Preparation method and application of COS-dsRNA nano nucleic acid pesticide for preventing and treating apple ring spot

Abstract

The invention provides a preparation method of a COS-dsRNA nano nucleic acid pesticide for preventing and treating apple ring rot, which comprises the following steps: carrying out in-vitro synthesis and purification on dsRNA of a key pathogenic effect gene BdNIS1 of targeted silence botryosphaeria dothidea to obtain dsRNA, and adding the dsRNA into a nano chitosan oligosaccharide aqueous solution to obtain the COS-dsRNA nano nucleic acid pesticide for preventing and treating botryosphaeria dothidea. The invention further provides application for preventing and treating apple ring spot. The exposed dsRNA and the nano chitosan oligosaccharide are compounded, the prepared COS-dsRNA nano nucleic acid pesticide has the effect of resisting the physalospora piricola, the expression quantity of pathogenic bacteria related target genes in the infection stage is obviously inhibited, and the infection capacity of the physalospora piricola is obviously reduced. The nano chitosan oligosaccharide is low in cost, easy to degrade in the environment, eco-friendly to the field and free of residues. Therefore, the RNAi nucleic acid pesticide has a wide application prospect in the field of prevention and control of physalospora piricola.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control, and particularly relates to a preparation method and application of a COS-dsRNA nano nucleic acid pesticide for preventing and controlling apple ring rot. Background Art

[0002] apple( Malusdomestica ) is one of the four world-famous fruits and one of the fruit varieties that my country exports the most foreign exchange. According to data from the National Bureau of Statistics, in 2022, my country's apple planting area was 29.3366 million mu, with a total output of 47.5718 million tons, accounting for 53.66% of the world's total apple production, with an output value of 228.71 billion yuan. Apple and its related industries occupy an important position in my country's agricultural economy and must be given high attention. Apple ring rot is one of the main diseases affecting the safe production of apples in my country. The pathogenic fungi of this disease are complex, mainly composed of the genus Botrytis ( Botryosphaeria dothidea ), Botrytis cinerea ( B. obtusa ), Botrytis cinerea ( B. stevensii ) and Botrytis cinerea ( B.kuwatsukai ) and other species, among which Botrytis cinerea ( B. dothidea ) is its main pathogen. B.dothidea Not only can it infect branches and trunks, causing rough bark or ulcers, severely weakening tree vigor and reducing fruit quality and quantity, it can also harm fruit during storage, causing it to rot and deteriorate, leading to serious economic losses. Currently, the prevention and control of apple ring rot mainly relies on the selection of excellent disease-resistant varieties, combined with scientific planting plans and the spraying of chemical pesticides for comprehensive prevention and control. However, the breeding cycle of apple varieties is long, difficult, and slow to show results. At the same time, the large-scale use of chemical agents not only pollutes the environment and enhances pathogen resistance, but also forms pesticide residues on the fruit, which enter the human body through the biological cycle and seriously threaten the health of consumers. Therefore, the development of new green, efficient, and low-residue biopesticides has become an indispensable path to achieving the sustainable development of modern agriculture.

[0003] In recent years, with the rapid development of RNAi technology, this technology has shown great application potential in the field of plant pathogen control. More and more cases have proved that it is feasible to use nucleic acid pesticides based on RNAi technology to control diseases. RNAi nucleic acid pesticides have been successfully used to control Fusarium graminearum ( Fusarium graminearum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), myrtle rust ( Austropuccinia psidii )、Botrytis cinerea( Botrytis cinerea ), Rice Sheath Blight Pathogen ( Rhizoctonia solani ), Aspergillus niger ( Aspergillus niger ) and Verticillium dahliae ( Verticillium dahliae ) and other fungi. B. dothidea ) , their sensitivity to RNAi, or the ability of pathogens to absorb exogenous double-stranded small RNA (dsRNA), is rarely reported. It is well known that RNAi primarily functions through dsRNA and siRNA. Naked dsRNA remains relatively stable only at low temperatures (<4°C), with its molecular stability significantly reduced in the wild. Exposed to the elements, naked dsRNA is susceptible to degradation by ultraviolet light, high temperatures, and the widespread nucleases found on plant surfaces, which can affect its efficacy. Therefore, developing RNAi-based nucleic acid pesticides based on dsRNA or siRNA, and enhancing their molecular stability through structural modifications or synergistic enhancers, will provide a breakthrough solution for the industrial application of a new generation of nucleic acid pesticides.

[0004] Chitosan oligosaccharide (COS) is an oligosaccharide derivative obtained by controlled degradation of chitosan. Its molecular structure consists of 2-8 glucosamine units connected by β-(1→4) glycosidic bonds. COS has the following remarkable properties: (1) excellent water solubility; (2) excellent transmembrane permeability; and (3) enhanced biological activity. These properties make COS show broad application prospects. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies in the above-mentioned prior art and provide a method for preparing a COS-dsRNA nanonucleic acid pesticide for preventing and treating apple ring rot and its application. The method compounds naked dsRNA with nano-chitosan oligosaccharides to prepare an RNAi-type nanonucleic acid pesticide that resists apple ring rot pathogens. The COS-dsRNA nanonucleic acid pesticide for preventing and treating apple ring rot of the present invention has an effect of resisting apple ring rot pathogens, and the expression level of pathogen-related target genes is significantly inhibited during the infection stage, significantly reducing the infection ability of apple ring rot pathogens. In addition, COS nanomaterials are easy to obtain and low in cost. At the same time, they are easily degraded in the environment, are eco-friendly to field fields, and leave no residue. Therefore, this RNAi-type nucleic acid pesticide has a very broad application prospect in the field of prevention and control of apple ring rot pathogens.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a COS-dsRNA nanopesticide for preventing and controlling apple ring rot, the method comprising: S1, targeted silencing of apple ring rot pathogen ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The dsRNA is synthesized in vitro and purified to obtain dsRNA; The apple ring rot pathogen ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The full-length nucleotide sequence of the apple ring rot pathogen ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The cDNA nucleotide sequence is shown as SEQ ID No. 2; the nucleotide sequence of the dsRNA is shown as SEQ ID No. 3; S2. Add the dsRNA obtained in S1 to the nano-chitosan oligosaccharide aqueous solution, and after mixing, obtain a COS-dsRNA nano-nucleic acid pesticide (named COS-dsBdNIS1) for preventing and controlling apple ring rot.

[0007] Preferably, the targeted silencing of apple ring rot pathogen in S1 ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The method for in vitro synthesis of dsRNA is: S101, extract apple ring rot pathogen ( Botryosphaeria dothidea ) total RNA, and reverse transcription was performed to obtain cDNA; S102. Based on the cDNA obtained in S1, PCR amplification was performed using primers T7-BdNIS1-F and T7-BdNIS1-R to obtain a PCR product. The amplified target gene fragment was recovered by electrophoresis on a 1.2% agarose gel to obtain a purified PCR product. The reaction system for PCR amplification was as follows: 1 μL of cDNA obtained in S1, 1 μL of primer T7-BdNIS1-F, 1 μL of primer T7-BdNIS1-R, 12.5 μL of KOD DNA polymerase, and ddH2O to make up to 25 μL; The reaction program for PCR amplification was as follows: 98°C for 3 min; 98°C for 10 sec, 58°C for 5 sec, and 68°C for 5 sec, for a total of 35 cycles; 68°C for 7 min, and hold at 16°C; The nucleotide sequence of the primer T7-BdNIS1-F is shown in SEQ ID No. 4; The nucleotide sequence of the primer T7-BdNIS1-R is shown in SEQ ID No. 5; SEQ ID No. 4-5 sequences all have T7 promoter added to the 5' end of the primer sequence.

[0008] S103, using the purified PCR product obtained in S102 as a template DNA, and using a T7 RNA Transcription Kit to synthesize naked dsRNA targeting the target gene fragment in vitro; Alternatively, the purified PCR product obtained in S102 is used with the HyperScribe™ T7 High Yield Cy3 / Cy5 RNA Labeling Kit to synthesize naked dsRNA containing a fluorescently labeled target gene fragment in vitro.

[0009] Preferably, when the T7 RNA Transcription Kit is used in S103, the reaction system is: 1 μL of T7 RNA Polymerase, 1 μL of RNase Inhibitor, 5 μL of 10× Transcription Buffer, 8 μL of NTP Mix, 1 μg of the purified PCR product obtained in S102, and DEPC H2O added to 50 μL; the reaction procedure is: 37°C for 4 hours, incubation at 72°C for 10 minutes, and then incubation at room temperature for 20 minutes; When using HyperScribe in S103 TM For the T7 High Yield Cy3 / Cy5 RNA Labeling Kit, the reaction system is: 2 μL of 10× Reaction Buffer, 2 μL of 20 mM ATP, 2 μL of 20 mM GTP, 2 μL of 20 mM CTP, 1.5 μL of 20 mM UTP, 1 μL of 10 mM Cy3-UTP, 1 μg of the purified PCR product obtained in S102, 2 μL of T7 RNA Polymerase Mix, and ddH2O to make up to 20 μL; the reaction procedure is: 37°C for 4 hours.

[0010] Preferably, the preparation method of the nano-chitosan oligosaccharide aqueous solution in S2 is: dissolving the nano-chitosan oligosaccharide in DEPC water and dispersing it by ultrasonic vibration.

[0011] Preferably, the final concentration of nano-chitosan oligosaccharide in the COS-dsRNA nano nucleic acid pesticide for preventing and treating apple ring rot in S2 is 0.005×10 -2 g / mL, and the final concentration of dsRNA was 0.005×10 -2 g / mL.

[0012] The present invention also provides the use of the COS-dsRNA nanoparticle nucleic acid pesticide for preventing and treating apple ring rot prepared by the above-mentioned preparation method, wherein the COS-dsRNA nanoparticle nucleic acid pesticide for preventing and treating apple ring rot is used to prevent and treat apple ring rot ( Botryosphaeria dothidea ).

[0013] Compared with the prior art, the present invention has the following advantages: 1. This invention, based on the green prevention and control of apple ring rot, utilizes RNAi technology to combine naked dsRNA with nano-chitosan oligosaccharides (COS) to produce an RNAi-based nanoparticle pesticide for the prevention and control of apple ring rot. This provides technical support for the green prevention and control of apple ring rot. Efficacy evaluation demonstrated that the COS-dsRNA nanoparticle pesticide for the prevention and control of apple ring rot was effective against the pathogen, significantly suppressing the expression of pathogen-related target genes during the infection phase and significantly reducing the infection capacity of the pathogen. Furthermore, COS nanomaterials, derived from the shells of crustaceans, are readily available and inexpensive. They also readily degrade in the environment, are eco-friendly for field use, and leave no residue. Therefore, this RNAi-based nanoparticle pesticide has broad application prospects in the prevention and control of apple ring rot.

[0014] 2. The COS-dsRNA nanonucleic acid pesticide prepared by the present invention for preventing and controlling apple ring rot has the effect of resisting apple ring rot fungus. Whether the nano-sized nucleic acid drug is applied to apple ring rot fungus or to apple fruits and branches, it can significantly inhibit the expression of pathogenic genes related to the pathogen, weaken the infection ability of apple ring rot fungus to a certain extent, and thus inhibit the expansion of apple ring rot lesions.

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The target silencing fungus for apple ring rot ( B. dothidea ) Key pathogenic effector genes BdNIS1 Gel electrophoresis of naked dsRNA (1.2% agarose gel, 120V, 28min).

[0017] Figure 2 In Example 2 of the present invention, Cy5 / Cy3-dsBdNIS1 labeled with fluorescein was used to incubate apple ring rot pathogen ( B. dothidea ) The absorption of fluorescently labeled dsRNA by pathogen hyphae was observed using a fluorescence microscope within 6 hours, and the mycelium at the corresponding treatment time points BdNIS1 Gene expression level.

[0018] Among them, (a) shows the absorption of dsRNA by the pathogen and the distribution of dsRNA in the mycelium cells when the mycelium of apple ring rot pathogen was incubated with Cy5-dsBdNIS1 for 6 hours; (b) shows the absorption of dsRNA by the pathogen and the distribution of dsRNA in the mycelium cells when the mycelium of apple ring rot pathogen was incubated with Cy3-dsBdNIS1 for 1-6 hours, and the distribution of dsRNA in the mycelium cells was observed every 1 hour; (c) shows the absorption of dsRNA in the mycelium of the pathogen at the corresponding time point in (b). BdNIS1Gene expression (** indicates significant difference at the P ≤ 0.01 level).

[0019] Figure 3 This figure shows the inhibitory effect of dsBdNIS1 on apple fruit ring rot and apple twig ring rot tested using two treatment methods in Example 2 of the present invention (Treatment A: incubating the mycelium of the apple ring rot pathogen with dsBdNIS1 for 6 hours before inoculation; Treatment B: spraying dsBdNIS1 directly onto the inoculation point on the fruit or twig and then inoculating the mycelium of the apple ring rot pathogen).

[0020] The statistical results for apple fruits are after 3 days of incubation, and the statistical results for apple branches are after 7 days of incubation. (a) is the incidence map of apple ring rot on fruits and branches; (b) is the statistical map of the area of ​​ring rot lesions and the interaction between apple ring rot pathogens and apple fruits. BdNIS1 Gene expression levels (** indicates significant differences at the P ≤ 0.01 level).

[0021] Figure 4 This is a diagram showing the coupling effect of nano-chitosan oligosaccharide (COS) on dsBdNIS1 in Example 2 of the present invention, and a diagram showing the inhibitory effect on apple ring rot on apple fruits when COS and dsBdNIS1 are applied in a 1:1 ratio.

[0022] Among them, (a) shows the coupling effect of COS on dsBdNIS1 at different concentration ratios; (b) and (c) show the inhibitory effect of CTS-dsBdNIS1 on apple ring rot tested by the TreatmentB method (** indicates significant difference at the P≤0.01 level, ns indicates no significant difference). DETAILED DESCRIPTION

[0023] Example 1 Apple ring rot fungus ( B.dothidea ) RNAi target gene, which has NIS1 (Necrosis-inducing secreted protein 1) domain, is the key pathogenic effect gene of apple ring rot pathogen identified. Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The gene sequence of B. dothidea The whole genome sequence and GFF3 gene annotation file were obtained through the correspondence between proteins and genes), from which a gene sequence with a length of 413 bp containing the key domain (NIS1) was selected ( BdNIS1Specific primers were designed based on the 55-467 bp region of the cDNA sequence (413 bp is SEQ ID No. 3) (T7 promoter was added to the 5' end of the primer).

[0024] BdNIS1 The nucleotide sequence of the cDNA sequence is shown in SEQ ID No. 2; The preparation method of the COS-dsRNA nanopesticide for preventing and treating apple ring rot in this embodiment is as follows: S1, targeted silencing of apple ring rot pathogen ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The dsRNA was synthesized in vitro and purified to obtain dsRNA (named dsBdNIS1); the apple ring rot pathogen ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The full-length nucleotide sequence of the apple ring rot pathogen is shown in SEQ ID No.1; Botryosphaeria dothidea Key pathogenic effector genes BdNIS1 The cDNA nucleotide sequence is shown in SEQ ID No. 2; the nucleotide sequence of the dsRNA is shown in SEQ ID No. 3; The targeted silencing of apple ring rot pathogen ( Botryosphaeria dothidea ) Key pathogenic effector genes BdNIS1 The method for in vitro synthesis of dsRNA is: S101, extract apple ring rot pathogen ( Botryosphaeria dothidea ) total RNA, and reverse transcription was performed to obtain cDNA; S102. Using the cDNA obtained in S1 as the amplification template, PCR amplification was performed with primers T7-BdNIS1-F and T7-BdNIS1-R (Table 1). The resulting PCR product was subjected to 1.2% agarose gel electrophoresis (120 V, 28 min). Subsequently, a universal DNA purification and recovery kit (Shanghai Huiling) was used to recover the amplified target gene fragment to obtain a purified PCR product. Table 1 Targeted effector genes of apple ring rot pathogen BdNIS1 Primers used for in vitro synthesis of dsRNA

[0025] The reaction system for PCR amplification was as follows: 1 μL of cDNA obtained in S1, 1 μL of primer T7-BdNIS1-F, 1 μL of primer T7-BdNIS1-R, 12.5 μL of KOD DNA polymerase, and ddH2O to make up to 25 μL; The reaction program for PCR amplification was as follows: 98°C for 3 min; 98°C for 10 sec, 58°C for 5 sec, and 68°C for 5 sec, for a total of 35 cycles; 68°C for 7 min, and hold at 16°C; S103, the purified PCR product obtained in S102 was used to TM T7 High Yield Cy3 / Cy5 RNA Labeling Kit (APE×BIO) for in vitro synthesis of naked dsRNA containing fluorescently labeled target gene fragments; The naked dsRNA synthesized in vitro with the fluorescently labeled target gene fragment is purified to obtain the fluorescein-labeled dsRNA; The reaction system is as follows: 10× Reaction Buffer 2μL, 20mM ATP 2μL, 20mM GTP 2μL, 20mM CTP 2μL, 20mM UTP 1.5μL, 10mM Cy3-UTP 1μL, 1μg of the purified PCR product obtained in S102, T7RNA Polymerase Mix 2μL, and ddH2O to make up to 20μL; The reaction procedure is: react at 37°C for 4 hours to form dsRNA.

[0026] Take 1µL of the reaction product, dilute it to 5µL with DEPC H2O, add 1µL of 6× Loading Buffer and mix well. Perform 1.2% gel electrophoresis to check the quality of dsRNA (qualified dsRNA is a single band of 413bp in size) Figure 1 The reverse transcribed dsRNA was recovered and stored at -20°C.

[0027] In the step S103 of this embodiment, the synthesis of dsRNA can also be dsRNA without fluorescein labeling, and the specific method is as follows: The purified PCR product obtained in S102 was used as template DNA to synthesize naked dsRNA targeting the target gene fragment in vitro using the T7 RNA Transcription Kit (Shanghai Bolang). The naked dsRNA targeting the target gene fragment is synthesized in vitro and purified to obtain dsRNA; The reaction system consisted of 1 μL of T7 RNA Polymerase, 1 μL of RNase Inhibitor, 5 μL of 10× Transcription Buffer, 8 μL of NTP Mix, and 1 μg of the purified PCR product obtained in S102, with DEPC H2O added to 50 μL. The reaction procedure was as follows: 37°C for 4 hours, incubation at 72°C for 10 minutes, and then incubation at room temperature for 20 minutes to form dsRNA. Take 1µL of the reaction product, dilute it to 5µL with DEPC H2O, then add 1µL of 6× Loading Buffer and mix well. Use 1.2% agarose gel electrophoresis (120V, 28min) to detect the synthesis of the transcription product (qualified dsRNA is a single band of 413bp in size, SEQ ID No. 3). Recover the transcribed dsRNA and store it at -20℃.

[0028] S2, dissolving nano-chitosan oligosaccharides in DEPC water, and dispersing by ultrasonic vibration to obtain nano-chitosan oligosaccharide aqueous solution; The dsRNA obtained in S1 was added to the obtained nano-chitosan oligosaccharide aqueous solution, and after mixing, a COS-dsRNA nano-nucleic acid pesticide for preventing and treating apple ring rot was obtained; the final concentration of nano-chitosan oligosaccharide in the COS-dsRNA nano-nucleic acid pesticide for preventing and treating apple ring rot was 0.005×10 -2 g / mL, and the final concentration of dsRNA was 0.005×10 -2 g / mL.

[0029] Example 2 This example is the application of the COS-dsRNA nanopesticide for preventing and treating apple ring rot prepared in Example 1. The COS-dsRNA nanopesticide for preventing and treating apple ring rot is used to prevent and treat apple ring rot ( Botryosphaeria dothidea ).

[0030] (1) Apple ring rot pathogen ( Botryosphaeria dothidea ) Absorption test of exogenous dsRNA Specific steps: (1) Use VIGSTool website (https: / / vigs.solgenomics.net / ) to predict BdNIS1 The best silencing site; (2) The fluorescein-labeled dsRNA synthesized in step S103 of Example 1 was diluted to 50 ng / μL with DEPC water. Mycelia at the edge of a rejuvenated apple ring rot pathogen colony on a PDA plate were immersed in the solution. The mixture was incubated at 25°C, 100 rpm, and protected from light for 6 h. During this period, a portion of the mycelia was collected every hour and observed under a Cy3 (λ = 550 nm) / Cy5 (λ = 649 nm) excitation light source to see if the fluorescent-labeled dsRNA had penetrated the pathogen's mycelia.

[0031] (3) The total RNA of mycelium within 1-6 hours of the above treatment was extracted using the Kangwei Century Ultrapure RNA Extraction Kit (CW0581S), and reverse transcribed using the All-in-One Script Rtpremix (with dsDNase) Kit (Ke Ermei) to obtain cDNA. Bd-Actin The key pathogenic effect genes of apple ring rot pathogen were detected by RT-qPCR technology after treatment with Cy5 / Cy3 fluorescent labeled dsRNA for 1-6h. BdNIS1 Target gene-specific primers were designed using DNAMAN (Table 2), and primer specificity was analyzed using Primer-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Primers were then ordered and synthesized on the Shanghai Bioengineering Online Primer Design website.

[0032] Table 2 RT-qPCR detection primers

[0033] like Figure 2 As shown, Figure 2 In Figures 2a and 2b, Bright is the microscope bright field channel, Cys (Cy5 / Cy3 fluorescence channel), and Merge is the fusion channel.

[0034] Figure 2 c is the expression level of BdNIS1 gene detected by real-time fluorescence quantitative PCR (RT-qPCR) at different time points after dsBdNIS1 treatment. The grouping treatments are: H2O treatment: (1) Pick the edge hyphae of the rejuvenated apple ring rot fungus colony on the PDA plate and immerse them in 500 μL of DEPC water. Incubate at 25°C, 100 rpm, in the dark for 1-6 hours. 6 treatments were performed in 1-hour increments. (2) Use the Kangwei Century Ultrapure RNA Extraction Kit (CW0581S) to extract the total RNA of the hyphae within 1-6 hours of the above treatment, and use the All-in-One Script Rtpremix (with dsDNase) Kit (Ke Ermei) to reverse transcribe and obtain cDNA for use.

[0035] Cy3 fluorescently labeled dsBdN1S1 treatment: (1) Pick the edge hyphae of the rejuvenated apple ring rot fungus colony on the PDA plate and immerse them in 500 μL of 50 ng / μL Cy3-dsBdNIS1. Incubate at 25°C, 100 rpm, in the dark for 1-6 hours. 6 treatments were performed in 1-hour increments. (2) Use the Kangwei Century Ultrapure RNA Extraction Kit (CW0581S) to extract the total RNA of the hyphae within 1-6 hours of the above treatment, and use the All-in-One Script Rtpremix (with dsDNase) Kit (Ke Ermei) to reverse transcribe and obtain cDNA for use.

[0036] RT-qPCR was used to detect the key pathogenic effect genes of apple ring rot pathogen after Cy3-dsBdNIS1 treatment for 1-6h. BdNIS1 The above H2O treatment 1-6h gene was used as a standard reference, and 2- △△Ct Conduct analysis.

[0037] The test results are as follows Figure 2 As shown in (a), 6 hours after treatment with Cy5-dsBdNIS1, Cy5 signals were observed within the mycelium. The Cy5 fluorescence signal was evenly distributed throughout the mycelium, with slightly stronger fluorescence at the mycelial septa than elsewhere. This suggests that after the apple ring rot pathogen absorbs dsRNA through the cell membrane and cell wall, it is primarily transported between cells through the pores in the septa.

[0038] To verify this conclusion, the dsRNA absorption by mycelium was further studied using Cy3-labeled dsRNA (dsBdNIS1-Cy3).

[0039] The test results are as follows Figure 2As shown in (b), after 1 hour of Cy3-dsBdNIS1 treatment, the Cy3 fluorescence signal was only observed near the hyphal septa, with a diffuse pattern. At 2 hours of treatment, a faint Cy3 fluorescence signal was observed within the hyphal cells. After 3 hours of treatment, more fluorescence signal appeared within the cells, and after 4 hours, the entire hyphal cell was completely filled. At 5 and 6 hours, the fluorescence signal within the hyphal cells became even more saturated and continuously increased. Strong fluorescence signals were observed near the hyphal septa at all treatment time points, suggesting that the apple ring rot pathogen may absorb dsRNA through the cell walls and membranes near the septa, gradually diffusing the dsRNA into the hyphal cells from the absorption point, ultimately filling the entire cell. Subsequently, the dsRNA can be transported through the pores in the hyphal septa to other adjacent cells, providing a crucial guarantee for sustained gene silencing. Figure 2 The arrows in (b) indicate the distribution location of Cy3-dsBdNIS1 inside the hyphae after the apple ring rot fungus absorbed the fluorescently labeled dsRNA (Cy3-dsBdNIS1).

[0040] extract Figure 2 (b) Total RNA from mycelia at the corresponding treatment time points was reverse transcribed and tested by RT-qPCR. BdNIS1 The experimental results are as follows Figure 2 (c) 2 hours after treatment BdNIS1 The expression level did not change significantly. After 3 hours, when dsBdNIS1 diffused into the cell, BdNIS1 The expression level of BdNIS1 The expression level of BdNIS1 In summary, the exogenously applied dsRNA can be efficiently absorbed by the apple ring rot pathogen and can significantly inhibit the expression of key pathogenic genes in the mycelium of the apple ring rot pathogen within a short period of time (within 4 hours).

[0041] (II) Inhibition of apple ring rot by naked dsBdNIS1 The naked dsBdNIS1 is the dsBdNIS1 synthesized using the T7 RNA Transcription Kit (Shanghai Bolang) in Example 1, that is, the naked dsBdNIS1 without a fluorescent label.

[0042] Specific steps: a.Apple fruit Step 1: Select healthy (no lesions, no insects) and uniformly sized Red Fuji apples picked from the same orchard. Wash them with tap water, then disinfect the surface with 75% alcohol. Place them in a cool and ventilated place to evaporate excess alcohol and dry excess water.

[0043] Step 2: Use a 5mm hole puncher to punch a 2-3mm deep hole in the middle of the treated apple (about 2cm from the fruit stem on the side of the fruit), and then apply 50μL of dsBdNIS1 into the hole.

[0044] Step 3: Use the apple ring rot pathogen isolated and preserved in our laboratory ( Botryosphaeria dothidea ) as the inoculum source, after it was rejuvenated on the PDA plate, the fungus was punched out at the edge of the colony with a 5mm puncher, and the fungus cake with the hyphae side inward was inoculated into the small hole in step 2, and cultured at 25℃ for 72h. The disease situation of each treatment group was observed and recorded by taking photos.

[0045] Step 4: Cut the tissue at the junction of the diseased and healthy parts of the fruit, and use the Quick RNA Isolation Kit (Huayueyang) to extract the total RNA of the above fruit tissue and reverse transcribe it.

[0046] Step 5: RT-qPCR reaction system was formed using 2×SYBR Greenq PCR Premix and Universal Real-time Fluorescence Quantitative PCR Kit (Kermei). BdNIS1 The gene expression level was used as a control to analyze the rot area and necrotic tissue of apple fruit after dsBdNIS1 treatment. BdNIS1 RT-qPCR was performed using a real-time fluorescence quantitative PCR system (Thermo Fisher Scientific Quant Studio 3). △△Ct analyze BdNIS1 The relative expression level.

[0047] b. Apple branches Straight and uniformly thick Red Fuji apple tree branches were selected from the same orchard, cut into 15 cm segments, rinsed with tap water, and surface disinfected with 75% alcohol. The branches were then placed in a cool and ventilated place to allow excess alcohol to evaporate.

[0048] The ends of the dried branch segments were sealed with paraffin to prevent dehydration of the branches, and then a small hole was punched in the middle of the branch using a 5 mm hole puncher, and then 50 μL of dsBdNIS1 was applied to the small hole.

[0049] The apple ring rot pathogen isolated and preserved in our laboratory ( Botryosphaeria dothidea ) as the inoculation source. After rejuvenation culture on PDA plates, cut the bacterial cake at the edge of the colony with a 5mm borer. Inoculate the cake with the mycelium side inward into the small hole in step 2. Culture at 25℃ with moisturizing for 7-10 days. Observe the disease situation of each treatment group and take photos for record.

[0050] The test results are as follows Figure 3 As shown in the figure, we adopted two treatment methods (Treatment A and Treatment B) to inoculate apple ring rot pathogen.

[0051] The groups are as follows: Treatment A: The mycelial blocks of the antibacterial fungus for apple ring rot were soaked in dsBdNIS1 for 6 hours before being inoculated onto apple branches and fruits (dsBdNIS1 in Treatment A in the figure). A mycelial block of the antibacterial fungus for apple ring rot, soaked in sterile H2O for the same period of time, was used as a positive control (Control in Treatment A in the figure).

[0052] Treatment B: dsBdNIS1 was sprayed directly onto the apple branches and fruits to be inoculated (dsBdNIS1 in Treatment B in the figure), and the mycelial blocks of apple ring rot pathogen were directly inoculated. Sterile H2O spraying was used instead of dsBdNIS1 spraying as a positive control (Control in Treatment B in the figure).

[0053] CK was the blank control, inoculated with sterile empty PDA agar blocks.

[0054] Treatment A and Treatment B were inoculated with apple ring rot pathogens, which could reduce the area of ​​rot or ulceration on apple fruits and branches. Total RNA was extracted from tissue samples at the junction of diseased and healthy apple fruits, and then reverse transcribed and tested by RT-qPCR. BdNIS1 The results showed that the expression level of apple ring rot pathogen in the sample tissues treated with Treatment A and Treatment B was BdNIS1 The gene expression levels decreased significantly. BdNIS1 With genes as targets, it is feasible to adopt spray-induced strategies for prevention and control.

[0055] Under Treatment A, the inhibition rate of apple fruit ring rot was about 50%, and the inhibition rate of branch ring rot was about 40%. BdNIS1 The gene expression level decreased by about 60%. Under Treatment B, the inhibition rates of apple fruit ring rot and branch ring rot were both about 40%, among which the fruit interaction-like tissue BdNIS1 Gene expression also decreased by about 60% ( Figure 3 a, Figure 3 b).

[0056] (III) Specific steps for modification of nanomaterials: Nano-chitosan oligosaccharide (COS): The mass concentration is 0.01×10-2 The preparation method of nano-chitosan oligosaccharide aqueous solution (mother liquor) with a concentration of 100 g / mL is as follows: dissolving nano-chitosan oligosaccharide in DEPC water and dispersing it by ultrasonic vibration.

[0057] (IV) Preparation of COS-dsRNA Nanopesticide for Controlling Apple Ring Rot Specific steps: Take 1.5mL of (3) with a mass concentration of 0.01×10 -2 g / mL aqueous solution of nano-chitosan oligosaccharide, 150 μg of dsRNA purified in step S1 of Example 1 was added thereto, and after thorough mixing, DEPC water was added to 3 mL. At this time, the working concentration of nano-chitosan oligosaccharide was 0.005×10 -2 g / mL, and the final concentration of dsRNA was 0.005×10 -2 g / mL, mix by inverting several times, and let it stand at room temperature for 3 min for efficacy evaluation.

[0058] (V) Testing the Coupling Effect of COS on dsRNA and Evaluation of the Disease Resistance Effect of COS-dsRNA Nanopesticide on Apple Fruit Specific steps: The purified dsRNA synthesized in step S1 of Example 1 was adjusted to 100 ng / μL for later use; The COS dispersed by ultrasonic vibration was adjusted to 1 times, 3 times, 5 times, 8 times and 10 times the concentration of dsRNA with DEPC water for later use; Mix equal volumes of COS and dsRNA at different concentrations (5 μL each, totaling 10 μL), then add 2 μL of 6× DNA Loading Buffer and mix thoroughly; Take 10 μL of the mixture and load it on 1.2% agarose gel electrophoresis to test the coupling effect, so as to screen the concentration at which the COS nanomaterial is completely coupled to dsRNA.

[0059] Select healthy (no lesions, no insects) and uniformly sized Red Fuji apples picked from the same orchard, wash them with tap water, then disinfect the surface with 75% alcohol, and place them in a cool and ventilated place to evaporate excess alcohol and dry excess water.

[0060] Use a 5mm hole puncher to punch a 2-3mm deep hole in the middle of the treated apple (about 2cm from the fruit stem on the side of the fruit), and then apply 50μL of COS-dsRNA nucleic acid drug into the hole.

[0061] The apple ring rot pathogen ( Botryosphaeria dothidea) was used as the inoculum source, and after it was rejuvenated on the PDA plate, the fungus was punched out at the edge of the colony with a 5mm puncher. The fungus cake with the mycelium side was inoculated into the small hole, and the culture was kept moist at 25℃ for 72h. The disease situation of each treatment group was observed and recorded by taking photos.

[0062] The groups are as follows: CK: inoculated blank sterile PDA agar block; Control: After spraying the inoculation site of apple fruit with 50 μL of sterile water, the edge hyphae of the rejuvenated apple ring rot pathogen colony was directly inoculated; dsBdNIS1: 50 μL of 0.005 × 10 -2 After spraying the inoculation site of apple fruit with dsBdNIS1 at a concentration of g / mL, the edge hyphae of the rejuvenated apple ring rot pathogen colony was directly inoculated; COS: 50 μL 0.005 × 10 -2 After spraying the inoculation point of apple fruit with a COS aqueous solution of 500 g / mL, the edge hyphae of the rejuvenated apple ring rot pathogen colony was directly inoculated; COS-dsBdNIS1: After spraying the inoculation site of apple fruit with 50 μL COS-dsBdNIS1, the marginal hyphae of the rejuvenated apple ring rot pathogen colony was directly inoculated, where COS:dsBdNIS1=1:1, that is, the final concentration of dsBdNIS1 in COS-dsBdNIS1 was 0.005×10 -2 g / mL, and the final concentration of COS was 0.005×10 -2 g / mL; The test results are as follows Figure 4 As shown in Figure a, naked dsBdNIS1 was mixed with COS at different mass ratios (1:1, 1:3, 1:5, 1:8, and 1:10). There was no significant shift in dsRNA banding, and no significant RNA deposition was observed in the sample-filled area. This indicates that COS does not have the ability to bind to dsRNA. However, chitosan oligosaccharides themselves can induce plant disease resistance and enhance plant resistance to pathogens. As a plant resistance inducer, COS can trigger immune defense responses in plants and enhance resistance to pathogens. Co-administration with dsBdNIS1 demonstrates a synergistic effect, without interfering with each other.

[0063] Based on this, we mixed dsBdNIS1 and COS in a mass ratio of 1:1 and evaluated the disease resistance effect on apple fruit. Figure 4 b. Figure 4 As shown in c, COS and dsBdNIS1 were mixed at a 1-fold mass ratio to treat apple fruits, and then inoculated with wild-type apple ring rot pathogens. The fruits were cultured at 25°C with a light:dark ratio of 12:12 for 3 days.

[0064] The control group consisted of a wild-type strain inoculated with H2O (positive control). CK and dsBdNIS1 served as negative controls, while COS and COS-dsBdNIS1 treatments reduced the area of ​​rot lesions on apple fruit to varying degrees compared to the positive control group. Only the COS-dsBdNIS1 treatment group showed significantly smaller lesion areas than the dsBdNIS1 negative control. The COS treatment group showed no significant difference from the dsBdNIS1 treatment alone. These results suggest that COS can induce resistance to ring rot in plants to a certain extent, and that the combined application of chitosan oligosaccharides (COS) and dsBdNIS1 at equal mass ratios can produce a synergistic effect.

[0065] The cross-cross method was used to measure and analyze the lesion area of ​​the diseased fruits in each treatment group. Figure 4 c) The lesion area of ​​the control group treated with bare dsBdNIS1 alone decreased by about 48%, the lesion area of ​​the control group treated with COS alone decreased by about 42%, and the lesion area of ​​the control group treated with COS-dsBdNIS1 together decreased by about 67%, which was significantly lower than that of the control group and the group treated with dsBdNIS1 and COS alone, and had the best control effect on apple ring rot.

[0066] Chitosan oligosaccharide (COS) is a low-molecular-weight oligosaccharide derived from chitosan through chemical or enzymatic degradation. It typically contains 2-20 glucosamine groups. Compared to chitosan, chitosan oligosaccharide has a smaller molecular weight, better water solubility, higher bioactivity, and broader potential applications. It is not only safe and non-toxic, but also acts as a broad-spectrum antimicrobial agent. In agricultural production, COS can be used as a plant growth regulator to induce systemic resistance in crops. It can also be used in seed coatings or foliar fertilizers to increase crop yields.

[0067] In the early stage of this invention, a key effector protein of apple ring rot pathogen BdNIS1 was identified and used as RNAi target to design and synthesize a targeted silencing BdNIS1 The dsRNA of the gene (dsBdNIS1) BdNIS1 It has a significant silencing effect, as shown by the following: after dsBdNIS1 treatment, the BdNIS1 Gene expression was significantly reduced. Treatment with dsBdNIS1 did not inhibit the growth of the apple ring rot pathogen, but significantly reduced its ability to infect apple hosts. Spraying dsBdNIS1 on apple fruit and branches also produced a degree of resistance to the pathogen within the sprayed area, significantly reducing its ability to infect apple ring rot.

[0068] Further experiments revealed that COS and dsBdNIS1 at a low ratio (1x) did not produce a significant coupling effect. However, a mixed application of the two exhibited a significant immediate effect against apple ring rot, attributed to the favorable properties of COS and the high sensitivity of the apple ring rot pathogen to dsRNA. This demonstrates the feasibility of combining COS with dsRNA targeting key effector genes of the apple ring rot pathogen for immediate control. On the one hand, COS induces systemic resistance in plants, enhancing their resistance to the pathogen. On the other hand, due to the high sensitivity of the apple ring rot pathogen to RNAi, it can efficiently absorb dsRNA near the hyphae and effectively silence key pathogenic target genes within a short period of time (within 6 hours), reducing the pathogen's infectivity. The two complement each other, each performing its own role, and have broad potential for green control of apple ring rot.

[0069] This invention, based on the green control of apple ring rot, utilizes RNAi technology to combine naked dsRNA with nano-chitosan oligosaccharides (COS) to create an RNAi-based nanoparticle pesticide for the prevention and control of apple ring rot. This provides technical support for the green control of apple ring rot. Efficacy evaluation demonstrated that the COS-dsRNA nanoparticle pesticide for the prevention and control of apple ring rot was effective against the pathogen, significantly suppressing the expression of pathogen-related target genes during the infection phase and significantly reducing the infection capacity of the pathogen. Furthermore, COS nanomaterials are readily available and inexpensive, derived from the shells of crustaceans. They are also easily degradable in the environment, are eco-friendly for field use, and leave no residue. Therefore, this RNAi-based nanoparticle pesticide has broad application prospects in the prevention and control of apple ring rot.

[0070] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a COS-dsRNA nanopesticide for preventing and treating apple ring rot, characterized in that: The method is: S1, targeted silencing of apple ring rot pathogen Botryosphaeria dothidea Key pathogenic effector genes BdNIS1 The dsRNA is synthesized in vitro and purified to obtain dsRNA; Psoralea corylifolia Botryosphaeria dothidea Key pathogenic effector genes BdNIS1 The full-length nucleotide sequence is shown in SEQ ID No. 1; Psoralea corylifolia Botryosphaeria dothidea Key pathogenic effector genes BdNIS1 The cDNA nucleotide sequence is shown in SEQ ID No. 2; the nucleotide sequence of the dsRNA is shown in SEQ ID No. 3; S2. Add the dsRNA obtained in S1 to the nano-chitosan oligosaccharide aqueous solution, and mix them to obtain the COS-dsRNA nano-nucleic acid pesticide for preventing and treating apple ring rot.

2. The method for preparing a COS-dsRNA nanopesticide for preventing and treating apple ring rot according to claim 1, characterized in that: Targeted silencing of Psoralea corylifolia as described in S1 Botryosphaeria dothidea Key pathogenic effector genes BdNIS1 The method for in vitro synthesis of dsRNA is: S101, Extraction of Apple Ring Rot Botryosphaeria dothidea Total RNA was reverse transcribed to obtain cDNA; S102. Based on the cDNA obtained in S1, PCR amplification was performed using primers T7-BdNIS1-F and T7-BdNIS1-R to obtain a PCR product. The amplified target gene fragment was recovered by electrophoresis on a 1.2% agarose gel to obtain a purified PCR product. The reaction system for PCR amplification was as follows: 1 μL of cDNA obtained in S1, 1 μL of primer T7-BdNIS1-F, 1 μL of primer T7-BdNIS1-R, 12.5 μL of KOD DNA polymerase, and ddH2O to make up to 25 μL; The reaction program for PCR amplification was as follows: 98°C for 3 min; 98°C for 10 sec, 58°C for 5 sec, and 68°C for 5 sec, for a total of 35 cycles; 68°C for 7 min, and hold at 16°C; The nucleotide sequence of the primer T7-BdNIS1-F is shown in SEQ ID No. 4; The nucleotide sequence of the primer T7-BdNIS1-R is shown in SEQ ID No. 5; S103, using the purified PCR product obtained in S102 as template DNA, using T7 RNA Transcription Kit to synthesize naked dsRNA targeting the target gene fragment in vitro; or using HyperScribe TM T7 High Yield Cy3 / Cy5 RNA Labeling Kit is used to synthesize naked dsRNA containing fluorescently labeled target gene fragments in vitro.

3. The method for preparing a COS-dsRNA nanopesticide for preventing and treating apple ring rot according to claim 2, characterized in that: When the T7 RNA Transcription Kit is used in S103, the reaction system is: 1 μL of T7 RNA Polymerase, 1 μL of RNase Inhibitor, 5 μL of 10× Transcription Buffer, 8 μL of NTP Mix, 1 μg of the purified PCR product obtained in S102, and DEPC HO to 50 μL; the reaction procedure is: 37°C for 4 hours, incubation at 72°C for 10 minutes, and then incubation at room temperature for 20 minutes; When the HyperScribe™ T7 High Yield Cy3 / Cy5 RNA Labeling Kit was used in S103, the reaction system was as follows: 2 μL of 10× Reaction Buffer, 2 μL of 20 mM ATP, 2 μL of 20 mM GTP, 2 μL of 20 mM CTP, 1.5 μL of 20 mM UTP, 1 μL of 10 mM Cy3-UTP, 1 μg of the purified PCR product obtained in S102, 2 μL of T7 RNA Polymerase Mix, and ddH2O was added to make up to 20 μL. The reaction procedure was: 37°C for 4 h.

4. The method for preparing a COS-dsRNA nanopesticide for preventing and treating apple ring rot according to claim 1, characterized in that: The preparation method of the nano-chitosan oligosaccharide aqueous solution in S2 is: dissolving the nano-chitosan oligosaccharide in DEPC water and dispersing it by ultrasonic vibration.

5. The method for preparing a COS-dsRNA nanopesticide for preventing and treating apple ring rot according to claim 1, characterized in that: The final concentration of nano-chitosan oligosaccharide in the COS-dsRNA nanopesticide for preventing and treating apple ring rot described in S2 is 0.005×10 -2 g / mL, and the final concentration of dsRNA was 0.005×10 -2 g / mL.

6. A use of a COS-dsRNA nanopesticide for preventing and treating apple ring rot prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The COS-dsRNA nano nucleic acid pesticide for preventing and controlling apple ring rot is used for preventing and controlling apple ring rot Botryosphaeria dothidea .

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