Inhibitory effect of FgCON7 and FgNDK1 gene silencing on Fusarium graminearum conidia. Prepare 500 ml of culture medium according to the PDB liquid medium instructions, and dispense it into 150 ml Erlenmeyer flasks, labeled 1, 2, 3, and 4 respectively. Add 0.5 ml of 20 ng / μL dsEGFP for internalization fluorescent labeling to flasks 1, 2, 3, and 4 respectively. Then add 0.5 ml of 20 ng / μL dsNDK1, dsCON7, and a mixture of their nucleic acids to flasks 2, 3, and 4 respectively. Incubate at 27°C and 140 rpm for 48 h on a shaker, and observe spore internalization and inhibition effects under a fluorescence inverted microscope. Results are shown below. Figure 5 The results successfully confirmed that spores can effectively internalize exogenous dsRNA in this system. Both dsCON7 and dsNDK1 can effectively inhibit spore growth and activity through the RNAi pathway, with the silencing of the dual-target group showing a stronger growth-inhibiting effect.

[0035] Example 7: Effects of FgCON7 and FgNDK1 gene silencing on hyphal mitochondrial morphology Liquid culture medium was prepared according to the YEPD medium instructions and dispensed into four 150 ml Erlenmeyer flasks, labeled 1, 2, 3, and 4. 0.5 ml of 20 ng / μL dsEGFP, dsNDK1, dsCON7, and a mixture of two nucleic acids were added to flasks 1, 2, 3, and 4, respectively. The flasks were incubated at 27 °C and 140 rpm for 12 h and stained with MitoTracker Red CMXRos. The results were then observed using a Nikon N-SIM laser confocal microscope. (See attached image). Figure 6Interference with the FgNDK1 gene can directly lead to the transformation of mitochondria from a healthy "wire-like" structure to an abnormal "fragmented" structure. The combined treatment of dsCON7 and dsNDK1 resulted in the most thorough disruption of mycelial homeostasis and mitochondrial structure.

[0036] Example 8: The preventive and inhibitory effects of FgCON7 and FgNDK1 gene silencing on Fusarium graminearum infection in wheat leaves. Prepare a 20 ng / μL mixture of dsNDK1, dsCON7, and two nucleic acids. Spray the leaves of 4-week-old wheat plants with a Fusarium graminearum inoculum solution (concentration 5 × 10⁻⁶). 4 After infection, water, dsNDK1, dsCON7, and a mixture of two nucleic acids were sprayed onto the bacteria. After incubation at 27 °C for 3 days, the control effect against *Fusarium graminearum* was observed in each group. A mixture of 20 ng / μL dsNDK1, dsCON7, and the two nucleic acids was prepared and added to *Fusarium graminearum* bacterial suspension (concentration 5 × 10⁻⁶). 4 After mixing to prepare a solution, it was sprayed onto the leaves of 4-week-old wheat plants. After incubation at 27 °C for 3 days, the inhibitory effect of each group on Fusarium graminearum was observed. Results are shown below. Figure 7 Exogenous spraying of 20 ng / μL dsRNA preparations effectively prevented (A) and inhibited (B) Fusarium graminearum infection of wheat leaves. The dsCON7 + dsNDK1 combination group showed excellent synergistic effects in both prevention and inhibition, with significantly better efficacy than any single nucleic acid group.

[0037] Example 9: The control effect of FgCON7 and FgNDK1 gene silencing on Fusarium graminearum infection in wheat ears. Prepare a mixture of 20 ng / μL dsNDK1, dsCON7, and two nucleic acids. Spray 4-week-old wheat ears with a Fusarium graminearum bacterial suspension (concentration of 5 × 10⁻⁶). 4 After infection, water, dsNDK1, dsCON7, and a mixture of two nucleic acids were sprayed onto the cells. After incubation at 27 °C for 3 days, the control effect against Fusarium graminearum was observed in each group. Results are shown below. Figure 8 Fungal infection causes wheat ears to become shorter and thinner due to necrosis and shriveling. The negative control group showed obvious typical symptoms of Fusarium head blight. Compared with the control group, the wheat ears in the two single-target groups maintained a large area of ​​green color, and the browning and necrotic areas were significantly reduced. The wheat ears in the dual-target experimental group were almost entirely healthy bright green, with no obvious lesions visible to the naked eye. The experiment shows that the combined dual-target treatment is significantly better than the traditional single-target strategy in inhibiting the disease and protecting the growth and development of wheat ears.

[0038] Example 10: Effects of FgCON7 and FgNDK1 gene silencing on the synthesis of the fungal toxin DON. Prepare 400 ml of TBI liquid culture medium according to the instructions, and dispense it into four 150 ml Erlenmeyer flasks. Prepare 20 ng / μL solutions of dsEGFP, dsNDK1, dsCON7, and a mixture of dsNDK1 and dsCON7. Add the above dsRNA solutions to the four Erlenmeyer flasks respectively, and incubate at 27 °C and 140 rpm for 7 days. Isolate mycelia and detect the DON content in each group according to the DON enzyme-linked immunosorbent assay kit instructions. Results are shown below. Figure 9 The positive control group showed that *Fusarium graminearum* possesses a strong toxin synthesis capacity in TBI liquid medium without specific gene silencing. Targeting the expression of FgCON7 or FgNDK1 effectively interferes with the fungal secondary metabolite synthesis pathway, significantly suppressing toxin production. The dual-target combined treatment group completely suppressed the DON toxin level to approximately 100 ppb, demonstrating the following core advantages.

[0039] In summary, this invention identified two pathogenicity-related genes, FgCON7 and FgNDK1, from *Fusarium graminearum*, and systematically verified their synergistic effects in regulating *Fusarium graminearum* growth, development, energy metabolism, toxin synthesis, and pathogenicity using RNAi technology. The dual-target strategy demonstrated superior synergistic effects compared to single-target strategies in inhibiting mycelial growth, conidia production, ATP synthesis, DON toxin production, and wheat plant infection. The dual-gene target combination and its application method provided by this invention offer important genetic resources and novel technical solutions for the green, efficient, and precise control of wheat scab, possessing significant agricultural application value and ecological security implications.

[0040] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The application of the combined FgCON7 and FgNDK1 genes in the preparation of agents for controlling wheat scab, characterized in that, dsRNA formulations targeting the FgCON7 and FgNDK1 genes were prepared separately, and the dsRNA formulations targeting the FgCON7 and FgNDK1 genes were simultaneously exogenously sprayed onto wheat plants. The gDNA sequence of the FgCON7 gene is shown in SEQ ID NO.1; the cDNA sequence of the FgCON7 gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the FgCON7 gene is shown in SEQ ID NO.3; the gDNA sequence of the FgNDK1 gene is shown in SEQ ID NO.4; the cDNA sequence of the FgNDK1 gene is shown in SEQ ID NO.5; and the amino acid sequence of the protein encoded by the FgNDK1 gene is shown in SEQ ID NO.

6.

2. A fungicide for controlling wheat scab, characterized in that, It is composed of dsRNA formulations of the FgCON7 and FgNDK1 genes.

3. The agent for controlling wheat scab according to claim 2, characterized in that: The concentration of the dsRNA formulations for both the FgCON7 and FgNDK1 genes was 20 ng / μL.

4. A method for controlling wheat scab, characterized in that, Includes the following steps: During the wheat growth process, the agent for controlling wheat scab as described in claim 2 or claim 3 is applied externally.

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