A pathogenic gene of an apple tree rot pathogen and its application and dsRNA

Abstract

This invention belongs to the field of genetic engineering technology and relates to a pathogenic gene of *V. mali*, the causal agent of apple tree rot, and its application and dsRNA. This invention discloses the pathogenic gene VmNRPS25 of *V. mali*, whose CDS region sequence is shown in SEQ ID NO: 1. Inhibiting the expression of the pathogenic gene VmNRPS25 in *V. mali* reduces the biomass of *V. mali* in the host plant and weakens the virulence of *V. mali*. This invention further constructs a dsRNA around the pathogenic gene VmNRPS25 of *V. mali*, which induces silencing of the pathogenic gene VmNRPS25 in *V. mali* through exogenous spraying, and can be used for the prevention and control of *V. mali* and its pathogenic plant diseases in host plants.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a pathogenic gene of apple tree rot fungus and its application and dsRNA. Background Technology

[0002] Apple tree canker, caused by the fungus *Valsa mali* (V. mali), can lead to tree death and orchard destruction in severe cases. Currently, control relies primarily on chemical pesticides, supplemented by agricultural and biological methods. However, the long-term use of chemical pesticides has resulted in numerous problems, including toxic side effects on humans and animals, pesticide residues, environmental pollution, and increased pathogen resistance. Therefore, there is an urgent need to develop new, highly effective, low-toxicity, and environmentally friendly pesticides.

[0003] dsRNA (double-stranded RNA) is part of the naturally occurring RNA interference (RNAi) process in plants, animals, and fungi. In RNAi, dsRNA molecules suppress gene expression by binding to a matching sequence of the target messenger RNA (mRNA). Therefore, dsRNA targeting key genes in pathogens can be synthesized in vitro and silencing can be induced by exogenous spraying. dsRNA can be introduced into animals and cells via injection, electroporation, and chemically mediated transfection. dsRNA preparation is a crucial step in RNAi. Currently, there are two main methods for dsRNA preparation: in vitro transcription and in vivo vector expression. In vitro transcription synthesizes dsRNA by attaching transcription start sites (such as T7, SP6, etc.) to both ends of the intended PCR product, first using RNA polymerase to transcribe single-stranded RNA, and then complementing it to form dsRNA. In contrast, for preparing large quantities of dsRNA, in vivo vector expression, i.e., constructing plasmids, is more economical and efficient for expression in bacteria. Hairpin-loop dsRNAs consist of two inversely complementary target sequence fragments with a single-stranded loop inserted in between. Constructing such DNA templates typically requires a three-step ligation process (three-step cloning). As a type of biopesticide, dsRNAs offer advantages over chemical pesticides, including environmental friendliness and a lower likelihood of inducing resistance. Their application in fungal control is an emerging and promising field. Summary of the Invention

[0004] The purpose of this invention is to design dsRNA agents targeting the pathogenic gene VmNRPS25 of *Aureobasidium cankerum*, aiming to provide new ideas and technical reserves for the green control of apple tree canker. To this end, this invention provides a pathogenic gene of *Aureobasidium cankerum*, its application, and dsRNA to address this need in the field.

[0005] On the one hand, the present invention relates to the application of the pathogenic gene VmNRPS25 of apple tree rot fungus in the control of plant diseases. The CDS region sequence of the pathogenic gene VmNRPS25 of apple tree rot fungus is shown in SEQ ID NO: 1. It inhibits the expression of the pathogenic gene VmNRPS25 of apple tree rot fungus in V. mali, reduces the biomass of V. mali in the host plant, and weakens the virulence of V. mali.

[0006] SEQ ID NO: 1 is as follows,

[0007]

[0008] Furthermore, in the application provided by the present invention, the non-conserved region of the pathogenic gene VmNRPS25 of the apple tree rot pathogen is shown in SEQ ID NO: 2.

[0009] SEQ ID NO: 2 is as follows,

[0010]

[0011] Furthermore, in the application provided by this invention, the host plant is apple, and the plant disease is apple tree rot.

[0012] On the other hand, the present invention relates to a dsRNA that inhibits the expression of the pathogenic gene VmNRPS25 of apple tree rot fungus, the CDS region sequence of which is shown in SEQ ID NO: 1.

[0013] Furthermore, in the dsRNA that inhibits the expression of the pathogenic gene VmNRPS25 of apple tree rot fungus provided by the present invention, the nucleotide sequence of the template DNA of the dsRNA is shown in SEQ ID NO: 2.

[0014] On the other hand, the present invention relates to the application of the dsRNA that inhibits the expression of the pathogenic gene VmNRPS25 of apple tree rot fungus in the preparation of V. mali control products.

[0015] Furthermore, in the application provided by this invention, the V. mali control product reduces the biomass of V. mali in the host plant.

[0016] Furthermore, in the application provided by this invention, the V. mali prevention product weakens the toxicity of V. mali.

[0017] Furthermore, in the application provided by the present invention, the pathogen causing the apple tree rot disease is V. mali.

[0018] On the other hand, the present invention relates to a method for preventing and controlling apple tree rot disease, comprising: the pathogen of apple tree rot disease is V. mali, and introducing dsRNA that inhibits the expression of the pathogenic gene VmNRPS25 of apple tree rot disease into V. mali.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] This invention utilizes dsRNA (double-stranded RNA) technology to induce the silencing of the pathogenic gene VmNRPS25 in *V. mali*, the causal agent of apple tree rot, thereby controlling apple tree rot. Compared to traditional chemical pesticides, dsRNA, as a biological pesticide, is environmentally friendly and less prone to developing resistance. This invention designs dsRNA targeting the specific pathogenic gene VmNRPS25 of *V. mali*, precisely targeting and inhibiting its expression, thereby effectively reducing the biomass of *V. mali* in the host plant, weakening its virulence, and achieving highly efficient control of apple tree rot. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the target gene knockout of the present invention.

[0023] Figure 2 This diagram shows the validation of VmNRPS25 gene knockout and reinstatement. In the diagram, A represents the four pairs of detections for the mutant: 03-8: wild-type V. mali strain; M: VmNRPS25 gene; G418: neo gene; K1: upstream fragment of the knockout cassette; K2: downstream fragment of the knockout cassette; B represents the Southern blot detection of the mutant; C represents the quantification of the VmNRPS25 gene; and D represents the PCR detection of the reinstatement strain.

[0024] Figure 3 Figure 1 shows the results of growth, sporulation, and pathogenicity testing of the ΔVmNRPS25 mutant. In the figure, A represents growth and sporulation; B represents growth data statistics; C represents sporulation data statistics; D represents pathogenicity testing of inoculated branches; E represents pathogenicity testing of inoculated leaves; F represents branch lesion data statistics; G represents leaf lesion data statistics; H represents fungal biomass in branch lesions; and I represents fungal biomass in leaf lesions.

[0025] Figure 4 The images show the electrophoretic detection of in vitro synthesized dsRNA. In the images, A represents the electrophoretic detection of GFP-dsRNA; and B represents the electrophoretic detection of VmNRPS25-dsRNA.

[0026] Figure 5 This image shows the results of laser confocal microscopy detection of GFP-dsRNA uptake by V. mali hyphae and protoplasm.

[0027] Figure 6 The growth detection results of V. mali after exogenous application of VmNRPS25-dsRNA are shown in the figure. In the figure, A is the result of plate test; B is the statistical result of colony diameter.

[0028] Figure 7The results of pathogenicity assays for V. mali after exogenous application of VmNRPS25-dsRNA are shown in the figure. A represents a photograph of the pathogenicity assay on branches; B represents the statistical results of lesion size in the branch pathogenicity assay; C represents the relative biomass of V. mali in the branch pathogenicity assay; D represents the relative expression level of VmNRPS25 in the branch pathogenicity assay; E represents a photograph of leaf lesions; F represents the statistical results of lesion size in the leaf lesion assay; G represents the relative biomass of V. mali in the leaf lesion assay; and H represents the relative expression level of VmNRPS25 in the leaf lesion assay. Detailed Implementation

[0029] The technical solution of the present invention will be described below with reference to embodiments; however, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0030] The test materials in the following examples were sourced from the following sources:

[0031] (1) Test culture medium and reagents

[0032] PDA medium (1L):

[0033] 200g of potatoes

[0034] 20g of glucose

[0035] 15g of agar

[0036] Peel and cut the potatoes into small pieces, boil them in double-distilled water for 30 minutes, then filter them through four layers of gauze into a 1L measuring cup, add glucose and stir well, bring the volume to 1L with double-distilled water, and autoclave at 121℃ for 21 minutes.

[0037] YEPD medium (1L):

[0038] 10g of yeast extract

[0039] 10g of tryptone

[0040] 20g of glucose

[0041] Dissolve in double-distilled water and bring the volume to 1L. Autoclave at 121℃ for 21 minutes.

[0042] TB3 medium (1L):

[0043] 3g of yeast extract

[0044] 3g of acid-hydrolyzed casein

[0045] 200g of sucrose

[0046] Dissolve in double-distilled water and bring the volume to 1L. Autoclave at 121℃ for 21 minutes.

[0047] 1.2M KCl solution:

[0048] KCl 89.5g

[0049] Dissolve in double-distilled water and bring the volume to 1L. Autoclave at 121℃ for 21 minutes.

[0050] STC buffer:

[0051] 200g of sucrose

[0052] 0.5M Tris-HCl (pH 8.0) 100mL

[0053] CaCl2·2H2O 7.35g

[0054] Dissolve in double-distilled water and bring the volume to 1L. Autoclave at 121℃ for 21 minutes.

[0055] PTC solution:

[0056] PEG 8000 200g

[0057] Dissolve in double-distilled water and bring the volume to 1L. Autoclave at 121℃ for 21 minutes.

[0058] (2) Test strains

[0059] Wild-type strain 03-8 of *V. mali*, the causal agent of apple tree rot, was isolated and preserved by the Fruit Tree Disease Pathogenic Biology and Integrated Management Research Team of the College of Plant Protection, Northwest A&F University. The ΔVmNRPS25 knockout mutant and the ΔVmNRPS25-C complement strain of *V. mali* were obtained and preserved by the same team.

[0060] (3) Test reagents

[0061] GFP-dsRNA and VmNRPS25-dsRNA (dsRNA agent) were synthesized and purified by the Fruit Tree Disease Pathogen Biology and Integrated Management Research Team of the College of Plant Protection, Northwest A&F University.

[0062] (4) Test materials

[0063] Healthy one-year-old branches of Fuji apple (Malus domestica borkh.cv. 'Fuji') were collected from Xintiandi Orchard in Yangling District, Xianyang City, Shaanxi Province to determine the pathogenicity of pathogens. Fuji apple tissue-cultured seedlings were grown into complete plants after approximately 30 days of alternating light and dark conditions at 25℃ for 16 / 8 hours. Leaves were used to determine the pathogenicity of pathogens.

[0064] (5) Test primers

[0065] The test primers are shown in Table 1 below, all of which were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0066] Table 1. Test primers

[0067]

[0068]

[0069] Example 1

[0070] This embodiment provides gene knockout in V. mali and the construction of VmNRPS25-dsRNA.

[0071] (1) Gene knockout

[0072] Reference Figure 1 The schematic diagram shows the design of specific primers for VmNRPS25 gene knockout: VmNRPS25-AF / AR, VmNRPS25-BF / BR, VmNRPS25-K1F / K1R, and VmNRPS25-K2F / K2R, as well as the detection primer G418-F / R for replacing the target gene, neo. Using genomic DNA from the wild-type V. mali strain 03-8 as a template, approximately 1500 bp fragments of the upstream (L) and downstream (R) segments of the VmNRPS25 gene were amplified. The full-length sequence of the neo gene was amplified using the pFL2 plasmid as a template. The L, R, and neo gene fragments were fused using a three-step double-joint PCR reaction to construct the VmNRPS25 gene knockout cassette.

[0073] Wild-type *V. mali* strain 03-8 was inoculated into 100 mL of YEPD medium and incubated at 25°C and 110 rpm for 48 h. Mycelia were then collected using sterile filter cloth. The mycelia were repeatedly rinsed with 1.2 M KCl solution and squeezed to remove most of the water. A compound enzymatic hydrolysate containing lysis enzyme and decomposition enzyme was prepared at a dosage of 10 mL of hydrolysate per 0.7 g of mycelia. The mycelia were placed in the hydrolysate and enzymatically hydrolyzed at 30°C and 100 rpm for approximately 2 h, until all protoplasmic spheres were visible under a microscope. The protoplasm was diluted to 1 × 10⁻⁶ with STC Buffer. 7After dispensing 200 μL of the knockout fragment per mL, add 5-10 μg of the knockout cassette fragment to the protoplasm. Incubate at room temperature for 20 min, then slowly add 1 mL of PTC buffer. Incubate for another 20 min, then add 5 mL of TB3 medium. Incubate at 25°C and 110 rpm for 36-48 h until visible mycelia appear. Transfer all the cultured mycelia to 100 mL of TB3 medium at approximately 50°C, and add 70 μg / mL of genimycin G418. Invert the plate after 12 h. Repeat this process after 3-4 days of incubation at 25°C in the dark. Pick transformants and plate them on PDA plates containing 70 μg / mL genimycin for further validation.

[0074] Wild-type *V. mali* strain 03-8 was inoculated into 100 mL of YEPD medium and incubated at 25°C and 110 rpm for 48 h. Mycelia were then collected using sterile filter cloth. The mycelia were repeatedly rinsed with 1.2 M KCl solution and squeezed to remove most of the water. A compound enzymatic hydrolysate containing lysis enzyme and decomposition enzyme was prepared at a dosage of 10 mL of hydrolysate per 0.7 g of mycelia. The mycelia were placed in the hydrolysate and enzymatically hydrolyzed at 30°C and 100 rpm for approximately 2 h, until all protoplasmic spheres were visible under a microscope. The protoplasm was diluted to 1 × 10⁻⁶ with STC Buffer. 7 After dispensing 200 μL of the knockout fragment per mL, add 5-10 μg of the knockout cassette fragment to the protoplasm. Incubate at room temperature for 20 min, then slowly add 1 mL of PTC buffer. Incubate for another 20 min, then add 5 mL of TB3 medium. Incubate at 25°C and 110 rpm for 36-48 h until visible mycelia appear. Transfer all the cultured mycelia to 100 mL of TB3 medium at approximately 50°C, and add 70 μg / mL of genimycin G418. Invert the plate after 12 h. Repeat this process after 3-4 days of incubation at 25°C in the dark. Pick transformants and plate them on PDA plates containing 70 μg / mL genimycin for further validation.

[0075] Genomic DNA from all mutants was analyzed by PCR using the VmNRPS25-MF / MR primers to screen for transformants lacking the target gene. Then, four primer pairs were used for PCR detection: VmNRPS25-MF / MR (to detect the absence of the target gene VmNRPS25), VmNRPS25-K1F / K1R (to detect downstream recombination fragments of the target gene), VmNRPS25-K2F / K2R (to detect upstream recombination fragments of the target gene), and G418-F / R (to detect neo gene insertion). Simultaneously, positive transformants were detected by Southern blot using digoxigenin-labeled DNA and a Roche detection kit.

[0076] More than 200 transformants were validated using PEG-mediated protoplast transformation, and one ΔVmNRPS25 knockout mutant was successfully obtained through four pairs of PCR detection, Southern blot, and quantitative verification. Figure 2 A, Figure 2 B, Figure 2 C).

[0077] (2) Gene complementation

[0078] The complement primer VmNRPS25-CF / CR was designed to amplify the full-length sequence of the target gene VmNRPS25 from 1000-1500 bp upstream of the start codon to before the stop codon. The amplified complemented target gene sequence was then combined with a linearized PDL2 vector and homologous recombinase to construct a recombinant vector carrying the target gene VmNRPS25. Protoplasm of the VmNRPS25 deletion mutant was prepared, and the recombinant protoplasm used for complementation was transformed into the protoplasm. Hygromycin was used for resistance selection, and then PCR detection was performed using primers VmNRPS25-5F and VmNRPS25-6R. Strains that tested correctly were identified as complemented strains of the VmNRPS25 deletion mutant.

[0079] Multiple ΔVmNRPS25-C complemented strains were obtained by PCR detection. Figure 2 D). Select one ΔVmNRPS25-C replacement strain for subsequent experiments.

[0080] (3) Growth and sporulation of the strain

[0081] Wild-type V. mali 03-8 and its mutant strain were inoculated onto PDA plates and activated at 25°C for 48 hours. Mycelial discs from the colony edges were punched using a 5mm diameter punch, and these discs from different strains were inoculated onto PDA plates. One group was incubated upside down at 25°C for 3 days, and colony morphology was observed; colony diameter was measured using the cross-crossing method. Another group was incubated at 25°C for 30 days, and sporulation was observed and photographed.

[0082] Colony growth assays revealed that the ΔVmNRPS25 mutant was indistinguishable from the wild-type 03-8 and ΔVmNRPS25-C strains, indicating that the absence of VmNRPS25 does not affect growth. Figure 3 A, Figure 3 B). However, the absence of VmNRPS25 resulted in approximately 57% of the sporulation yield of the wild type, significantly reducing sporulation output. Figure 3 A, Figure 3 C). This indicates that the absence of VmNRPS25 significantly affects sporulation.

[0083] (4) Pathogenicity detection

[0084] Healthy, intact, and uniformly sized one-year-old Fuji apple branches were collected from the orchard. The branches were cut into 12cm sections and disinfected with a 0.6% sodium hypochlorite solution before use. Activated *V. mali* wild-type 03-8 and mutant strains were extracted using a 5mm punch. Mycelial cakes were then picked up with a sterile needle and applied upside down to the wounds on the branches. The inoculated apple branches were placed in trays with water at the bottom, sealed with plastic wrap, and incubated at 25℃ for 3 days. The diameter of the lesions was measured and photographed. Leaves from Fuji apple tissue culture seedlings were inserted onto 1% water agar plates. Activated wild-type 03-8 and mutant strains were extracted using a 5mm punch. Mycelial cakes were picked up with a sterile needle and applied upside down to the wounds on the leaves. After incubation at 25℃ for 36 hours, photographs were taken and the lesion area was statistically analyzed using ImageJ. All experiments were performed in triplicate (three biological replicates and three technical replicates).

[0085] Three days after inoculating apple branches and leaves with wild-type V. mali 03-8, ΔVmNRPS25 mutant, and ΔVmNRPS25-C strain, it was found that branches and leaves inoculated with ΔVmNRPS25 mutant only developed the disease at the inoculation point, showing a significant reduction in pathogenicity compared to the wild type. The pathogenicity of ΔVmNRPS25-C strain was no different from that of wild-type 03-8 strain, indicating that the change in pathogenicity is caused by the absence of VmNRPS25. Figure 3 D, Figure 3 E, Figure 3 F, Figure 3 G).

[0086] (5) Fungal biomass determination

[0087] Genomic DNA was extracted from the mycelium of *V. mali*, the causal agent of apple tree rot, using a polysaccharide-polyphenol plant genomic DNA extraction kit. The genomic DNA concentrations were diluted to 100, 75, 50, 20, 10, and 5 ng / μL, respectively. DNA was extracted from branches and leaves inoculated with wild-type *V. mali* 03-8, the ΔVmNRPS25 mutant, and the ΔVmNRPS25-C complement strain. The DNA from each treatment group was diluted to the same concentration. The Ct values ​​of *V. mali* VmG6PDH in each DNA solution were detected using quantitative real-time PCR. A regression curve was constructed by plotting the Ct values ​​of the *V. mali* genomic DNA on the x-axis and the DNA solution concentration (Log10) on the y-axis, and the regression equation was calculated. The Ct values ​​of each group were substituted into the regression equation to calculate the DNA concentration in the sample, and the relative biomass was calculated. The biomass assay was repeated three times.

[0088] Fungal biomass measurements revealed that the fungal biomass of branches and leaves inoculated with the ΔVmNRPS25 mutant was significantly lower than that of the wild-type 03-8 and ΔVmNRPS25-C strains. Figure 3 H, Figure 3 I). In summary, VmNRPS25 is an important virulence factor of V. mali and a potential drug target site.

[0089] Example 2

[0090] This embodiment provides the construction and application effects of VmNRPS25-dsRNA.

[0091] (1) In vitro synthesis of fluorescently labeled GFP-dsRNA

[0092] First, a template containing the T7 promoter was amplified. Using primers GFP-T7F / GFP-R and GFP-F / GFP-T7R, fragments with the T7 promoter at the 5' end of the sense strand and the antisense strand, respectively, were amplified and used as templates. Fluorescein-labeled GFP-dsRNA was transcribed using T7 RNAploymerase and Fluorescein RNA Labeling Mix. The PCR reaction system is shown in Table 2. The reaction was carried out at 37°C for 2 hours, followed by the addition of 2 μL of DNase I and incubation at 37°C for 15 minutes. Finally, 2 μL of 0.2 MEDTA was added to stop the reaction.

[0093] Table 2. Reaction system for in vitro synthesis of fluorescein-labeled GFP-dsRNA

[0094]

[0095]

[0096] The dsRNA of the universal gene GFP was successfully synthesized in vitro, with a band size of 720 bp. Figure 4 A).

[0097] (2) In vitro synthesis of VmNRPS25-dsRNA

[0098] Fragments with the T7 promoter at the 5' end of the sense strand and the antisense strand were amplified using primers VmNRPS25-T7F / VmNRPS25-R and VmNRPS25-F / VmNRPS25-T7R, respectively. These fragments were then synthesized in vitro using the Novizan T7 RNAi Transcription Kit. The PCR reaction system is shown in Table 3. The reaction was carried out at 37°C for 2 hours. The synthesized dsRNA was treated with Dnase I / RNase T1 and purified to remove residual DNA template and single-stranded RNA. Further purification of the purified dsRNA was performed using magnetic beads. Finally, the absorbance of the product A260 was measured to determine its concentration, and the product was stored at -20°C.

[0099] Table 3. Reaction system for in vitro synthesis of dsRNA

[0100] Components volume DNase I 1μL RNase T1 2μL 10x Transcription buffer 2μL Transcription template 1 1-4μL Transcription template 2 1-4μL <![CDATA[RNA free H2O]]> Add to 20μL

[0101] dsRNA with a band size of 489 bp in the non-conserved region of the target gene VmNRPS25 was successfully synthesized in vitro. Figure 4 B).

[0102] (3) Confocal microscopy observation of the uptake of dsRNA by V. mali hyphae and protoplasts

[0103] Wild-type *V. mali* 03-8 mycelial pellets were inoculated onto glass slides containing PDA medium. 20 μL of 50 ng / μL fluorescein-labeled GFP-dsRNA was added to the mycelial pellets. The mixture was incubated at 25°C for 12 h, and before observation, it was treated with 75 U nuclease (MNase) at 37°C for 30 min. Fluorescence signals were analyzed using an LSM880 confocal microscope. Alternatively, fluorescein-labeled GFP-dsRNA was added to *V. mali* mycelium YEPD liquid medium. After 48 h of incubation, the mycelium was collected and protoplasts were prepared. After MNase treatment for 30 min, fluorescence signals were analyzed using an LSM880 confocal microscope.

[0104] To demonstrate whether *V. mali* hyphae can absorb dsRNA, fluorescein-labeled GFP-dsRNA was applied to *V. mali* hyphae grown on PDA medium. After incubation for 12 h, followed by treatment with MNase for 30 min, fluorescence signal accumulation was observed within the *V. mali* hyphae. Figure 5 A). To further confirm that dsRNA enters the cells of *V. mali*, rather than in the space between the cell membrane and cell wall, GFP-dsRNA labeled with fluorescein was added to *V. mali* mycelium in YEPD liquid medium and cultured for 48 hours. After preparing protoplasts and treating them with MNase, the fluorescence signal within the *V. mali* protoplasts was still clearly visible. Figure 5 B). This indicates that the hyphae and protoplasts of V. mali can absorb dsRNA.

[0105] (4) Application effect of VmNRPS25-dsRNA

[0106] 20 μL of GFP-dsRNA and VmNRPS25-dsRNA at a concentration of 50 ng / μL were spread onto PDA plates using a 5 mm punch. Activated wild-type 03-8 mycelial pellets were collected and inoculated onto the PDA plates coated with dsRNA. After incubation at 25°C upside down for 2 days, colony morphology was observed, and colony diameter was measured using the cross-hatching method. 20 μL of GFP-dsRNA and VmNRPS25-dsRNA at a concentration of 50 ng / μL were respectively applied to wounds on apple branches and leaves. Wild-type 03-8 mycelial pellets with diameters of 5 mm and 2 mm were then inoculated at the same locations where dsRNA was applied. After incubation at 25°C for 3 days and 36 hours respectively, the diameter of the lesions was measured and photographed. All experiments were performed in triplicate (three biological replicates and three technical replicates).

[0107] DNA was extracted from branches and leaves of *V. mali* wild-type 03-8 co-inoculated with GFP-dsRNA and VmNRPS25-dsRNA, respectively, and the DNA from each treatment group was diluted to the same concentration. The fungal biomass of different treatments was determined using quantitative real-time PCR. The disease-health interface of branches co-inoculated with GFP-dsRNA, VmNRPS25-dsRNA, and *V. mali* wild-type 03-8 was flash-frozen in liquid nitrogen and ground into powder. RNA was extracted from the samples according to the instructions of the Plant Tissue Total RNA Extraction Kit (Plant RNA Extraction Kit 3.0, Huayueyang), and the obtained RNA was stored at -80℃ for later use. The RNA was reverse transcribed into cDNA using a reverse transcription kit (Thermo Scientific RevertAid First Strand cDNA Synthesis Kit), and stored at -20℃ for later use. Quantitative real-time PCR was performed using *V. mali* rot pathogen VmG6PDH as an internal control, utilizing 2... -ΔΔCt The formula was used to calculate the gene expression level of VmNRPS25 in different samples. The gene expression assay was performed using three biological weights and repeated three times.

[0108] 20 μL of GFP-dsRNA and VmNRPS25-dsRNA at a concentration of 50 ng / μL were separately spread onto PDA plates. Activated *V. mali* wild-type 03-8 bacterial discs were then punched using a 5 mm diameter punch and inoculated onto the PDA plates coated with dsRNA. Results showed no significant difference in growth between the different treatments. Figure 6 A, Figure 6B) indicates that exogenous application of VmNRPS25-dsRNA does not affect the vegetative growth of V. mali mycelia. 20 μL of GFP-dsRNA and VmNRPS25-dsRNA at a concentration of 50 ng / μL were applied exogenously to the wound surfaces of apple branches and leaves, respectively. Then, V. mali wild-type 03-8 mycelial cakes were inoculated at the same locations on the branches and leaves where dsRNA was applied. Pathogenicity results showed that, compared to wild-type 03-8 co-inoculated with GFP-dsRNA, wild-type 03-8 co-inoculated with VmNRPS25-dsRNA exhibited smaller lesion areas. Figure 7 A, Figure 7 B, Figure 7 E, Figure 7 F). Fungal biomass assay results showed that the biomass of *V. mali* after exogenous application of VmNRPS25-dsRNA was significantly lower than that after application of GFP-dsRNA. Figure 7 C, Figure 7 G). qRT-PCR analysis revealed that, compared to exogenous application of GFP-dsRNA, exogenous application of VmNRPS25-dsRNA significantly reduced the expression level of VmNRPS25 in V. mali. Figure 7 D, Figure 7 In summary, exogenous application of VmNRPS25-dsRNA inhibited the expression of VmNRPS25 in V. mali, reduced the biomass of V. mali, and weakened the virulence of V. mali.

[0109] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Inhibition of the pathogenicity gene of Valsa mali VmNRPS25 application in the control of plant diseases, characterized in that, The pathogenic gene of the apple tree rot fungus VmNRPS25 The CDS region sequence is shown in SEQ ID NO: 1, and the plant disease is *Pseudomonas aeruginosa* (Apple black rot fungus). Valsa mali , V.mali Apple tree rot caused by ) inhibition V.mali The pathogenic gene of the apple tree rot fungus described inside VmNRPS25 The expression of [something] is reduced in the host plant. V.mali biomass, weaken V.mali Its toxicity.

2. Inhibition of the pathogenicity gene of Valsa mali VmNRPS25 the dsRNA expressed in the preparation of V.mali for use in the control of products, characterized in that, The pathogenic gene of the apple tree phytophthora cactorum VmNRPS25 The CDS region sequence of the pathogenic gene is shown as SEQ ID NO:

1.

3. The pathogenic gene of Valsa mali according to claim 2, which is inhibited. VmNRPS25 the dsRNA expressed in the preparation of V. mali for use in the control of products, characterized in that, The nucleotide sequence of the template DNA of the dsRNA is shown as SEQ ID NO: 2.

Images