Method for inducing rice to resist rice blast and bacterial blight by graphene

Abstract

The invention discloses a method for inducing rice to resist rice blast and bacterial blight by using graphene, and belongs to the technical field of plant disease prevention and control. A 10-50 ppm graphene solution is applied in the three-leaf stage of a rice variety Yuenongsimian. Then respectively inoculating magnaporthe oryzae (bacterial strain Guy11) and bacterial blight (bacterial strain PXO99), culturing in an environment with the temperature of 28-32 DEG C and the humidity of 95-100% after inoculation, recording disease symptoms and analyzing the disease-resistant effect. The graphene treatment obviously delays the disease time of the rice blast (postponed by 48 hours), effectively reduces the length of the scab of the rice blast and the bacterial blight (reduced by 50-80%), and successfully induces the enhancement of the resistance of the rice to the rice blast and the bacterial blight. The method is easy and convenient to operate, low in cost and remarkable in effect, meanwhile, use of chemical bactericides is avoided, and the method is environmentally friendly and suitable for disease prevention and control and disease resistance improvement in large-scale rice planting.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, in particular to research on rice disease prevention and control, and specifically to a method for inducing rice resistance to rice blast and bacterial blight by using graphene materials. Background Art

[0002] Rice is one of the important food crops for human survival.

[0003] Rice blast, commonly known as fire blast or head-knocking blast, is one of the three major diseases of rice. It can infect multiple parts of the plant, such as leaves, stems and ears, throughout the entire growth period. The manifestations of rice blast are diverse. According to the different periods and parts of the affected area, it can be divided into seedling blast, leaf blast, node blast, ear neck blast and grain blast. Among them, ear neck blast poses the most serious threat to yield. Seedling blast often occurs before the three-leaf stage and is caused by seed bacteria. The base of the diseased seedlings is gray-black, and the upper part turns brown and curls up and dies; leaf blast often occurs from tillering to jointing stage, and in severe cases can cause the field to appear "burned". Rice blast occurs to varying degrees in both northern and southern rice-growing areas. Bacterial leaf blight is also one of the main diseases of rice. Its symptoms initially appear as small yellow-green spots on the tips and edges of leaves, which then expand along the veins into pale or yellow-brown stripes, eventually causing the leaves to turn gray and die. The affected plants are prone to lodging, and the rate of unfruitfulness of rice ears also increases. Studying the disease resistance mechanism of rice to rice blast and bacterial blight, and exploring effective prevention and control strategies or breeding disease-resistant varieties have important scientific value and practical significance.

[0004] With the rapid development of nanobiotechnology, the application of nanomaterials in agricultural production has gradually attracted attention, including increasing crop yields, optimizing varieties and enhancing stress resistance. Graphene, as a carbon-based non-toxic nanomaterial, has been widely used in many fields by virtue of its unique physicochemical properties, such as high specific surface area, high carrier mobility, excellent stability, high-efficiency photothermal conversion performance and non-toxicity. However, the potential of graphene in plant disease prevention and control has not been fully developed. The inventors of the present invention have unexpectedly discovered in experiments on rice disease resistance and its induction mechanism for many years that the resistance of rice to rice blast and bacterial blight can be significantly improved by graphene treatment. Therefore, the method of graphene-induced rice resistance to rice blast and bacterial blight provided by the present invention can reveal a new application prospect of graphene in inducing plant disease resistance. Summary of the invention

[0005] The object of the present invention is to provide a method for improving the resistance of rice to rice blast and bacterial blight, which has the characteristics of simple operation, low cost and significant effect, and can enhance the disease resistance of rice in a short time, thereby improving the yield and economic benefits of rice.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] A method for improving rice resistance to rice blast and bacterial blight based on graphene treatment, comprising the following steps: at the three-leaf stage of rice, apply a graphene solution with a concentration of 10-50 ppm by spraying. After applying the graphene solution, inoculate Magnaporthe oryzae by the spray inoculation method, and inoculate Xanthomonas oryzae pv. oryzae by the leaf-clipping inoculation method. After inoculation, place the rice plants in an environment with a temperature of 28-32 °C and a humidity of 95-100%, until the disease symptoms are stable; evaluate the disease resistance effect by measuring the lesion length and calculating the disease onset time.

[0008] Further, before inoculating Magnaporthe oryzae, adjust the spore concentration to 1×10 5 CFU / mL, and before inoculating Xanthomonas oryzae pv. oryzae, adjust the OD 600 value of the bacterial suspension to 0.5.

[0009] Further, the graphene solution is prepared by diluting graphene quantum dots (GQDs) to 50 ppm.

[0010] Further, the seedling height at the three-leaf stage of the rice is about 10 cm, and the stage with vigorous plant growth is selected for graphene solution treatment.

[0011] Preferably, the rice variety is Yuenong Simiao.

[0012] Advantages of the present invention: The present invention utilizes the unique properties of graphene materials (such as enhancing cell wall defense, inducing the expression of disease-resistant genes, etc.) to provide a green and efficient rice disease resistance improvement scheme. Compared with traditional pesticides, the method adopted in the present invention is more environmentally friendly and can be widely applied to rice disease resistance improvement and agricultural production. In addition, compared with the prior art, the operation of the present invention is simple and suitable for large-scale farmland management; the dosage of graphene materials is small and the cost is low; the effect of improving rice disease resistance is significant, and it can rapidly enhance the resistance during the epidemic period of rice blast and bacterial blight; it is green and environmentally friendly, meeting the sustainable concept of modern agricultural development. Description of the Drawings

[0013] Figure 1 Shows the effect of graphene solution treatment on rice resistance to rice blast. Among them, (A) shows the disease symptoms after inoculating Magnaporthe oryzae (strain Guy11) on the rice leaves of the control group and the graphene solution treatment group; (B) shows the statistical analysis chart of the quantitative determination of the lesion length after 48 hours of infection.

[0014] Figure 2 Shows the effect of graphene solution treatment on rice resistance to bacterial blight. Among them, (A) shows the disease symptoms after inoculating Xanthomonas oryzae pv. oryzae (strain PXO99) on the rice leaves of the control group and the graphene solution treatment group; (B) shows the statistical analysis chart of the quantitative determination of the lesion length after 48 hours of infection. Detailed Implementation Modes

[0015] The following are specific embodiments of the present invention, which further illustrate the technical solutions of the present invention. However, the protection scope of the present invention is not limited to this embodiment. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0016] Example 1: Preparation and Treatment of Graphene Solution

[0017] Place 2 grams of citric acid in a Teflon-lined autoclave and heat it in an oven at 210 °C for 6 hours. At high temperature, the reactants react to form a dark brown solid mixture, commonly known as graphene quantum dots (GQDs). After the reaction is completed, take out the autoclave and let it cool naturally to room temperature. Subsequently, dissolve the obtained dark brown solid (GQDs) in deionized water to 10 mL to form a graphene solution. Then, quickly adjust the pH value of the graphene solution to 7.0 by rapidly adding an appropriate amount of NaOH solution. To remove large particle impurities, centrifuge the solution at 12,000 rpm for 10 minutes and discard the precipitate. The supernatant is further diluted with deionized water to a total volume of 200 mL. The concentration of the obtained graphene solution is 10 mg / mL, and it is stored in a 4 °C refrigerator for later use.

[0018] Before treating the graphene solution, the following routine work needs to be done:

[0019] (1) Preparation of the planting land: Through agricultural techniques such as mechanical plowing, fertilization, and irrigation, ensure that the paddy field is flat, the soil is loose and fertile, and the weeds and stubble are cleared. When applying the base fertilizer, add 50 kg of compound fertilizer per mu (the content of potassium sulfate and nitrate nitrogen is not less than 45%), and finally level the field surface.

[0020] (2) Seedling stage management: Through reasonable temperature control and water and fertilizer management during the seedling stage, ensure the healthy growth of the seedlings. When transplanting the seedlings, choose a sunny day, ensure that the seedlings are shallowly inserted and evenly distributed, and irrigate and fertilize in a timely manner after transplanting.

[0021] Treatment of rice plants at the three-leaf stage:

[0022] Dilute the graphene solution with deionized water to a concentration of 50 ppm to treat the rice plants. The spraying method can be used to treat the plants at the three-leaf stage of rice. The spraying method is to evenly spray the diluted graphene quantum dot solution with a concentration of 10 - 50 ppm on the surface of the rice leaves to ensure that the leaves of all plants can come into contact with the graphene solution. The treated rice plants can effectively resist the damage of rice blast and bacterial blight. Through scientific treatment, the rice plants can significantly delay the onset time, reduce the disease loss, and increase the yield in the subsequent disease challenges.

[0023] Example 2: Preparation and Inoculation of Rice against Bacterial Blight and Rice Blast

[0024] (1) Preparation and Inoculation of Magnaporthe oryzae (Strain Guy11)

[0025] First, inoculate Magnaporthe oryzae (strain Guy11) onto a medium containing 20 g / L potato dextrose agar (PDA) and culture it in an incubator at a constant temperature of 28 °C for 7 days. After the colonies mature, rinse and filter with sterile water to collect the fungal spores. Then, use a microscopic counter to adjust the spore concentration to 1×10 5 spores / mL. Using the spray inoculation method, evenly spray the bacterial solution on the surface of the rice leaves to ensure that the leaves of each rice plant can fully contact the pathogen. After inoculation, place the rice plants in a constant temperature environment of 28 - 32 °C with a humidity controlled at 95 - 100% to ensure suitable growth and infection conditions for the pathogen.

[0026] (2) Preparation and Inoculation of Xanthomonas oryzae pv. oryzae (Strain PXO99)

[0027] Xanthomonas oryzae pv. oryzae (strain PXO99) is first inoculated onto a medium containing 10 g / L nutrient agar (NA) and cultured in an incubator at a constant temperature of 28 °C for 48 hours. After the colonies form, collect the bacteria with sterile water. Use a spectrophotometer (OD 600 value) to measure the bacterial concentration and adjust the OD 600 value to 0.5 for standby.

[0028] In the experiment, Xanthomonas oryzae pv. oryzae (strain PXO99) usually adopts the leaf - clipping inoculation method. Use scissors dipped in the bacterial solution to cut the tips of the rice leaves to ensure that the pathogen invades the rice plant tissues through the cut wounds. This inoculation method can effectively simulate the infection of the pathogen under natural conditions and enhance the reliability of the experimental results. After inoculation, the rice plants need to be placed in an environment with a temperature of 28 - 32 °C and a humidity of 95 - 100% to promote the infection and spread of the pathogen. The above - mentioned pathogen preparation and inoculation methods can ensure the accuracy and reliability of the experiment.

[0029] Example 3: Detection of Disease Resistance Effect

[0030] (1) Detection of Rice Blast Resistance Effect: After inoculating Magnaporthe oryzae, continuously observe the disease occurrence of the rice. After the disease symptoms are stable, record the disease occurrence and disease severity of each group of plants. The experimental results show that ( Figure 1) Treatment with graphene solution significantly enhanced the resistance of rice to rice blast. In the rice blast resistance experiment, the appearance time of rice blast symptoms in the rice plants treated with graphene solution was delayed by 48 hours. Compared with the control group, an obvious disease delay effect was shown. At the same time, the lesion length in the graphene solution treatment group was significantly reduced, indicating an inhibitory effect on the expansion of rice blast. After the rice treated with graphene solution was inoculated with Magnaporthe oryzae, the lesion length was reduced by about 50%-80% compared with the untreated rice, showing strong disease resistance.

[0031] (2) Detection of the disease resistance effect against bacterial blight: In the bacterial blight resistance experiment, after the rice plants treated with graphene solution were infected with Xanthomonas oryzae pv. oryzae ( Figure 2 ), the lesion length was significantly shorter than that of the control group, indicating that graphene solution could effectively slow down the expansion of the lesion. In addition, the treatment with graphene solution significantly delayed the disease progression of bacterial blight, and the lesion length of the treatment group was significantly shortened, showing a concentration-dependent relationship. This result indicates that the treatment with graphene solution can effectively improve the resistance of rice to bacterial blight.

[0032] The above experimental results show that the treatment with graphene solution can not only delay the disease progression of the above-mentioned diseases, but also effectively reduce the severity of the diseases, providing a technically promising means for improving the disease resistance of rice.

[0033] As mentioned above, it is only the optimal implementation mode in the present invention, but the protection scope of the present invention is not limited thereto. Any transformation or replacement that can be understood and conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for inducing rice resistance to rice blast and bacterial blight using graphene, characterized in that: The following steps are included: When rice was in the three-leaf stage, a graphene solution with a concentration of 10-50ppm was applied by spraying. After the graphene solution was applied, rice blast was inoculated by spray inoculation, and white leaf blight was inoculated by leaf cutting inoculation. After inoculation, the rice plants were placed in an environment with a temperature of 28-32℃ and a humidity of 95-100% until the disease symptoms stabilized; the disease resistance effect was evaluated by recording the length of the lesions and the time of onset.

2. The method of inducing rice resistance to rice blast and bacterial blight using graphene according to claim 1, characterized in that: Before inoculation of rice blast fungus, the spore concentration was adjusted to 1×10 5 / mL, the bacterial suspension OD was adjusted before inoculation of the bacterial blight pathogen 600 value to 0.

5.

3. The method for inducing rice resistance to rice blast and bacterial blight using graphene according to claim 1, characterized in that: The graphene solution is prepared by diluting graphene quantum dots (GQDs) to 50 ppm.

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