A DNA pesticide, its preparation method and application

Abstract

The application belongs to the technical field of biological control of agriculture, and particularly relates to a DNA pesticide as well as a preparation method and application thereof. The application provides a DNA pesticide, which comprises a nano-carrier and a genome of a fungal virus; the fungal virus is a plant pathogenic fungal virus, and can cause pathogenicity decline of a host fungus. The application uses the nano-carrier to load the genomic DNA of the fungal virus, so as to deliver the genomic DNA of the fungal virus to a plant pathogenic fungus, make the genomic DNA of the fungal virus replicate, transcribe and translate in the plant pathogenic fungus, and form a mature virus, thereby causing pathogenicity decline of the plant pathogenic fungus, and achieving the prevention and treatment of fungal diseases.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biological control technology, specifically relating to a DNA pesticide, its preparation method, and its application. Background Technology

[0002] According to statistics from the Food and Agriculture Organization of the United Nations, global crop losses due to diseases reach 20-40% annually, with fungi causing over 80% of crop diseases. Fungal diseases not only reduce crop yields but also damage the quality of agricultural products. The mycotoxins produced when infecting crops pose a serious threat to human and animal health. Currently, the control of plant diseases mainly relies on chemical pesticides, but long-term use of chemical pesticides leads to the high frequency of pesticide-resistant strains and environmental pollution. Therefore, it is imperative to vigorously develop safe alternative control measures.

[0003] Fungal viruses are a class of viruses that infect fungi, causing severe growth restriction and decreased pathogenicity in host fungi, thus representing a potential resource for biocontrol. However, the complex nutritional compatibility of fungal communities limits the spread of viruses among fungi. Currently, live mycelial preparations made from attenuated strains of fungal viruses can be used to control plant fungal diseases. However, the short shelf life of live mycelial preparations restricts their application. Therefore, there is an urgent need in this field to innovate application technologies to overcome this transmission limitation, achieve disease control, and promote the development of sustainable disease management strategies. Summary of the Invention

[0004] The purpose of this invention is to provide a DNA pesticide, its preparation method, and its application. The DNA pesticide can deliver fungal viral DNA to pathogenic fungi, causing it to replicate, transcribe, translate, and form mature viruses within the fungi, thereby inducing the decline of pathogenicity in highly virulent strains and achieving the control of fungal diseases.

[0005] This invention provides a DNA pesticide comprising a nanocarrier and genomic DNA of a fungal virus; the fungal virus is a plant pathogenic fungal virus that can cause a decline in the pathogenicity of the host fungus.

[0006] As a preferred embodiment, the mass ratio of the nanocarrier to the genomic DNA of the fungal virus is 10:1 to 100:1.

[0007] As a preferred embodiment, the fungal virus includes at least one of the following: Sclerotinia sclerotiorum attenuated viral-associated DNA virus 1 (SsHADV-1), Fusarium graminearum three-component virus type 1 (FgGMTV1), Botrytis cinerea DNA virus BGDaV1, Botrytis cinerea DNA virus BGDaV2, and Soybean pseudostem spot circular DNA virus type 1.

[0008] As a preferred embodiment, the nanocarrier comprises at least one of the following: mesoporous silica nanoparticles, carbon nanotubes, carbon quantum dots, and chitosan.

[0009] The present invention also provides a method for preparing the DNA pesticide described above, comprising the following steps: mixing the nanocarrier and the genomic DNA of the fungal virus, incubating, and obtaining the DNA pesticide.

[0010] As a preferred embodiment, the incubation is a rotary incubation; the rotation speed is 30~60 r / min; and the incubation time is 30~60 min.

[0011] The present invention also provides the application of the DNA pesticide described in the above scheme in inhibiting fungal infection.

[0012] This invention also provides the application of the DNA pesticide described above in the prevention and control of fungal diseases.

[0013] As a preferred embodiment, the fungal disease includes at least one of the following: sclerotinia rot, Fusarium head blight, gray mold, and twig blight.

[0014] The present invention also provides a method for preventing and controlling fungal diseases, comprising the following steps: spraying the DNA pesticide described in the above scheme onto the plant.

[0015] Beneficial Effects: This invention provides a DNA pesticide comprising a nanocarrier and genomic DNA of a fungal virus; the fungal virus is a plant pathogenic fungal virus that can cause a decline in the pathogenicity of the host fungus. The nanocarrier of this invention possesses good environmental compatibility, degradability, and low cytotoxicity, and can load and protect nucleic acid molecules; the fungal virus can infect plant pathogenic fungi, transforming highly pathogenic strains into attenuated strains, thus reducing their pathogenicity. This invention utilizes a nanocarrier to load the genomic DNA of a fungal virus, delivering the genomic DNA of the fungal virus to the plant pathogenic fungus, allowing it to replicate, transcribe, and translate within the pathogenic fungus to form a mature virus, leading to a decline in the pathogenicity of the pathogenic fungus and its spread within the pathogenic fungal population, thereby achieving the control of fungal diseases.

[0016] This invention also provides a method for preparing DNA pesticides, comprising the following steps: mixing a nanocarrier with the genomic DNA of a fungal virus, incubating, and obtaining the DNA pesticide. The preparation method of this invention is simple to operate, and the nanocarrier can effectively protect the DNA of the fungal virus from DNase degradation, providing a longer window period for the application of DNA pesticides in the field.

[0017] This invention also provides the application of DNA pesticides in inhibiting fungal infection and in controlling fungal diseases. The DNA pesticides described in this invention can transfect fungi, causing them to replicate, transcribe, and translate within the fungi to form mature viruses, inducing a decline in the pathogenicity of highly virulent strains, thus achieving in vitro infection of fungal viral genomic DNA.

[0018] This invention also provides a method for controlling fungal diseases, comprising the following steps: spraying the DNA pesticide onto the plant. Spraying the DNA pesticide onto the plant enables the delivery of fungal viral DNA into the fungal cells, thereby inducing a decline in the pathogenicity of highly virulent strains, establishing an effective biocontrol barrier on the plant surface, and achieving the control of fungal diseases through the spread of the fungal virus within the pathogenic fungal population. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 The images show the results of packaging SsHADV-1 with the nanomaterial MON-NH2. A shows the loading ratio of SsHADV-1 genomic DNA in MON-NH2 packaging, indicating that a 10:1 packaging ratio fully loads the genomic DNA of SsHADV-1. B shows the DNA degradation results, with "-" indicating no exogenous DNase I addition and "+" indicating exogenous DNase I addition. C is a scanning electron microscope image of SsHADV-1 genomic DNA after MON-NH2 packaging; the left image shows the unpackaged nanomaterial with many pores, while the right image shows the fully loaded nanomaterial with no pores after packaging. Figure 2 Figures showing the colony morphology and growth rate of *Sclerotinia sclerotiorum* treated with MON-NH2@SsHADV-1; where A is the colony morphology of *Sclerotinia sclerotiorum* and B is the mycelial growth rate of *Sclerotinia sclerotiorum*. Figure 3 Sclerotium sclerotiorum strains delivered with MON-NH2@SsHADV-1 Rep The relative expression level of genes; Figure 4 A graph showing the detection of CP protein of SsHADV-1 in *Sclerotium sclerotiorum* strains treated with MON-NH2@SsHADV1. Figure 5 Horizontal propagation of SsHADV-1 in *Sclerotinia sclerotiorum* strains treated with MON-NH2@SsHADV1; where A shows the morphology of *Sclerotinia sclerotiorum* strains in confrontation culture on PDA; and B shows the detection of the SsHADV-1 Rep gene in the progeny of the recipient strain after confrontation culture. Figure 6Figure showing the stability of DNA carried by the MON-NH2@SsHADV-1 formulation in Arabidopsis thaliana; Figure 7 The MON-NH2@SsHADV-1 formulation was used to prevent sclerotinia thaliana disease; where A represents the disease incidence of Arabidopsis leaves 48 h after inoculation with Sclerotinia thaliana; and B represents the area of ​​lesions on Arabidopsis leaves (48 hpi). Figure 8 Results of using MON-NH2@SsHADV-1 formulation to prevent Sclerotinia sclerotiorum rot in rapeseed; A shows the disease incidence 48 h after inoculation of rapeseed cotyledons with Sclerotinia sclerotiorum; B shows the expansion of lesions on rapeseed cotyledons. Figure 9 The results of spraying MON-NH2@SsHADV-1 formulation to prevent sclerotinia stem rot in rapeseed; where A represents the growth status of rapeseed 60 h after inoculation; B represents the growth status of rapeseed 7 days after inoculation; and C represents the survival rate of rapeseed. Figure 10 The study investigated the use of MON-NH2@SsHADV-1 formulation to treat sclerotinia sclerotinia disease in Arabidopsis thaliana. A represents the disease incidence of Arabidopsis plants in vivo inoculated with Sclerotinia sclerotinia 1–7 days after treatment; B represents the area of ​​lesions on Arabidopsis leaves after spraying; and C represents the survival rate of the treated Arabidopsis plants. Figure 11 A map showing the regional distribution of rapeseed experimental plots during the full bloom stage in the field; where A is the natural distribution map of rapeseed plots during the full bloom stage in the field, and the red area is the treatment area of ​​this experiment; B is the distribution of field plots; Figure 12 The results of MON-NH2@SsHADV-1 formulation in controlling sclerotinia stem rot in rapeseed in the field; where A represents the number of diseased rapeseed plants in each group; and B represents the disease incidence rate. Figure 13 Figure 1 shows the effect of spraying MON-NH2@SsHADV-1 on rapeseed seedlings; A shows the growth status of rapeseed 14 days after treatment; B and C show the hypocotyl and fresh weight of rapeseed in each treatment, respectively. Detailed Implementation

[0021] This invention provides a DNA pesticide comprising a nanocarrier and genomic DNA of a fungal virus; the fungal virus is a plant pathogenic fungal virus that can cause a decline in the pathogenicity of the host fungus. This invention utilizes a nanocarrier to load the genomic DNA of a fungal virus, delivering the genomic DNA of the fungal virus to the plant pathogenic fungus, causing it to replicate, transcribe, and translate within the pathogenic fungus to form a mature virus, leading to a decline in the pathogenicity of the pathogenic fungus and achieving the control of plant fungal diseases.

[0022] Unless otherwise specified, all raw materials involved in this invention are obtained through conventional commercial methods.

[0023] Fungal viruses are a class of viruses that obligately parasitize fungi. Based on nucleic acid type, fungal viruses can be divided into fungal RNA viruses and fungal DNA viruses, with fungal RNA viruses accounting for over 90%. Some fungal viruses can severely restrict the growth of host fungi and reduce their pathogenicity. Preparing fungal viruses into live mycelial preparations allows them to spread among pathogenic fungi and is used to control plant fungal diseases. The nucleic acids of fungal viruses can also achieve the same function. Delivering the nucleic acid of a fungal virus to plant pathogenic fungi allows it to replicate, transcribe, and translate within the pathogen, forming a mature virus that reduces the pathogenicity of the fungus, thus controlling plant fungal diseases. However, RNA is unstable in the environment and easily degrades, while DNA is more stable due to its double helix structure. Furthermore, in the preparation process, DNA can be obtained in large quantities through PCR amplification, while RNA requires reverse transcription, which is complex and costly. Therefore, using the genomic DNA of fungal viruses as a biocontrol resource has the advantages of structural stability and low preparation cost. Currently, several fungal DNA viruses that can weaken the pathogenicity of pathogenic fungi and can be used as biological control resources have been discovered, such as Sclerotinia sclerotiorum attenuated viral DNA virus 1 (SsHADV-1), Fusarium graminearum three-component virus type 1 (FgGMTV1), Botrytis cinerea DNA virus BGDaV1, Botrytis cinerea DNA virus BGDaV2, and Soybean pseudostem spot circular DNA virus type 1 (DsCDV1). The inventors discovered that the hypovirulence-associated DNA virus 1 (SsHADV-1) of Sclerotinia sclerotiorum has strong infectivity and can overcome the limitation of viral horizontal transmission caused by mycelial nutritional incompatibility, and can spread efficiently between strains with different nutritional affinity groups; its virus particles can directly infect the mycelium of Sclerotinia sclerotiorum (see Yu X, et al. A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(18): 8387-92).Fusarium graminearum tricomponent virus type 1 (FgGMTV1) can reduce the fungus's ability to infect host plants such as wheat by interfering with host virulence-related pathways (such as the expression of DON toxin synthesis gene clusters, the secretion of cell wall degrading enzymes, and the formation of infection structures); and can also lead to slower mycelial growth rate, abnormal colony morphology, and reduced sporulation (see Li PF, et al. A tripartite ssDNA mycovirus from a plant pathogenic fungus is infectious ascloned DNA and purified virions. Science Advances, 2020, 6(14): eaay9634). BGDaV1 and BGDaV2, isolated from the plant pathogen Botrytis cinerea, can weaken the virulence of the host pathogenic fungus and reduce its pathogenicity (see Khalifa ME, and MacDiarmid RM. A mechanically transmitted DNA mycovirus is targeted by the defence machinery of its host, Botrytiscinerea. Viruses, 2021, 13(7): 1315). Soybean stalk fungus circular DNA virus type 1 (DsCDV1) can significantly weaken the growth of the host fungus and eliminate its virulence (see Wang X, et al. A circular single-stranded DNA mycovirus infects plants and confers broad-spectrum fungal resistance. Molecular Plant, 2024, 17(6): 955-71). The nutritional compatibility of fungal communities is complex and diverse, and nutritional incompatibility is common among different strains, making hyphae difficult to fuse and preventing normal cytoplasmic communication, thus greatly limiting the horizontal transmission and utilization of fungal viruses among different strains. This invention uses a nanocarrier to load fungal virus DNA, which can deliver the genomic DNA of the fungal virus to plant pathogenic fungi (not limited to a certain nutritional affinity type), causing it to replicate, transcribe, translate and form a mature virus in the pathogenic fungi, resulting in a decline in the pathogenic fungi's pathogenicity and achieving the prevention and control of fungal diseases.

[0024] In a specific embodiment of the present invention, the selection of SsHADV-1 genomic DNA to construct DNA pesticides is merely illustrative and does not constitute a limitation on the scope of protection of the present invention.

[0025]

[0026] The genomic DNA of fungal viruses lacks infectivity and cannot effectively invade fungal cells. Therefore, this invention selects nanocarriers to load the genomic DNA of fungal viruses for delivery to pathogenic fungi. Nanocarriers are characterized by small particle size, large specific surface area, and controllable structure. They can efficiently load nucleic acids through electrostatic adsorption and physical encapsulation, protecting them from environmental degradation. They also possess good biocompatibility and cell penetration, facilitating targeted delivery and controlled release, significantly improving the stability, delivery efficiency, and applicability of nucleic acids. Numerous types of nanocarriers exist, such as mesoporous silica nanoparticles, carbon nanotubes, carbon quantum dots, and chitosan. In this specific embodiment, mesoporous silica nanoparticles were chosen as the nanocarrier for subsequent research, which does not constitute a limitation on the scope of protection of this invention.

[0027] Mesoporous silica nanoparticles are inorganic nanomaterials with good environmental compatibility, biodegradability, and low cytotoxicity. They can also load and protect large amounts of nucleic acid molecules, making them suitable for constructing nucleic acid delivery systems. As one embodiment, the mesoporous silica nanoparticles include mesoporous organic silica; the mesoporous organic silica has an extremely high specific surface area and a large pore volume, enabling it to load DNA and form a mesoporous nanoparticle-nucleic acid complex. As one embodiment, the pore size of the mesoporous organic silica is 20-80 nm. In specific embodiments of the present invention, the pore size of the mesoporous organic silica can be any value within the range of 20-80 nm, such as 20, 30, 40, 50, 60, 70, or 80 nm. In specific embodiments, the applicant has found that the 50 nm mesoporous organic silica nanocarrier MON-NH2 can efficiently load the genomic DNA of the extracellularly prepared virus SsHADV-1, forming a MON-NH2@SsHADV-1 complex. In one embodiment, the mesoporous organic silica comprises amino-based mesoporous silica nanoparticles (MON-NH2). Furthermore, the mesoporous organic silica of this invention optimizes the structure of traditional MSNs by introducing organic components. Amino-mediated electrostatic adsorption allows direct binding to negatively charged nucleic acids such as DNA, concentrating nucleic acid molecules from a loose, dispersed state into nanoparticles, forming small-volume, regularly shaped nanogene carrier complexes. While retaining its advantageous properties, this significantly improves the dispersion stability, controllable degradation performance, biocompatibility, and environmental responsiveness of the material system, which is beneficial for loading nucleic acids.

[0028] In a specific embodiment of this invention, SsHADV-1 genomic DNA was loaded onto mesoporous organic silica, establishing a cell-free delivery system for viral nucleic acid to control sclerotinia rot in rapeseed. This system overcomes the key bottleneck of fungal viral genomic DNA lacking infectivity and being unable to directly cross fungal cell walls and membranes to enter cells and restore the infection chain, while also addressing application constraints such as the easy degradation of nucleic acids outside cells and insufficient field persistence. The results of the embodiments show that the DNA pesticide can deliver SsHADV-1 genomic DNA to the plant pathogenic fungus *Sclerotinia sclerotiorum*, causing it to replicate, transcribe, and translate within *Sclerotinia sclerotiorum* to form a mature virus. This leads to a decline in the pathogenicity of *Sclerotinia sclerotiorum*, and the virus spreads within the *Sclerotinia sclerotiorum* population through SsHADV-1, thus achieving the control of sclerotinia rot.

[0029] In one embodiment, the mass ratio of the nanocarrier to the genomic DNA of the fungal virus is 10:1 to 100:1. In specific embodiments of the present invention, the mass ratio of the nanocarrier to the genomic DNA of the fungal virus is any ratio within the range of 10:1 to 100:1, such as 10:1, 15:1, 16:1, 20:1, 25:1, 30:1, 33:1, 35:1, 40:1, 45:1, 50:1, 60:1, 65:1, 70:1, 80:1, 90:1, or 100:1. Within this ratio range, the nanocarrier can be fully loaded with DNA and can effectively protect the DNA from DNase degradation.

[0030] In this invention, the DNA pesticide can be referred to as a nanoparticle-nucleic acid complex or a nanonucleic acid preparation.

[0031] The present invention also provides a method for preparing the DNA pesticide described above, comprising the following steps: mixing the nanocarrier and the genomic DNA of the fungal virus, incubating, and obtaining the DNA pesticide.

[0032] The types of nanocarriers described in this invention have been explained above and will not be repeated here.

[0033] This invention does not impose any special limitation on the source of the genomic DNA of the fungal virus; any publicly available sequence may be used. These publicly available sequences have been discussed above and will not be repeated here. As one embodiment, the genomic DNA of SsHADV-1 is isolated from *Sclerotinia sclerotiorum* strain DT-8. As another embodiment, the genomic DNA of SsHADV-1 is obtained using a rolling circle amplification method.

[0034] In a specific embodiment of the present invention, mesoporous silica nanoparticles (mesoporous organic silica) are used as a carrier to load fungal viral genomic DNA. As one embodiment, the mesoporous organic silica is mixed with water and ultrasonically dispersed to obtain a mesoporous organic silica suspension. As one embodiment, the mass-to-volume ratio of the mesoporous organic silica to water is 1 mg:1 mL; as one embodiment, the ultrasonic power is 40 kHz; the ultrasonic temperature is 25°C; and the ultrasonic time is 15 min. The ultrasonic dispersion of the present invention facilitates the complete suspension of the mesoporous silica, which is beneficial for subsequent loading of fungal viral genomic DNA.

[0035] This invention involves mixing a mesoporous organic silica suspension with genomic DNA of a fungal virus and incubating the mixture to obtain the DNA pesticide. As one embodiment, the incubation is a rotational incubation; as another embodiment, the rotation speed is 30-60 r / min. In specific embodiments of this invention, the rotation speed can be any value within the range of 30-60 r / min, for example, 30, 35, 40, 50, or 60 r / min. The rotational incubation of this invention allows the mesoporous organic silica (especially MON-NH2) to bind more fully to the free nucleic acids in the system, improving loading efficiency. As one embodiment, the preferred incubation temperature is 4°C. At 4°C, the degradation of nucleic acids not bound to MON-NH2 can be prevented.

[0036] As one implementation method, the incubation time is 30-60 min. In a specific embodiment of the present invention, the incubation time can be any value within the range of 30-60 min, such as 30, 35, 40, 45, 50, 55, or 60 min. Within this incubation time, mesoporous organic silica (especially MON-NH2) can bind more fully to the free nucleic acids in the system, improving loading efficiency. Compared to other methods, 60 min yields better results in terms of transfection efficiency and loading rate.

[0037] This invention also provides the application of the DNA pesticide described above in inhibiting fungal infection. As one embodiment, the fungi include *Sclerotinia sclerotiorum*, *Fusarium graminearum*, *Botrytis cinerea*, and *Pseudomonas aeruginosa*. The DNA pesticide of this invention can deliver the genomic DNA of a fungal virus to the pathogenic fungus, causing it to replicate, transcribe, and translate within the pathogenic fungus to form a mature virus, leading to a decline in the pathogenicity of the fungus and thus inhibiting fungal infection.

[0038] This invention also provides the application of the DNA pesticide described above in the control of fungal diseases. As one embodiment, the fungal diseases include sclerotinia rot, Fusarium head blight, gray mold, and branch blight. This invention uses a nanocarrier to load fungal viral genomic DNA to construct a DNA pesticide, achieving "stable protection—efficient delivery—intrafungal replication—attenuated virulence and transmissibility" of fungal viral genomic DNA, establishing a cell-free delivery technology system for the control of fungal diseases, used for the prevention and control of plant fungal diseases.

[0039] This invention also provides a method for controlling sclerotinia stem rot, comprising the following steps: spraying the DNA pesticide described in the above scheme onto the plants. The DNA pesticide of this invention acts directly on the pathogenic fungi that infect plants and can be used to control fungal diseases caused by pathogenic fungi. This invention establishes a holistic application chain around "delivery—replication—virus formation—attenuation—disease prevention": fungal virus genes and related protein expression are detected within the pathogenic fungi, and it is demonstrated that after applying the DNA pesticide, the formed virus has the ability to spread and can transform highly pathogenic strains into attenuated strains. Finally, indoor and field trial efficacy data support its actual control effect on fungal diseases. As one embodiment, the spraying sites include plant leaves and stems; the spraying amount is 22.7 L / acre.

[0040] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0041] To clearly explain the DNA pesticide, in a specific embodiment of the present invention, the DNA pesticide is prepared using mesoporous silica nanoparticles and SsHADV-1 genomic DNA, but this does not constitute a limitation on the scope of protection of the present invention.

[0042] Example 1 DNA pesticide preparation and characterization The *Sclerotinia sclerotiorum* strain DT-8, isolated from sclerotia of diseased rapeseed stems infected with *Sclerotinia sclerotiorum* in Jinpen Town, Datong Lake, Yiyang City, Hunan Province, carries the DNA virus SsHADV-1. It exhibits weak pathogenicity to rapeseed and other hosts. *Sclerotinia sclerotiorum* strain DT-8 is published in the literature [Yu X, Li B, Fu YF, et al. A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus]. Proceedings of the National Academy of Sciences of the United States of America , 2010, 107(18): 8387-92】.

[0043] Preparation of amino-mesoporous silica nanoparticles (MON-NH2): 10 mg of MON-NH2 (purchased from Shaanxi Xingbei Aike Biotechnology Co., Ltd., catalog number X-NJ-50) was ultrasonically dispersed in 10 mL of pure water to prepare a MON-NH2 suspension with a concentration of 1000 mg / L. The ultrasonic parameters were 40 kHz, 25 °C, and 15 min for uniform dispersion.

[0044] Strain strain DT-8 was cultured on PDA medium. After 6-8 days of culture, genomic DNA was extracted from the attenuated virulent strain DT-8 using the high-salt CTAB method. DNA from SsHADV-1 was recovered via gel extraction and used as a template for rolling circle amplification. The amplified product was analyzed using a single restriction enzyme site on the SsHADV-1 genome. Xho I enzyme digestion and T4 DNA ligase cyclization yielded a large amount of SsHADV-1 genomic DNA (SEQ ID NO.1).

[0045] System: Nuclease-free ddH2O 4 µL, Phi29 MAX DNA Polymerase Reaction Buffer (10×) 1 µL, random primers (100 µM) 2.5 µL, SsHADV-1 DNA (1 ng / µL) 1 µL, total volume 8.5 µL.

[0046] Procedure: Incubate at 95°C for 3 min in a preheated PCR instrument, then immediately cool on ice for 5 min. Add 1 µL of dNTPs (10 mM) and 0.5 µL of Phi29 MAX DNA Polymerase, vortex to mix, and then briefly centrifuge to collect the mixture. Incubate overnight at 30°C. After incubation, maintain at 65°C for 10 min to inactivate the Phi29 MAX DNA Polymerase.

[0047] (1) To investigate the binding ability of MON-NH2 vector to the SsHADV-1 DNA prepared above, the mass ratio of MON-NH2 to DNA was set to 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, 16:1, 20:1 and 50:1 respectively. After mixing, the two were blown and incubated at 30~60 r / min for 1 h at 4℃.

[0048] Meanwhile, the MON-NH2 group and the SsHADV-1 group were set up as controls, and the specific operation was as follows: MON-NH2 group: 1 mL MON-NH2 suspension (1000 mg / L); SsHADV-1 group: 100 ng SsHADV-1 genomic DNA; After incubation, each solution was centrifuged at 12000 r / min. The supernatant was then subjected to agarose gel electrophoresis to detect the free DNA in the supernatant. The results are as follows: Figure 1 As shown in Figure A. The results indicate that ( Figure 1 (A) As the MON-NH2:DNA mass ratio increases, the brightness of the unloaded and free DNA bands in the supernatant gradually decreases, and the content of free SsHADV-1 genomic DNA gradually decreases. Under the conditions of a mass ratio of 10:1 and higher, complete loading of SsHADV-1 DNA is achieved.

[0049] (2) The protective effect of MON-NH2 on DNA MON-NH2-SsHADV-1 group: The nanomaterial MON-NH2 was quantified to 1000 ng and incubated with 10 ng SsHADV-1 genomic DNA (MON-NH2:DNA=100:1), 12.5 ng SsHADV-1 genomic DNA (MON-NH2:DNA=80:1), 25 ng SsHADV-1 genomic DNA (MON-NH2:DNA=40:1), 50 ng SsHADV-1 genomic DNA (MON-NH2:DNA=20:1) and 100 ng SsHADV-1 genomic DNA (MON-NH2:DNA=10:1), respectively, using the same method as above.

[0050] Meanwhile, the MON-NH2 group and the SsHADV-1 group were set up as controls, and the specific procedures were as follows: MON-NH2 group: 1 mL MON-NH2 suspension (1000 mg / L); SsHADV-1 group: 100 ng SsHADV-1 genomic DNA; After incubation, each solution was centrifuged at 12000 r / min, and the precipitate was washed three times with ddH2O to obtain the precipitate for later use. Each group was further divided into two subgroups. One subgroup was digested with DNase I (TransGen Biotech, GD201-01) for 30 min, and then SDS was added to a final concentration of 0.5% to inactivate DNase I and depolymerize and release DNA wrapped around the MON-NH2 surface, in order to evaluate the protective effect of MON-NH2 on DNA. The other subgroup did not add DNase I. Results are shown below. Figure 1 In the case of DNase I, "-" indicates that no exogenous DNase I was added, and "+" indicates that DNase I was added. According to... Figure 1As shown in Figure B, the band brightness of the "-" and "+" bands in each lane of the MON-NH2-SsHADV-1 group was consistent, while the "+" band in the SsHADV-1 group disappeared. This indicates that under all mass ratios (100:1, 80:1, 40:1, 20:1, and 10:1), MON-NH2 can effectively protect SsHADV-1 DNA from DNase I degradation. At a ratio of 10:1, MON-NH2 can effectively protect viral nucleic acid from degradation by DNase I in the environment, providing more stable and durable protection for SsHADV-1 DNA preparations during transportation, storage, and practical application.

[0051] (3) Scanning electron microscopy was used to observe the unloaded DNA nanocarrier MON-NH2 and the MON-NH2 loaded with SsHADV-1 genomic DNA prepared in step (2) with MON-NH2:DNA = 10:1. The results are as follows: Figure 1 As shown in Figure C. Scanning electron microscopy analysis revealed that the average particle size of the DNA-free nanocarrier MON-NH2 was 50 nm. The surface was convex, and the spheres exhibited a typical mesoporous and hollow structure with obvious pore distribution on the surface, demonstrating good dispersibility and a visible porous and loose structure. Figure 1 (C) After loading SsHADV-1 genomic DNA, the surface of the MON-NH2 spheres lost its translucency, the black color at the edge of the spheres became significantly darker, and the pores were clearly blocked, indicating that the genomic DNA of the viral SsHADV-1 was successfully loaded onto the surface of the MON-NH2 spheres. Figure 1 (C)

[0052] Example 2 The SsHADV-1 genomic DNA delivered by MON-NH2 invades Sclerotium sclerotiorum hyphae cells and replicates stably.

[0053] Preparation of the nano-nucleic acid formulation MON-NH2@SsHADV-1: 1000 ng of MON-NH2 prepared in Example 1 was dissolved in water to prepare a MON-NH2 solution with a concentration of 1000 ng / µL; 100 ng of genomic DNA of SsHADV-1 prepared in Example 1 was dissolved in water to prepare a DNA solution with a concentration of 100 ng / µL; the MON-NH2 solution and the DNA solution were mixed at a mass ratio of 1:1 (i.e., MON-NH2:DNA = 10:1), and incubated at 4°C with shaking at 30~60 r / min for 1 h to obtain the nano-nucleic acid formulation MON-NH2@SsHADV-1, which is the DNA pesticide of the present invention, for subsequent experiments.

[0054] (1) Strain morphology Sclerotium sclerotiorum strain DT-8VF: This strain, obtained by detoxifying the attenuated strain DT-8 through hyphal tips, exhibits strong pathogenicity against hosts such as rapeseed. In this study, DT-8VF was selected as the recipient strain, as published in the literature [Yu X, et al. Ageminivirus-related DNA mycovirus that confers hypovirulence to a plantpathogenic fungus. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(18): 8387-92]. Tender hyphae of DT-8VF, continuously activated for 2-3 generations, were processed using a homogenizer at 45 Hz for 1 min to obtain a hyphal suspension.

[0055] DT-8VF mycelial suspension was mixed with water, MON-NH2 (1000 mg / L), SsHADV-1 genomic DNA (100 ng / µL), and the nanonucleic acid preparation MON-NH2@SsHADV-1 at a volume ratio of 1:1 and incubated at 4°C with shaking at 30 r / min for 2 h. The co-incubated mycelia were spread on 1 / 4 concentration PDA plates (containing Cef resistance, 100 µg / mL) and incubated at 20°C in the dark for 3 days to obtain transfected strains. The strains obtained from the above treatment were named Mock-F1 (mixed with water), MON-F1 (mixed with MON-NH2), and Viral DNA-F1 (mixed with SsHADV-1 genomic DNA), respectively. The three strains obtained from the nanonucleic acid preparation treatment were named MON-V2, MON-V4, and MON-V5, respectively. The above-mentioned strains were cultured separately on 1 / 2 concentration PDA medium at 20℃, with strain DT-8 carrying SsHADV-1 as a positive control. The morphology of the strains was observed, and the results are as follows: Figure 2 As shown in Figure A.

[0056] Refer to [Zhang L, et al. A novel virus that infecting hypovirulent strainXG36-1 of plant fungal pathogen Sclerotinia sclerotiorum [Virus Journal, 2009, 6: 96-104] Method: The mycelial growth rate of each group was measured, and the results are as follows. Figure 2 As shown in B and Table 1.

[0057] Table 1. Mycelial growth rate of each group

[0058] according to Figure 2 It was found that strains MON-V4, MON-V5, and MON-V2 grew slowly, with significant pigment accumulation, and their morphology was consistent with strain DT-8. In contrast, the *Sclerotinia sclerotiorum* strains treated with water, MON-NH2, and SsHADV-1 genomic DNA showed normal morphology. This indicates that the *Sclerotinia sclerotiorum* strains treated with MON-NH2@SsHADV-1 exhibited morphological characteristics consistent with strain DT-8 after infection with SsHADV-1.

[0059] (2) RNA detection RNA was extracted from each *Sclerotinia sclerotiorum* strain in step (1). 5 µg of each strain was mixed with 1 µL of Oligo d(T)18, and RNase-free water was added to a final volume of 8 µL. The mixture was incubated at 65°C for 5 min, followed by rapid cooling on ice for 2–3 min. 10 µL of 2 × ES Reaction Mix, 1 µL of EasyScripe RT Enzyme Mix, and 1 µL of gDNA remover were added, mixed thoroughly, and incubated at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 s to obtain cDNA.

[0060] With Sclerotium Actin Detection using specific primers Actin-ZF and Actin-ZR Actin The expression of SsHADV-1 was used as an internal reference. Rep Gene-specific primers BF1-REP0904 and BR1-REP0904 target *Sclerotinia sclerotiorum* strains. Rep Gene expression was analyzed by qPCR. The primer sequences used are as follows: Actin-ZF (SEQ ID NO.2): 5'-CTGGAAGATTGACTGGCGGTTTG-3'; Actin-ZR (SEQ ID NO.3): 5'-AGCACCAGAGGAGCACCAGTTT-3'; BF1-REP0904 (SEQ ID NO.4): 5'-TACTACTGGCTCTGTAATC-3'; BR1-REP0904 (SEQ ID NO. 5): 5'-TGTCTGTTAAGGAATTGTATAG-3'.

[0061] The qPCR reaction system consisted of: 10 µL of 2×PerfectStart Green qPCR SuperMix, 0.4 µL each of 10 µM PrimerF / R, 1 µL of cDNA diluted 5 times, and RNase-free water to a final volume of 20 µL.

[0062] Reaction procedure: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 40 cycles; 55~95℃, increasing by 0.5℃ per cycle, for 5 s, and collecting fluorescence signals. Store at 16℃ for 1 min.

[0063] In the positive control DT-8 strain Rep The expression level is set to 1, and through 2 -△△CT The relative accumulation of virus in mycelium was calculated using a method with four technical replicates for each strain. The test results were as follows: Figure 3 As shown.

[0064] The results showed that strains MON-V4, MON-V5, and MON-V2... Rep The average relative expression levels were 0.1451, 0.3796, and 0.1944, respectively, while Mock-F1, MON-F1, and Viral DNA-F1 showed almost no expression. This indicates that the SsHADV-1 genomic DNA delivered by MON-NH2@SsHADV-1 can undergo gene transcription after invading Sclerotinia sclerotiorum cells.

[0065] (3) Protein detection Western blot analysis was performed using an SsHADV-1 capsid protein-specific antibody (published in the literature [Yu X, Li B, Fu YF, et al. A geminivirus-related DNA mycovirus that confers hypovirulence to a plantpathogenic fungus. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(18): 8387-92]). The results are as follows: Figure 4 As shown.

[0066] After color development, SsHADV-1 protein blots were observed in strains DT-8, MON-V2, MON-V4, and MON-V5 in the 35 kDa and 40 kDa range. No hybridization signal was observed in strains treated with water, SsHADV-1 nucleic acid, or MON-NH2. Figure 4 This indicates that after the SsHADV-1 genomic DNA delivered by MON-NH2@SsHADV-1 enters the Sclerotium sclerotiorum cell, the gene can be transcribed and can utilize the host's cellular environment for protein translation.

[0067] (4) Strain confrontation The strains selected in step (1) were used as donor strains and subjected to confrontation culture with DT-8VF (recipient strain), and the specific groups are as follows: ①DT-8VF: DT-8VF group: Inoculate strains Mock-F1 and DT-8VF from step (1) into petri dishes and culture them respectively; ②MON-F1:DT-8VF group: Inoculate strains MON-F1 and DT-8VF from step (1) into petri dishes and culture them respectively; ③ DNA-F1:DT-8VF group: Viral DNA-F1 and DT-8VF strains from step (1) were inoculated into culture dishes and cultured. ④DT-8: DT-8 group: The DT-8 strain was inoculated twice into a petri dish and cultured. ⑤ DT-8:DT-8VF group: Strains DT-8 and DT-8VF were inoculated into petri dishes and cultured. ⑥MON-V4: MON-V4 group: Inoculate the strain MON-V4 from step (1) twice in a petri dish and culture it; ⑦MON-V4:DT-8VF group: Inoculate strains MON-V4 and DT-8VF from step (1) into petri dishes and culture them respectively; ⑧MON-V5:DT-8VF group: Inoculate the strains MON-V5 and DT-8VF from step (1) into petri dishes and culture them respectively; ⑨MON-V2:DT-8VF group: Inoculate the strains MON-V2 and DT-8VF from step (1) into petri dishes and culture them.

[0068] The mycelial blocks on the left side of each group above represent donor strains, while the mycelial blocks on the right side represent recipient strains. Figure 5 After 3 days of confrontation culture (A), the growth status of each group of strains was observed, and the results are as follows: Figure 5 As shown in Figure A. Strains were picked from the recipient strain side and cultured in fresh PDA medium, encoded by SsHADV-1. Rep Gene-specific primers REP-5F and REP-6R were used to detect the presence and reactivity of SsHADV-1 in the recipient strain DT-8VF by PCR and RT-PCR, respectively. Rep Gene expression, in Sclerotinia sclerotiorum ActinDetection using specific primers actin-qF2 and actin-qR4 Actin The expression is internal reference.

[0069] The primer sequences are shown below: REP-5F (SEQ ID NO.6): 5'-TCACATGACTTTCGACTTCCACGC-3'; REP-6R (SEQ ID NO.7): 5'-CCGGCGCAGCCGATATGGATAAT-3'; actin-qF2 (SEQ ID NO.8): 5'-GAGCTGTTTTCCCTCTCCATTGTC-3'; actin-qR4 (SEQ ID NO. 9): 5'-GACGACACCGTGCTCGATTGG-3'.

[0070] The reaction system consisted of 10 µL of 2×PCR Mix, 1 µL each of 10 µM Primer F / R, 1 µL of template (RT-PCR used cDNA as template, PCR used genomic DNA 1 ng / µL as template), and RNase-free water to a final volume of 15 µL.

[0071] The reaction procedure was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, 32 cycles; 72℃ extension for 5 min; 16℃ for 2 min, 1% agarose gel electrophoresis detection segment.

[0072] Test results as follows Figure 5 As shown in Figure B, regardless of whether PCR or RT-PCR was used, no bands were observed in the progeny strains of the recipient strains confronting the water-treated strain (DT-8VF:DT-8VF), the MON-NH2-treated strain (MON-F1:DT-8VF), or the SsHADV-1 nucleic acid-treated strain (DNA-F1:DT-8VF). However, the progeny strains of the recipient strains confronting strains MON-V2 (MON-V2:DT-8VF), MON-V4 (MON-V4:DT-8VF), and MON-V5 (MON-V5:DT-8VF) showed bands consistent with those observed in the case of strain DT-8 (DT-8:DT-8VF). This indicates that SsHADV-1 carried by the MON-NH2@SsHADV-1 transfected strain can horizontally spread to new strains and replicate and transcribe.

[0073] In summary, the SsHADV-1 genomic DNA delivered by MON-NH2@SsHADV-1 can enter Sclerotinia sclerotiorum, where it replicates, transcribes, and translates to form a mature virus. The new virus can be transmitted to the highly pathogenic strain DT-8VF through contact, enabling it to acquire SsHADV-1 and exhibit degenerative phenomena such as slow growth.

[0074] Example 3: The MON-NH2@SsHADV-1 complex increases DNA stability in plants. To determine the stability of the viral genomic DNA carried by MON-NH2@SsHADV-1 in plants, Arabidopsis thaliana seedlings of 21 days age and uniform growth were randomly divided into 4 groups: Mock group: Spray with water, 2 mL / plant, 4 plants per treatment, cultured in a culture room, and recorded as Mock; MON-NH2: The procedure is the same as the Mock group, except that the spraying of water is replaced with spraying of 1000 mg / L MON-NH2; SsHADV-1 genomic DNA: The procedure was the same as the Mock group, except that the spraying of water was replaced with spraying of 0.1 g / L ssHADV-1 genomic DNA, denoted as Viral DNA; MON-NH2@SsHADV-1: The operation is the same as the Mock group, except that the spraying of water is replaced by spraying MON-NH2@SsHADV-1 prepared in Example 2, which is denoted as MON-SsHADV-1; On days 5 and 10 after spraying, treated leaves were cut and placed in 2 mL Eppendorf tubes filled with sterile water. The tubes were ultrasonically cleaned for 1 min, immersed in 75% ethanol for 1 min, and rinsed three times with sterile water. Nucleic acid DNA was extracted from the leaf tissue. Primers REP-5F (SEQ ID NO. 6) and REP-6R (SEQ ID NO. 7) were used to target SsHADV-1. Rep The gene was detected by PCR, following the same procedure as in Example 2, and the results are as follows. Figure 6 As shown.

[0075] The results showed that on day 5, SsHADV-1 was undetectable in the Mock group and the MON-NH2 group, while it was detectable in the SsHADV-1 genomic DNA group and the MON-NH2@SsHADV-1 formulation group. On day 10, SsHADV-1 was undetectable in the Mock group, the MON-NH2 group, and the SsHADV-1 genomic DNA group, while it was still detectable in four plant samples from the MON-NH2@SsHADV-1 formulation treatment group. Figure 6This indicates that the stability of viral nucleic acid is increased after being loaded with the nanomaterial MON-NH2, giving the MON-NH2@SsHADV-1 formulation a longer window period for field application.

[0076] Example 4: Prevention of Sclerotinia stem rot using MON-NH2@SsHADV-1 formulation (1) The leaf inoculation method of Sclerotinia sclerotiorum was used, and the method is described in [Yu X, et al. A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(18): 8387-92] to determine the preventive effect of MON-NH2@SsHADV-1 on Arabidopsis thaliana sclerotiorum disease.

[0077] Leaves from Arabidopsis thaliana seedlings of 21 days age and with uniform growth were randomly divided into 4 groups, with 6 leaves per treatment group as replicates. The specific groupings are as follows: Mock group: Spray with water, 10 μL / plant, air dry naturally, and then inoculate with mycelial blocks of Sclerotinia sclerotiorum DT-8VF strain, 1 mycelial block / leaf, denoted as Mock; the mycelial block diameter is 5 mm, and the strain is the outermost mycelium that has been activated and is growing normally. MON-NH2 group: The procedure was the same as the Mock group, except that the spraying of water was replaced with spraying of 1000 mg / L MON-NH2; Viral DNA group: The procedure is the same as the Mock group, except that the water is replaced with 0.1 g / L SsHADV-1 genomic DNA spray. MON-NH2@SsHADV-1 group: The operation is the same as the Mock group, except that the water is replaced with MON-NH2@SsHADV-1 prepared in Example 2, which is also referred to as MON-SsHADV-1; After inoculation, the leaves were cultured under moist conditions. The diameter of each lesion was statistically analyzed using ImageJ software at 24 h, 48 h, and 72 h. Graphs were generated using GraphPad Prism 8 software, and one-way ANOVA was performed using IBM SPSS Statistics 24. P <0.01, indicating a significant difference in lesion area between treatments a and b. Disease incidence on rapeseed cotyledons 48 h after inoculation, and lesion area results are shown below. Figure 7 As shown.

[0078] At 48 h after inoculation, the average lesion area in the Mock treatment group was 1.079 cm². 2 The MON-NH2 treatment group had a diameter of 1.039 cm. 2 The viral DNA treatment group had a diameter of 1.313 cm. 2 The MON-SsHADV-1 treatment group had a diameter of 0.398 cm⁻¹. 2 ,exist P The area of ​​lesions at the <0.01 level was significantly lower than that in the other three treatment groups. Figure 7 The results indicate that spraying the MON-NH2@SsHADV-1 formulation can prevent Arabidopsis thaliana sclerotinia disease, with a control efficacy of 63.11%.

[0079] (2) The in vitro leaf inoculation method of Sclerotinia sclerotiorum was used to determine the preventive effect of MON-NH2@SsHADV-1 on Sclerotinia sclerotiorum rot in rapeseed. Isolated rapeseed cotyledons were used as the subjects. The specific grouping and operation were the same as in step (1), except that: water, MON-NH2, SsHADV-1 genomic DNA, and MON-NH2@SsHADV-1 preparation were respectively added to the isolated rapeseed cotyledons at 10 μL / leaf. After natural air drying, mycelial blocks of Sclerotinia sclerotiorum DT-8VF strain were inoculated, one mycelial block / leaf, with 6 leaves per treatment as replicates. The leaves were kept moist and cultured. After 48 h, the diameter of the lesions was statistically analyzed using ImageJ software, and graphs were generated using GraphPad Prism 8 software. One-way ANOVA was performed using IBM SPSS Statistics 24. P <0.05, where "ns" indicates no significant difference. "This indicates a significant difference, as shown in the results." Figure 8 As shown.

[0080] The results showed that the average lesion area of ​​rapeseed leaves in the Mock treatment group was 0.7145 cm². 2 The MON-NH2 treatment group had a concentration of 0.7037 cm⁻¹. 2 The viral DNA treatment group had a diameter of 0.7335 cm⁻¹. 2 The MON-SsHADV-1 treatment group had a diameter of 0.2170 cm. 2 ,exist P The level <0.05 was significantly lower than that of the other three control groups ( Figure 8 The results indicate that spraying the MON-NH2@SsHADV-1 formulation can prevent sclerotinia stem rot in rapeseed, with a control efficacy of 69.63%.

[0081] (3) The preventive effect of MON-NH2@SsHADV-1 on sclerotinia rot in rapeseed was determined by inoculating live leaves of Sclerotinia sclerotiorum. Rapeseed seedlings aged 7 days were used as subjects. The specific grouping and operation were the same as in step (1), except that: water, MON-NH2, SsHADV-1 genomic DNA, and MON-NH2@SsHADV-1 preparation were sprayed, 2 mL / pot, 4 seedlings / pot, with each treatment repeated 3 times. The seedlings were air-dried naturally. Three days later, DT-8VF mycelial suspension (OD2000) was inoculated. 600 =0.5), sprayed onto rapeseed seedlings, 0.5 mL / plant, and cultured under moist conditions. The growth status of each rapeseed group was recorded 60 h and 7 d after inoculation with DT-8VF mycelial suspension, and the survival rate of the rapeseed was measured daily. The results are as follows: Figure 9 As shown in Table 2, Table 2 presents the rapeseed survival rate data for 1, 2, 5, and 7 days after inoculation.

[0082] The results showed that at 60 h, lesions had expanded to the hypocotyl in the Mock, MON-NH2, and SsHADV-1 genomic DNA treatment groups, with overall plant collapse and small-area infection spread; lesions appeared on the leaves of the MON-NH2@SsHADV-1 formulation treatment group. At 7 days, the average survival rate of rapeseed in the Mock group was 12.0%, the MON-NH2 group was 18.7%, the SsHADV-1 genomic DNA group was 24.0%, while the MON-SsHADV-1 formulation treatment group had a survival rate of 60.0%, significantly higher than the other three groups. Figure 9 This indicates that the MON-NH2@SsHADV-1 formulation has a good preventive effect against sclerotinia stem rot in rapeseed.

[0083] Table 2. Rapeseed survival rate (%)

[0084] Example 5: Treatment of Sclerotinia stem rot with MON-NH2@SsHADV-1 (DNA pesticide) formulation The therapeutic effect of MON-NH2@SsHADV-1 on sclerotinia disease was investigated by spraying Arabidopsis thaliana infected with Sclerotinia sclerotiorum. The specific procedure was as follows: 21-day-old Arabidopsis thaliana seedlings with uniform growth were inoculated with mycelial blocks of Sclerotinia sclerotiorum DT-8VF strain, one mycelial block per leaf, three leaves per plant. After 24 hours of moist incubation, the leaves were randomly divided into four groups, with five replicates per treatment group. The mycelial block setup was the same as in Example 4, and the specific groupings were as follows: Mock group: The initial lesion area was 0.426 cm². 2 Spray with clean water, 2 mL / plant, and culture in a humidified culture room. This is called Mock. MON-NH2 group: The initial lesion area was 0.416 cm².2 The procedure was the same as the Mock group, except that the spraying of water was replaced with spraying of 1000 mg / L MON-NH2; Viral DNA group: The average area of ​​the initial lesion was 0.461 cm². 2 The procedure was the same as the Mock group, except that the spraying of water was replaced with spraying of 0.1 g / L SsHADV-1 genomic DNA. MON-SsHADV-1 group: The average area of ​​the initial lesion was 0.414 cm². 2 The operation is the same as the Mock group, except that the spraying of water is replaced with spraying of MON-NH2@SsHADV-1 prepared in Example 2; Plant growth and survival rates were recorded at 1, 3, 5, and 7 days after spraying. Three days after spraying, the size of lesions was measured using ImageJ software, and graphs were generated using GraphPad Prism 8 software. One-way ANOVA was performed using IBM SPSS Statistics 24. P <0.01, there was a significant difference in lesions between treatments using the uppercase letters A and B, as shown in the results. Figure 10 As shown in Table 3. The results showed that after 72 h, the average lesion area in the Mock group, MON-NH2, and SsHADV-1 genomic DNA group was 1.11 cm². 2 1.03 cm 2 and 1.10 cm 2 The lesion area in the MON-NH2@SsHADV-1 treatment group was 0.53 cm². 2 The killing effect of Sclerotinia sclerotiorum on plants under different treatments was continuously observed. On day 3, the survival rates of Mock, MON-NH2, SsHADV-1 genomic DNA, and MON-NH2@SsHADV-1 formulations were 100.00%, 77.78%, 80.56%, and 100%, respectively. On day 5, 58.33% of Mock plants were infected with Sclerotinia sclerotiorum in the leaf core, while 47.22% and 52.78% of the MON-NH2 and SsHADV-1 genomic DNA groups had lesions extending to the plant core, respectively. In contrast, 22.23% of the MON-NH2@SsHADV-1 formulation treatment group had Sclerotinia sclerotiorum infection in the plant core. On day 7… At time d, the entire plant in the Mock group was infected by Sclerotinia sclerotiorum mycelium and died. The survival rates of the MON-NH2 and SsHADV-1 genomic DNA groups were 16.67% and 22.22%, respectively. The plants in these groups were in poor growth condition and did not produce new leaves. In contrast, 63.89% of the plants in the MON-NH2@SsHADV-1 treatment group still had healthy leaves and normally growing leaf cores. Figure 10This indicates that the MON-NH2@SsHADV-1 preparation has a good therapeutic effect on sclerotinia stem rot.

[0085] Table 3. Survival rate of Arabidopsis thaliana (%)

[0086] Example 6: Control of Sclerotinia stem rot in rapeseed using MON-NH2@SsHADV-1 formulation To further determine the control effect of MON-NH2@SsHADV-1 formulation against sclerotinia stem rot in rapeseed, a field trial was conducted in the spring of 2025 using the rapeseed variety Huayouza 62 at the full flowering stage in the experimental field of Huazhong Agricultural University in Wuhan, Hubei Province. The specific groupings are as follows: Mock group: spray with clean water, 26.7 L / acre, recorded as Mock; MON-NH2 group: The procedure was the same as the Mock group, except that the spraying of water was replaced with spraying of 1000 mg / L MON-NH2; Viral DNA group: The procedure is the same as the Mock group, except that the spraying of water is replaced with spraying of 0.1 g / L SsHADV-1 genomic DNA; MON-SsHADV-1 group: The operation is the same as the Mock group, except that the spraying of water is replaced by spraying MON-NH2@SsHADV-1 prepared in Example 2; DT-8 group: The operation is the same as the Mock group, except that the spraying of water is replaced with spraying of DT-8 mycelial suspension (OD). 600 =0.5) Three cells were processed per cell, each cell measuring 1.5 m × 5 m, with cells randomly distributed. See [link / reference]. Figure 11 Three days later, rapeseed leaves from the same location were inoculated with mycelial blocks (5 mm) of *Sclerotinia sclerotiorum* DT-8VF. Eight plants were randomly inoculated per treatment, and the plants were bagged to retain moisture and allow for disease development. The disease incidence was assessed seven days later. Results showed that the disease incidence in the three plots of the Mock treatment group was... , and The average incidence rate was 95.83%; the incidence rate in the MON-NH2 treatment group was... , and The average incidence rate was 83.3%; the incidence rate in the SsHADV-1 genomic DNA treatment group was... , and The average incidence rate was 95.83%; the incidence rate in the MON-SsHADV-1 treatment group was... , and The average incidence rate was 41.67%, and the control efficacy was 56.52%; the incidence rate in the DT-8 mycelial suspension treatment group was... , and The average incidence rate was 16.67%, and the control efficacy was 82.60%. This indicates that the MON-NH2@SsHADV-1 formulation has a good control effect on sclerotinia stem rot in rapeseed under natural field conditions.

[0087] Thirty days after spraying, during the rapeseed growth and pod development stage, the occurrence of naturally occurring sclerotinia stem rot in the field was investigated. Sixteen rows of rapeseed plants were randomly selected from each plot for the survey, and the number of naturally infected plants was counted. Field disease incidence and rate are shown in [link to relevant data]. Figure 12 The disease incidence rates in the Mock, MON-NH2, Viral DNA, MON-NH2@SsHADV-1, and DT-8 groups were 23.48%, 21.14%, 23.72%, 12.27%, and 8.31%, respectively. Compared to Mock, the MON-NH2@SsHADV-1 formulation showed a control efficacy of 50.47%; the positive control DT-8 showed a control efficacy of 64.62%. Figure 12 This further demonstrates the effectiveness of the MON-NH2@SsHADV-1 formulation in controlling sclerotinia stem rot in rapeseed in the field.

[0088] Example 7: Safety Verification of MON-NH2@SsHADV-1 Using rapeseed as a representative plant, the safety of MON-NH2@SsHADV-1 was verified. The specific procedures are as follows: Seven-day-old rapeseed seedlings were randomly divided into four groups, with three replicates in each group. The specific groupings are as follows: Mock group: Sterile ultrapure water was sprayed onto the surface of 7-day-old rapeseed plants, 2 mL / pot, 15 rapeseed seedlings / pot; MON-NH2 group: The procedure was the same as the Mock group, except that 1000 mg / L MON-NH2 was sprayed. Viral DNA group: The procedure is the same as the Mock group, except that 0.1 g / L SsHADV-1 genomic DNA is sprayed. MON-SsHADV-1 group: The operation is the same as the Mock group, except that MON-NH2@SsHADV-1 prepared in Example 2 is sprayed, which is denoted as MON-SsHADV-1; After treatment, the rapeseed plants were placed in a plant culture room for normal growth. After 14 days, the hypocotyl length and fresh weight of each group were recorded. GraphPad Prism 8 was used for plotting, and IBM SPSS Statistics 24 was used for one-way ANOVA. P<0.05, where "ns" indicates no significant difference, results are as follows. Figure 13 As shown.

[0089] The results showed that after 14 days, the average hypocotyl lengths of plants in the CK treatment were 6.645 cm, 6.702 cm, and 6.677 cm, with average fresh weights of 0.245 g, 0.248 g, and 0.2189 g, respectively; the average hypocotyl lengths of plants in the MON-NH2 treatment were 6.3955 cm, 6.4815 cm, and 6.5755 cm, with average fresh weights of 0.227 g, 0.236 g, and 0.242 g, respectively; the average hypocotyl lengths of plants in the SsHADV-1 nucleic acid treatment were 6.489 cm, 6.538 cm, and 6.681 cm, with average fresh weights of 0.227 g, 0.253 g, and 0.248 g, respectively; and the average hypocotyl lengths of plants in the MON-NH2@SsHADV-1 treatment were 6.547 cm, 6.717 cm, and 6.496 cm, with average fresh weights of 0.239 g, 0.248 g, and 0.2189 g, respectively. g, 0.249 g, and 0.230 g. The four treatments of rapeseed seedlings showed differences in fresh weight and hypocotyl length. P The difference was not statistically significant at the <0.05 level. This indicates that the application of the MON-NH2@SsHADV-1 formulation had no significant effect on the growth of rapeseed seedlings.

[0090] In summary, this invention is the first to apply nanocarrier technology to the utilization of biocontrol resources of the fungal virus SsHADV-1, providing a potential approach to address the problems of low transmission efficiency and poor environmental stability in traditional fungal virus applications. By protecting the genomic DNA of the fungal virus through nanocarriers, non-contact cross-hyphae delivery of the fungal virus is achieved, providing a new approach for the green control of plant diseases.

[0091] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A DNA pesticide, characterized in that, It includes a nanocarrier and the genomic DNA of a fungal virus; the fungal virus is a plant pathogenic fungal virus that can cause a decline in the pathogenicity of the host fungus.

2. The DNA pesticide according to claim 1, characterized in that, The mass ratio of the nanocarrier to the genomic DNA of the fungal virus is 10:1 to 100:

1.

3. The DNA pesticide according to claim 1, characterized in that, The fungal viruses include at least one of the following: Sclerotinia sclerotiorum attenuated viral-associated DNA virus 1 (SsHADV-1), Fusarium graminearum three-component virus type 1 (FgGMTV1), Botrytis cinerea DNA virus BGDaV1, Botrytis cinerea DNA virus BGDaV2, and Soybean pseudostem spot circular DNA virus type 1.

4. The DNA pesticide according to claim 1, characterized in that, The nanocarrier includes at least one of the following: mesoporous silica nanoparticles, carbon nanotubes, carbon quantum dots, and chitosan.

5. The method for preparing the DNA pesticide according to any one of claims 1 to 4, characterized in that, The process includes the following steps: mixing the nanocarrier and the genomic DNA of the fungal virus, incubating them, and obtaining the DNA pesticide.

6. The preparation method according to claim 5, characterized in that, The incubation is a rotational incubation; the rotation speed is 30~60 r / min; the incubation time is 30~60 min.

7. The application of the DNA pesticide according to any one of claims 1 to 4 in inhibiting fungal infection.

8. The application of the DNA pesticide according to any one of claims 1 to 4 in the prevention and control of fungal diseases.

9. The application according to claim 8, characterized in that, The fungal diseases include at least one of the following: sclerotinia rot, Fusarium head blight, gray mold, and twig blight.

10. A method for preventing and controlling fungal diseases, characterized in that, The method includes the following steps: spraying the DNA pesticide according to any one of claims 1 to 4 onto the plant.

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