Preparation method and application of E52-dsRNA-NC nucleic acid nano pesticide for preventing and treating corn southern rust disease

Abstract

The invention belongs to the technical field of plant disease prevention and treatment, and relates to a preparation method and application of an E52-dsRNA-NC nucleic acid nano pesticide for preventing and treating corn southern rust disease. The preparation method comprises the following steps: carrying out in-vitro synthesis and purification on dsRNA of a key pathogenic effect gene E52 of target silence puccinia polypoda, so as to obtain dsRNA, and adding the dsRNA into a nano chitin colloidal dispersion system, so as to form the nano chitin colloidal dispersion. The invention also provides an application for preventing and treating southern rust of corn. Compared with the prior art, the dsRNA-NC nucleic acid nano pesticide has the advantages that the exposed dsRNA is combined with the nano chitin, the prepared dsRNA-NC nucleic acid nano pesticide has the effect of resisting the puccinia polystachys, the expression quantity of pathogenic bacteria related target genes in the infection stage is obviously inhibited, and the infection capacity of the puccinia polystachys is obviously reduced. The nano chitin is derived from shells of crustacean organisms, is low in cost, easy to degrade in the environment, eco-friendly to a field, free of residues and wide in application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology and relates to nucleic acid pesticides for controlling southern rust disease of corn. Background Technology

[0002] Southern rust of maize, caused by *Puccinia polysora*, is a typical airborne fungal disease, primarily affecting tropical and subtropical maize-growing areas and a significant cause of high and stable maize yields. The pathogen overwinters mainly in year-round maize-growing areas in southern my country, spreading via urediniospores in summer. These urediniospores travel long distances from south to north, carried by air currents, wind, and rain, eventually infecting maize plants. The disease primarily damages maize leaves and leaf sheaths; during large-scale outbreaks, husks and tassels can also be infected, exhibiting obvious symptoms. The occurrence and spread of southern rust are highly correlated with environmental conditions. Suitable temperatures of 24–28°C combined with high humidity in the field significantly accelerate the spread of the disease, representing the core environmental trigger for outbreaks. In recent years, affected by global climate change, its suitable growing areas have continued to expand to high latitude regions. Now it has become a common and serious disease in the main summer maize producing areas of Huang-Huai-Hai Plain in my country. When the disease occurs severely, maize plants will show signs such as whole plant drying and premature aging. The yield reduction in severely affected fields can exceed 20%, which poses a serious threat to the safe production of maize in my country.

[0003] Currently, my country mainly adopts a comprehensive control approach for southern rust of maize, combining the breeding of disease-resistant varieties with the application of chemical pesticides. However, this model has revealed many prominent defects and limitations in practical field applications. On the one hand, the breeding of disease-resistant maize varieties is characterized by long cycles, high technical difficulties, and slow effectiveness in field application, making it difficult to quickly respond to the actual needs of disease control during outbreaks. On the other hand, large-scale and high-frequency spraying of chemical pesticides not only easily pollutes and damages the agricultural ecological environment, such as soil and water bodies, but also induces resistance in many rust fungi, leading to a gradual decline in the control effect of pesticides. Furthermore, pesticide residues accumulate in maize kernels and plants, and after entering the human body through the food chain, they pose a potential threat to human health. Against this backdrop, the development of green, efficient, low-residue, and environmentally friendly new biological pesticides has become a core direction and inevitable choice for breaking through the current bottlenecks in the control of southern rust of maize and promoting the sustainable development of modern agriculture. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for preparing E52-dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] In the first aspect, this application provides a method for preparing E52-dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize, the steps of which are as follows:

[0007] S1. The in vitro synthesis and purification of the dsRNA (E52-dsRNA) of the key pathogenic effect gene E52 of Puccinia polysora yielded the corresponding dsRNAs. The cDNA nucleotide sequence of the key pathogenic effect gene E52 of Puccinia polysora is shown in SEQ ID No. 3. The nucleotide sequence of the E52-dsRNA is shown in SEQ ID No. 4.

[0008] S2. Add the E52-dsRNA obtained in step S1 to the nano-chitin colloidal dispersion system, and after mixing, obtain the dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize.

[0009] The method for in vitro synthesis of dsRNA targeting and silencing the key pathogenic effector gene E52 of the multistalked rust fungus Puccinia polysora, as described in S1, is as follows:

[0010] S101. Extract total RNA from maize leaves carrying uredinia of Puccinia polysora and reverse transcribe to obtain cDNA.

[0011] S102. Based on the E52 gene cDNA sequence, PCR amplification was performed using primers T7-E52-F and T7-E52-R to obtain the PCR product. The amplified target gene fragment was recovered by 1.2% agarose gel electrophoresis to obtain the purified PCR product.

[0012] The PCR amplification reaction system was as follows: 2 μL of cDNA obtained from S101, 2 μL of primer T7-E52-F, 2 μL of primer T7-E52-R, 25 μL of 2 × KeyPo Master Mix (Dye Plus), and ddH2O to a final volume of 50 μL.

[0013] The PCR amplification reaction program was as follows: 98℃ for 30 sec; 98℃ for 10 sec; 57℃ for 5 sec; 72℃ for 10 sec, for a total of 35 cycles; 72℃ for 1 min, then hold at 16℃.

[0014] The nucleotide sequence of the primer T7-E52-F is shown in SEQ ID No. 7;

[0015] The nucleotide sequence of the primer T7-E52-R is shown in SEQ ID No. 8;

[0016] S103. Using the purified PCR product obtained in S102 as template DNA, dsRNA targeting the E52 gene fragment was synthesized in vitro using the T7 RNATranscription Kit.

[0017] The nucleotide sequence of the cDNA of the E52 gene of *Russula multifiliis* in step (1) is shown in SEQ ID No. 3; the nucleotide sequence of the dsRNA is shown in SEQ ID No. 4.

[0018] In step (2) above, the final concentrations of nano-chitin and dsRNA in E52-dsRNA-NC nucleic acid nanopesticide are the same.

[0019] The final concentration of the aforementioned nano-chitin was 100-150 mg / L, and the final concentration of dsRNA was 100-150 mg / L.

[0020] The aforementioned southern rust disease of corn is caused by the rust fungus Puccinia polysora.

[0021] Secondly, the present invention provides an E52-dsRNA-NC nucleic acid nanopesticide prepared using the above-described method.

[0022] In the above-mentioned E52-dsRNA-NC nucleic acid nanopesticide, E52-dsRNA binds to nano-chitin.

[0023] Thirdly, this invention provides the application of the aforementioned E52-dsRNA-NC nucleic acid nanopesticide in the control of southern rust disease in maize. The concentration of the aforementioned E52-dsRNA-NC nucleic acid nanopesticide used is 100-150 mg / L.

[0024] The present invention has the following beneficial effects:

[0025] 1. In vitro synthesis and purification of dsRNA targeting and silencing the key pathogenic gene E52 of *Puccinia polysora*, followed by addition to a nanochitin (NC) colloidal dispersion system. This invention also provides an application for controlling southern rust of maize. This invention combines naked dsRNA with nanochitin to prepare a dsRNA-NC nucleic acid nanopesticide that exhibits anti-*Puccinia polysora* activity. The expression levels of the pathogen-related target gene are significantly inhibited during the infection stage, resulting in a significant reduction in the infectivity of *Puccinia polysora*. Nanochitin, derived from the shells of crustaceans, is inexpensive, readily degradable in the environment, eco-friendly in the field, and leaves no residue. Therefore, this RNAi-based nucleic acid pesticide has broad application prospects in the control of *Puccinia polysora*.

[0026] 2. This invention takes the green control of southern rust in maize as its basic starting point. Based on RNAi technology, it combines naked dsRNA with NC to prepare RNAi-based nucleic acid nanopesticides resistant to southern rust, providing technical support for the green control of *Hemiptera chinensis*. Efficacy evaluation demonstrates that the dsRNA-NC nucleic acid nanopesticide of this invention effectively controls southern rust in maize. The expression levels of target genes related to *Hemiptera chinensis* are significantly inhibited during the infection stage, resulting in a significant reduction in the infectivity of *Hemiptera chinensis*. Furthermore, chitin, derived from the shells of crustaceans, is readily available, inexpensive, and readily degradable in the environment, making it eco-friendly and residue-free. Therefore, this RNAi-based nucleic acid pesticide has a very broad application prospect in the control of *Hemiptera chinensis*. Attached Figure Description

[0027] 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.

[0028] Figure 1 The images are gel electrophoresis diagrams; where A is a Flu-EGFP-dsRNA gel electrophoresis diagram; B is an E52-dsRNA gel electrophoresis diagram; and C is an EGFP-dsRNA gel electrophoresis diagram.

[0029] Figure 2 A diagram illustrating the effect of conjugating EGFP-dsRNA to nanochitin (NC).

[0030] Figure 3 This describes the absorption of exogenous dsRNA by *Russula multifiliis*.

[0031] Figure 4 The diagram shows the inhibitory effect of E52-dsRNA-NC on southern rust of maize; where A is the phenotype of southern rust of maize; B is the expression level of the E52 gene in *Russula multifiliis*; C is the biomass of *Russula multifiliis* (** indicates significant difference at P < 0.01); D is the mycelium diagram of *Russula multifiliis*; and E is a statistical graph of the mycelium coverage area of ​​*Russula multifiliis*. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example 1

[0035] Specific primers were designed based on the EGFP cDNA sequence (with the T7 promoter added to the 5' end of the primers) to amplify an EGFP fragment of 326 bp (340–665 bp) in length by PCR.

[0036] S1. In this Example 1, the fluorescently labeled dsRNA (Flu-EGFP-dsRNA) used to detect the ability of Puccinia polysora to absorb exogenous dsRNA was synthesized in vitro and purified to obtain dsRNA.

[0037] The in vitro synthesis method of the fluorescently labeled dsRNA (Flu-EGFP-dsRNA) used to detect the ability of Puccinia polysora to absorb exogenous dsRNA is as follows:

[0038] S101. Based on the EGFP-containing plasmid stored in the laboratory, PCR amplification was performed using primers T7-EGFP-F and T7-EGFP-R (Table 1) to obtain PCR products. The PCR products were subjected to 1.2% agarose gel electrophoresis, and then the amplified target gene fragment was recovered using a universal DNA purification and recovery kit (Novizan) to obtain purified PCR products.

[0039] The nucleotide sequence of the primer T7-EGFP-F is shown in SEQ ID No. 5; the nucleotide sequence of the primer T7-EGFP-R is shown in SEQ ID No. 6; the T7 promoter has been added to the 5' end of both SEQ ID No. 5 and No. 6 sequences.

[0040] Table 1. Primers used for detecting the in vitro synthesis of exogenous EGFP-dsRNA by *Russula multifiliis*.

[0041]

[0042] The PCR amplification reaction system was as follows: 1 μL of plasmid containing EGFP, 2 μL of primer T7-EGFP-F, 2 μL of primer T7-EGFP-R, 25 μL of 2 × KeyPo Master Mix (Dye Plus), and ddH2O to a final volume of 50 μL.

[0043] The PCR amplification reaction program was as follows: 98℃ for 30 sec; 98℃ for 10 sec; 57℃ for 5 sec; 72℃ for 10 sec, for a total of 35 cycles; 72℃ for 1 min, then hold at 16℃.

[0044] S102. Using the Fluorescein RNA Labeling Mix Kit, the purified PCR product obtained in S101 was used to synthesize naked dsRNA containing a fluorescently labeled EGFP gene fragment in vitro; the reaction system was as follows:

[0045] 10 × Reaction Buffer 2 μL, Fluorescein RNA Labeling Mix Kit 2 μL, purified PCR product obtained from S101 1 μg, T7 RNA Polymerase Mix 2 μL, ddH2O to make up to 20 μL;

[0046] The reaction procedure is as follows: react at 37℃ for 3 h, incubate at 72℃ for 10 min, and then incubate at 25℃ for 20 min;

[0047] Take 1 µL of the reaction product, dilute it 10-fold with DEPC·H2O, add 1 µL of 5 × Loading Buffer and mix well. Perform 1.2% gel electrophoresis to check the quality of dsRNA (the correct dsRNA size is a single band of 326 bp, i.e., ...). Figure 1 -A), purified in vitro transcribed dsRNA, stored at -80℃.

[0048] S2. The target gene E52 of Puccinia polysora RNAi is a key pathogenic gene for southern maize rust. The cDNA sequence of the key pathogenic gene E52 of Puccinia polysora was obtained by bioinformatics. Based on the E52 cDNA sequence, specific primers (with the T7 promoter added to the 5' end of the primers) were designed, and the E52 fragment with a length of 308 bp (42–350 bp) was amplified by PCR.

[0049] The in vitro synthesis method for targeting and silencing the key pathogenic effector gene E52 of Puccinia polysora is as follows:

[0050] S103. Extract total RNA from maize leaves carrying spore masses of Puccinia polysora and reverse transcribe to obtain cDNA.

[0051] S104. Based on the cDNA obtained in S103, PCR amplification was performed using primers T7-E52-F and T7-E52-R (Table 2) to obtain PCR products. The amplified target gene fragments were then recovered by 1.2% agarose gel electrophoresis and purified using a universal DNA purification and recovery kit (Novizan) to obtain purified PCR products.

[0052] The nucleotide sequence of primer T7-E52-F is shown in SEQ ID No. 7; the nucleotide sequence of primer T7-E52-R is shown in SEQ ID No. 8; both SEQ ID No. 7 and No. 8 sequences have a T7 promoter added to the 5' end of the primer sequence.

[0053] Table 2 Primers used for in vitro synthesis of dsRNA targeting the effector gene E52 of *Rust hygroscopicus*.

[0054]

[0055] The PCR amplification reaction system was as follows: 1 μL of cDNA obtained from S103, 2 μL of primer T7-E52-F, 2 μL of primer T7-E52-R, 25 μL of 2 × KeyPo Master Mix (Dye Plus), and ddH2O to a final volume of 50 μL.

[0056] The PCR amplification reaction program was as follows: 98℃ for 30 sec; 98℃ for 10 sec; 57℃ for 5 sec; 72℃ for 10 sec, for a total of 35 cycles; 72℃ for 1 min, then hold at 16℃.

[0057] S105. Using the purified PCR product obtained in S104, naked dsRNA was synthesized in vitro using the T7 RNA Transcription Kit; the reaction system was as follows:

[0058] 1 μL T7 RNA Polymerase, 1 μL RNase Inhibitor, 2 μL T7 Transcription Buffer, 2 μL 100 mM rATP, 2 μL 100 mM rGTP, 2 μL 100 mM rCTP, 2 μL 100 mM rUTP, 1 μg of purified PCR product obtained from S104, and DEPC·H2O to a final volume of 20 μL.

[0059] The reaction procedure is as follows: react at 37℃ for 3 h, incubate at 72℃ for 10 min, and then incubate at 25℃ for 20 min;

[0060] Take 1 µL of the reaction product, dilute it 10-fold with DEPC·H2O, add 1 µL of 5 × Loading Buffer and mix well. Perform 1.2% gel electrophoresis to check the quality of dsRNA (correct dsRNA is a single band of 308 bp). Figure 1 -B), purified in vitro transcribed dsRNA, stored at -80℃.

[0061] S3. Disperse nano-chitin (NC) in DEPC·H2O and sonicate to obtain an NC colloidal dispersion system. Add the dsRNA obtained in S1 and S2 to the obtained NC colloidal dispersion system and mix thoroughly to obtain Flu-EGFP-dsRNA-NC for detecting the ability of *Heterophytic rust* to absorb exogenous dsRNA and E52-dsRNA-NC for controlling southern rust of maize.

[0062] In step S3 above, the final concentration of NC in the dsRNA-NC nucleic acid nanopesticide for controlling southern rust of corn can be in the range of 100-150 mg / L, and the final concentration of dsRNA can also be in the range of 100-150 mg / L.

[0063] Screening of the mass concentration of dsRNA conjugated with nano-chitin (NC): In Example 1, the purified EGFP-dsRNA synthesized in step S1 was adjusted to 250 mg / L for later use; the NC colloidal dispersion obtained in S3 was diluted with DEPC·H2O, and the mass concentration of NC was adjusted to 250 mg / L, 500 mg / L, 750 mg / L, 1250 mg / L, and 2500 mg / L, respectively.

[0064] Mix NC colloidal dispersions of different concentrations with EGFP-dsRNA solutions of equal volume (2 μL each, 4 μL total), then add 2 μL of 5× DNA Loading Buffer and mix thoroughly. Take 6 μL of the mixture and load it onto a 1.2% agarose gel for electrophoresis to check the coupling effect, in order to screen the mass concentration ratio when NC is completely coupled with dsRNA.

[0065] The results are as follows Figure 2 As shown, when naked EGFP-dsRNA is mixed with NC at different mass concentration ratios, the coupling ability of NC to naked EGFP-dsRNA molecules is continuously enhanced as the proportion of NC increases. This is manifested by the increasing amount of precipitate around the sample well. When the mass ratio of naked dsRNA to NC reaches 1:5, naked EGFP-dsRNA can be completely coupled (i.e., 100% coverage).

[0066] Example 2

[0067] This embodiment describes the application of the dsRNA-NC nucleic acid nanopesticide prepared in Example 1 for controlling southern rust of maize. The dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize is used to control Puccinia polysora.

[0068] (a) Tests on the ability of Puccinia polysora to absorb exogenous dsRNA

[0069] Specific steps:

[0070] (1) Add Flu-EGFP-dsRNA to the NC colloidal dispersion system with a mass concentration of 250 mg / L, so that the final concentration of NC is 125 mg / L and the final concentration of dsRNA is 125 mg / L. Mix by inverting several times and let stand at room temperature for 3 min.

[0071] Specific experimental steps:

[0072] Fresh urediniospores of *Russula multifiliis* were collected and evenly sprinkled on the surface of Flu-EGFP-dsRNA and Flu-EGFP-dsRNA-NC solutions, respectively, allowing them to come into contact with air (*Russula multifiliis* urediniospores do not germinate under anaerobic conditions). They were then incubated at 25°C in the dark for 3 h. The entry of Flu-EGFP-dsRNA into the *Russula multifiliis* urediniospores and germ tubes was observed under a fluorescence microscope.

[0073] The results are as follows Figure 3As shown, no green fluorescence signal was observed in *Rust floribunda* treated with Flu-EGFP-dsRNA, while green fluorescence signals were observed in both urediniospores and germ tubes of *Rust floribunda* treated with Flu-EGFP-dsRNA-NC. The results indicate that *Rust floribunda* cannot absorb naked dsRNA, but can absorb dsRNA coupled with NC.

[0074] (II) Detection of the efficacy of NC-conjugated E52-dsRNA against southern rust in maize

[0075] (1) Preparation of NC colloidal dispersion system

[0076] Specific steps:

[0077] The preparation method of NC colloidal dispersion with a mass concentration of 200-300 mg / L is as follows: 2-3 mg of nano-chitin is added to 10 mL of DEPC·H2O and dispersed by ultrasonic vibration.

[0078] (2) Application of dsRNA-NC nucleic acid nanopesticides for the prevention and control of southern rust in maize

[0079] Specific steps:

[0080] The E52-dsRNA obtained after purification in step S105 of Example 1 was diluted with DEPC·H2O to a concentration of 200-300 mg / L. An equal volume of the 200-300 mg / L E52-dsRNA solution and 200-300 mg / L NC colloidal dispersion were mixed, and an appropriate amount of Tween was added. The mixture was inverted several times to obtain an E52-dsRNA-NC nucleic acid nanopesticide with a concentration of 100-150 mg / L. The mixture was then allowed to stand at room temperature for 3 min for efficacy evaluation.

[0081] Fresh urediniospores of *Russula multiflora* were collected and prepared into a spore suspension (concentration 1×10⁻⁶). 5 ), 20 μL of spore suspension was inoculated onto healthy two-leaf stage B73 maize leaves. After the inoculated plants were cultured in the dark at 25°C and 90% relative humidity for 24 h, E52-dsRNA-NC nucleic acid nanopesticide at a concentration of 125 mg / L was sprayed onto the maize leaves of the treatment group using a sprayer.

[0082] The specific grouping arrangements are as follows:

[0083] H2O: Blank control group, each leaf was sprayed with 20 μL of sterile H2O;

[0084] EGFP-dsRNA-NC: Negative control group, each leaf was sprayed with 20 μL of EGFP-dsRNA-NC;

[0085] E52-dsRNA-NC: Treatment group, each leaf was sprayed with 20 μL of E52-dsRNA-NC;

[0086] Both control and treatment plants continued to be cultured in a growth chamber at 25°C and 90% relative humidity (12 h / 12 ​​h light / dark cycle). Samples were taken 3 and 5 days after inoculation, and disease development was observed 10 days after inoculation. Photos were taken and samples were collected.

[0087] Total RNA was extracted from leaves of the control and treatment groups 3 days after inoculation using the Trizol method, and cDNA was obtained by reverse transcription using the HiScript II Reverse Transcriptase (Glycerol-free) kit (Novizan). The PP-tub-Actin gene, which is stably expressed at different growth and development stages of *Russula multitubatus*, was used as an internal control, and the expression level of the key pathogenic gene E52 of *Russula multitubatus* was detected by RT-qPCR.

[0088] WGA staining

[0089] Specific experimental steps:

[0090] Five days after inoculation, maize leaves from the control group and the treatment group were collected and placed in 5 ml centrifuge tubes. The tissues were decolorized with a decolorizing solution (glacial acetic acid: anhydrous ethanol = 1:1) until the leaves turned white and no chlorophyll was visible.

[0091] Clear the decolorized leaf tissue with chloral hydrate for 1-2 days; rinse the cleared sample twice with 50% ethanol for 15 min each time; rinse once or twice with distilled water for 10 min each time.

[0092] The sample was transferred into a 1 M potassium hydroxide solution and placed in a boiling water bath for 20-30 minutes to soften the leaf tissue and facilitate the entry of the fluorescent dye into the mesophyll cells.

[0093] Rinse 1-2 times with distilled water, 10 min each time; then transfer the sample to 50 mM Tri-HCl (pH 7.0-7.4) and soak for 30 min.

[0094] Stain with 20 ug / ml WGA staining solution in the dark for at least 10 minutes (the time can be long because the staining is specific and there will be no background staining); recover the WGA staining solution (the staining solution can be stored at 4℃ for a long time); rinse with distilled water 2-3 times, 10 minutes each time; transfer to 50% glycerol for storage.

[0095] Observe and photograph under a fluorescence microscope.

[0096] Total DNA was extracted from leaves of the control and treatment groups 10 days after inoculation using DNA extraction buffer and diluted to 200 ng / μL. The PP-tub-Actin gene, which is stably expressed at different growth and development stages of *Russula multitubatus*, and the ZmUbi-Actin gene, which is stably expressed at different growth and development stages of maize, were used as internal controls. The biomass of *Russula multitubatus* was detected by RT-qPCR.

[0097] qPCR primers were designed using the Nanjing GenScript Biotech Co., Ltd. website (https: / / www.genscript.com.cn / ) and synthesized by ordering from the Shanghai Sangon Biotech Online Primer Design website.

[0098] Table 3 Primers for RT-qPCR detection

[0099]

[0100] The results are as follows Figure 4 As shown in (AE), there were no significant differences in lesion area, E52 gene expression level, biomass of *Russula multifiliis*, and the area covered by its infecting hyphae between H2O treatment (blank control) and EGFP-dsRNA-NC treatment (negative control). Compared with the blank control and negative control, E52-dsRNA-NC treatment showed a significant reduction in lesion area, a significant downregulation of E52 gene expression level, a significant decrease in *Russula multifiliis* biomass, and a significant reduction in the area covered by *Russula multifiliis* infecting hyphae.

[0101] This invention targets the key effector protein E52 of *Hemiberlesia lataniae*, and designs and synthesizes dsRNA (E52-dsRNA) targeting the E52 gene based on RNAi technology. This dsRNA has a significant silencing effect on E52. Meanwhile, NC nanomaterials themselves carry a positive charge, allowing them to bind to the fungal cell wall and exhibit broad-spectrum antibacterial effects. Furthermore, E52-dsRNA carries a negative charge. When NC and E52-dsRNA are mixed, it is found that NC can efficiently couple with E52-dsRNA through electrostatic interactions (E52-dsRNA:NC = 1:5). After binding with NC, E52-dsRNA is easily absorbed by *Hemiberlesia lataniae*, efficiently silencing its key effector gene E52 within the pathogen, effectively "disarming" the pathogen and reducing its pathogenicity. The combined application of NC and E52-dsRNA allows for synergistic effects, working both externally and internally to achieve broad-spectrum and precise control of *Hemiberlesia lataniae*, demonstrating significant application potential.

[0102] Specifically, using nanomaterials to conjugate and coat dsRNA can prevent its degradation in the natural environment (such as soil and plant surface) or within organisms (such as fungal extracellular matrix), thus protecting the effective amount of applied dsRNA. Spraying E52-dsRNA-NC onto maize leaves significantly reduced the lesion area of ​​southern rust in maize, and significantly downregulated the expression level of the E52 gene in the pathogen, resulting in a significant reduction in its infectivity to maize.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an E52-dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize, characterized in that, The steps are as follows: (1) Using the cDNA of the E52 gene of *Russula multifiliis* as a template, E52-dsRNA targeting the E52 gene fragment was synthesized in vitro; nano-chitin was dispersed in DEPC water and ultrasonically vibrated to obtain an NC colloidal dispersion system. (2) Add the dsRNA from step (1) to the NC colloidal dispersion system and mix to obtain E52-dsRNA-NC nucleic acid nanopesticide.

2. The method for preparing dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize according to claim 1, characterized in that: The nucleotide sequence of the E52 gene cDNA of *Russula multifiliis* in step (1) is shown in SEQ ID No.

3.

3. The preparation method of the dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize according to claim 1, characterized in that: The nucleotide sequence of the dsRNA in step (1) is shown in SEQ ID No.

4.

4. The preparation method of the E52-dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize according to claim 3, characterized in that: In step (2), the final concentrations of nano-chitin and dsRNA in the E52-dsRNA-NC nucleic acid nanopesticide are the same.

5. The method for preparing the E52-dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize according to claim 4, characterized in that: The final concentration of the nano-chitin is 100-150 mg / L, and the final concentration of the dsRNA is 100-150 mg / L.

6. The method for preparing the E52-dsRNA-NC nucleic acid nanopesticide for controlling southern rust of maize according to any one of claims 1-5, characterized in that: The southern rust disease of maize is caused by the rust fungus Puccinia polysora.

7. The E52-dsRNA-NC nucleic acid nanopesticide prepared using the method described in claim 6.

8. The E52-dsRNA-NC nucleic acid nanopesticide according to claim 7, characterized in that: In the E52-dsRNA-NC nucleic acid nanopesticide, E52-dsRNA binds to nano-chitin.

9. The application of the E52-dsRNA-NC nucleic acid nanopesticide according to claim 7 or 8 in the prevention and control of southern rust disease in maize.

10. The application according to claim 9, characterized in that: The concentration of the E52-dsRNA-NC nucleic acid nanopesticide used is 100-150 mg / L.

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