Application of linalool and method for preventing and treating field rice leaf streak

Abstract

The invention belongs to the technical field of plant protection and biological prevention and treatment, and particularly relates to application of linalool and a method for preventing and treating rice bacterial streak in a field. The invention discloses a method for preventing and treating rice bacterial streak in a field. The method comprises the following steps: mixing linalool, tween-80 and water to prepare a solution; the rice is treated regularly. In the solution, the final concentration of the Tween-80 is 0.1 mg / L, and the concentration of the linalool is 0.675 mg / L-0. 900 mg / L. The rice is treated from the tillering stage to the heading stage of rice growth. According to the application disclosed by the invention, a bacteriostatic test proves that the linalool has a relatively strong inhibiting effect on the rice bacterial streak and can be used for preventing and treating the rice bacterial streak.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant protection and biological control, and particularly relates to application of linalool and a method for preventing and treating rice bacterial leaf streak. BACKGROUND

[0002] Rice bacterial leaf streak (hereinafter referred to as "leaf streak") is a plant disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Xanthomonas oryzae pv. oryzicola It is an important quarantine disease of rice in China, and has the characteristics of outbreak, prevalence and destructiveness, which seriously threatens the yield and quality of rice.

[0003] High temperature and high humidity are the main environmental conditions for the occurrence and prevalence of rice bacterial leaf streak. Generally, the leaf streak disease is most serious during the tillering and heading stages of rice. The disease mainly occurs in tropical and subtropical rice-growing areas. With the global warming, the planting of hybrid rice and the popularization of southward transplanted rice, the disease area is gradually expanding. In China, the disease mainly occurs in the Yangtze River Basin, South China and Central South China. In recent years, the disease has shown a trend of aggravation. Generally, the disease can reduce the yield of rice by 10% to 25%, and in severe cases, the yield can be reduced by 40% to 60%, and even the yield can be reduced by 40% to 60% in severe cases. In the current production, chemical fungicides are still the main measure for preventing and treating rice bacterial leaf streak. However, long-term use of chemical fungicides will pollute the environment. Some fungicides may have a long residual period, pollute the soil and water, or have a negative impact on non-target organisms, disrupt the ecological balance, and even enter the human body through the food chain, posing a threat to human health. At the same time, long-term use of the same fungicide can lead to drug resistance of the pathogenic microorganism, thereby reducing the control effect of the fungicide on the pathogenic microorganism.

[0004] Therefore, it is urgent to develop a new environment-friendly biological fungicide for rice bacterial leaf streak. SUMMARY

[0005] In order to solve the problems in the prior art, the application provides a method for preventing and treating rice bacterial leaf streak in the field and application of linalool. The bacteriostatic test proves that linalool has a strong inhibitory effect on rice bacterial leaf streak bacteria, and can prevent and treat rice bacterial leaf streak.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions: One of the purposes of the application is to protect the application of linalool in preventing and treating rice bacterial leaf streak.

[0007] In the application, the linalool used can be directly purchased from ordinary market products or obtained by using conventional extraction methods in the art.

[0008] Linalool is a natural substance with a mixed woody and floral aroma, primarily found in plants such as bergamot, rosewood, sandalwood, jasmine, and Dendrobium officinale. Studies have reported that linalool possesses sedative, hypnotic, analgesic, and anti-inflammatory effects. This study found that linalool exhibits strong inhibitory activity against rice bacterial leaf streak causal agent.

[0009] Furthermore, in this application, the concentration of linalool is 0.375 mg / L to 0.675 mg / L; more preferably 0.675 mg / L.

[0010] The second objective of this invention is to protect a method for controlling rice leaf streak in the field, which uses linalool or uses linalool as the sole active ingredient for the control of bacterial leaf streak in rice in the field.

[0011] Furthermore, the method for controlling rice leaf streak in the field includes the following steps: mixing linalool, Tween-80 and water to prepare a solution; and treating the rice regularly.

[0012] Furthermore, in the method for controlling rice leaf streak in the field, the final concentration of Tween-80 in the solution is 0.1 mg / L.

[0013] Furthermore, in the method for controlling rice leaf streak in the field, the concentration of linalool in the solution is 0.675 mg / L - 0.900 mg / L; more preferably 0.900 mg / L.

[0014] Furthermore, in the aforementioned method for controlling rice leaf streak in the field, the rice is treated during the period from the peak tillering stage to the heading stage; more preferably, during the peak tillering stage.

[0015] Furthermore, in the aforementioned method for controlling rice leaf streak in the field, when controlling bacterial leaf streak in rice, a drone is used to spray the pesticide solution, with 3L~7L of solution sprayed per acre (specifically, 3L, 4L, 5L, 6L, 7L, etc.).

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (i) This invention provides the use of linalool in the prevention and control of plant diseases. Linalool is a natural substance that is widely found in the plant kingdom. The raw materials are widely available and environmentally friendly.

[0017] (II) Through the study of the inhibitory effect of linalool on the bacterial leaf streak of rice and its field control effect, it was found that at the treatment concentrations of 0.375 mg / L and 0.675 mg / L, the inhibition rate of linalool on the bacterial leaf streak of rice was 90.10% and 100%, respectively, which were significantly better than the control agent kasugamycin (28.32% and 78.81%).

[0018] (III) Inhibitory effect of linalool on rice bacterial leaf streak fungus: The inhibitory activity of linalool increases with increasing concentration. The EC50 of linalool on the fungus... 50 The values ​​were 0.1744 mg / L, and the inhibitory activity was significantly better than that of the control drug, kasugamycin (EC). 50 The value was 0.5887 mg / L. Linalool showed good control efficacy against bacterial leaf streak of rice. The control efficacy of 0.900 mg / L linalool against bacterial leaf streak of rice was 89.93%, which was significantly higher than that of the control agent 0.900 mg / L kasugamycin (68.22%).

[0019] (iv) Linalool has an inhibitory effect on alkaline phosphatase in rice bacterial leaf streak cells, and can also act on the cell wall to increase cell wall permeability. Attached Figure Description

[0020] Figure 1 The graph shows the inhibitory effect of linalool on bacterial streak bacteria at different concentrations. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, 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.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] In this invention, some conventional operating equipment, devices and components have been omitted or only briefly described.

[0025] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0026] In this application, unless otherwise specified, % refers to its mass percentage content.

[0027] Example 1: 1. Test materials 1.1 Test Reagents Linalool was purchased from Jiangxi Xinsen Natural Plant Oil Co., Ltd.

[0028] 1.2 Culture medium NA solid medium (NA), NB liquid medium (NB), and Tween-80 were all domestically produced analytical grade.

[0029] 1.3 Test pathogens (1) Isolation of pathogens In 2023, rice leaf samples with typical symptoms of bacterial leaf streak and bacterial ooze were collected from naturally occurring fields. Then, under aseptic conditions, the bacterial ooze from the samples was streaked and purified on NA medium. The single colony morphology of the isolated bacterial strain on NA medium was consistent with that of the bacterial leaf streak pathogen reported in the standard (GB / T28099-2011). (2) Pathogen identification The experiment further extracted genomic DNA from the pathogen, performed PCR amplification and sequencing using specific primers for *Bacillus streakus*, the causal agent of rice bacterial leaf streak. The amplified sequence was compared with the sequence of *Bacillus streakus* in the NCBI GenBank database. The sequence of the isolated strain showed 100% similarity to the sequence of the *Bacillus streakus* strain in the GenBank database (accession number: AY395713.1). Therefore, the bacterial strain isolated in this experiment is *Bacillus streakus*. Xanthomonas oryzae pv. oryzicola ).

[0030] The isolated bacterial leaf streak pathogen was stored at -20℃ for later use.

[0031] The isolation and identification of bacterial leaf streak in rice are both existing technologies.

[0032] 1.4 Rice varieties tested The Wushan Simiao rice variety, susceptible to bacterial leaf streak, was selected.

[0033] 2. Experimental Methods and Results 2.1 Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of linalool against bacterial streak causal agent The MIC was determined using the agar dilution method. Under aseptic conditions, linalool and Tween-80 were filtered through a bacterial filter to obtain sterile linalool and Tween-80, respectively. Mix 3 g of sterile linalool with 1 mL of Tween-80 to prepare a 750 mg / L mixed solution. Add 50 μL, 70 μL, 90 μL, 100 μL, 120 μL, 150 μL, and 200 μL of this solution to 100 mL of NA medium to prepare seven drug-containing media with final concentrations of 0.375 mg / L, 0.525 mg / L, 0.675 mg / L, 0.750 mg / L, 0.900 mg / L, 1.125 mg / L, and 1.500 mg / L, respectively. Pour the NA medium into petri dishes. The final concentrations of the control drug, kasugamycin, were also 0.375 mg / L, 0.525 mg / L, 0.675 mg / L, 0.750 mg / L, 0.900 mg / L, 1.125 mg / L, and 1.500 mg / L. mg / L, with drug-free culture medium containing 0.25% Tween-80 as a control, OD was obtained by pipetting. 600 80 μL of *Pseudomonas streakae* with a concentration of 1 was evenly spread onto a medium containing NA (nitric acid), with each concentration replicated five times. The medium was incubated in the dark at 28°C for 6 days, after which colony growth was observed. Results are shown below. Figure 1 See Table 1.

[0034] Table 1. Growth of *Lysimachia streak* under different concentrations of linalool treatment.

[0035] Note: —. No colonies; +. Less than 20 single colonies; ++. 20~50 colonies; +++, 100~200 single colonies; ++++. A large number of colonies; +++++. A large number of colonies.

[0036] Table 1 shows that linalool has a good inhibitory effect on *Pseudomonas streakus*. At a concentration of 0.375 mg / L, the inhibitory effect of linalool on *Pseudomonas streakus* was visible to the naked eye compared with the control group. Therefore, 0.375 mg / L was determined as the MIC value of linalool against *Pseudomonas streakus* (significantly lower than the MIC value of the control agent kasugamycin, 0.675 mg / L). When the concentration of linalool reached 0.900 mg / L, no visible colonies grew on the plate. Therefore, 0.900 mg / L was determined as the MBC value of linalool against *Pseudomonas streakus* (significantly lower than the MBC value of the control agent kasugamycin, 1.500 mg / L). 2.2 Determination of antibacterial effect The inhibitory effect of linalool on rice leaf streak pathogen was determined by turbidimetric assay. The OD value was used as the metric.600 A concentration of 1 was used as the inoculum for *Strombus streakus*. NB medium was used to prepare bacterial suspensions with concentration gradients of 0 mg / L, 0.1875 mg / L, 0.375 mg / L, 0.525 mg / L, 0.675 mg / L, 0.750 mg / L, 0.900 mg / L, 1.125 mg / L, and 1.500 mg / L. Each treatment was incubated on a shaker at 28 °C and 180 rpm for 48 h. The OD600 value of the bacterial suspensions was measured using a spectrophotometer, and the average value was calculated. Each concentration treatment was repeated three times.

[0037] Antibacterial rate (%) = (Control OD value - Treatment OD value) / Control OD value × 100. The results are shown in Table 2.

[0038] Table 2. Inhibition rate of rice leaf streak pathogen under different concentrations of linalool treatment (%)

[0039] As shown in Table 2, at concentrations of 0.375 mg / L and 0.675 mg / L, linalool inhibited rice leaf streak pathogens at rates of 90.10% and 100%, respectively, both significantly higher than the inhibition rates of the control agent kasugamycin (28.32% and 78.81%).

[0040] Table 3. Virulence of linalool against rice leaf streak pathogen

[0041] As shown in Table 3, linalool has strong inhibitory activity against rice leaf streak pathogen. The inhibitory activity increases with the increase of linalool concentration, with an EC50 value of 0.1744 mg / L. The inhibitory activity is significantly higher than that of the control agent kasugamycin (EC50 value 0.5887 mg / L).

[0042] 2.3 Effects of linalool on intracellular and extracellular alkaline phosphatase (AKP) activity in rice streak pathogen cells / (King's units / g) Linalool solution was added to the suspension of *Strombus streakus* cultured to the logarithmic growth phase to prepare bacterial solutions containing concentration gradients of 0, 0.1875, 0.375, 0.675, and 1.125 mg / L. Each treatment was placed on a shaker and cultured at 28 ℃ and 180 rpm for 0 h, 24 h, and 48 h, respectively. (1) Centrifuge and collect the bacterial cells, add the reagent extracted from the AKP kit, homogenize on ice, centrifuge at 4 ℃, 8000 rpm / min for 10 min, collect the supernatant, place the supernatant at -20 ℃ for testing, and then measure the intracellular AKP activity value according to the steps of the AKP assay kit. The measurement results are shown in Table 4. (2) Centrifuge and collect the supernatant; then measure the extracellular AKP activity value according to the steps of the AKP assay kit. The measurement results are shown in Table 5.

[0043] Table 4. Effects of linalool on intracellular AKP activity in *Pseudomonas streakae*.

[0044] Table 4 shows that linalool inhibited the intracellular AKP activity of *Strombus streakus*, and the inhibitory effect increased with increasing linalool concentration and treatment time. At a linalool concentration of 0.900 mg / L for 48 h, the intracellular AKP activity of *Strombus streakus* was as low as 0.50, significantly lower than the control agent kasugamycin (1.51). Since the main function of AKP is to provide energy metabolism and substance transport support for bacteria, the results of this experiment indicate that linalool can inhibit the transport of bacterial energy metabolism substances by inhibiting the intracellular AKP activity of *Strombus streakus*, thereby achieving a bacteriostatic effect.

[0045] Table 5. Effects of linalool on extracellular AKP activity of *Strombus streakus*

[0046] Table 5 shows that the extracellular AKP activity of *Strombus stingans* increased with increasing linalool concentration and treatment time. At a linalool concentration of 1.125 mg / L for 48 h, the extracellular AKP activity reached as high as 2.93, significantly higher than the control agent kasugamycin (1.56). Under normal cell conditions, AKP activity is undetectable outside the cell. When the cell wall is damaged, its permeability increases, allowing AKP between the cell wall and cell membrane to easily leak out. Therefore, the results of this experiment indicate that linalool can induce cell wall damage in *Strombus stingans*, increasing cell wall permeability and leading to AKP leakage into the extracellular space, thereby achieving an antibacterial effect.

[0047] 2.4 Field control effect of linalool on rice leaf streak The field control efficacy of linalool against rice leaf streak was tested using a spray method. The susceptible variety, Jingang 30, was selected for the experiment. Rice was planted in a rice experimental field infected with bacterial leaf streak to ensure the disease fully developed.

[0048] The experiment consisted of 7 treatments, as detailed below: The pesticides and dosages used in this experiment were: 0.675 mg / L, 0.750 mg / L, and 0.900 mg / L linalool solution (containing 0.1 mg / L Tween-80), and 0.675 mg / L, 0.750 mg / L, and 0.900 mg / L kasugamycin. Each treatment was sprayed with water as a control. The pesticide solutions were applied using an agricultural drone. The experiment consisted of 10 different treatments, as detailed below: Treatment 1: Spray 0.675 mg / L, 0.750 mg / L, and 0.900 mg / L linalool solution during the peak tillering and booting stages of rice; Treatment 2: Spray 0.675 mg / L, 0.750 mg / L, and 0.900 mg / L linalool solution at the end of the tillering stage and the heading stage of rice; Treatment 3: Spray 0.675 mg / L, 0.750 mg / L, and 0.900 mg / L kasugamycin at the peak tillering and booting stages of rice; Treatment 4: Spray 0.675 mg / L, 0.750 mg / L, and 0.900 mg / L kasugamycin at the end of the tillering stage and the heading stage of rice. Treatment 5: Water was used as a control. Each treatment was replicated in triplicate. Twenty-one days after application, leaf disease incidence was investigated, disease index was calculated, and control efficacy was evaluated.

[0049] Rice leaf streak disease severity index grading standards and calculation methods: Grade 0: No disease; Grade 1: Lesions less than 0.5 cm in size; Grade 3: Lesions between 0.5 and 3 cm in size; Grade 5: Lesions between 3 and 6 cm in size; Grade 7: Lesions between 6 and 10 cm in size; Grade 9: Lesions greater than 10 cm in size.

[0050] Disease index = [∑(number of diseased leaves at each level × representative value at each level) ( / total number of leaves surveyed × highest representative value)] × 100 Relative prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100 The results of field efficacy trials are shown in Table 6: Table 6. Field efficacy test results of linalool against bacterial leaf streak in rice.

[0051] Table 5 shows that linalool has a good control effect on bacterial leaf streak of rice in the field. During the tillering and booting stages, spraying with 0.900 mg / L linalool solution has the best control effect on leaf streak, with a control efficacy of 89.93%, which is significantly better than the control agent kasugamycin (68.22%).

[0052] In summary, the linalool provided by this invention has a significant inhibitory effect on the growth of bacterial leaf streak in rice; it inhibits the activity of alkaline phosphatase within the cells of the leaf streak bacterium, and simultaneously increases cell wall permeability; it can effectively control bacterial leaf streak in rice, with a field control efficacy of up to 89.93%. Linalool is a natural plant product, readily available, environmentally friendly, and highly safe for humans and livestock.

[0053] The above embodiments are merely preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any modifications, refinements, or combinations made under the main design and principles of the present invention, which solve technical problems consistent with the present invention, are considered to be within the protection scope of the present invention.

Claims

1. Application of linalool in the control of bacterial leaf streak in rice.

2. The application according to claim 1, characterized in that: The concentration of linalool is 0.375 mg / L to 0.675 mg / L.

3. The application according to claim 1, characterized in that: The concentration of linalool was 0.675 mg / L.

4. A method for controlling bacterial leaf streak disease of rice in the field, characterized in that: This method uses linalool or linalool as the sole active ingredient for the control of bacterial leaf streak in rice in the field.

5. A method for controlling bacterial leaf streak of rice in the field according to claim 4, characterized in that... Includes the following steps: Prepare a solution by mixing linalool, Tween-80, and water; treat the rice regularly.

6. A method for controlling bacterial leaf streak of rice in the field according to claim 5, characterized in that: The final concentration of Tween-80 in the solution is 0.1 mg / L.

7. A method for controlling bacterial leaf streak of rice in the field according to claim 7, characterized in that: The concentration of linalool in the solution is 0.675 mg / L - 0.900 mg / L.

8. A method for controlling bacterial leaf streak of rice in the field according to claim 5, characterized in that: The concentration of linalool in the solution is 0.900 mg / L.

9. A method for controlling bacterial leaf streak of rice in the field according to claim 5, characterized in that: The rice was treated during the period from the peak tillering stage to the heading stage.

10. A method for controlling bacterial leaf streak of rice in the field according to claim 5, characterized in that: The rice was treated during the peak tillering stage of its growth.

Images