Application of RPB1-M15 protein in prevention and treatment of plant diseases

Abstract

The invention relates to application of RPB1-M15 protein in prevention and treatment of plant diseases, the protein is separated from fermentation supernate of talaromyces aureoflavus and is used for inhibiting at least one of fusarium oxysporum, fusarium moniliforme and pestalotiopsis neoplasm, and then the RPB1-M15 protein can be used for inhibiting at least one of the fusarium oxysporum, the fusarium moniliforme and the pestalotiopsis neoplasm through the bacteriostatic action on the fusarium oxysporum, the fusarium moniliforme and the pestalotiopsis neoplasm. The RPB1-M15 protein can be used for preventing and treating tomato fusarium wilt, rice bakanae disease and strawberry leaf spot disease, has an ideal prevention and treatment effect on the plant diseases, has no pollution to the environment and no toxicity to human and livestock, and can be used for preparing medicines for preventing and treating various plant diseases caused by fusarium oxysporum, fusarium moniliforme and / or pestalotiopsis neoplasm.

Description

Technical Field

[0001] The invention relates to the technical field of plant control, and relates to an application of RPB1-M15 protein in controlling plant diseases, in particular to controlling tomato wilt, rice seedling blanching disease and strawberry leaf spot. Background Art

[0002] Plant diseases refer to abnormal plant growth and development or physiological dysfunction caused by biological factors (such as pathogens, viruses, nematodes, etc.) or non-biological factors (such as environmental stress, nutritional imbalance, etc.). They are one of the main threats to crop growth, leading to reduced crop yields, reduced crop growth quality, and in severe cases, crop death, often causing serious losses to my country's national economy and agricultural production. Among them, diseases caused by pathogens are particularly common and serious. Various pathogens invade plants through different pathways, use plant nutrients to grow and reproduce rapidly, and produce toxins to destroy plant cell structures and metabolic functions, ultimately leading to various plant disease symptoms, such as yellowing and wilting of leaves, and fruit rot.

[0003] In addition, continuous cropping all year round can easily lead to soil nutrient deficiency and pathogen enrichment, further increasing the incidence of plant diseases. Among them, the increase in the number of pathogens such as Fusarium oxysporum, Fusarium moniliforme, and Neodiscus polytrichosporium is one of the main reasons for the continuous cropping problem. These pathogens have a wide range of hosts and can infect economic crops such as tomatoes, bananas, rice, strawberries, cotton, and potatoes, causing these crops to suffer from wilt, seedling blight, or leaf spot, causing serious damage to crop growth. In severe cases, it directly causes crop death and threatens my country's grain production.

[0004] Fusarium oxysporum is a soil-borne pathogenic fungus with a worldwide distribution and a wide host range. It can cause more than 100 plant wilt and root rot diseases in melons, solanaceae, bananas, cotton, legumes and flowers, such as tomato wilt, sweet potato stem rot, pepper root rot, banana wilt, soybean root rot, etc. Fusarium moniliforme affects plant growth through the gibberellins and fusarium toxins it produces. It is often distributed in the roots, stems, ears, seeds and soil of plants such as rice, corn, cotton, sugarcane, citrus, onions, etc., causing plant diseases such as rice seedling disease, peanut root rot, corn wilt, corn top rot, garlic bulb disease, etc. Pestalotiopsis neofusicola, as an asexual endophytic fungus of the Deuteromycetes, is a well-known plant pathogen that can infect and cause serious diseases of a variety of economic crops, such as strawberry leaf spot, blueberry ring spot, blueberry branch blight, pine red blight, guava ring spot, etc.

[0005] At present, the main methods of plant disease prevention and control include agricultural prevention, physical prevention, chemical prevention, research and development of new varieties, and biological prevention. However, agricultural prevention is limited by seasons and regions and has the defect of slow prevention and control; physical prevention is difficult to operate, has high cost investment, and has a limited prevention and control range; chemical prevention easily causes crops to develop drug resistance, and there are problems such as environmental pollution and high pesticide residues in crops; the research and development of new varieties has a long research and development cycle, high research and development costs, and is easily affected by social economy. Biological control is currently the focus and hot spot of plant disease prevention and control research and development because it is pollution-free to the environment, safe for humans and animals, and not easy to develop resistance. It can effectively reduce the number of diseases, has significant prevention and control effects, is low in price, and has good economic and social benefits. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a RPB1-M15 protein to provide a new option for plant disease prevention and control.

[0007] The present invention is achieved through the following technical scheme: an application of RPB1-M15 protein in preventing and controlling plant diseases, wherein the amino acid sequence of the RPB1-M15 protein is shown in SEQ ID NO.2.

[0008] Compared with the prior art, the RPB1-M15 protein provided by the present invention has a good antibacterial effect on plant pathogens such as Fusarium oxysporum, Fusarium moniliforme and Neodiscus polytrichosporum, and has the potential to inhibit a variety of plant diseases caused by Fusarium oxysporum, Fusarium moniliforme and / or Neodiscus polytrichosporum, and is non-polluting to the environment and non-toxic to humans and animals. Conventional chemical pesticides require a high concentration of 50 mg / mL of carbendazim to achieve its good prevention effect and are prone to environmental pollution. Therefore, the RPB1-M15 protein described in the present invention can be used to prepare drugs for preventing and controlling a variety of plant diseases caused by Fusarium oxysporum, Fusarium moniliforme and / or Neodiscus polytrichosporum, which provides a new type of biocontrol protein for preventing and controlling Fusarium oxysporum, Fusarium moniliforme and / or Neodiscus polytrichosporum.

[0009] Furthermore, the plant diseases include plant diseases caused by at least one pathogen selected from the group consisting of Fusarium oxysporum, Fusarium moniliforme and Neodiscus polytrichosporium.

[0010] Furthermore, the plant diseases include tomato wilt caused by Fusarium oxysporum. The present invention first discovered that the RPB1-M15 protein has a good antibacterial effect on Fusarium oxysporum that causes tomato wilt, wherein the antibacterial rate of 2 mg / mL RPB1-M15 on Fusarium oxysporum is 53.6%, which provides a new biocontrol protein for preventing and controlling tomato wilt.

[0011] Furthermore, the plant diseases include rice seedling blight caused by Fusarium moniliforme. The present invention also discovered for the first time that the RPB1-M15 protein has a good antibacterial effect on Fusarium moniliforme that causes rice seedling blight, wherein the antibacterial rate of 2 mg / mL RPB1-M15 on Fusarium moniliforme is 48.5%, which provides a new type of biocontrol protein for preventing and controlling rice seedling blight.

[0012] Furthermore, the plant diseases include strawberry leaf spot caused by Neodiscus hirsutus. The present invention also discovered for the first time that the RPB1-M15 protein has a good antibacterial effect on Neodiscus hirsutus that causes strawberry leaf spot, wherein the antibacterial rate of 2 mg / mL RPB1-M15 on Neodiscus hirsutus is 65.7%, which provides a new type of biocontrol protein for the prevention and control of Neodiscus hirsutus.

[0013] Furthermore, the present invention provides a fungicide, which contains the above-mentioned RPB1-M15 protein.

[0014] Furthermore, the present invention provides a method for preventing and controlling plant diseases caused by plant pathogens, comprising the following steps: applying 5-10 mL of 0.02 mg / mL RPB1-M15 protein solution to each plant when the plants are planted; and applying 2-5 mL of 0.02 mg / mL RPB1-M15 protein solution to each plant by drip irrigation every 10-20 days during the growth period of the plants.

[0015] Furthermore, the preparation method of the above-mentioned RPB1-M15 protein includes the following steps: liquid fermentation of T. aureus for 72 hours to obtain conidia liquid; centrifuging the conidia liquid to obtain the supernatant, adding 314 grams of ammonium sulfate per 1L of supernatant, centrifuging to obtain the precipitate, and obtaining the RPB1-M15 protein.

[0016] Furthermore, the strain of the golden basket fungus is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20241164.

[0017] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The SDS-PAGE gel electrophoresis diagram of crude protein precipitated by 50% ammonium sulfate (separation gel concentration is 12%); wherein, M: mArker, 1: fermentation supernatant of Talaromyces aureus M15; 2: 50% ammonium sulfate precipitated protein;

[0019] Figure 2 This is the total ion current of the secondary mass spectrometry of RPB1-M15 protein;

[0020] Figure 3It is the agarose gel electrophoresis diagram of the RPB1-M15 protein gene cloning product;

[0021] Figure 4 This is the result of the amino acid sequence alignment of RPB1-M15 proteins;

[0022] Figure 5 is the antibacterial activity of RPB1-M15 protein against Fusarium oxysporum; among them, Figure 5 a: Fusarium oxysporum + sterile water (blank control); Figure 5 b: Fusarium oxysporum + 50 mg / mL carbendazim (positive control); Figure 5 c: Fusarium oxysporum + 2 mg / mL RPB1-M15 protein.

[0023] Figure 6 The application effect of RPB1-M15 protein in tomato cultivation; Figure 6 The left side shows the effect of the experimental group (RPB1-M15). Figure 6 The right side shows the effect of the blank control group.

[0024] Figure 7 is the antibacterial activity of RPB1-M15 protein against Fusarium moniliforme; among them, Figure 7 a: Fusarium moniliforme + sterile water (blank control); Figure 7 b: Fusarium moniliforme + 50 mg / mL carbendazim (positive control); Figure 7 c: Fusarium moniliforme + 2 mg / mL RPB1-M15 protein.

[0025] Figure 8 This is the application effect of RPB1-M15 protein in rice cultivation.

[0026] Fig. 9 is the antibacterial activity of RPB1-M15 protein against Neodiscus polytrichosporium; Fig. 9 a: Neodiscus polytrichomonas + sterile water (blank control); Fig. 9 b: Neodiscus polytrichosporium + 50 mg / mL carbendazim (positive control); Fig. 9 c: Neodiscus polytrichosporum + 2 mg / mL RPB1-M15 protein.

[0027] Fig.10 The application effect of RPB1-M15 protein in strawberry cultivation; Fig.10 a: Effect of experimental group (RPB1-M15), Fig.10 b: Effect of blank control group. DETAILED DESCRIPTION

[0028] Traditional means of crop disease control include agricultural control, physical control, chemical control, and the development of new disease-resistant varieties. However, traditional disease control methods have problems such as high control costs, environmental pollution, easy to produce drug resistance, and impact on human health. In addition, with the change of disease types and distribution, it is easy to have incomplete control, and traditional disease control methods may affect the quality of crops and thus affect the market competitiveness of crops. Biological control uses organisms to control the spread of pathogenic microorganisms and affect the occurrence of diseases. Because biological control has the advantages of no pollution to the environment, safety for humans and animals, high control effect, long sustainability, and strong adaptability, it is widely used in agricultural production, forest protection, urban greening and other fields. In the field of green and healthy planting of crops and disease prevention and control, biological control using the antagonistic effect of organisms or their metabolites on pathogens has become a new way to control plant diseases.

[0029] There are many pathogens that cause crop diseases. Among them, Fusarium oxysporum, Fusarium moniliforme, and Neodiscus polytrichosporium have a wide host range and are prone to cause cropping problems. Fusarium oxysporum is the pathogen that causes tomato wilt. After being infected with Fusarium oxysporum, tomato plants develop poorly, leaves wilt and necrotize, vascular bundles become lesions and turn brown, and the plants lose water, eventually leading to the death of tomato plants, causing significant losses to tomato production. Rice, as an important food crop in my country, is often attacked by pathogens such as Fusarium moniliforme during its growth. After rice plants are infected with Fusarium moniliforme, the internodes of the plants elongate, the stems gradually turn brown and rot, and the rice plants eventually die, often causing serious losses to my country's national economy and agricultural production. Neodiscus polytrichosporium is a well-known plant pathogen that can cause strawberry leaf necrosis and fruit rot during the growth process, and can cause a variety of injured plants to become diseased and necrotic, resulting in a reduction in strawberry production.

[0030] Based on this, the purpose of the present invention is to provide a more efficient biological control means for preventing and treating Fusarium oxysporum, Fusarium moniliforme and Neodisc multi-hairy spore. The present invention uses the fermentation supernatant of multiple bacteria to carry out antibacterial experiments on Fusarium oxysporum, Fusarium moniliforme and Neodisc multi-hairy spore, and finds that the fermentation supernatant of golden basket fungus M15 has good antibacterial effect on Fusarium oxysporum, Fusarium moniliforme and Neodisc multi-hairy spore, and there is no report on the inhibitory effect of golden basket fungus M15 on Fusarium oxysporum, Fusarium moniliforme and Neodisc multi-hairy spore at home and abroad. Based on this, the present invention further separates and purifies the substances in the fermentation supernatant of golden basket fungus M15, aiming to find substances that play an antibacterial role, and provides a new pollution-free, pollution-free green fungicide for preventing and treating Fusarium oxysporum, Fusarium moniliforme and Neodisc multi-hairy spore.

[0031] The present invention will be further described in detail below in conjunction with specific examples, which are only used to illustrate the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0032] Example 1

[0033] In this example, the fermentation supernatant of Talaromyces aureus M15 was separated and purified to screen out the RPB1-M15 protein that inhibits the growth of Fusarium oxysporum, Fusarium moniliforme and Polytrichomonas neodiscus. The method for preparing the RPB1-M15 protein from Talaromyces aureus M15 includes the following steps:

[0034] S101: Culture Talaromyces aurantiacus M15 on a PDA (potato dextrose agar) plate at 28°C for 7 days. Take a 5mm diameter disc of the Talaromyces aurantiacus colony edge and place it in 250mL of liquid PDA, culture at 30°C, 220rpm, and 72h to obtain conidia liquid. Among them, Talaromyces aurantiacus M15 is an ascomycete fungus belonging to the genus Ascomycetes of the Mucoraceae family, with the Latin name Talaromyces aurantiacus, which is deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: M20241164, and the deposit date is June 6, 2024.

[0035] S102: The conidia liquid was centrifuged at 4°C and 6000 r / min for 10 min, and the supernatant was taken. 314 g of ammonium sulfate was added to every 1 L of supernatant. The mixture was allowed to stand at 4°C for 4 h, and then centrifuged at 4°C and 9000 r / min for 30 min. The precipitate was collected to obtain protein A. 200 mg of protein A could be prepared per liter of fermentation liquid.

[0036] S103: The precipitated protein A was dissolved in Tris-HCl buffer (0.02 M, pH 7.6), collected in a 1.5 mL centrifuge tube and stored at 4° C. for later use.

[0037] S104: taking part of the protein for SDS-PAGE gel electrophoresis experiment, specifically comprising the following steps:

[0038] (1) Preparation of electrophoresis gel: After the separation gel is prepared, mix it thoroughly, inject it into two glass plates, add deionized water to seal the gel (use deionized water to seal the gel when the concentration is less than 10%, and use isopropanol to seal the gel when the concentration is greater than 10%), let it stand at room temperature for 30 minutes, and after the gel is coagulated, pour out the water (or isopropanol) and aspirate it; after the concentrated gel is prepared, inject it into the middle of the plate in the same way, slowly insert the comb, let it stand at room temperature for 30 minutes, take it out after the gel is coagulated, put the gel plate into the electrophoresis tank, and slowly add the electrode buffer;

[0039] Specifically, the separation gel and concentrated gel used are configured as follows:

[0040] 12% separation gel (15 mL): 4.9 mL water; 6 mL 30% acrylamide solution; 3.8 mL separation gel buffer (1.5 M Tris-HCl pH = 8.8); 0.15 mL 10% SDS; 0.15 mL 10% ammonium persulfate; 0.006 mL TEMED.

[0041] 5% stacking gel (5 mL): 3.4 mL water; 0.83 mL 30% acrylamide solution; 0.63 mL stacking gel buffer (1.0 M Tris-HCl pH=6.8); 0.05 mL 10% SDS; 0.05 mL 10% ammonium persulfate; 0.005 mL TEMED.

[0042] (2) Sample treatment: The sample was mixed with 5×SDS-PAGE loading buffer in a ratio of 4:1, incubated in a metal bath for 5 min, with a loading volume of 20 μL-50 μL and 5 μL of protein mArker loaded.

[0043] (3) Electrophoresis: When the samples are in the concentration gel, a constant voltage of 100 V is used for electrophoresis; when the samples are in the separation gel, a constant voltage of 180 V is used for electrophoresis. The electrophoresis can be stopped when the bromophenol blue approaches the edge of the separation gel.

[0044] (4) Staining: After electrophoresis, remove the gel sheet, carefully peel off the gel and place it in a staining dish. After adding the staining solution, place the staining dish on a decolorizing shaker and stain for 15 minutes.

[0045] (5) Destaining: Place the stained gel in a destaining solution and destain it on a destaining shaker. Change the destaining solution several times until the protein bands are clear.

[0046] Wherein, the solution used in the SDS-PAGE gel electrophoresis process is prepared as follows:

[0047] 30% acrylamide solution: Prepared with (purchased) deionized water, containing 29.2% (w / v) acrylamide and 0.8% (w / v) N,N'-methylene acrylamide. Filter with filter paper, place in a brown bottle, and store at 4°C away from light for later use.

[0048] Separation gel buffer (2M Tris-HCl pH=8.8): Dissolve 24.2 g Tris base in 50 mL water, adjust the pH to 8.8 with HCl (about 4 mL), make up to 100 mL, and place at room temperature for use.

[0049] Stacking gel buffer (0.3 M Tris-HCl pH = 6.8): Dissolve 3.6 g Tris base in 50 mL water, adjust the pH to 6.8 with HCl (about 1.8 mL), make up to 100 mL, and place at room temperature for use.

[0050] 10% ammonium persulfate (AP): Prepare to a concentration of 10% (w / v) with deionized water and store at 4°C until use.

[0051] 10× electrode buffer: 30g Tris, 144g glycine, 10g SDS, dissolved in water, do not stir too vigorously to avoid bubbles, dilute to 1000mL, the pH of the solution should be 8.3 or above, dilute (1×) before use, and place at room temperature for later use.

[0052] 50% (v / v) glycerol: Prepare glycerol to a concentration of 50% (w / v) with deionized water and place at room temperature for later use.

[0053] 10% (w / v) SDS: 10 g SDS was dissolved in 100 mL deionized water and kept at room temperature for later use (prepared and used at 68°C for dissolution).

[0054] 1% (w / v) bromophenol blue: 0.1 g bromophenol blue was dissolved in 10 mL deionized water, filtered and placed at room temperature for later use.

[0055] 5×SDS-PAGE Loading Buffer: 0.3M Tris-HCl (pH=6.8) stacking gel buffer 8.5mL, 50% glycerol 8.0mL, 10% SDS 8.0mL, 0.1% (w / v) bromophenol blue 10mL, β-mercaptoethanol 2.0mL, add distilled water to a total volume of 40mL.

[0056] Staining solution: 45% methanol, 10% glacial acetic acid, 45% deionized water, 0.25% (w / v) Coomassie Brilliant Blue (CommAssise Blue R250), filtered (0.45 μm filter) and placed at room temperature for use.

[0057] Decolorizing solution: 5% methanol, 7.5% glacial acetic acid, 87.5% deionized water, set aside at room temperature.

[0058] TEMED (tetramethylethylenediamine): stock solution.

[0059] See also Figure 1 , Figure 1 The SDS-PAGE gel electrophoresis results of protein A prepared by T. aureus M15 are shown. The molecular weight of the purified protein A is about 32KD.

[0060] Example 2

[0061] This example identifies purified protein A, including the following steps:

[0062] The purified protein bands obtained by SDS-PAGE gel electrophoresis in Example 1 were cut with a sterile scalpel, placed in a 1.5 mL centrifuge tube, and sent to Shanghai Ouyi Biotechnology Co., Ltd. for LC-MS / MS mass spectrometry identification, and the data obtained by LC-MS / MS were searched and compared with the database.

[0063] See also Figure 2 , Figure 2 The total ion current of the secondary mass spectrometry of the purified protein is shown. In addition, the comparison results of the data obtained by LC-MS / M show that the protein A separated and purified by the present invention corresponds to the molecular weight of RNAbinding proteins with SequenCe ID (sequence number) AEY85240.1, so it is speculated that the protein A is one of the RNA binding proteins, and the protein A is named RPB1-M15 protein.

[0064] Example 3

[0065] In this example, the coding gene of the purified RPB1-M15 protein is cloned to determine the amino acid sequence and coding gene sequence of the RPB1-M15 protein, including the following steps:

[0066] The genome of T. aurea M15 was extracted using the SP FungAl DNA Kit from OMEGA Biotech and used as template DNA. The specific primers RPB1-F1843: ATTTYGAYGGTGAYGARATGAAC and RPB1-R3096: GRACRGTDCCRTCATAYTTRACC were used to amplify the gene coding frame and protein sequence of the purified protein. The specific procedure for PCR amplification was 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s; 52°C renaturation for 30 s; 72°C extension for 1 min, 35 cycles; 72°C extension for 10 min; 4°C storage for 10 min. The PCR product was verified by agarose gel electrophoresis.

[0067] See also Figure 3 , Figure 3 The agarose gel electrophoresis diagram of the gene cloning product is shown. The target band sample was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing, and the nucleotide sequence of the RPB1-M15 protein is shown in SEQ ID NO.1.

[0068] Example 4

[0069] This implementation translates the nucleotide sequence obtained by gene cloning into an amino acid sequence and performs a homology comparative analysis of the amino acid sequence, including the following steps:

[0070] The ORF open coding frame of the nucleotide sequence obtained by gene cloning was analyzed to obtain the amino acid sequence shown in SEQ ID NO.2 in the sequence table; the amino acid sequence was compared through NCBI to find the protein with the highest homology, and a multiple sequence alignment was performed with the amino acid sequence of the protein using DNAMAN software.

[0071] NCBI alignment results show that the amino acid sequence of RPB1-M15 protein is 97.85% homologous to the largest subunit of RNA polymerase II (SequenCe ID: WNV63755.1). Figure 4 , Figure 4 The multiple sequence alignment results of the amino acid sequence of RPB1-M15 protein and the amino acid sequences of other RNA polymerase II largest subunits are shown.

[0072] Example 5

[0073] This example studies the antibacterial activity of the purified RPB1-M15 protein against Fusarium oxysporum, comprising the following steps:

[0074] The purified RPB1-M15 was dissolved in 0.02 M Tris-HCl (pH=7.6) buffer to prepare a 2 mg / mL RPB1-M15 solution.

[0075] Using the plate confrontation method, take the Fusarium oxysporum cake in the center of the PDA culture medium, place 3 sterilized filter papers at 15mm away from the cake and every 120°, add 20μL RPB1-M15 solution on the sterilized filter paper respectively; put the PDA plate in a 30℃ incubator for 5 days. In addition, sterile water and 50mg / mL carbendazim were used as blank control and positive control.

[0076] Among them, the formula of PDA solid culture medium is: 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 20% potato extract (200g potato), 1.5% agar, a total of 1L.

[0077] See also Figure 5 a-5c, which show the antibacterial activity experiment of RPB1-M15 protein against Fusarium oxysporum and the control experiment results. Figure 5 As shown in a-5c, the RPB1-M15 protein has an antibacterial effect on Fusarium oxysporum. Since Fusarium oxysporum can cause a variety of plant wilt and root rot diseases, such as tomato wilt, sweet potato stem rot, pepper root rot, banana wilt, soybean root rot, etc., the RPB1-M15 protein has the potential to inhibit plant wilt and root rot caused by Fusarium oxysporum and can be used to prepare drugs for preventing and controlling plant wilt and root rot.

[0078] Since Fusarium oxysporum is the main pathogen causing tomato wilt, tomato wilt was taken as an example to further verify the inhibitory effect of RPB1-M15 protein on plant wilt.

[0079] Example 6

[0080] This example studies the application effect of RPB1-M15 protein in tomato cultivation.

[0081] The experiment was conducted in a tomato greenhouse at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. The tomato wilt disease caused by Fusarium oxysporum in the greenhouse was serious, and the death rate of tomatoes last season reached more than 45%.

[0082] The experimental group includes 3 rows, each row has 50 tomato seedlings, and the specific experimental process is as follows: dilute the RPB1-M15 obtained in Example 1 to a final concentration of 0.02 mg / mL, fully mix the soil with the 0.02 mg / mL RPB1-M15 protein solution, and make the soil moisture 80%-90%, and place the mixture for 5 days, transplant the tomato seedlings into the soil mixed with the RPB1-M15 protein solution for field planting, and after field planting, 0.02 mg / mL RPB1-M15 protein solution is drip-irrigated to the root system, and 100 mL of protein solution is used for each row of tomato seedlings. After field planting, every 10 days, 0.02 mg / mL RPB1-M15 protein solution is drip-irrigated to the root system, and 100 mL of protein solution is used for each row of tomato seedlings. The number of surviving trees is calculated during the tomato fruiting period, and the survival rate is calculated.

[0083] The blank control group included 3 rows, with 50 tomato seedlings in each row. The blank control group used water instead of the RPB1-M15 protein solution, and the other conditions were the same as those of the experimental group and were carried out in parallel with the experimental group.

[0084] Among them, the mortality rate = the number of dead plants / the total number of plants × 100%, and the survival rate = the number of surviving plants / the total number of plants × 100%.

[0085] See also Figure 6 , Figure 6 shows the results of tomato cultivation, by Figure 6 It can be seen that the final survival rate of the 150 tomatoes in the experimental group was 94%, and the survival rate of the blank control group was 71%. The use of the RPB1-M15 protein of the present invention significantly improves the survival rate of tomatoes and reduces the mortality rate of tomatoes.

[0086] Example 7

[0087] In this example, the antibacterial activity of the purified RPB1-M15 protein against Fusarium moniliforme was tested, comprising the following steps:

[0088] The purified RPB1-M15 protein was dissolved in 0.02 M Tris-HCl (pH=7.6) buffer to prepare a 2 mg / mL RPB1-M15 protein solution.

[0089] Using the plate confrontation method, take the Fusarium moniliforme cake in the center of the PDA culture medium, place 3 sterilized filter paper pieces at 15mm away from the cake and every 120°, add 20μL RPB1-M15 protein solution or 20μL 50mg / mL carbendazim to each sterilized filter paper piece; put the culture medium in a 30℃ incubator for 5 days. In addition, sterile water and 50mg / mL carbendazim were used as blank control and positive control.

[0090] See also Figure 7a-7c, which are the results of the antibacterial activity experiment of RPB1-M15 protein against Fusarium moniliforme and the control experiment. Figure 7 As shown in a-7c, RPB1-M15 protein has an antibacterial effect on Fusarium moniliforme. Fusarium moniliforme is often distributed in the roots, stems, ears, seeds and soil of plants such as rice, corn, cotton, sugarcane, citrus, onion, etc., causing a variety of plant diseases, such as rice seedling blight, peanut root rot, corn wilt, corn top rot, garlic bulb disease, etc. Therefore, RPB1-M15 protein has the potential to inhibit plant diseases caused by Fusarium moniliforme.

[0091] Fusarium moniliforme is the main pathogen that causes rice seedling disease. Therefore, rice seedling disease is taken as an example to further verify the inhibitory effect of RPB1-M15 protein on plant diseases caused by Fusarium moniliforme.

[0092] Example 8

[0093] This example studies the application effect of RPB1-M15 protein in rice cultivation.

[0094] The experiment was conducted in a rice field at the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. In this field, the rice seedling blight caused by Fusarium moniliformis was serious when planting rice. In the last season, the necrosis rate of rice reached 53%, posing a serious threat to rice production.

[0095] The experimental group includes 3 rows, each row has 100 rice seedlings, and the specific experimental process is as follows: the rice seedlings are transplanted into the rice field for planting, the RPB1-M15 protein obtained in Example 1 is diluted to a final concentration of 0.02 mg / mL, and 0.02 mg / mL RPB1-M15 protein solution is used for spraying after planting, and 500 mL of protein solution is used for each row of rice. After planting, 0.02 mg / mL RPB1-M15 protein solution is used to spray rice every 20 days, and 500 mL of protein solution is used for each row of rice. The number of surviving plants is calculated at the peak fruiting period, and the survival rate is calculated.

[0096] The blank control group included 3 rows, with 100 rice seedlings in each row. The blank control group used water instead of the RPB1-M15 protein solution, and the other conditions were the same as those of the experimental group and were carried out in parallel with the experimental group.

[0097] Among them, the mortality rate = the number of dead plants / the total number of plants × 100%, and the survival rate = the number of surviving plants / the total number of plants × 100%.

[0098] See also Figure 8 , Figure 8 As a result of rice cultivation, Figure 8It can be seen that the final survival rate of the 300 rice plants in the experimental group was 95%, and the survival rate of the blank control group was 71%. Therefore, the use of the RPB1-M15 protein of the present invention significantly improves the survival rate of rice and reduces the mortality rate of rice.

[0099] Example 9

[0100] In this example, the antibacterial activity experiment of the purified RPB1-M15 protein against Neodiscus polytrichosporium was conducted, comprising the following steps:

[0101] The purified RPB1-M15 protein was dissolved in 0.02 M Tris-HCl (pH=7.6) buffer to prepare a 2 mg / mL RPB1-M15 protein solution.

[0102] Using the plate confrontation method, take a new P. sphaerocephala cake in the center of the PDA culture medium, place 3 sterilized filter paper pieces at 15 mm away from the cake and every 120°, add 20 μL RPB1-M15 protein solution or 20 μL 50 mg / mL carbendazim to each sterilized filter paper piece; place the culture medium in a 30°C incubator for 5 days. In addition, sterile water and 50 mg / mL carbendazim were used as blank and positive controls.

[0103] Among them, the formula of PDA solid culture medium is: 20g glucose, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 20% potato extract (200g potato), 1.5% agar, a total of 1L.

[0104] See also Fig. 9 a-9c, which are the results of the antibacterial activity experiment of RPB1-M15 protein against Neodiscus polytrichosporium and the control experiment. Fig. 9 a-9c shows that RPB1-M15 protein has an antibacterial effect on Neodiscus hirsutus. Neodiscus hirsutus is a well-known plant pathogen species that causes strawberry leaf spot, blueberry ring spot, blueberry branch blight, pine red blight, guava ring spot, etc. Therefore, RPB1-M15 protein has the potential to inhibit plant diseases caused by Neodiscus hirsutus.

[0105] Since Neodiscus is the main pathogen causing strawberry leaf spot, strawberry leaf spot was taken as an example to further verify the inhibitory effect of RPB1-M15 protein on plant diseases caused by Neodiscus.

[0106] Example 10

[0107] This example studies the application effect of RPB1-M15 protein in strawberry cultivation.

[0108] The experiment was conducted in the experimental teaching base of South China Agricultural University in Zengcheng District, Guangzhou City, Guangdong Province. In this plot, due to the continuous cultivation of strawberries, pathogens accumulated in the soil, causing strawberries to wilt and die. Among them, strawberry leaf spot caused by Neodiscus hirsutus was the most serious, with a mortality rate of 49% in the last season.

[0109] Experimental group: 3 rows, 100 trees per row, transplant the strawberry seedlings into the soil for planting. Dilute the RPB1-M15 protein obtained in Example 1 to a final concentration of 0.02 mg / mL, and use 0.02 mg / mL RPB1-M15 protein solution for irrigation after planting, with an irrigation volume of 1 L. Thereafter, use 0.02 mg / mL RPB1-M15 protein solution to drip irrigate the root system every 10 days, use 200 mL of protein solution for each row of strawberry seedlings, calculate the number of surviving trees at the peak fruiting period, and calculate the survival rate.

[0110] Blank control group: 3 rows, 100 trees in each row. The blank control group used water instead of RPB1-M15 protein solution. The other conditions were the same as those of the experimental group and were carried out in parallel with the experimental group.

[0111] Among them, the mortality rate = the number of dead plants / the total number of plants × 100%, and the survival rate = the number of surviving plants / the total number of plants × 100%.

[0112] Fig.10 shows the results of strawberry cultivation, by Fig.10 It can be seen that the final survival rate of the 300 strawberries in the experimental group was 96%, and the survival rate of the blank control group was 67%. The use of the RPB1-M15 protein of the present invention greatly improves the survival rate of strawberries and reduces the mortality rate of strawberries.

[0113] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0114] (1) The low concentration RPB1-M15 protein of the present invention has a good antibacterial effect on Fusarium oxysporum, Fusarium moniliforme and Neopseudomonas, while the conventional chemical pesticides require a high concentration of 50 mg / mL of carbendazim to achieve its good prevention effect and easily cause environmental pollution and other problems. It has the potential to inhibit various plant diseases caused by Fusarium oxysporum, Fusarium moniliforme and / or Neopseudomonas, and can be used to prepare drugs for preventing and controlling various plant diseases caused by Fusarium oxysporum, Fusarium moniliforme and / or Neopseudomonas.

[0115] (2) The present invention first discovered that the RPB1-M15 protein separated and purified from the protein of T. aurea has a good inhibitory effect on Fusarium oxysporum, the pathogen causing tomato wilt, Fusarium moniliforme, the pathogen causing rice seedling blight, and Neopseudomonas aeruginosa, the pathogen causing strawberry leaf spot. In the antibacterial activity experiment, the inhibition rates of 2 mg / mL RPB1-M15 protein on Fusarium oxysporum, Fusarium moniliforme, and Neopseudomonas aeruginosa were 53.6%, 48.5%, and 65.7%, respectively, and the antibacterial effect was good. During the plant cultivation process, the inhibition rates of long-term interval application of RPB1-M15 protein solution on tomato wilt caused by Fusarium oxysporum, rice seedling blight caused by Fusarium moniliforme, and strawberry leaf spot caused by Neopseudomonas aeruginosa were 79.31%, 82.76%, and 87.88%, respectively.

[0116] (3) The RPB1-M15 protein is environmentally friendly and harmless to humans and animals, which provides a new type of safe biocontrol protein for the prevention and control of various plant diseases such as tomato wilt, rice seedling blight, and strawberry leaf spot.

[0117] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. An application of RPB1-M15 protein in preventing and controlling plant diseases, characterized in that: The amino acid sequence of the RPB1-M15 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that: The plant diseases include plant diseases caused by at least one pathogenic fungus selected from the group consisting of Fusarium oxysporum, Fusarium moniliforme and Neodiscoideum polytrichosporium.

3. The application according to claim 2, characterized in that: The plant diseases include tomato wilt caused by Fusarium oxysporum.

4. The application according to claim 2, characterized in that: The plant diseases include rice bakanae disease caused by Fusarium moniliforme.

5. The application according to claim 2, characterized in that: The plant diseases include strawberry leaf spot caused by Neodiscus hirsutus.

6. A fungicide, characterized in that: The bactericidal agent comprises the RPB1-M15 protein as claimed in claim 1.

7. The bactericide according to claim 6, characterized in that: The fungicide is applied to the plants by root dipping or root irrigation.

8. A method for preventing and controlling plant diseases caused by plant pathogens, characterized in that: The method comprises the following steps: applying 5-10 mL of 0.02 mg / mL RPB1-M15 protein solution to each plant when the plants are planted; and applying 2-5 mL of 0.02 mg / mL RPB1-M15 protein solution to each plant by drip irrigation every 10-20 days during the growth period of the plants.

9. The method for preparing the RPB1-M15 protein according to claim 1, characterized in that: The method comprises the following steps: fermenting the liquid of Talaromyces chrysanthemi for 72 hours to obtain conidia liquid; centrifuging the conidia liquid, adding 314g of ammonium sulfate to each 1L of supernatant, centrifuging to obtain a precipitate, and obtaining RPB1-M15 protein.

10. The method for preparing the RPB1-M15 protein according to claim 9, characterized in that: The strain of the golden basket fungus is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20241164.

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