Application of methyl o-methylamino benzoate in prevention and control of bacterial leaf streak of rice

Abstract

The application discloses application of methyl o-methylamino benzoate in prevention and control of bacterial leaf streak of rice. The application finds that methyl o-methylamino benzoate can inhibit growth and biofilm formation of bacterial leaf streak of rice, reduce pathogenicity of the pathogen, and inhibit infection of the pathogen to rice, and can be used for prevention and control of the bacterial leaf streak of rice and diseases caused by the pathogen. Methyl o-methylamino benzoate naturally exists in citrus leaf oil and peel oil, and is widely used for preparation of orange oil, peach, grape, grapefruit and other essences, and has no toxic and harmful effects on the environment, human bodies and plants. In addition, the methyl o-methylamino benzoate has a very low use amount in prevention and control of the bacterial leaf streak of rice, can play an obvious bacteriostatic effect at a concentration of 3 mg / mL, and can completely achieve prevention and control of the bacterial leaf streak of rice at a concentration of 5 mg / mL, has a low risk of drug resistance, can be used for green prevention and control of the bacterial leaf streak of rice, and has a high application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology and pesticides. More particularly, it relates to the application of methyl o-tolylcarbamate in preventing and controlling bacterial leaf streak of rice. BACKGROUND

[0002] Rice is the most important food crop in the world, and more than half of the world's population relies on rice as their main food. Bacterial leaf streak (BLS, simply referred to as streak disease) of rice is the second largest bacterial disease of rice in China and even in the world, caused by Xanthomonas oryzae pv. oryzicola (Xoc), and is also one of the important quarantine diseases in China. The disease is transmitted by seed-borne, has the characteristics of suddenness, outbreak, and difficulty in eradication, and causes more than 40% yield reduction in severe cases. In recent years, due to the influence of comprehensive factors such as climate change, frequent typhoons and heavy rains, off-site seed multiplication, intensive seed exchange, and more susceptible varieties, the damage of bacterial leaf streak of rice is becoming more and more serious in China and even in the world, and has become an important bacterial disease threatening the safety of rice production in the rice-growing areas of South China and the Yangtze River Basin.

[0003] The currently registered control agents for bacterial leaf streak are very limited and generally have low control effect, and excessive use of pesticides in control practice has a greater impact on food production safety. Therefore, it is of great value and significance to develop green pesticides for controlling bacterial leaf streak of rice, which are harmless to human health and safety, friendly to the environment, and have low risk of drug resistance. SUMMARY

[0004] The present application aims to overcome the shortcomings of the current bacterial leaf streak control agents, which generally have low control effect, require large amounts of use, and have high risk of drug resistance, and to provide a green pesticide for controlling bacterial leaf streak of rice, which is harmless to human health and safety, friendly to the environment, and has low risk of drug resistance.

[0005] The first object of the present application is to provide the application of methyl o-tolylcarbamate in preventing and controlling bacterial leaf streak of rice.

[0006] The second object of the present application is to provide a method for preventing and controlling bacterial leaf streak of rice.

[0007] The above objects of the present application are achieved by the following technical solutions:

[0008] The present application found that methyl o-tolylcarbamate can inhibit the growth of Xoc and inhibit the formation of biofilm, thereby reducing the pathogenicity of Xoc. Leaf experiments and pot experiments have proved that methyl o-tolylcarbamate has an effect on preventing and controlling bacterial leaf streak of rice.

[0009] Therefore, the present application claims the following solutions:

[0010] Application of methyl o-methylamino benzoate in preventing and controlling bacterial leaf streak of rice.

[0011] Application of methyl o-methylamino benzoate in preparing products for preventing and controlling bacterial leaf streak of rice.

[0012] Application of methyl o-methylamino benzoate in inhibiting growth of bacterial leaf streak pathogen of rice.

[0013] Application of methyl o-methylamino benzoate in preparing products for inhibiting growth of bacterial leaf streak pathogen of rice.

[0014] Application of methyl o-methylamino benzoate in inhibiting biofilm formation of bacterial leaf streak pathogen of rice.

[0015] Application of methyl o-methylamino benzoate in preparing products for inhibiting biofilm formation of bacterial leaf streak pathogen of rice.

[0016] Application of methyl o-methylamino benzoate in inhibiting pathogenicity of bacterial leaf streak pathogen of rice.

[0017] Application of methyl o-methylamino benzoate in preparing products for inhibiting pathogenicity of bacterial leaf streak pathogen of rice.

[0018] Application of methyl o-methylamino benzoate in inhibiting infection of bacterial leaf streak pathogen of rice.

[0019] Application of methyl o-methylamino benzoate in preparing products for inhibiting infection of bacterial leaf streak pathogen of rice.

[0020] Application of methyl o-methylamino benzoate in preventing and controlling diseases caused by bacterial leaf streak pathogen of rice.

[0021] Application of methyl o-methylamino benzoate in preparing products for preventing and controlling diseases caused by bacterial leaf streak pathogen of rice.

[0022] In addition, the present application also provides a method for preventing and controlling bacterial leaf streak of rice, wherein a methyl o-methylamino benzoate solution is used to treat rice plants.

[0023] To ensure the effect, preferably, the concentration of the methyl o-methylamino benzoate solution is not less than 3 mg / mL.

[0024] More preferably, to ensure the effect and stability, the concentration of the methyl o-methylamino benzoate solution is not less than 5 mg / mL.

[0025] Specifically preferably, the concentration of the methyl o-methylamino benzoate solution can be selected as 3-10 mg / mL.

[0026] More preferably, the concentration of the methyl o-methylamino benzoate solution can be selected as 5-10 mg / mL.

[0027] As an alternative embodiment, the method of treatment is spraying the rice plants with a methyl o-methylamino benzoate solution.

[0028] The present application has the following beneficial effects:

[0029] The present application has the following beneficial effects:

[0030] Methyl o-methylamino benzoate naturally exists in citrus leaf oil and peel oil, and is widely used in the preparation of orange oil, orange flower, peach, grape, grapefruit, etc. The type of essence has no obvious toxic effect on the environment, human body and plants. In addition, the use amount of methyl o-methylamino benzoate in the prevention and control of rice bacterial leaf streak is very low, and obvious bacteriostatic effect is exerted at a concentration of 3 mg / mL, and the prevention and control of rice bacterial leaf streak can be realized at a concentration of 5 mg / mL. It is speculated that the prevention and control effect is achieved by inhibiting the formation of pathogenic bacterial biofilm to inhibit the pathogenic bacteria, which is more conducive to the construction of microecological balance, has low drug resistance risk, can be used in the green prevention and control of rice bacterial leaf streak, and has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Methyl o-methylamino benzoate has bacteriostatic effect on rice bacterial leaf streak (A is the bacterial cell density of rice bacterial leaf streak bacterial suspension after 24h culture of different treatments; B is the growth of rice bacterial leaf streak in test tube; C is the growth result of rice bacterial leaf streak bacterial suspension on the plate).

[0032] Figure 2 Methyl o-methylamino benzoate has bacteriostatic effect on rice bacterial leaf streak (A is the bacterial cell density of rice bacterial leaf streak bacterial suspension after 24h culture of different treatments; B is the growth of rice bacterial leaf streak in test tube; C is the growth result of rice bacterial leaf streak bacterial suspension on the plate).

[0033] Figure 3 Methyl o-methylamino benzoate has bacteriostatic effect on rice bacterial leaf streak (A is the bacterial cell density of rice bacterial leaf streak bacterial suspension after 24h culture of different treatments; B is the growth of rice bacterial leaf streak in test tube; C is the growth result of rice bacterial leaf streak bacterial suspension on the plate).

[0034] Figure 4The results of the influence of methyl o-methylamino benzoate on the biofilm formation of X. oryzae (A: biofilm staining images of different treatment groups; B: quantitative detection results of biofilm production of different treatment groups). DETAILED DESCRIPTION

[0035] The present application is further illustrated by the following description in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] The strain of X. oryzae used in the following examples is GDXc267, which is a strain C18 (SSSSRSS) from Guangdong Maoming, a strong dominant pathogenic type in South China.

[0038] The rice variety is Wushan Siliao (WSSM).

[0039] The strain GDXc267 of X. oryzae and the rice variety Wushan Siliao (WSSM) are disclosed in the article by the inventors: Feng Aiqing, Wang Congying, Su Jing, Feng Jinqi, Chen Kailing, Lin Xiaopeng, Chen Bing, Liang Meiling, Yang Jianyuan, Zhu Xiaoyuan, Chen Shen. Development of a new rice bacterial leaf streak resistant line and analysis of its agronomic traits. Chinese Journal of Rice Science, 2023, 37(6): 587-596.

[0040] Methyl o-methylamino benzoate (MMA), CAS No.: 85-91-6, purchased from Shanghai McLean Biochemical Technology Co., Ltd., with product number: D806419-25g; the structural formula is:

[0041]

[0042] The formula of NA (Nutrient Agar) medium: peptone 6g, beef extract 3g, sucrose 12g, agar 18g, water to 1L, pH=7.0.

[0043] The formula of NB (Nutrient Broth) liquid medium: peptone 6g, beef extract 3g, sucrose 12g, water to 1L, pH=7.0.

[0044] Example 1 Methyl o-methylamino benzoate has antibacterial effect on X. oryzae

[0045] (I) Experimental method

[0046] Methyl-m-aminobenzoate (MMA) was prepared into a stock solution with a concentration of 800 mg / mL using dimethyl sulfoxide (DMSO).

[0047] The Xanthomonas oryzae was cultured on NA solid medium, and after 2 days of culture, the bacteria were washed with sterile water and placed in 50 mL centrifuge tubes with a quantitative concentration of OD 600 equal to 1.0, obtaining a Xanthomonas oryzae bacterial suspension.

[0048] The treatment groups with different concentrations of MMA were set up as follows:

[0049] The MMA stock solution was added to the Xanthomonas oryzae bacterial suspension to obtain a final concentration of 0, 0.5, 1, 3, 5 and 10 mg / mL of MMA in the Xanthomonas oryzae bacterial suspension, preparing Xanthomonas oryzae bacterial suspensions containing different concentrations of MMA.

[0050] The CK group and the 1% DMSO group were set up as follows:

[0051] CK group: Xanthomonas oryzae bacterial suspension;

[0052] 1% DMSO group: DMSO was added to the Xanthomonas oryzae bacterial suspension to obtain a final concentration of 1% DMSO in the Xanthomonas oryzae bacterial suspension.

[0053] The Xanthomonas oryzae bacterial suspensions of different treatments were incubated at 28°C and 200 rpm for 24 h, with 3 replicates for each treatment. The OD 600 values after 24 h were measured, the growth curves were drawn, and the growth of Xanthomonas oryzae in the test tubes under the action of MMA was observed and photographed.

[0054] Then, 2 μL of bacterial solution was taken from each Xanthomonas oryzae bacterial suspension with different concentrations of MMA after 24 h of incubation and dropped on NA solid medium plates for 2 days of culture, and photographs were taken and recorded.

[0055] (B) Experimental results

[0056] The bacterial cell density of the Xanthomonas oryzae bacterial suspension after 24 h of incubation of different treatments is shown in Figure A, and the results show that as the concentration of MMA increases, the OD 600 value of the Xanthomonas oryzae bacterial solution decreases, indicating that MMA has a significant inhibitory effect on the growth of Xanthomonas oryzae. Figure 1 The growth of Xanthomonas oryzae in the test tubes of different treatments is shown in Figure B, and the results show that as the concentration of MMA increases, the growth of Xanthomonas oryzae in the test tubes decreases, indicating that MMA has a significant inhibitory effect on the growth of Xanthomonas oryzae.

[0057] Figure 1 ​As shown in Figure B, it can be seen from the figure that the liquid culture medium gradually becomes clear as the MMA concentration increases.

[0058] The growth results of rice bacterial leaf streak fungal suspensions under different treatments on plates are as follows: Figure 1 As shown in Figure C, the results indicate that at an MMA concentration of 3 mg / mL, OD 600 The value has decreased by about 80%. At an MMA concentration of 5 mg / mL, the OD... 600 The value was close to 0, but after 2 days of plate culture, a small number of colonies grew, and 5 mg / mL was better than 3 mg / mL. However, when the MMA concentration was 10 mg / mL, the OD... 600 The value is close to 0, and after plate culture, almost no colony growth can be observed.

[0059] In summary, the results indicate that MMA has a significant inhibitory effect on the growth of bacterial leaf streak pathogens in rice.

[0060] Example 2: Methyl o-aminobenzoate inhibits the pathogenicity of rice bacterial leaf streak pathogen on rice.

[0061] (I) Experimental Methods

[0062] To determine whether MMA can inhibit the pathogenicity of rice bacterial leaf streak, MMA was applied to rice leaves, and the pathogen was inoculated into the rice leaves through a hole-punching method.

[0063] Method of inoculation by punching holes: Use a puncher to punch holes on both sides of the midrib of the rice leaf for inoculation. Gently press the hole with the puncher to inoculate the suspension of rice bacterial leaf streak (preparation method is the same as in Example 1) into the intercellular spaces of the leaf tissue.

[0064] The MMA application method is spraying: spray rice seedlings with an MMA solution of a final concentration of 5 mg / mL.

[0065] Five treatment groups were set up: Xoc group (positive control group), Xoc+DMSO group (negative control group), Water group (control group), -24h group and 0h group;

[0066] Xoc group (positive control group): Inoculated only with rice bacterial leaf streak;

[0067] Xoc+DMSO group (negative control group): Inoculated with rice bacterial leaf streak causal agent and 1% DMSO;

[0068] Water group (control group): Water was used instead of bacterial suspension for inoculation;

[0069] -24h group: MMA was applied 24 hours before inoculation with bacterial leaf streak of rice;

[0070] Group 0h: MMA was applied simultaneously with inoculation with bacterial leaf streak of rice.

[0071] Each treatment was set up with 3 replicates, with 5 seedlings planted in each replicate, and 3 leaves punched in each seedling. The length of the lesions was investigated 15 days after inoculation, and 5 lesions were randomly selected from each replicate to measure the length of the lesions.

[0072] (II) Experimental Results

[0073] The effects of MMA on the pathogenicity of rice bacterial leaf streak causal agent are as follows: Figure 2 As shown, the results indicate that applying MMA at a concentration of 5 mg / mL 24 hours in advance or simultaneously with inoculation with bacterial leaf streak of rice significantly inhibited the formation of lesions, demonstrating that MMA can inhibit the pathogenicity of bacterial leaf streak of rice and achieve the effect of preventing and controlling bacterial leaf streak disease.

[0074] Example 3: Methyl o-aminobenzoate can effectively control bacterial leaf streak in rice.

[0075] (I) Experimental Methods

[0076] To further clarify the control effect of MMA on bacterial leaf streak of rice, rice seedlings were treated with MMA and the bacterial leaf streak pathogen was inoculated onto the seedlings by spraying. The experiment was conducted to test whether MMA had a control effect on bacterial leaf streak of rice in a pot experiment.

[0077] The MMA application method is spraying: spray the rice seedlings with an MMA solution with a final concentration of 5 mg / mL.

[0078] Methods for potted plant spray inoculation and disease severity statistics: Five-week-old rice seedlings were sprayed with bacterial suspension for inoculation. The OD of the inoculated bacterial suspension was measured. 600 Value = 1.0. 20 mL of bacterial suspension was sprayed onto each pot. Five treatment groups were set up as in Example 2 (Xoc group, Xoc+DMSO group, Water group (control group), -24h group, and 0h group). After inoculation, water was sprayed regularly to maintain humidity above 90%. Disease incidence was investigated after 15 days. Ten rice seedlings were planted in each pot, and each treatment was replicated three times. The disease grade of each plant was recorded, with the percentage of leaf area covered by lesions as the indicator: Grade 0, no symptoms; Grade 1, lesions covering less than 1% of leaf area; Grade 3, lesions covering 1%–5% of leaf area; Grade 5, lesions covering 6%–25% of leaf area; Grade 7, lesions covering 26%–50% of leaf area; Grade 9, lesions covering 51%–100% of leaf area.

[0079] (II) Experimental Results

[0080] The prevention and control effect of MMA on bacterial leaf streak of rice in a pot experiment is shown in Table 1. Figure 3 As shown in Table 1, the results show that the concentration of 5 mg / mL of MMA can significantly inhibit the occurrence of bacterial leaf streak of rice when applied 24 hours in advance or at the same time as the inoculation of bacterial leaf streak of rice, and the disease levels are mainly at levels 1, 3 and 5, among which the total proportion of levels 1 and 3 reaches 80%, and the plants show disease resistance. The disease levels of the control group are mainly at levels 7 and 9, and the total proportion of the two levels is more than 80%, and the plants show disease susceptibility, indicating that 5 mg / mL of MMA can effectively prevent and control the development of bacterial leaf streak of rice.

[0081] Example 4 Methyl o-methylamino benzoate inhibits the formation of biofilm of Xanthomonas oryzae pv. oryzae

[0082] (I) Experimental method

[0083] Biofilm production detection: The formation of biofilm was detected by using a 96-well plate. The bacterial suspension of Xanthomonas oryzae pv. oryzae was adjusted to OD 600 value = 1.0, and two treatment groups were set, which were denoted as Xoc group and Xoc+MMA group.

[0084] Xoc group: 99 μL of NB liquid medium was added to three wells of a 96-well plate, and 1 μL of bacterial suspension of Xanthomonas oryzae pv. oryzae (OD 600 = 1.0) was added to three wells of the 96-well plate, respectively;

[0085] Xoc+MMA group: 98.375 μL of NB liquid medium was added to three wells of a 96-well plate, and 1 μL of bacterial suspension of Xanthomonas oryzae pv. oryzae (OD 600 = 1.0) was added to three wells of the 96-well plate, respectively, and 0.625 μL of MMA stock solution (final concentration of 5 mg / ml) was added.

[0086] The two treatment groups were cultured at 28°C and 150 rpm for 2 days, the culture solution was removed, 150 μL of 0.1% (w / v) crystal violet was added to the wells for staining at room temperature for 20 min, the staining solution was removed, and each well of the 96-well plate was washed with water for three times, dried, and 200 μL of 95% ethanol was added to dissolve the crystal violet of the colored bacterial biofilm cells. The biofilm content was determined by an enzyme marker at 595 nm, and each treatment was repeated three times.

[0087] (II) Experimental results

[0088] The results of the effect of MMA on the biofilm formation of Xanthomonas oryzae pv. oryzae are shown in Table 2. Figure 4As shown, the results show that the biofilm content of Xoc+MMA group is significantly lower than that of Xoc group, which indicates that MMA can inhibit the formation of biofilm of Xoc, reduce the toxicity of Xoc, and thus achieve the effect of effectively preventing and controlling bacterial leaf streak of rice.

[0089] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Use of methyl o-methylamino benzoate in the prevention and treatment of bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzae strain GDXc267.

2. Use of methyl o-methylamino benzoate in the preparation of a product for the prevention and treatment of bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzae strain GDXc267.

3. Use of methyl o-methylamino benzoate in the inhibition of the growth of Xanthomonas oryzae pv. oryzae, or in the preparation of a product for the inhibition of the growth of Xanthomonas oryzae pv. oryzae, wherein the strain of Xanthomonas oryzae pv. oryzae is GDXc267.

4. Use of methyl o-methylamino benzoate in the inhibition of biofilm formation of Xanthomonas oryzae pv. oryzae, or in the preparation of a product for the inhibition of biofilm formation of Xanthomonas oryzae pv. oryzae, wherein the strain of Xanthomonas oryzae pv. oryzae is GDXc267.

5. Use of methyl o-methylamino benzoate in the inhibition of pathogenicity of Xanthomonas oryzae pv. oryzae, or in the preparation of a product for the inhibition of pathogenicity of Xanthomonas oryzae pv. oryzae, wherein the strain of Xanthomonas oryzae pv. oryzae is GDXc267.

6. Use of methyl o-methylamino benzoate in the inhibition of the infection of rice by Xanthomonas oryzae pv. oryzae, wherein the strain of Xanthomonas oryzae pv. oryzae is GDXc267.

7. Use of methyl o-methylamino benzoate in the preparation of a product for the inhibition of the infection of rice by Xanthomonas oryzae pv. oryzae, wherein the strain of Xanthomonas oryzae pv. oryzae is GDXc267.

8. Use of methyl o-methylamino benzoate in the prevention and treatment of diseases caused by Xanthomonas oryzae pv. oryzae, or in the preparation of a product for the prevention and treatment of diseases caused by Xanthomonas oryzae pv. oryzae, wherein the strain of Xanthomonas oryzae pv. oryzae is GDXc267.

9. A method for controlling bacterial leaf streak of rice, characterized by, A rice plant is treated with a solution of methyl o-methylamino benzoate, wherein the concentration of the solution is not less than 3 mg / mL, and wherein the bacterial leaf streak of rice is caused by Xanthomonas oryzae pv. oryzae strain GDXc267.

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