A siRNA nucleic acid pesticide targeting the DICER2 gene to control rice blast fungus.

Abstract

This invention provides an siRNA nucleic acid pesticide targeting the DICER2 gene to control rice blast fungus, belonging to the field of biotechnology. The siRNA molecule can delay and inhibit the formation of rice blast fungus appressoria, thereby reducing the pathogenicity of the fungus and providing a new method for controlling rice blast. When used in combination with tricyclazole or clotrimazole, it has a synergistic effect. The siRNA molecule targeting the DICER2 gene is prepared into a nucleic acid pesticide and sprayed on rice leaves, effectively reducing rice blast lesions and their area, thus enabling its use in rice blast control. Furthermore, it avoids the cumbersome process of cultivating genetically modified crops and addresses consumer concerns.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an siRNA nucleic acid pesticide that targets the DICER2 gene to control rice blast fungus. Background Technology

[0002] Rice blast, caused by the fungus *Magnaporthe oryzae*, is one of the most destructive rice diseases worldwide, resulting in a loss of approximately 10% to 30% of global rice yields annually. The fungus can infect multiple parts of the rice plant, and depending on the stage or tissue in which the disease occurs, it can be classified into seedling blast, node blast, leaf blast, and neck blast, with neck blast being the leading cause of yield loss. Besides rice, *M. oryzae* can also infect more than 50 other grasses, including economically and agriculturally important crops such as wheat (*Triticum spp.*), barley (*Hordeum vulgare*), maize (*Zea mays*), and weeds surrounding rice paddies.

[0003] The negative impacts of traditional pest control methods make the creation of new, green protection strategies crucial. Among these, RNA interference (RNAi) technology has shown great potential in the green control of pests and diseases. RNAi is a conserved gene regulation mechanism in eukaryotes, and the Dicer gene of the RNA III polymerase family plays an important role in the RNAi pathway. RNAi technology mainly refers to the process of target mRNA molecule degradation mediated by non-coding small interfering RNA (siRNA) molecules (a type of 20-24 bp double-stranded RNA molecule). After long double-stranded RNA (dsRNA) enters the cell, the RNase III nuclease Dicer, with the participation of ATP, cleaves the dsRNA into 21-23 bp siRNA. Subsequently, the siRNA binds to proteins such as Dicer and Argonaute to form the RNA-induced silencing complex (RISC). Finally, the RISC specifically binds to mRNA with a sequence complementary to the siRNA and cleaves the mRNA, thereby terminating translation and ultimately achieving gene silencing. Spray-induced gene silencing (SIGS) is an RNAi strategy that involves directly spraying dsRNA or siRNA onto plants, allowing plant pathogens to absorb the exogenously applied RNA and subsequently silence target genes. Currently, SIGS has been effectively applied to the control of pathogen infections in both monocot and dicotyledonous plants, and is also used for crop protection. SIGS technology is simple to operate, suitable for field applications, and easy for agricultural producers to implement. However, the known target genes and siRNAs capable of controlling rice blast are still limited.

[0004] The DICER gene family first appeared in the early evolution of eukaryotes and has independently replicated and diversified in organisms such as plants, animals, and fungi, consistent with the origin and evolution of multicellular organisms. Its expression is regulated by a complex gene regulatory network. As a member of the RNase III family, DICER enzymes mainly participate in the recognition and processing of dsRNA, specifically promoting double-stranded RNA breaks to form single-stranded small siRNAs or miRNAs, thereby inducing sequence-specific gene silencing. Rice blast fungus mainly contains two DICER genes, DICER1 and DICER2. Studies have found that knockout of the DICER2 gene affects the accumulation of siRNAs in the RNAi pathway of rice blast fungus, but whether the DICER2 gene is related to the pathogenicity of rice blast fungus remains unknown.

[0005] In summary, there is an urgent need to develop new target genes and siRNAs for the prevention and control of rice blast fungus. Summary of the Invention

[0006] This invention provides an siRNA nucleic acid pesticide targeting the DICER2 gene to control rice blast fungus, belonging to the field of biotechnology. The siRNA molecule can delay and inhibit the formation of rice blast fungus appressoria, thereby reducing the pathogenicity of the fungus and providing a new method for controlling rice blast. When used in combination with tricyclazole or clotrimazole, it has a synergistic effect. The siRNA molecule targeting the DICER2 gene is prepared into a nucleic acid pesticide and sprayed on rice leaves, effectively reducing rice blast lesions and their area, thus enabling its use in rice blast control. Furthermore, it avoids the cumbersome process of cultivating genetically modified crops and addresses consumer concerns.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] On one hand, the present invention provides an siRNA for controlling rice blast fungus, the siRNA targeting and silencing the Dicer2 gene of rice blast fungus, the Dicer2 gene containing the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing.

[0009] This invention first screened seven key genes involved in the growth and development of rice blast fungus through antibacterial experiments. It was found that when the Dicer2 gene of rice blast fungus was targeted and silenced, the formation rate of appressorium of rice blast fungus was significantly reduced. At the same time, knocking out the Dicer2 gene of rice blast fungus also led to slow growth of rice blast fungus and a reduction in the number of spores produced. These experimental results indicate that the Dicer2 gene plays an important role in the morphogenesis and growth and development of rice blast fungus. Therefore, subsequent research will focus on this target gene.

[0010] In order to further screen for the siRNA that best targets and silences the Dicer2 gene and inhibits rice blast fungus, this invention designed 12 pairs of siRNAs (siDicer2).

[0011] Specifically, the siRNA comprises any one or more of the sequences shown in the sequence listing: SEQ ID NO: 28-29, SEQ ID NO: 30-31, SEQ ID NO: 32-33, SEQ ID NO: 34-35, SEQ ID NO: 36-37, SEQ ID NO: 38-39, SEQ ID NO: 40-41, SEQ ID NO: 42-43, SEQ ID NO: 44-45, SEQ ID NO: 8-9, SEQ ID NO: 46-47, and SEQ ID NO: 48-49.

[0012] Next, based on the off-target probabilities predicted by the website, four pairs of siRNAs with the lowest off-target probabilities were selected. Specifically, the siRNAs comprise any one or more of the nucleotide sequences shown in the SEQ ID NO: 32-33, SEQ ID NO: 36-37, SEQ ID NO: 38-39, and SEQ ID NO: 8-9. Experimental verification showed that all four pairs of siRNAs could inhibit the morphogenesis of rice blast fungus.

[0013] Preferably, the siRNA comprises SEQ ID NO: 8~9 shown in the sequence listing, namely siDicer2-10 as described in this invention.

[0014] After 4 h of incubation with siDicer2 molecule and rice blast fungus, compared with siDicer2-3, siDicer2-5 and siDicer2-6, the formation of rice blast fungus appressoria with siDicer2-10 molecule was inhibited and the morphology was smaller, indicating that siDicer2-10 had the most obvious inhibitory effect.

[0015] On the other hand, the present invention provides a nucleic acid pesticide for controlling rice blast fungus, the nucleic acid pesticide comprising the above-mentioned siRNA.

[0016] Furthermore, the working concentration range and working time of the siRNA are 1 ~ 25 μM and 4 ~ 24 h, respectively.

[0017] On the other hand, the present invention provides a composition for controlling rice blast fungus, the composition comprising the above-mentioned siRNA.

[0018] Furthermore, the composition also contains substances that inhibit melanin formation and / or inhibit fungal cell wall formation in rice blast fungus. In some embodiments, the substances alone cannot affect the morphogenesis and appressorium formation of rice blast fungus and need to be used in conjunction with siRNA targeting the Dicer2 gene to achieve the purpose of controlling rice blast fungus.

[0019] In some specific embodiments, the compound that inhibits melanin formation in rice blast fungus is tricyclazole; in other embodiments, the substance that forms the fungal cell wall is clotrimazole.

[0020] Specifically, the combination of siDicer2-10 molecules with tricyclazole and the combination of siDicer2-10 molecules with clotrimazole have a synergistic effect, and the combination of siDicer2-10 + clotrimazole is preferred.

[0021] On the other hand, the present invention provides a method for preventing and controlling rice blast fungus, wherein the method refers to spraying a solution containing the above-mentioned siRNA or the above-mentioned composition onto rice plants or leaves.

[0022] On the other hand, the present invention provides the use of the Dicer2 gene as a target gene for the control of rice blast fungus, the Dicer2 gene comprising the nucleotide sequence shown in SEQ ID NO: 1.

[0023] On the other hand, the present invention provides the use of siRNA for preparing reagents that interfere with the development or pathogenicity of rice blast fungus appressoriums, wherein the siRNA is an siRNA that targets and silences the Dicer2 gene of rice blast fungus.

[0024] The beneficial effects of this invention include:

[0025] 1. This invention utilizes RNAi technology to investigate the effects of several key genes in the growth and development of rice blast fungus on the formation of rice blast fungus appressorium. After comparison, the effect of targeting and silencing the Dicer2 gene was the best, thus providing a new target gene that plays a role in the control of rice blast fungus.

[0026] 2. In this invention, by screening siRNAs that target and silence the Dicer2 gene, a pair of siRNAs with the best inhibitory effect on rice blast fungus were obtained, namely siDicer2-10 (SEQ ID NO: 8 ~ SEQ ID NO: 9).

[0027] 3. This invention also confirms that the combination of siDicer2-10 with tricyclazole and the combination of siDicer2-10 with clotrimazole have a synergistic effect in controlling rice blast fungus, with the combination of siDicer2-10 with clotrimazole being preferred;

[0028] 4. The siRNA molecule (siDicer2) against rice blast fungus provided by this invention can be prepared as a nucleic acid pesticide and sprayed on rice leaves, avoiding the cumbersome process of cultivating genetically modified crops and the concerns of consumers, thus providing a new option for the prevention and control of rice blast. Attached Figure Description

[0029] 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.

[0030] Figure 1 The effect of siRNAs targeting different genes of rice blast fungus on the appressorium formation rate of rice blast fungus was investigated. The differences in data were analyzed by t-test, and the results showed that P < 0.0001. DDW and siTubulin-1236 were used as blank control and negative control, respectively.

[0031] Figure 2 The effect of siRNAs targeting different genes of rice blast fungus on the morphology of rice blast fungus appressorium. The scale bar is 20 μm. DDW and siTubulin-1236 were used as blank control and negative control, respectively.

[0032] Figure 3 The effects of knocking out different Dicer genes on the growth of rice blast fungus;

[0033] Figure 4 Schematic diagram of the distribution of siRNA targeting the Dicer2 gene (siDicer2);

[0034] Figure 5 The inhibitory effects of different siDicer2 molecules on rice blast fungus, where the scale bar = 20 μm; control refers to co-incubation with rice blast fungus spores in sterile water;

[0035] Figure 6 Rice blast fungus appressorium formation rate after treatment with different concentrations of siDicer2-10 for different durations;

[0036] Figure 7 The effects of different treatment times with siDicer2-10 and / or tricyclazole on the appressorium of rice blast fungus, among which, Figure 7 In the figure, A represents the morphology of rice blast fungus appressoria after different treatments, with a scale bar of 20 μm. Figure 7B in the figure represents the statistical graph of rice blast fungus appressorium formation rate after different treatments. The t-test was used to analyze the significant differences. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns indicates no significant difference.

[0037] Figure 8 The effects of different treatment times with siDicer2-10 and / or clotrimazole on the appressorium of rice blast fungus, among which, Figure 8 In the figure, A represents the morphology of rice blast fungus appressoria after different treatments, with a scale bar of 20 μm. Figure 8 B in the figure represents the statistical graph of rice blast fungus appressorium formation rate after different treatments. The t-test was used to analyze the significant differences. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns indicates no significant difference.

[0038] Figure 9 Effects of siDicer2-10 spray on the pathogenicity of rice blast fungus. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the rice blast fungus used in this invention is Guy11, and the siRNA was directly synthesized by a biotechnology company (Shanghai Jierui). Unless otherwise specified, the materials and reagents used are commercially available.

[0041] Example 1: Screening of target genes for the control of rice blast fungus

[0042] To obtain new target genes for the control of rice blast fungus, this embodiment screened key genes regulating the growth and development of rice blast fungus (Chs1 (MGG_01802), Chs6 (MGG_13013), Dicer1 (MGG_01541), Dicer2 (MGG_12357, SEQ ID NO: 1), Hox7 (MGG_12865), Pex5 (MGG_10840), Pex19 (MGG_00971)). The specific steps are as follows:

[0043] 1.1 siRNA Design: siRNAs targeting the above-mentioned genes were designed according to the methods described on the siRNA design website (http: / / siDirect2.rnai.jp). Then, combining bioinformatics and gene data analysis, the possibility of targeting other genes of the rice genome and rice blast fungus was reduced, resulting in siRNAs (siChs1-2298, siChs6-3473, siDicer1-3010, siDicer2-10, siHox7-1, siHox7-2, siPex5-557, siPex5-947, siPex5-1350, siPex19-319, siPex19-884, siPex19-692), with specific sequences shown in Table 1. The designed siRNA double-stranded molecules were directly synthesized by a biotechnology company (Shanghai Jierui). The synthesized dry powder was dissolved in water to a final concentration of 10 μM, aliquoted, and stored. For short-term use, store at -20℃; for long-term storage, store at -80℃.

[0044] Table 1. siRNA sequences targeting different genes

[0045]

[0046] 1.2 Collection of rice blast fungus spore suspension: Wild-type rice blast fungus (Guy11) plates grown for 8-10 days were selected. Mycelia were scraped from the surface using a sterile, disposable spreader moistened with sterile water. The liquid collected by the spreader was filtered through three layers of sterile lens paper; the filtrate was the spore suspension. The concentration of the spore suspension was then adjusted to 2 × 10⁻⁶ using a hemocytometer. 5 The sample was prepared at 100 cells / mL and then diluted with a 0.3% gelatin solution to a concentration of 1×10⁻⁶. 5 per mL.

[0047] 1.3 Co-incubation of siRNA with rice blast fungus: The Gelbond hydrophobic membrane induced to form appressoria was pre-treated with RNase-free enzyme (i.e., soaked overnight in 0.1% DEPC water). After soaking, the hydrophobic membrane was rinsed three times with sterile water and then wiped with 70% ethanol prepared with nuclease-free water. The wiped hydrophobic membrane was then placed in a humidification box. The siRNA and rice blast fungus spore solution were mixed at a volume ratio of 1:1, with a final concentration of siRNA of 5 μM. The mixture containing siRNA and rice blast fungus spores (approximately 20 μL) was then spot-inoculated into the center of the treated hydrophobic membrane. After incubation for 4 h, the formation of appressoria was observed and recorded. A blank control (DDW group) and a negative control (siTubulin-1236 group) were set up, with 3 replicates for each experimental group.

[0048] The results are as follows Figure 1As shown, compared with the blank control (DDW group), negative control (siTubulin-1236 group), and siRNAs targeting other genes, the siRNA targeting the Dicer2 gene of rice blast fungus (siDicer2-10) significantly reduced the proportion of rice blast fungus appressorium formation; that is, compared with the control, the rice blast fungus appressorium formation rate in the siDicer2-10 group decreased by about 24%. Simultaneously, the spore morphology of rice blast fungus was observed 4 h after treatment. Figure 2 Compared with the control and siRNAs targeting other genes, siDicer2-10 treatment of rice blast fungus resulted in abnormally elongated germ tubes, smaller appressorium morphology, and a shallower melanin layer in the appressorium. This indicates that the exogenously added siDicer2-10 targeting the Dicer2 gene of rice blast fungus can inhibit the morphogenesis of rice blast fungus appressorium, suggesting that the Dicer2 gene can serve as a target gene for the control of rice blast fungus. Notably, silencing the Dicer1 gene, which is homologous to the Dicer2 gene, did not significantly differ the formation rate of rice blast fungus from the control, indicating that not all homologous genes of Dicer2 can serve as targets for the control of rice blast fungus.

[0049] To further verify the possibility of the Dicer2 gene as a target gene for controlling rice blast fungus, this embodiment knocked out two Dicer genes (Dicer1 and Dicer2) in rice blast fungus, either separately or simultaneously. The relevant sequences of the two genes were retrieved from the NCBI database according to their gene numbers, thereby constructing knockout vectors with different resistance levels. The knockout vectors were then introduced into rice blast fungus using Agrobacterium-mediated transformation to obtain mutants of the relevant genes.

[0050] The knockout results show ( Figure 3 Knocking out the Dicer1 gene alone had little or no effect on the growth of rice blast fungus, consistent with the above results. Conversely, knocking out the Dicer2 gene inhibited the growth of rice blast fungus and also reduced its spore production. Furthermore, knocking out the Dicer2 gene on the basis of the Dicer1 gene knockout mutant revealed that the growth of rice blast fungus was slow and the spore production was reduced. These results further confirm that Dicer2 plays an important role in the growth of rice blast fungus.

[0051] In summary, this embodiment screened a target gene, Dicer2, that regulates the growth of rice blast fungus and can be used to control rice blast fungus.

[0052] Example 2: Screening of siRNAs targeting the Dicer2 gene

[0053] Based on the results of Embodiment 1 above, it is shown that the Dicer2 gene is an effective target gene for controlling rice blast fungus. Therefore, in order to select the siRNA with the best targeting and silencing effect on the Dicer2 gene, this embodiment designed siRNA (siDicer2) targeting the full-length target silencing region of the Dicer2 gene, resulting in 12 pairs of siRNAs (including siRNA siDicer2-10 involved in Embodiment 1). The design principles of the siRNAs are as described in Embodiment 1, and the specific sequences are shown in Table 2, with the positions as follows. Figure 4 As shown; then, based on the website design results, four siRNA sequences with the highest non-off-target effects (siDicer2-5, siDicer2-3, siDicer2-6, siDicer2-10) were selected for antibacterial experiments (see Example 1 for specific experimental steps). The effect of siDicer2 on rice blast fungus was observed and recorded after 4 hours, and the results are as follows. Figure 5 As shown in the figure, at 4 h, compared with the control, the morphology (including appressoria and germ tubes) of rice blast fungus treated with siDicer2 showed varying degrees of abnormalities, further demonstrating the effectiveness of the Dicer2 gene as a target for the control of rice blast fungus. More specifically, compared with other siDicer2s, the rice blast fungus treated with siDicer2-10 showed the most severe inhibition of appressoria formation, the smallest appressoria size, the most obvious germ tube elongation, and the smallest spore body, indicating that siDicer2-10 is the siRNA molecule with the best effect in targeting the Dicer2 gene; correspondingly, the effect of siDicer2-6 was only slightly better than the control group, so siDicer2-10 was used as an example for subsequent experiments.

[0054] In summary, siDicer2-5, siDicer2-3, siDicer2-6, and siDicer2-10 can all be used to control rice blast fungus. The order of effectiveness from best to worst is: siDicer2-10 > siDicer2-3 > siDicer2-5 > siDicer2-6. Therefore, siDicer2-10 is the best choice.

[0055] Table 2. siRNA sequences targeting the Dicer2 gene

[0056]

[0057] Example 3: Effect of siDicer2-10 concentration on appressorium of rice blast fungus

[0058] Based on the results of Examples 1 and 2, this example selects siDicer2-10 as the research object to investigate the effects of different concentrations of siRNA on the formation of appressorium of rice blast fungus. The siRNA molecule dissolution and preservation methods are the same as described in Example 1.

[0059] In this embodiment, siDicer2-10 was first prepared into a stock solution with an initial concentration of 25 μM, and then mixed with 1×10 5 Equal volumes of rice blast fungus spore suspension (prepared as described in Example 1) were mixed to form siRNA at a final concentration of 12.5 μM. The siRNA was then spot-inoculated onto the center of a hydrophobic membrane and incubated for 4 h, 8 h, and 24 h, respectively. The formation of appressorium was observed and recorded. The results are as follows: Figure 6 As shown.

[0060] After 4 hours of incubation with *Oryza sativa* blast fungus using siDicer2-10, the vast majority of *Oryza sativa* blast fungus failed to form appressoria. After 8 hours of co-incubation, compared with the control, siDicer2-10 still effectively inhibited the formation of *Oryza sativa* appressoria, with an appressoria formation rate of approximately 50%, lower than the 80% of 5 μM. Figure 7 By 24 hours, rice blast fungus appressoria gradually formed, and the inhibitory effect of siDicer2-10 gradually weakened. The results of this example show that when the concentration of siDicer2-10 is increased to 12.5 μM, the inhibitory effect on rice blast fungus can be maintained for more than 8 hours.

[0061] Similar conclusions were obtained when the above experiments were performed using siDicer2-5, siDicer2-3, siDicer2-6, and siDicer2-10.

[0062] Example 4: Effect of siDicer2-10 in combination with other compounds on appressorium formation of rice blast fungus

[0063] To further improve the inhibitory efficiency of siDicer2-10 on the morphogenesis of rice blast fungus appressorium and prolong its duration of action, this embodiment combines siDicer2-10 with different chemical substances (tricyclazole and clotrimazole) to screen for the optimal combination. Tricyclazole primarily inhibits the formation of melanin in rice blast fungus; clotrimazole, an azole drug, mainly acts on the fungal cell wall.

[0064] In this embodiment, 4 μL of a 10 μg / μL tricyclazole solution (aaddin) or 1 μL of a 1 mg / mL clotrimazole solution (source leaf) was added to 1 mL of spore solution, and then mixed with an equal volume of siDicer2-10, resulting in a final concentration of 5 μM for siDicer2-10. The prepared spore mixture was then spot-inoculated onto a hydrophobic membrane, and the formation of rice blast fungus appressoria was observed at 4 h, 8 h, 20 h, and 24 h. The detailed procedure was the same as described in Example 1, and the results were as follows. Figures 7-8 As shown; this experiment also included a control group that received only tricyclazole / clotrimazole or siDicer2-10.

[0065] Depend on Figure 7 It is known that the use of tricyclazole alone does not affect the morphology and formation of rice blast fungus. This is because, at appropriate concentrations, tricyclazole can only inhibit melanin formation in rice blast fungus and weaken the mechanical function of appressorium by blocking melanin synthesis, but it cannot destroy the morphology of rice blast fungus. After treatment with siDicer2-10, regardless of whether tricyclazole was added, the appressorium formation rate of rice blast fungus was significantly reduced at 4 h and 8 h compared with the control, which is consistent with the results of Examples 1-2. Compared with siDicer2-10 treatment alone, the appressorium formation rate of rice blast fungus was significantly reduced after 4 h of treatment with tricyclazole and siDicer2-10 mixture. As the incubation time increased to 8 h and 20 h, although there was no significant difference in the appressorium formation rate, at 20 h, compared with the blank control (DDW), tricyclazole group, and siDicer2-10 group, the rice blast fungus appressorium treated with tricyclazole + siDicer2-10 group showed a higher degree of morphological and differentiation abnormality and formed less melanin layer. Figure 7 These results indicate that although tricyclazole itself does not inhibit the growth of rice blast fungus, its combined use with siDicer2-10 enhances the inhibitory effect of siDicer2-10 and prolongs its duration of action. This may be because tricyclazole can inhibit the formation of melanin in the appressorium of rice blast fungus, thereby reducing the resistance to siRNA (siDicer2-10) entering the fungus. Therefore, the combination of tricyclazole and siDicer2-10 molecules has a synergistic effect, further illustrating that melanin is an influencing factor in RNA entry into rice blast fungus.

[0066] Depend on Figure 8It is evident that, similar to tricyclazole, clotrimazole alone does not affect the growth of rice blast fungus; however, the synergistic effect of clotrimazole is better than that of tricyclazole. Specifically, at the same time points (4 h, 8 h, 20 h, 24 h), the clotrimazole + siDicer2-10 combination showed significantly stronger inhibitory effects on the morphogenesis and formation rate of rice blast fungus than the clotrimazole group, the siDicer2-10 group, and the tricyclazole + siDicer2-10 group. Furthermore, the clotrimazole + siDicer2-10 group effectively inhibited the growth of rice blast fungus for more than 24 h, and after 24 h of co-incubation with rice blast fungus, the appressorium formation rate was only about 60%, indicating that the inhibitory effect of this combination remains strong. Based on existing experience, the effect time of this combination can be as high as 48 hours. It is also worth noting that in this example, the working concentration of clotrimazole was 0.5 μg / mL, while the working concentration of tricyclazole was 10 μg / μL. Although the working concentration of clotrimazole is much lower than that of tricyclazole, the effect of the former combined with siDicer2-10 is significantly better than that of the latter, further highlighting the superiority of the combined use of clotrimazole and siDicer2-10.

[0067] In summary, although neither tricyclazole nor clotrimazole alone can affect the growth of rice blast fungus, both substances can enhance the inhibitory effect of siDicer2-10 on rice blast fungus and prolong its duration of action to varying degrees. Clotrimazole is preferred when used in combination with siDicer2-10.

[0068] Similar conclusions were obtained when the above experiments were performed using siDicer2-5, siDicer2-3, siDicer2-6, and siDicer2-10.

[0069] Example 5: The control effect of siDicer2-10 on rice blast disease

[0070] This embodiment uses a spray method to investigate whether siDicer2-10 can affect the pathogenicity of rice blast fungus to rice. The specific operation is as follows:

[0071] 5.1 Rice cultivation: Soak and germinate rice seeds (CO-39) in advance. Select rice seeds with uniform growth status and sow 22-25 seeds per pot. When the rice grows to the three-leaf and one-heart stage, cover the rice plants with plastic bags to prevent liquid from overflowing during spraying and to keep them moist.

[0072] 5.2 Collection and acquisition of rice blast fungus spore liquid: as described in Example 1.

[0073] 5.3 Pathogenicity Experiment of Rice Leaves: An equal volume of siDicer2-10 molecular solution and rice blast fungus spore suspension were mixed, with a final siRNA concentration of 5 μM. 3 mL of the siRNA and spore mixture was sprayed onto each rice plant. Sterile water was used as a control. The mixtures of different treatments were incubated at room temperature for 2 h. Before use, the cells were vortexed to ensure adhesion. The plants were then placed in an incubator and cultured at 22℃ in the dark for 2 days, followed by a 10 h:12 h light:dark cycle at 25℃. The disease incidence on rice leaves was observed, and leaf samples were collected to statistically analyze the disease results. The results are shown below. Figure 9 As shown.

[0074] The results are as follows Figure 9 As shown, compared with the control, the size of lesions on rice leaves was significantly reduced after treatment with siDicer2-10, indicating that the pathogenicity of rice blast fungus spores to rice leaves was significantly reduced. The results of this example demonstrate that siDicer2-10, targeting the key gene DICER2 of rice blast fungus, can control rice blast, providing a theoretical and experimental basis for SIGS control of rice blast fungus.

[0075] Similar conclusions were obtained when the above experiments were performed using siDicer2-5, siDicer2-3, siDicer2-6, and siDicer2-10.

[0076] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A siRNA for controlling rice blast fungus, characterized in that, The siRNA is SEQ ID NO: 8~9 as shown in the sequence listing.

2. A nucleic acid pesticide for controlling rice blast fungus, characterized in that, The nucleic acid pesticide comprises the siRNA as described in claim 1.

3. A composition for controlling rice blast fungus, characterized in that, The composition comprises the siRNA as described in claim 1.

4. The composition according to claim 3, characterized in that, The composition also contains substances that inhibit melanin formation in rice blast fungus and / or inhibit fungal cell wall formation.

5. A method for controlling rice blast fungus, characterized in that, The method refers to spraying a solution containing the siRNA as described in claim 1 or the composition as described in any one of claims 3 to 4 onto rice plants.

6. Dicer2 The use of genes as target genes for designing siRNA to control rice blast fungus is characterized by, The Dicer2 The gene contains the nucleotide sequence shown in SEQ ID NO: 1, and the siRNA is shown in SEQ ID NO: 8-9.

7. The use of siRNA in the preparation of reagents that interfere with the development or pathogenicity of rice blast fungus appressoriums, characterized in that, The siRNA is the sequence shown in the sequence listing as SEQ ID NO: 8~9.

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