Application of quercetin in prevention and control of rice bacterial streak

Abstract

The invention discloses application of quercetin in prevention and control of rice bacterial streak. Researches find that the quercetin can inhibit the growth of the rice bacterial streak pathogen, inhibit the movement ability of the rice bacterial streak pathogen, reduce the pathogenicity of the rice bacterial streak pathogen and inhibit the rice bacterial streak pathogen from infecting rice, and can be used for preventing and controlling the rice bacterial streak pathogen and diseases caused by the rice bacterial streak pathogen. Quercetin naturally exists in plants such as citrus fruits, onions, green tea and the like, has no obvious toxic effect on the environment, human bodies and the plants, is low in drug resistance risk, is green and environment-friendly, and conforms to the development direction of sustainable agriculture. Besides, the using amount of quercetin in prevention and control of the rice bacterial streak is low, the quercetin shows a remarkable antibacterial effect under the concentration of 1.5 mg / mL, meanwhile, the rice bacterial streak can be effectively prevented and controlled under the concentration, and the quercetin has good application potential and prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and pesticides. More specifically, it relates to the application of quercetin in preventing and controlling bacterial leaf streak of rice. Background Art

[0002] Rice is an important cereal widely cultivated in the tropics of Asia, and more than half of the world's population uses rice as the main food source. However, bacterial leaf streak (BLS) of rice is seriously threatening rice production. This disease is caused by the infection of Xanthomonas oryzae pv. Oryzicola (Xoc), which is one of the important quarantine diseases in China and the second largest bacterial disease of rice worldwide after bacterial blight.

[0003] Bacterial leaf streak of rice has been found in many provinces in China, mainly including the rice-growing areas in South China and the middle and lower reaches of the Yangtze River. In recent years, due to the influence of various factors such as climate change, frequent typhoons and heavy rains, the expansion of seed propagation areas, and the extensive cultivation of susceptible varieties, the spread and damage of bacterial leaf streak have been further aggravated, becoming a key bacterial disease threatening the safety of rice production in the rice-growing areas of South China and the Yangtze River Basin.

[0004] Bacterial leaf streak of rice mainly damages the leaves of rice. In the initial stage, small water-soaked short stripe spots appear on the leaf surface of diseased plants, and gradually develop into longitudinal stripe spots, which are translucent when observed against the light; in severe cases, the whole leaf turns yellow and withered, and even turns reddish-brown. When bacterial leaf streak of rice is severe, the yield of rice fields can be reduced by more than 40%. Due to the sudden and explosive characteristics of bacterial leaf streak and the difficulty in curing it after onset, the prevention and control of bacterial leaf streak of rice is particularly crucial.

[0005] At present, the types of pesticides registered for preventing and controlling bacterial leaf streak of rice are limited, and the prevention and control effects are generally not ideal. In order to achieve the prevention and control goals, chemical pesticides are often overused in actual applications, which not only increases the production cost but also poses a threat to the safety of food production. Therefore, developing drugs for preventing and controlling bacterial leaf streak of rice that are harmless to human health, environmentally friendly, and have a low risk of drug resistance has important value and significance for the prevention and control of bacterial leaf streak of rice. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies of the existing drugs for preventing and controlling bacterial leaf streak of rice, such as generally low prevention and control efficacy and high risk of drug resistance, and provides a green drug for preventing and controlling bacterial leaf streak of rice that is safe and harmless to human health, environmentally friendly, and has a low risk of drug resistance.

[0007] The first object of the present invention is to provide the application of quercetin in preventing and controlling bacterial leaf streak of rice.

[0008] The second object of the present invention is to provide a method for preventing and controlling bacterial leaf streak of rice.

[0009] The above object of the present invention is achieved by the following technical solutions:

[0010] The present invention discovers through research that quercetin can inhibit the growth of Xanthomonas oryzae pv. oryzicola, and inhibit its motility, thereby reducing the pathogenicity of Xanthomonas oryzae pv. oryzicola. Moreover, both leaf experiments and pot experiments prove that quercetin has the effect of preventing and controlling bacterial leaf streak of rice.

[0011] Therefore, the present invention claims the following solutions:

[0012] Use of quercetin in preventing and controlling bacterial leaf streak of rice.

[0013] Use of quercetin in preparing a product for preventing and controlling bacterial leaf streak of rice.

[0014] Use of quercetin in inhibiting the infection of rice by Xanthomonas oryzae pv. oryzicola.

[0015] Use of quercetin in preparing a product for inhibiting the infection of rice by Xanthomonas oryzae pv. oryzicola.

[0016] Use of quercetin in inhibiting Xanthomonas oryzae pv. oryzicola.

[0017] Use of quercetin in preparing a product for inhibiting Xanthomonas oryzae pv. oryzicola.

[0018] Use of quercetin in inhibiting the growth of Xanthomonas oryzae pv. oryzicola.

[0019] Use of quercetin in preparing a product for inhibiting the growth of Xanthomonas oryzae pv. oryzicola.

[0020] Use of quercetin in inhibiting the motility of Xanthomonas oryzae pv. oryzicola.

[0021] Use of quercetin in preparing a product for inhibiting the motility of Xanthomonas oryzae pv. oryzicola.

[0022] Use of quercetin in inhibiting the pathogenicity of Xanthomonas oryzae pv. oryzicola.

[0023] Use of quercetin in preparing a product for inhibiting the pathogenicity of Xanthomonas oryzae pv. oryzicola.

[0024] Use of quercetin in preventing and controlling diseases caused by Xanthomonas oryzae pv. oryzicola.

[0025] Use of quercetin in preparing a product for preventing and controlling diseases caused by Xanthomonas oryzae pv. oryzicola.

[0026] In addition, the present invention also provides a method for preventing and controlling rice bacterial leaf streak disease, which involves treating rice plants with a quercetin solution.

[0027] To ensure the effect, preferably, the concentration of the quercetin solution is not less than 1.5 mg / mL.

[0028] More preferably, to ensure the effectiveness and stability of the effect, the concentration of the quercetin solution is not less than 3 mg / mL.

[0029] As an alternative implementation, the treatment method is to spray the rice plants with the quercetin solution.

[0030] The present invention has the following beneficial effects:

[0031] Through research, the present invention has found that quercetin can inhibit the growth of Xanthomonas oryzae pv. oryzicola, inhibit the motility of Xanthomonas oryzae pv. oryzicola, reduce the pathogenicity of Xanthomonas oryzae pv. oryzicola, and inhibit the infection of rice by Xanthomonas oryzae pv. oryzicola, and can be used to prevent and control Xanthomonas oryzae pv. oryzicola and the diseases caused by it.

[0032] Quercetin is widely present in plants such as citrus fruits, onions, and green tea. It has no obvious toxic effects on the environment, human body, and plants. It has a low risk of drug resistance, is green and environmentally friendly, and conforms to the development direction of sustainable agriculture. In addition, the amount of quercetin used in the prevention and control of rice bacterial leaf streak disease is relatively low. It shows a significant antibacterial effect at a concentration of 1.5 mg / mL, and can effectively prevent and control rice bacterial leaf streak disease at this concentration, having good application potential and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shows the antibacterial effects of quercetin on Xanthomonas oryzae pv. oryzicola and Bacillus amyloliquefaciens.

[0034] Figure 2 Shows the results of the effect of quercetin on the motility of Xanthomonas oryzae pv. oryzicola (Figure A shows the colony phenotypes of Xanthomonas oryzae pv. oryzicola in NA plates of different treatment groups; Figure B shows the measurement results of the colony diameters of different treatment groups).

[0035] Figure 3 Shows the results of the effect of quercetin on the pathogenicity of Xanthomonas oryzae pv. oryzicola on rice (Figure A shows the lesion phenotypes of rice leaves of different treatment groups; Figure B shows the statistical results of the lesion lengths of rice leaves of different treatment groups).

[0036] Figure 4 Shows the prevention and control effect of quercetin on rice bacterial leaf streak disease in a pot experiment (Figure A shows the phenotypes of rice seedlings of different treatment groups; Figure B shows the disease index of different treated rice seedlings). DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0039] The strain of the bacterial leaf streak pathogen of rice used in the following examples is GDXc267. The strain is from Maoming, Guangdong, and belongs to the strong dominant pathogenic strain C18 (SSSSRSS) in South China.

[0040] The Bacillus amyloliquefaciens LM-1 strain has been disclosed in the inventor's article "Liang M, Feng A, Wang C, Zhu X, Su J, Xu Z, Yang J, Wang W, Chen K, Chen B, Lin X, Feng J, Chen S. Bacillus amyloliquefaciens LM-1 Affects Multiple Cell Biological Processes in Magnaporthe oryzae to Suppress Rice Blast. Microorganisms. 2024 Jun 20;12(6):1246."

[0041] The rice variety is Wushan Simiao (WSSM).

[0042] The strain GDXc267 of the bacterial leaf streak pathogen of rice and the rice variety Wushan Simiao (WSSM) have been disclosed in the inventor's article "Feng Aiqin, Wang Congying, Su Jing, Feng Jinqi, Chen Kailing, Lin Xiaopeng, Chen Bing, Liang Meiling, Yang Jianyuan, Zhu Xiaoyuan, Chen Shen. Creation of New Strains Resistant to Bacterial Leaf Streak of Rice and Analysis of Their Agronomic Traits [J]. Chinese Journal of Rice Science, 2023, 37(6): 587-596."

[0043] Quercetin (QU), CAS No.: 117-39-5, was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the product number is: SQ8030; the structural formula is:

[0044]

[0045] The formula for NA (Nutrient Agar) solid medium: 5 g of peptone, 3 g of beef extract, 10 g of sucrose, 15 g of agar, 1 g of yeast extract, made up to 1 L with water, pH = 7.0.

[0046] 0.3% NA agar plate formula: 5 g of peptone, 3 g of beef extract, 10 g of sucrose, 3 g of agar, 1 g of yeast extract, made up to 1 L with water, pH = 7.0.

[0047] NB (Nutrient Broth) liquid medium formula: 5 g of peptone, 3 g of beef extract, 10 g of sucrose, 1 g of yeast extract, made up to 1 L with water, pH = 7.0.

[0048] LB (Luria-Bertani) medium formula: 10 g of peptone, 5 g of yeast extract, 5 g of sodium chloride, 15 g of agar, made up to 1 L with water, pH = 7.0.

[0049] Example 1 Quercetin has an antibacterial effect on Xanthomonas oryzae pv. oryzicola (plate confrontation)

[0050] (I) Experimental method

[0051] Quercetin (QU) was prepared into a stock solution with a concentration of 33 mg / mL using absolute ethanol (ET). ET)

[0052] Xanthomonas oryzae pv. oryzicola (Xoc) was cultured on NA solid medium. After 2 days of culture, the bacteria were washed with sterile water and placed in a 50 mL centrifuge tube, and the concentration was adjusted to an OD 600 = 1.0 bacterial suspension.

[0053] Bacillus amyloliquefaciens was cultured on LB solid medium. After 1 day of culture, the bacteria were washed with sterile water and placed in a 50 mL centrifuge tube, and the concentration was adjusted to an OD 600 = 1.0 bacterial suspension.

[0054] Treatment groups with different concentrations of QU were set up:

[0055] The QU stock solution was added to the bacterial suspensions of Xanthomonas oryzae pv. oryzicola and Bacillus amyloliquefaciens respectively, so that the final concentrations of QU in the two bacterial suspensions were 0, 0.38, 0.75, 1.0, 1.5, and 3 mg / mL, respectively, to prepare bacterial suspensions of Xanthomonas oryzae pv. oryzicola and Bacillus amyloliquefaciens containing different concentrations of QU.

[0056] CK group and CK+ET group were set up:

[0057] CK group: Bacterial suspension of Xanthomonas oryzae pv. oryzicola, bacterial suspension of Bacillus amyloliquefaciens;

[0058] CK+ET group: ET was added to the bacterial suspension of Xanthomonas oryzae pv. oryzicola so that the final concentration of ET in the bacterial suspension of Xanthomonas oryzae pv. oryzicola was 4%.

[0059] Take 2 μL of the bacterial suspension of Xanthomonas oryzae pv. oryzicola from each group and drop it on the NA solid medium plate for 2 days of culture, then take pictures and record.

[0060] Take 2 μL of the Bacillus amyloliquefaciens bacterial suspension from each group and drop it on the LB solid medium plate for 1 day of culture, then take pictures and record.

[0061] (II) Experimental results

[0062] The growth results of the bacterial suspensions of Xanthomonas oryzae pv. oryzicola and Bacillus amyloliquefaciens with different treatments on the plate are as Figure 1 shown. The results show that when the concentration of QU is 0.75 and 1.0 mg / mL, the colony growth of Xanthomonas oryzae pv. oryzicola begins to slow down. When the concentration of QU is 1.5 mg / mL, there is a small amount of colony growth, while when the concentration of QU is 3 mg / mL, almost no colonies can be observed growing on the plate. The colony growth of Bacillus amyloliquefaciens LM-1 is not affected at different concentrations of QU.

[0063] In summary, the results show that QU has a significant inhibitory effect on the growth of Xanthomonas oryzae pv. oryzicola, but has no inhibitory effect on the growth of Bacillus amyloliquefaciens.

[0064] Example 2 Inhibition of the motility formation of Xanthomonas oryzae pv. oryzicola by quercetin

[0065] (I) Experimental method

[0066] Prepare the bacterial suspension of Xanthomonas oryzae pv. oryzicola. The preparation method is the same as that in Example 1.

[0067] Motility detection: Adjust the bacterial suspension of Xanthomonas oryzae pv. oryzicola to be tested to OD 600 = 1.0, and set up a control group and a treatment group:

[0068] Xoc group: Bacterial suspension of Xanthomonas oryzae pv. oryzicola (OD 600 = 1.0).

[0069] ET group: Add ET to the bacterial suspension of Xanthomonas oryzae pv. oryzicola to make the final concentration of ET in the bacterial suspension of Xanthomonas oryzae pv. oryzicola 4%.

[0070] Xoc + QU (0.38 mg / mL) group: Take 98.84 μL of the bacterial suspension of Xanthomonas oryzae pv. oryzicola and add it to a centrifuge tube, and then add 1.15 μL of the 33 mg / mL QU mother liquor (final concentration is 0.38 mg / mL).

[0071] Xoc + QU (0.75 mg / mL) group: 97.72 μL of the bacterial suspension of Xanthomonas oryzae pv. oryzicola was added to a centrifuge tube, and 2.27 μL of the 33 mg / mL QU stock solution (final concentration: 0.75 mg / mL) was added.

[0072] Xoc + QU (1.0 mg / mL) group: 96.96 μL of the bacterial suspension of Xanthomonas oryzae pv. oryzicola was added to a centrifuge tube, and 3.03 μL of the 33 mg / mL QU stock solution (final concentration: 1.0 mg / mL) was added.

[0073] Xoc + QU (1.5 mg / mL) group: 95.45 μL of the bacterial suspension of Xanthomonas oryzae pv. oryzicola was added to a centrifuge tube, and 4.55 μL of the 33 mg / mL QU stock solution (final concentration: 1.5 mg / mL) was added.

[0074] 2 μL of the bacterial solution from each group was pipetted and inoculated onto a 0.3% NA agar plate, then placed in an incubator at 28 °C. After 24 h, the motility of each strain was observed and photographed, and three replicates were made for each strain.

[0075] (II) Experimental results

[0076] The results of the effect of quercetin on the motility of Xanthomonas oryzae pv. oryzicola are as Figure 2 shown. The results indicate that the colony diameters of Xanthomonas oryzae pv. oryzicola under the treatments of 0.75 mg / mL and 1.5 mg / mL QU were significantly smaller than those of the Xoc group and the ET group. Especially, the colony diameter was even smaller under the treatment of 1.5 mg / mL QU, indicating that QU decreased the motility of Xanthomonas oryzae pv. oryzicola.

[0077] Example 3 Quercetin can inhibit the pathogenicity of Xanthomonas oryzae pv. oryzicola on rice (hole punching inoculation)

[0078] (I) Experimental method

[0079] To determine whether QU can inhibit the pathogenicity of Xanthomonas oryzae pv. oryzicola on rice, QU was applied to rice leaves, and Xanthomonas oryzae pv. oryzicola was inoculated onto rice leaves by the hole punching inoculation method.

[0080] Method of hole punching inoculation: Holes were punched and inoculated on both sides of the midrib of rice leaves using a hole puncher, and the bacterial suspension of Xanthomonas oryzae pv. oryzicola (the preparation method of the bacterial suspension was the same as in Example 1) was gently pressed into the intercellular spaces of the leaf tissue with the hole puncher.

[0081] The method of applying QU was by spraying: A QU solution with a final concentration of 1.5 mg / mL was sprayed on rice seedlings.

[0082] Five treatment groups were set up, namely the Xoc group, the ET group, the -24h group, the 0h group, and the +24h group;

[0083] Xoc group: inoculated with Xanthomonas oryzae pv. oryzicola;

[0084] ET group: inoculated with a mixture of Xanthomonas oryzae pv. oryzicola and 4% ET;

[0085] -24h group: QU was applied 24 h before inoculation with Xanthomonas oryzae pv. oryzicola;

[0086] 0h group: QU was applied simultaneously with the inoculation of Xanthomonas oryzae pv. oryzicola.

[0087] +24h group: QU was applied 24 h after inoculation with Xanthomonas oryzae pv. oryzicola.

[0088] Three replicates were set for each treatment, 5 seedlings were planted in each replicate, and 2 leaves of each seedling were punched. The length of the disease spots was investigated 15 days after inoculation, and 5 disease spots were randomly selected from each replicate to measure the length of the disease spots.

[0089] (II) Experimental results

[0090] The results of the effect of QU on the pathogenicity of Xanthomonas oryzae pv. oryzicola on rice are as Figure 3 shown. The results showed that the rice in the Xoc group and the ET group would form thin stripe disease spots, showing high susceptibility, and the average length of their disease spots exceeded 6 cm. However, QU at a concentration of 1.5 mg / mL, when applied 24 h in advance, simultaneously with the inoculation of Xanthomonas oryzae pv. oryzicola, or 24 h after inoculation, could significantly inhibit the formation of disease spots, and the average length of the disease spots was about 0.9 cm, showing resistance. This indicates that QU can inhibit the pathogenicity of Xanthomonas oryzae pv. oryzicola on rice and achieve the effect of controlling bacterial leaf streak disease.

[0091] Example 4 Quercetin can effectively prevent and control bacterial leaf streak disease of rice (spray inoculation)

[0092] (I) Experimental method

[0093] To further clarify the prevention and control effect of QU on bacterial leaf streak disease of rice, rice seedlings were treated with QU, and Xanthomonas oryzae pv. oryzicola was inoculated onto the rice seedlings by the spray inoculation method to test whether QU has a prevention and control effect on bacterial leaf streak disease in the pot experiment.

[0094] The method of applying QU adopted the spray method: a QU solution with a final concentration of 1.5 mg / mL was sprayed on the rice seedlings.

[0095] Pot spray inoculation and disease grade statistical method: 5-week-old rice seedlings were inoculated with the spray bacterial suspension, and the OD of the inoculated bacterial suspension 600= 1.0, 20 mL of the bacterial suspension was sprayed per pot. Five treatment groups (Xoc group, ET group, -24 h group, 0 h group, and +24 h group) were set up in the manner of Example 3. After inoculation, water was sprayed regularly to keep the humidity above 90%. The disease incidence was investigated after 15 days. Ten rice seedlings were set in each pot, and each treatment was repeated 3 times. The disease grade of each plant was recorded. The disease grade was based on the percentage of the leaf area occupied by the lesions: grade 0, no disease symptom; grade 1, the lesion area accounted for less than 1% of the leaf area; grade 3, the lesion area accounted for 1% - 5% of the leaf area; grade 5, the lesion area accounted for 6% - 25% of the leaf area; grade 7, the lesion area accounted for 26% - 50% of the leaf area; grade 9, the lesion area accounted for 51% - 100% of the leaf area.

[0096] (II) Experimental results

[0097] The control effect of QU on bacterial leaf streak of rice in the pot experiment was as Figure 4 shown. The results showed that bacterial leaf streak occurred in rice plants under the treatments of the Xoc group and the ET group. However, applying QU 24 h before inoculation, at the same time of inoculation, or 24 h after inoculation could significantly inhibit the occurrence of bacterial leaf streak of rice. The disease index was about 15%. While the Xoc group and the ET group showed high susceptibility, and their average disease index exceeded 60%. This indicated that 1.5 mg / mL of QU could effectively control the occurrence of bacterial leaf streak of rice.

[0098] The above embodiments are the preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of quercetin in the prevention and control of rice bacterial leaf streak disease.

2. Application of quercetin in the preparation of products for preventing and controlling rice bacterial leaf streak disease.

3. Use of quercetin in inhibiting rice leaf streak pathogenic bacteria, or in preparing a product for inhibiting rice leaf streak pathogenic bacteria.

4. Use of quercetin in inhibiting oxysporum leaf streak of rice, or in preparing a product for inhibiting oxysporum leaf streak of rice.

5. Use of quercetin in inhibiting the growth of oxysporum leaf streak on rice, or in preparing a product for inhibiting the growth of oxysporum leaf streak on rice.

6. Use of quercetin in inhibiting the motility of oxysporum leaf streak pathogenicum, or in preparing a product for inhibiting the motility of oxysporum leaf streak pathogenicum.

7. Use of quercetin in inhibiting the pathogenicity of Pathogen Pseudomonas syringae pv. oryzae, or in preparing a product for inhibiting the pathogenicity of Pathogen Pseudomonas syringae pv. oryzae.

8. Use of quercetin in preventing and controlling diseases caused by Psoralea oryzae var. oryzae, or in preparing products for preventing and controlling diseases caused by Psoralea oryzae var. oryzae.

9. A method for preventing and controlling rice bacterial leaf streak disease, characterized in that: Rice plants were treated with quercetin solution.

10. The method according to claim 9, characterized in that: The concentration of the quercetin solution is not less than 1.5 mg / mL.

Images