DsRNA fragment for preventing and treating fusarium graminearum as well as composition and application of dsRNA fragment

Abstract

The invention discloses a dsRNA fragment for preventing and treating fusarium graminearum as well as a composition and application of the dsRNA fragment. The DNA corresponding to the dsRNA is a DNA fragment which has any continuous 20 bp or more than 20 bp with fusarium graminearum gene CHS3b and MGV1 transcripts, and the sequence consistency is greater than 90%. The dsRNA provided by the invention can efficiently interfere with the pathogenicity of fusarium graminearum, can be developed into a nucleic acid pesticide which plays a bactericidal function based on RNA interference, and can be used for preventing and treating plant scab and stem rot.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a dsRNA fragment for preventing and controlling Fusarium graminearum and a composition and application thereof. Background Art

[0002] Fusarium graminearum is a plant pathogenic fungus belonging to the subphylum Ascomycota. It is the main pathogen causing crop diseases such as wheat fusarium head blight and corn stem rot. The pathogen can cause serious damage to crops, not only resulting in reduced yields, but also accumulating mycotoxins, threatening food safety. With global warming and the emergence of drug resistance, the prevention and control of this pathogen is facing tremendous pressure. Current control mainly relies on the use of chemical fungicides and the cultivation of resistant varieties. However, there are few resistant varieties, and with the repeated use of chemical fungicides, the pollution of soil, water and air and the multidrug resistance of most pathogenic fungi are intensifying. Therefore, finding alternative technologies and biopesticides has become an urgent need for modern green agricultural production.

[0003] The development of efficient and green pest control products is of great significance to ensuring food production safety and addressing chemical pesticide resistance. Nucleic acid pesticides are innovative plant protection products that use RNA interference (RNAi) technology to control pests and diseases. Nucleic acid pesticides, especially those based on dsRNA (double-strand RNA), can specifically bind to the messenger RNA of specific genes in target organisms, thereby silencing the expression of the corresponding genes, thereby interfering with the normal growth of the target organisms and their harm to the host plants. dsRNA molecules are cut into small fragments in the cell, which bind to complementary mRNA, causing mRNA degradation or translation blockage, thereby achieving gene silencing.

[0004] Nucleic acid pesticides can be designed precisely for specific genes of harmful organisms, so their targets are very specific and will not affect non-target organisms, which helps protect the ecological environment and beneficial insects. In addition, since nucleic acid pesticides mainly act on the RNA level, they do not involve protein synthesis, and RNA is easily degraded in the environment, so their residue and pollution problems are relatively small, which meets the public's demand for green pesticides. As an emerging plant protection product, nucleic acid pesticides have advantages such as high specificity, green safety, and low R&D costs, and have made significant progress in the prevention and control of pests, pathogens and viruses. With the deepening of research and the advancement of technology, nucleic acid pesticides are expected to play a more important role in future agricultural production.

[0005] At present, nucleic acid pesticides have achieved remarkable effects on many pests, such as diamondback moth, corn rootworm, soybean stink bug, whitefly, etc. By spraying a solution containing specific dsRNA, the growth and reproduction of pests can be effectively inhibited. However, nucleic acid pesticides are relatively rare for the prevention and control of fungal diseases, such as fusarium head blight and stem base rot. The dsRNA-based foliar spray-induced gene silencing technology (SIGS) has the advantages of simple application and good safety. After dsRNA is sprayed on the leaves, when the pathogen infects the crop, it can be directly absorbed by contact, or it can be absorbed by the crop and then processed into siRNA and indirectly transported to the fungus to achieve a silencing effect. In the field test, the dsRNAs and their compositions provided by the present invention were sprayed to effectively prevent and control wheat fusarium head blight. Therefore, the present invention has obvious technical innovation and great application value, and can be used as a main component for the development of new biological pesticides. Summary of the invention

[0006] The purpose of the present invention is to provide a dsRNA fragment for controlling Fusarium graminearum and a composition and application thereof.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a dsRNA fragment for controlling Fusarium graminearum, wherein the DNA corresponding to the dsRNA is a fragment of Fusarium graminearum ( Fusarium graminearum )Gene CHS3b Transcript or gene MGV1 Any continuous DNA fragment of 20 bp or more in the transcript with a sequence identity greater than 90%.

[0008] Among them, genes CHS3b The nucleotide sequence of the transcript is: A) the nucleotide sequence shown in SEQ ID NO: 1; B) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence expresses a protein with the same function; C) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO: 1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or D) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of A), B) or C) and expresses a protein with the same function; Gene MGV1 The nucleotide sequence of the transcript is: a) the nucleotide sequence shown in SEQ ID NO: 2; b) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 2 and the nucleotide sequence expresses a protein with the same function; c) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO: 2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or d) A nucleotide sequence that has more than 90% homology with the nucleotide sequence of a), b) or c) and expresses a protein with the same function.

[0009] Further, it is a target gene of Fusarium graminearum CHS3b dsRNA1, the corresponding DNA sequence of which is shown in SEQ ID NO: 3; or, Fusarium graminearum gene MGV1 The dsRNA2, whose corresponding DNA sequence is shown in SEQ ID NO:4.

[0010] In a second aspect, the present invention provides a composition for controlling Fusarium graminearum, wherein the composition contains dsRNA1 and / or dsRNA2.

[0011] In a third aspect, the present invention provides biological materials containing the dsRNA fragment or its corresponding DNA fragment, wherein the biological materials include but are not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0012] In a fourth aspect, the present invention provides use of the dsRNA fragment, the composition or the biological material in preventing and controlling Fusarium graminearum or plant diseases caused thereby.

[0013] In a fifth aspect, the present invention provides use of the dsRNA fragment, the composition or the biological material in preparing a formulation for preventing and controlling Fusarium graminearum or plant diseases caused thereby.

[0014] Furthermore, the plant diseases include plant head blight and stem base rot.

[0015] In a sixth aspect, the present invention provides a pesticide for preventing and controlling Fusarium graminearum or plant diseases caused thereby, wherein the active ingredient is the dsRNA fragment or the composition or the biological material.

[0016] In a seventh aspect, the present invention provides a method for preventing and controlling Fusarium graminearum or plant diseases caused thereby, comprising spraying the dsRNA fragment or the composition or the biological material or the pesticide on plants.

[0017] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The dsRNA of the present invention can effectively interfere with the pathogenicity of Fusarium graminearum, can be developed into a nucleic acid pesticide that exerts a bactericidal function through RNA interference, and can be used to prevent and control plant fusarium head blight and stem base rot.

[0018] By using the preferred dsRNA fragments and compositions thereof of the present invention, spore germination and hyphae extension of Fusarium graminearum can be inhibited, and its infection and harm to plants can be significantly reduced, achieving a control efficiency of more than 90% after spraying. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The extraction and purification of dsRNA in a preferred embodiment of the present invention: M, DNA ladder; 1-3, three dsRNA fragments of different sizes obtained by TRIzol extraction; 4-6, dsRNA fragments 1-3 after digestion with DNase I and RNase A and purification.

[0020] Figure 2 The temperature stability of dsRNA in a preferred embodiment of the present invention: dsCHS3b was incubated at 4, 25, 37, 50, 60, 70, 80, 90, 95°C for 12 h and then detected by electrophoresis.

[0021] Figure 3 The pH stability of dsRNA in a preferred embodiment of the present invention is as follows: dsCHS3b was incubated at pH 1-14 for 12 hours and then detected by electrophoresis.

[0022] Figure 4 In a preferred embodiment of the present invention, dsRNA inhibits the germination of Fusarium graminearum spores: the Fusarium graminearum spores were added with different dsRNAs and observed 12 hours later.

[0023] Figure 5 In a preferred embodiment of the present invention, dsRNA inhibits hyphal extension of Fusarium graminearum: After dsRNA is applied to different plates, Fusarium graminearum is cultured for 7 days for observation.

[0024] Figure 6 Microscopic observation of Fusarium graminearum hyphae after adding different dsRNAs in the preferred embodiment of the present invention.

[0025] Figure 7 This is the inhibitory effect of dsRNA on the infection of wheat leaves by Fusarium graminearum in a preferred embodiment of the present invention.

[0026] Figure 8 This is the inhibitory effect of dsRNA on Fusarium graminearum infection of wheat ears in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0027] The present invention aims to provide a nucleic acid pesticide and a composition thereof for resisting Fusarium graminearum. The key to fungal control based on RNAi is to select target genes that are indispensable for fungal survival. CHS3b Encodes chitin synthase, involved in fungal cell wall synthesis. MGV1 Encoding mitogen-activated protein kinase, it is important for plant infection, DON production and sexual reproduction. In view of this, the present invention selects these two genes of Fusarium graminearum as silencing targets. The dsRNAs of these two genes can be synthesized in vitro or intracellularly using recombinant microorganisms, and have the nucleotide composition in SEQ ID NO: 1 and 2. Any fragment greater than 20bp of these two dsRNAs is used as an effective ingredient, and any other substance can be added to prevent and control Fusarium graminearum in the form of a composition. Using the preferred dsRNA fragments and compositions of the present invention, the germination of Fusarium graminearum and the extension of mycelium can be inhibited, and its infection and harm to plants can be significantly reduced, and the prevention and control efficiency can reach more than 90% after spraying.

[0028] The present invention adopts the following technical solution: The present invention provides two dsRNA fragments and compositions thereof that can prevent and control Fusarium graminearum from infecting plants. The nucleic acid fragments and compositions thereof are the following dsRNAs and substances containing any of their components: 1) dsRNAs composed of nucleotides shown in SEQ ID NOs: 1 and 2; 2) dsRNAs in which the nucleotide residues of the dsRNAs composed of nucleotides shown in SEQ ID NOs: 1 and 2 are replaced and / or deleted and / or modified by one or more nucleotide residues; 3) a composition that can prevent and control Fusarium graminearum from infecting plants, characterized in that it contains dsRNAs composed of nucleotides shown in SEQ ID NOs: 1 and 2 or fragments greater than 20 bp.

[0029] Furthermore, the dsRNA fragment of the present invention has the nucleotide composition shown in SEQ ID NO: 1 or 2.

[0030] The present invention also provides a series of dsRNA compositions comprising any consecutive 20 nucleotides of SEQ ID NO: 1 or 2.

[0031] The present invention also provides a composition comprising any consecutive identical 20-nucleotide fragments of these two dsRNAs.

[0032] Furthermore, the dsRNA is used to interfere with Fusarium graminearum.

[0033] Preferably, the dsRNAs corresponding to SEQ ID NO: 3 and 4 and compositions thereof are used to control plant head blight and stem base rot caused by Fusarium graminearum.

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0035] Example 1 Acquisition of target gene 1. Chemical synthesis Fusarium graminearum Fusarium graminearum PH-1 gene CHS3b Transcript (NCBI locus_tag: FGSG_10116, 3408 bp) and MGV1 The transcript (locus_tag: FGSG_10313, 1810bp) was used as a template to synthesize RNA (which could be siRNA or dsRNA) corresponding to DNA with a consistency of more than 90% for any consecutive 20bp of the two genes by chemical synthesis.

[0036] 2. PCR amplification Using cDNA of Fusarium graminearum as template, we selected genes CHS3b and MGV1 The gene sequence of any length of 200-800 bp (as shown in SEQ ID NO:3 and 4) was designed, and specific primers were designed to obtain the target gene fragment by PCR amplification technology. The target fragment was purified using a gel recovery purification kit and detected by electrophoresis using 0.8% agarose gel. The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5min; then 95℃ 30s, 55℃ 30s, 72℃ 120s, for a total of 35 cycles; finally 72℃ extension for 5min to obtain a specific PCR product of the expected size.

[0037] Example 2 In vitro enzymatic synthesis of dsRNA Use any target gene in Example 1 as a template for in vitro transcription, perform BLAST comparison on the NCBI website to analyze its specificity, and reselect if the specificity is not good. Design and synthesize two pairs of primers, and add the T7 promoter sequence (5′-TAATACGACTCACTATAGGG-3′) to the 5' end of the two primers. Use each pair of primers to perform PCR to synthesize DNA templates, and prepare the PCR system in an RNAse free centrifuge tube according to the following composition (Table 1): Table 1 PCR system preparation

[0038] After mixing, vortex centrifuge to the bottom of the tube, put it into the PCR instrument, and amplify it according to the following conditions: 95℃ pre-denaturation for 5min, then 95℃ 30s, 55℃ 30s, 72℃ 120s, a total of 35 cycles; finally, extend at 72℃ for 5min to obtain a specific PCR product of the expected size. Then prepare 100µL in vitro transcription system according to Table 2: Table 2 Preparation of dsRNA in vitro synthesis system

[0039] Note: DTT is dithiothreitol.

[0040] Centrifuge the reagent to the bottom of the tube and incubate at 37℃ for 120min for transcription. Add 5µL DNase I and incubate at 37℃ for 30min to degrade the template. Add an equal volume of phenol:chloroform (volume ratio 1:1), vortex for 30s, centrifuge at 12000rpm for 15 min, and transfer the upper liquid phase to a new 1.5 ml microcentrifuge tube. Add 1 / 10 volume of sodium acetate (3 mol / L, pH5.2) and 2.5 volumes of anhydrous ethanol to the liquid phase, vortex to mix, and place at -20℃ for more than 30 min to precipitate the product. After centrifugation and discarding the supernatant, add 1ml of 70% ethanol to wash the precipitate (4℃, 12000rpm, 30min), then open the centrifuge tube and place it for several minutes to allow the ethanol to evaporate. Dissolve the dsRNA precipitate in a certain volume of nuclease-free water, and use Nanodrop to measure the concentration and purity of RNA. Finally, the centrifuge tube was placed on a metal bath at 95°C for 1 min, and naturally cooled to room temperature to allow the two chains to renature into a dsRNA solution with a concentration of 0.5 μmol / L, and then stored at -80°C for later use.

[0041] Alternatively, the dsRNA can be prepared using a commercial dsRNA in vitro synthesis kit according to the instructions.

[0042] Example 3 Intracellular synthesis of dsRNA using recombinant bacteria 1. Construction of dsRNA synthetic recombinant bacteria The L4440 vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) contains a pair of T7 promoters in opposite directions and is widely used as a dsRNA expression vector. The target gene fragment obtained above was subcloned, double-enzyme digested according to the reaction system in Table 3, and connected to the L4440 vector according to the reaction system in Table 4 to obtain a recombinant vector.

[0043] Table 3 DNA double enzyme digestion reaction system

[0044] Note: QuickCut TMKpn I, QuickCut TM Bgl II and 10×QuickCut Buffer are restriction endonucleases and buffers purchased from TAKARA Co., Ltd., Japan.

[0045] Then, the vector L4440-CHS or L4440-MGV containing the target gene fragment was introduced into Escherichia coli HT115 ( Escherichia coli HT115, E. coli HT115, purchased from Hangzhou Baosai Biotechnology Co., Ltd.). The bacteriophage λDE3-encoded RNA polymerase is integrated into the genome of Escherichia coli HT115. Under the induction of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG), a large amount of T7 RNA polymerase can be expressed. T7 RNA polymerase binds to the T7 promoter and mediates the transcription of two complementary mRNA chains from the downstream DNA sequence, which then anneal in vivo to form dsRNA according to the principle of complementary base pairing.

[0046] Table 4 Enzymatic ligation reaction system

[0047] The recombinant bacteria constructed E. coli HT115 / L4440-CHS or E. coli A single colony of HT115 / L4440-MGV was streaked onto LB solid medium with a final concentration of 100 μg / ml of ampicillin and cultured at 37°C for 12 hours. Three single colonies were randomly selected for PCR, and the PCR-positive colonies were cultured in liquid. Then the plasmid was extracted and sequenced to confirm that the sequence was correct and without mutation. Glycerol tubes were prepared for the recombinant bacteria that were sequenced correctly and stored at -80°C.

[0048] 2. Recombinant bacteria fermentation and induction The recombinant bacteria constructed E. coli HT115 / L4440-CHS or E. coli A single colony of HT115 / L4440-MGV was inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml ampicillin and cultured at 150 rpm at 37°C for 12 h. The culture was transferred to 500 mL of fresh LB liquid medium (containing ampicillin at a final concentration of 100 μg / ml) at a 1% inoculum volume and cultured at 37°C until OD 600 When the concentration reaches 0.6, add IPTG to a final concentration of 0.8 mM to the culture medium for induction, and continue shaking culture at 25°C for 4-16 hours.

[0049] Example 4 Extraction and preparation of dsRNA 1. dsRNA extraction and purification Extract dsRNA from the induced bacteria according to the total RNA extraction method. Take 1 mL of bacteria, centrifuge at 12000rpm for 1min, and discard the supernatant. Resuspend the bacterial pellet with 1 mL RNA isolater and let it stand for 5 min to fully lyse the bacteria. Then add 200 µL of chloroform to the bacterial lysate, vortex vigorously for 30 s, and let it stand at 4 °C for 5 min. Centrifuge for 15 min (12000 rpm, 4°C), carefully transfer the supernatant to a new sterile, enzyme-free 2 mL centrifuge tube and add an equal volume of isopropanol, mix by inversion, and let it stand at 4 °C for 10 min. Centrifuge for 15 min, discard the supernatant, and wash the precipitate three times with 75% ethanol. Place the centrifuge tube in a clean bench to dry for 5~10 min until the alcohol is completely evaporated. Dissolve the precipitate with 50 µL RNase-free H20. Digest the extracted total RNA with DNase I and RNase A according to Table 5 to remove double-stranded DNA and single-stranded RNA. Then use an RNA extraction kit to purify, eliminate proteins and small molecule nucleic acids, and obtain purified dsRNA.

[0050] Table 5 Enzymatic degradation reaction system

[0051] 2. dsRNA concentration determination and purity analysis The purified dsRNA is dissolved in RNase Free dH2O and the absorbance at 230, 260, and 280 nm is measured using a Nanodrop or full-wavelength microplate reader or other equipment with UV absorption measurement function. The dsRNA concentration is calculated based on the conversion factor of each absorbance at 260 nm of 47 ng / µL. When A260 / A280 is between 1.8-2.0 and A260 / A230 is between 2.1–2.4, it indicates high purity, and a ratio less than 1.8 indicates protein residue.

[0052] 3. dsRNA molecular weight analysis The purity and molecular weight of dsRNA can be evaluated by agarose gel electrophoresis. The concentration of extracted and purified dsRNA is adjusted to 10-50 ng / µL, and 3 µL is mixed with the loading buffer containing the fluorescent dye Gelred and added to the gel loading well. Use a commercial DNA ladder as a reference, perform constant voltage electrophoresis at 80 V for 40 minutes, and then use a gel imager to obtain the electrophoresis image. The electrophoresis results of the extraction and purification of dsRNA containing dsCHS3b (SEQ ID NO: 3) and dsMGV1 (SEQ ID NO: 4) are shown in Figure 2. Figure 1As shown. By referring to the DNA ladder of known molecular weight, the molecular weight of the target dsRNA can be roughly determined, and the purity can be partially reflected from the specificity of the electrophoresis band.

[0053] 4. Determination of dsRNA temperature and pH stability The purified dsRNA was incubated at 4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, and 95 °C for 12 h. The dsRNA at different temperatures was subjected to agarose gel electrophoresis to detect its integrity. The disappearance or weakening of the band indicated that it was unstable. Figure 2 As shown, dsCHS3b has good temperature stability and does not degrade within 12 h below 80 °C.

[0054] The purified dsRNA was placed in a solution of pH = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and incubated for 12 h. The dsRNA at different pH levels was subjected to agarose gel electrophoresis to detect its integrity. Figure 3 As shown, dsCHS3b has good pH stability and does not degrade within 12 h at pH 4-12.

[0055] Example 5 Inhibition of Fusarium graminearum by dsRNAs Use a cork punch to take three fresh Fusarium graminearum cakes from the edge of the PDA plate and transfer them to a conical flask containing 100 mL of carboxymethyl cellulose (CMC) and culture at 28 °C and 180 rpm for three days. Then, filter using four layers of sterile gauze, discard the gauze, centrifuge the filtrate and resuspend it in sterile water to obtain a spore suspension of Fusarium graminearum. Add 100 µL of PDB medium to a 2 mL sterile, enzyme-free centrifuge tube and add the Fusarium graminearum spore suspension to a final concentration of 10 5 spores / mL. Then, 5 µL of dsCHS3b or dsMGV1 at a concentration of 1 µg / µL was added to the culture medium. After incubation at 28 °C for 12 h, light observation showed that dsCHS3b inhibited the germination of Fusarium graminearum hyphae ( Figure 4 ).

[0056] Prepare sterile PDA and SNA plates, evenly spread 200 µL of dsRNA solution (20 ng / µL) on the plate surface, place a piece of Fusarium graminearum PH-1 cake in the center of the plate, and culture it in a constant temperature incubator at 28 °C. After a period of culture, observe the changes in colony diameter and pick hyphae to observe morphological changes under an optical microscope. Figure 5As shown in the figure, after 7 days of cultivation, the diameter of the bacterial cake on the plate coated with dsCHS3b and dsMGV1 was smaller than that of the control, indicating that dsRNA can inhibit the extension of Fusarium graminearum hyphae. The hyphae on the plate were picked and observed under an optical microscope ( Figure 6 ), compared with the control, the hyphal septa disappeared after dsCHS3b treatment.

[0057] Example 6 dsRNAs for the prevention and control of fusarium head blight Three wounds were evenly made on the leaves of four-week-old wheat 'Xinmai 27' using a sterilized syringe, and 50 μL of dsRNA solution (20 ng / μL) containing 0.05% surfactant Silwet L-77 (purchased from Beijing Biolab Technology Co., Ltd.) was added dropwise using a pipette. After 12 h, 30 μL of Fusarium graminearum PH-1 spore suspension (10 5 The wheat was grown at room temperature. Seven days after inoculation, the leaves were collected and photographed to observe the size of the lesions. Figure 7 As shown in the figure, compared with the water treatment, the dsCHS3b and dsMGV1 treatment groups reduced the lesion area caused by Fusarium graminearum on wheat leaves by 65.98% and 60.43%, respectively, indicating that exogenously applied dsRNA can control the infection of Fusarium graminearum on leaves.

[0058] Take wheat ears at the flowering stage and spray the dsRNA solution (5 µg) containing Silwet L-77 onto the detached wheat ears using a 5 mL spray bottle. 12 h after spraying, 10 µL of Fusarium graminearum spore suspension was injected into each wheat ear. The inoculated wheat ears were placed on a tray filled with water to maintain humidity. 14 days after fungal inoculation, the wheat ears were Figure 8 As shown, compared with water spraying, the dsCHS3b and dsMGV1 treatment groups had very few hyphae on the wheat ears, indicating that exogenously applied dsRNA can control the infection of Fusarium graminearum on wheat ears.

[0059] In the field, a dsRNA spraying experiment (600 mg / 30 L per mu) was carried out on the wheat ears in the flowering stage using a sprayer. Photos were taken 3 weeks after the first spraying, the fusarium disease index was calculated, and the prevention effect was calculated. After the wheat matured, the wheat grains were harvested, and the content of vomitoxin (DON) in the wheat grains with different treatments was detected according to the instructions of the vomitoxin ELISA detection kit. The statistical results of the prevention effect are shown in Table 6. The effective rate of one spraying is >50%, and the effective rate of two sprayings is >90%. The DON content test showed that after spraying dsCHS3b and dsMGV1 twice, the toxin content was reduced to below the national limit standard (1000 ppb), with a reduction of more than 90%.

[0060] Table 6 Statistics on the efficacy of spraying dsRNA in the field to control wheat scab

[0061] From the above experimental results, it can be seen that dsCHS3b and dsMGV1 have significant fungicidal effects on Fusarium graminearum, and can prevent and control Fusarium graminearum infection-induced fusarium head blight in the field, and the prevention effect is significantly higher than that of mainstream chemical fungicides. This shows that dsCHS3b and dsMGV1, as the main components of new pesticides, have good application prospects.

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A dsRNA fragment for controlling Fusarium graminearum, characterized in that: The DNA corresponding to the dsRNA is related to Fusarium graminearum ( Fusarium graminearum )Gene CHS3b Transcript or gene MGV1 Any continuous DNA fragment of 20 bp or more in the transcript with a sequence identity greater than 90%; Among them, genes CHS3b The nucleotide sequence of the transcript is: A) the nucleotide sequence shown in SEQ ID NO: 1; B) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence expresses a protein with the same function; C) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO: 1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or D) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of A), B) or C) and expresses a protein with the same function; Gene MGV1 The nucleotide sequence of the transcript is: a) the nucleotide sequence shown in SEQ ID NO: 2; b) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 2 and the nucleotide sequence expresses a protein with the same function; c) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO: 2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or d) A nucleotide sequence that has more than 90% homology with the nucleotide sequence of a), b) or c) and expresses a protein with the same function.

2. The dsRNA fragment according to claim 1, characterized in that It targets the gene of Fusarium graminearum CHS3b The dsRNA1, whose corresponding DNA sequence is shown in SEQ ID NO:

3.

3. The dsRNA fragment according to claim 1, characterized in that It targets the gene of Fusarium graminearum MGV1 The dsRNA2, whose corresponding DNA sequence is shown in SEQ ID NO:

4.

4. A composition for controlling Fusarium graminearum, characterized in that: The composition contains dsRNA1 and / or dsRNA2; Wherein, the dsRNA1 is the same as that described in claim 2, and the dsRNA2 is the same as that described in claim 3.

5. A biological material containing the dsRNA fragment or the corresponding DNA fragment according to any one of claims 1 to 3, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

6. Use of the dsRNA fragment according to any one of claims 1 to 3, the composition according to claim 4, or the biological material according to claim 5 in controlling Fusarium graminearum or plant diseases caused thereby.

7. Use of the dsRNA fragment according to any one of claims 1 to 3, the composition according to claim 4, or the biological material according to claim 5 in the preparation of a preparation for controlling Fusarium graminearum or plant diseases caused thereby.

8. The use according to claim 6 or 7, characterized in that: The plant diseases include plant head blight and stem base rot.

9. A pesticide for preventing and controlling Fusarium graminearum or plant diseases caused by it, characterized in that: The active ingredient is the dsRNA fragment according to any one of claims 1 to 3, the composition according to claim 4, or the biological material according to claim 5.

10. A method for controlling Fusarium graminearum or plant diseases caused by it, characterized in that: The dsRNA fragment according to any one of claims 1 to 3, the composition according to claim 4, the biological material according to claim 5, or the pesticide according to claim 9 is sprayed on the plants.

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