6ba for use in the preparation of a formulation for enhancing the resistance of a plant to blast

Abstract

The application discloses application of 6BA in preparation of a preparation for enhancing the rice blast resistance of plants, wherein the plants are rice, and spraying the preparation containing 6BA with a proper concentration on the surface of the rice can significantly improve the resistance of the rice to the Magnaporthe grisea; researches show that 6BA can enhance the resistance of the rice to the Magnaporthe grisea by increasing the expression amount of the disease resistance genes OsPR5, OsPAL, OsWRKY45 and OsWRKY76 and increasing the activity of the antioxidant enzymes SOD, CAT and PAL, and the application provides a new thought for the research on the rice blast resistance.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to the application of 6BA in the preparation of formulations to enhance plant resistance to rice blast. Background Technology

[0002] 6BA, or 6-benzylaminopurine (N-(Phenylmethyl)-9H-purin-6-amine), with the molecular formula C12H11N5, is the first artificially synthesized plant growth regulator with effects similar to cytokinins. Studies have found that spraying appropriate concentrations of 6BA during plant growth and development can regulate related processes within the plant, such as fatty acid metabolism, thus balancing protein metabolism and further improving the plant's physiological state. Simultaneously, the increased chlorophyll content enhances oxygen transport, significantly improving photosynthetic capacity. Research also indicates that metabolic and physiological deficiencies caused by magnesium deficiency, such as magnesium deficiency-related plant nutrient deficiencies, can be alleviated by applying a certain amount of 6BA. Furthermore, 6BA can act as a cytokinin, promoting plant cell division. Appropriate spraying of certain concentrations of 6BA can significantly increase the chlorophyll and soluble protein content in the leaves of plants such as cotton, enhancing the plant's ability to scavenge reactive oxygen species, maintaining a high level of photosynthetic pigments in the leaves, and delaying senescence.

[0003] 6-BA can act as an inducible regulatory factor to enhance plant resistance to harsh environments. For example, under low temperatures, 6-BA can significantly increase the proportion of unsaturated acids and chlorophyll content in plants by increasing enzyme activity, inhibiting cell membrane lipid peroxidation, and protecting the integrity and stability of membrane structure and function, thus greatly enhancing the plant's cold resistance induced by low temperatures. Simultaneously, applying a certain concentration of 6-BA under drought stress can reduce leaf water loss by preventing a decrease in photosynthesis in seedlings, altering the composition of water under drought conditions, increasing the content of bound water in plants, and improving the total water content, free water content, and growth and metabolism of seedlings, thereby enhancing the plant's drought resistance. Furthermore, under salt stress, 6-BA, as an exogenous substance, can accelerate the formation and accumulation of new substances in plants, increasing the germination rate, germination index, and vigor index of seedlings under salt stress. At the same time, excess free radicals in plants can be cleared under the regulation of 6-BA, thereby mitigating the harm of salt stress and enhancing plant salt tolerance.

[0004] With the increasing global population, the demand for food will continue to grow, leading to an expansion of the planting area for rice, a crucial crop. Conidia produced by *Blastomyces oryzae* are the primary source of infection for rice blast disease, which is one of the most devastating diseases affecting rice yields. Currently, rice blast is controlled using resistant varieties, chemical agents, and cultivation practices, with breeding resistant varieties being the most economical and effective measure. However, *Blastomyces oryzae* strains typically mutate very rapidly, causing newly introduced resistant varieties to lose or reduce their resistance after 3-5 years of application. Existing rice blast control agents mostly act directly on the *Blastomyces oryzae*, requiring spraying, which is inconvenient, toxic, and environmentally polluting, with less than ideal results. Currently, there is a lack of agents that can effectively enhance rice seedling resistance to blast while being convenient and pollution-free. A search revealed no reports on the role of 6BA in improving rice blast resistance. Summary of the Invention

[0005] This invention is the first to discover that external spraying of 6BA can significantly improve the resistance of rice seedlings to rice blast, and further studies the disease resistance mechanism of 6BA in rice blast resistance, providing new ideas and approaches for the research on rice blast resistance.

[0006] The specific technical solution of the present invention is as follows:

[0007] The first aspect of this invention provides the use of 6BA in the preparation of formulations for enhancing plant resistance to rice blast.

[0008] Preferably, in the above application, the formulation containing 6BA is sprayed onto the plant surface.

[0009] In one embodiment of the present invention, the plant is a rice seedling, and the optimal 6BA concentration for induction treatment of rice seedlings is 10 mg / L.

[0010] The experimental data of this invention further demonstrate that formulations containing 6BA enhance the resistance of rice to rice blast through at least the following pathways:

[0011] a) Induces upregulation of disease resistance-related gene expression;

[0012] b) Increase the content and / or activity of antioxidant enzymes.

[0013] Specifically, the disease-resistance-related genes include OsPR5, OsPAL, OsWRKY45, and OsWRKY76, and the antioxidant enzymes include SOD (superoxide dismutase), CAT (catalase), and PAL (phenylalanine ammonia-lyase). After induction treatment with a preparation containing 6BA, the content of MDA (malondialdehyde) increased significantly, and the activities of SOD, CAT, and PAL were significantly enhanced.

[0014] A second aspect of the present invention provides an formulation for enhancing plant resistance to rice blast, one of the active ingredients of which is 6BA.

[0015] Preferably, in the above-described formulation, the dosage form of the formulation is a liquid formulation.

[0016] The third aspect of this invention provides a method for preventing and controlling rice blast disease, specifically: when rice is not infected with the disease, an induction treatment containing 6BA is used to enhance the resistance of rice plants to rice blast fungus, thereby playing a role in preventing and controlling rice blast disease.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention is the first to discover that external spraying of 6BA can induce resistance in plants to rice blast fungus, thereby significantly improving the plant's resistance to rice blast and achieving the goal of controlling rice blast. Furthermore, experimental data shows that the appropriate concentration of 6BA is a key factor affecting the induction effect; for example, for rice seedlings, the optimal 6BA concentration is 10 mg / L, while concentrations that are too high or too low will significantly reduce the control effect.

[0019] This invention conducts an in-depth study on the disease resistance mechanism of 6BA in rice blast resistance. The results show that 6BA improves rice resistance to rice blast by increasing the expression levels of disease resistance genes OsPR5, OsPAL, OsWRKY45, and OsWRKY76, as well as increasing the activity of antioxidant enzymes SOD, CAT, and PAL, thus providing new insights for the study of rice blast resistance. Attached Figure Description

[0020] Figure 1 This is a comparison of the resistance of rice seedlings to rice blast under different concentrations of 6BA induction in Example 1 of the present invention.

[0021] Figure 2 This is a graph showing the changes in antioxidant enzyme activity in rice seedlings after 6BA induction treatment in Example 2 of the present invention.

[0022] Figure 3This is a graph showing the analysis of the expression levels of disease resistance-related genes in rice seedlings after 6BA induction treatment in Example 2 of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term "comprising" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusion.

[0025] The rice variety used in the following examples is Hwayoung, and the rice blast fungus used is strain JC2. Other main reagents and instruments are shown in Tables 1 and 2, respectively.

[0026] Table 1. Experimental Reagent Information

[0027]

[0028] Table 2.2 Experimental Apparatus

[0029]

[0030] Example 1

[0031] This example uses rice seedlings to demonstrate that appropriate concentrations of 6BA formulations can induce enhanced resistance in rice to rice blast fungus. The specific experimental procedure is as follows:

[0032] (1) Cultivation of rice seedlings.

[0033] Seed germination: Select an appropriate amount of rice seeds with similar shape and size. First, soak them in 75% C2H5OH for 30 seconds to disinfect them. Then, rinse the seeds with sterile water 4-5 times. Next, soak them in 20% NaClO for 40 minutes to disinfect them (shake the glass petri dish every 10 minutes). Then, wash the seeds thoroughly with sterile water about 10 times until there is no pungent odor. Place the washed seeds evenly in a petri dish and soak them in clean water for 24 hours at a temperature of 28℃ to allow the seeds to enter the germination state.

[0034] Filter paper preparation: After 24 hours, wash the HY seeds with clean water 3-4 times. Cut the filter paper to the size of the petri dish, place two sheets of filter paper in each petri dish, and then place the HY seeds on the filter paper. Add a little water to the petri dish and germinate at 28℃.

[0035] Sowing seeds: After 48 hours, wash the germinated HY seeds with clean water and select the best-growing seedlings to transplant onto the hydroponic box. The HY seeds that have not yet germinated will continue to grow in the petri dish. Once the HY seeds have germinated, transplant them onto the hydroponic box.

[0036] Change the water and wash the roots: During the seedling growth process, carefully rinse the roots of the seedlings with clean water every 4-5 days, clean the hydroponic box and change the water.

[0037] Nutrient solution change: After the rice seedlings have grown for about 10 days, prepare the nutrient solution for the rice, add it to the hydroponic box, and carefully rinse the roots of the seedlings. Maintain the temperature at 28℃. Continue to wash the roots and change the nutrient solution every 5-7 days.

[0038] Seedling stage completed: Seedlings are cultivated to the stage of three leaves and one bud, with the fourth leaf just emerging.

[0039] (2) Cultivation of rice blast fungus.

[0040] Prepare 150ml of PDA medium and 350ml of PDB medium: Weigh 100g of washed and peeled potatoes, slice them thinly, boil them until soft, and filter them while hot through two layers of gauze. Pour the filtrate into beakers containing 3g and 7g of sucrose respectively, adding 150ml and 350ml of liquid medium (add 2.75g of agar to the beaker containing 3g of sucrose). Stir well with a glass rod, and immediately dispense into four 250ml Erlenmeyer flasks, label them, and sterilize. After sterilization, pour the hot PDA medium into glass petri dishes in a laminar flow hood, filling them to two-thirds full. This will yield six bottles of PDA medium and three bottles of PDB liquid medium.

[0041] Preparation of tomato-oat culture medium: After juicing tomatoes, filter the juice through three layers of gauze and take 200ml. Pour this into a beaker containing 18g of agar and 18g of soluble starch (dissolve the soluble starch in a small amount of water first). Cook 40g of oats until soft, filter the supernatant into the beaker, and bring the volume to 1L. Stir well with a glass rod, then add an alkaline solution (NaOH) to adjust the pH to neutral. Finally, dispense the mixture into seven 250ml Erlenmeyer flasks, sterilize, and pour into 30 agar plates.

[0042] Activation of rice blast fungus: The selected rice blast fungus is variety JC2. In a clean bench, two filter paper discs carrying the fungal strain were inoculated onto the cooled PDA medium using sterilized tweezers. The medium was then incubated in the dark at 28°C for 5-6 days. To prevent the filter paper discs from falling off, the culture dish was placed upright on the first day of incubation, and then inverted for further incubation.

[0043] Shaking culture: Using a sterilized inoculation needle in a clean bench, pick up a white, translucent rice blast fungus colony from the edge of the activated rice blast fungus colony and inoculate it into PDB medium. Place the culture in a shaker and culture at 28°C and 180 rpm for 5-6 days.

[0044] Sporulation culture: After the shake flask culture is completed, a large number of dark brown mycelial clusters can be observed. Use a pipette to draw two 800 μL suspensions from the shake flask culture and spread them onto a tomato-oat medium. Then, use a sterilized spreader to disperse the mycelium. Finally, remove the filtrate. Incubate at 28°C under natural light for about 5 days. When there are a large number of white, fluffy colonies on the medium, gently scrape the tomato-oat medium with a sterile cotton swab to ensure even spore dispersion and better sporulation results.

[0045] (3) 6BA was used to induce rice seedlings.

[0046] Prepare 50 ml of each of 5 mg / L, 10 mg / L, and 20 mg / L 6BA solutions. Two days before inoculation, spray these three concentrations of 6BA solutions evenly onto rice seedlings. Take another hydroponic box and spray it with an equal amount of sterile water (this is a blank control). Label the boxes and place them in a 28℃ greenhouse for cultivation. When inducing 6BA, first perform a 24-hour dark treatment, and then cultivate rice seedlings under natural light conditions.

[0047] (4) Inoculation.

[0048] Add 2 ml of sterile water to the culture medium that has completed sporulation. Gently scrape the spores with a sterile cotton swab and pour the spores into a conical flask filtered through three layers of gauze to generate a spore suspension. First, use a pipette to draw a small amount of the filtrate onto a hemocytometer and level it. Adjust the microscope and the field of view until there are 30-40 viable spores in the visible field of view. Add an appropriate amount of gelatin to the spore suspension to ensure that the spores can remain firmly attached to the rice leaves. Place rice seedlings that have been induced with 6BA for two days and a pot of seedlings that have not been induced with 6BA in an incubator. Add an appropriate amount of water to the bottom and spray the spores evenly onto each leaf of the seedlings. Take another pot of seedlings that have not been induced with 6BA and place it in another incubator without inoculation. Cover the incubator with plastic wrap and filter cloth, and finally cover it with a black cloth for 24 hours of dark treatment. Remove the black cloth.

[0049] (5) Rice phenotypic identification.

[0050] After inoculation, rice seedlings were first treated in the dark for 24 hours, and then cultured for 5 to 7 days under 12-hour light and 12-hour dark conditions before resistance identification was performed. The specific method was as follows: Rice seedlings induced and inoculated with 6BA and treated with sterile water in the incubator were cut off with clean scissors, the rice leaves were unfolded, and pasted onto clean, blank A4 paper. The phenotypic results could be printed out using a printer, and the A4 paper was stored in the refrigerator. The rice blast resistance level was evaluated according to Table 3.

[0051] Table 3 Evaluation Criteria for Rice Blast Resistance Levels

[0052]

[0053] Based on the above experimental procedure, rice seedlings were induced using different concentrations of 6BA solution (5 mg / L, 10 mg / L, 20 mg / L). Two days after external spraying induction, seedlings were inoculated with rice blast fungus. Resistance was assessed 5–7 days after inoculation. Results are as follows: Figure 1 As shown, there was no significant difference in disease susceptibility between the control group rice seedlings and rice seedlings induced by a 5 mg / L 6BA solution. The lesions were larger than 3 mm in diameter, with dark brown edges, and tended to expand into patches, indicating high susceptibility (level 5). Rice leaves induced by a 10 mg / L 6BA solution showed a small number of lesions with diameters of 0.5-1 mm, indicating moderate resistance (level 2). Rice lesions induced by a 20 mg / L 6BA solution were spindle-shaped and merged into patches, indicating susceptibility (level 4). These results indicate that a 10 mg / L 6BA solution can induce enhanced resistance in rice.

[0054] Example 2

[0055] This example, based on Example 1, further explores the disease resistance mechanism of 6BA in rice blast resistance, specifically including the following steps:

[0056] (1) Determination of antioxidant enzyme activity.

[0057] Forty-eight hours after inoculation, three rice seedlings each were cut from the 10 mg / L 6BA-treated seedlings and the seedlings inoculated only, and labeled. They were then refrigerated at -80°C. The refrigerated rice seedling samples were removed and placed in a ceramic mortar (pre-cooled with liquid nitrogen). Liquid nitrogen was quickly added, and the samples were ground into powder. The powdered samples were then transferred to centrifuge tubes, labeled, and quickly placed in liquid nitrogen for refrigeration. Each centrifuge tube was labeled, and the activities of each antioxidant enzyme were measured according to the instructions of the antioxidant enzyme kit. Significance analysis was performed, and the results are as follows: Figure 2 As shown.

[0058] from Figure 2It can be seen that, compared with the control group, after induction treatment with 10 mg / L 6BA solution, the MDA content increased significantly, the activities of SOD, CAT and PAL were significantly enhanced, and the POD activity showed a slight increasing trend, but no significant change.

[0059] Antioxidant enzymes participate in the degradation of excess hydrogen peroxide and other reactive oxygen species in plant cells, reducing damage to plants. The biochemical activity of this series of enzymes is positively correlated with rice resistance to rice blast, which proves that 6BA can induce rice to develop resistance to rice blast fungus.

[0060] (2) Determination of disease resistance-related genes.

[0061] Forty-eight hours after inoculation, three rice seedlings each were cut from the 10 mg / L 6BA-treated and the inoculated rice seedlings alone, and marked and placed in a -80°C refrigerator. The mortar and pestle were pre-cooled with liquid nitrogen, and the chilled rice seedling samples were placed in the mortar, with an appropriate amount of liquid nitrogen added and quickly ground into powder.

[0062] RNA extraction: RNA was extracted from rice leaves using the TransZol method. Rice HY samples (JC2 inoculated for 48 h) and rice HY samples (JC2 inoculated for 48 h and induced for two days with 10 mg / L 6BA) were ground in liquid nitrogen. 0.1 g of each sample was placed in a 1.5 ml centrifuge tube (EP tube). 1 mL of Trizol lysis solution was added and quickly mixed with the sample. The mixture was allowed to stand at room temperature for 5 min. The mixture was then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was transferred to a new EP tube, and 200 μL of chloroform was added. The mixture was vigorously shaken for 15 s and allowed to stand at room temperature for 3 min. The centrifuge was then set to 4 °C and centrifuged rapidly at 12000 rpm for 15 min. 400 μL of the supernatant was gently added to a new EP tube. 400 μL of C3H8O was then added and gently... Invert the EP tube and mix the sample, incubate at 25°C for 10 min; centrifuge at 12000 rpm at 4°C for 10 min and discard the supernatant; add 1 mL of 75% ethanol to the precipitate and gently shake to rinse the RNA precipitate; centrifuge at 12000 rpm at 4°C for 1 min and discard the supernatant, then centrifuge again for a short time, then aspirate the remaining liquid and air dry for 5 min; add 40 μL of DEPC water (inactivated) to dissolve the precipitate, and place on ice after dissolution.

[0063] Fluorescence quantitative PCR: The extracted RNA was reverse transcribed to generate cDNA. Using a TaKaRa kit, 2 μL of cDNA was added to a 10 μL system (final primer concentration was 0.3 mmol / L, 5 μL of SYBR Green (2×)). The fluorescence signal was detected in real time in a Bio-rad Fluorescent PCR device according to the instruction manual. All primers in this system were annealed at 60 °C for 1 min for a total of 40 cycles.

[0064] This study analyzed seven disease resistance-related genes: OsPR1a, OsPR5, OsPAL, OsWRKY28, OsWRKY45, OsWRKY62, and OsWRKY76. Rice actin was used as an internal reference gene. Cellular copy number of the cDNA template was corrected, and the relative expression levels of the genes were calculated using a 22T / T ratio. -△△Ct Methods. The primer sequences involved in this example are shown in Table 4.

[0065] Table 4 Primers for Real-Time Quantitative PCR

[0066]

[0067]

[0068] The results are as follows Figure 3 As shown: Compared with the control group, spraying with 6BA two days before inoculation with rice blast fungus and taking samples 48 hours after inoculation showed a significant increase in the expression levels of disease resistance-related genes OsPR5, OsPAL, OsWRKY45, and OsWRKY76. The expression levels of OsPR1a and OsWRKY28 were not significantly different from the control, while the expression level of OsWRKY62 did not change significantly.

[0069] In summary, by externally spraying 6BA onto the leaves of rice seedlings to induce resistance, followed by inoculation with rice blast pathogens, with no 6BA induction serving as a control, rice seedlings treated with appropriate concentrations of 6BA showed fewer lesions and increased resistance to rice blast. The results exploring the mechanism of induced resistance indicated that 6BA treatment increased the activities of antioxidant enzymes SOD, CAT, and PAL in rice seedlings to varying degrees, increased MDA content, and significantly increased the expression levels of disease resistance-related genes OsPR5, OsPAL, OsWRKY45, and OsWRKY76. This demonstrates that under appropriate concentrations of 6BA, rice resistance to rice blast pathogens is enhanced, meaning that 6BA can induce resistance.

[0070] The above description is a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

The application of 1.6BA in enhancing plant resistance to rice blast is characterized by, The preparation containing 6BA was sprayed onto the surface of the plant; the concentration of 6BA in the preparation was 10 mg / L; the plant was a rice seedling.

2. The application according to claim 1, characterized in that, The formulation enhances plant resistance to rice blast through at least the following pathways: a) Induces upregulation of disease resistance-related gene expression; b) Increase the content and / or activity of antioxidant enzymes.

3. The application according to claim 2, characterized in that, The disease resistance-related genes include OsPR5 , OsPAL , OsWRKY45 , OsWRKY76 .

4. The application according to claim 2, characterized in that, The antioxidant enzymes include SOD, CAT, and PAL.

5. A method for preventing and controlling rice blast, characterized in that, Rice was induced to enhance its resistance to rice blast fungus by using a formulation containing 6BA; in the formulation, the concentration of 6BA was 10 mg / L.

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