Application of tryptophol in prevention and treatment of corn bacterial stem rot

Abstract

The invention relates to application of tryptophol in prevention and treatment of corn bacterial stem rot and relates to the technical field of biological prevention and control, the pathogenic bacterium of the corn bacterial stem rot is Dickeya zeae, it is found for the first time that tryptophol can inhibit the mobility of the Dickeya zeae, reduce the activity of cell wall degrading enzyme and relieve the disease degree, and the tryptophol can inhibit the activity of the Dickeya zeae, so that the tryptophol can be used for preventing and treating the corn bacterial stem rot. The tryptophol can effectively reduce the pathogenicity of tryptophol, but has no adverse effect on the cell viability and form of the corn dibasic bacteria, which indicates that the effect of tryptophol on the corn dibasic bacteria is realized by inhibiting the pathogenicity of bacteria instead of killing bacteria, so that tryptophol acts on the corn bacterial stem rot, the drug resistance is not easy to generate, and the tryptophol can be used for treating the corn bacterial stem rot. The tryptophol is expected to become a substitute of antibiotics and is used for preventing and treating the corn bacterial stem rot, the diseases can be effectively controlled, the selective pressure of drug resistance development can be relieved, and a new solution is provided for sustainable prevention and treatment of the corn bacterial stem rot.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control, and specifically relates to the application of tryptophol in controlling corn bacterial stalk rot. Background Art

[0002] Corn bacterial stalk rot is a soil-borne disease caused by Dickeya zeae, which can cause a 21% to 98.8% reduction in crop yield when severe. This disease has been reported in the United States, Canada, China, India, and Africa, etc., and poses a major threat in tropical and subtropical corn-growing regions. Currently, chemical fungicides and disease-resistant varieties are common means for controlling plant bacterial diseases. However, both of these methods have certain limitations. On the one hand, the genetic research on corn bacterial diseases is relatively scarce, resulting in slow progress in breeding disease-resistant varieties; on the other hand, the extensive use of toxic chemicals such as chemical fungicides not only harms the ecological environment but may also prompt pathogens to develop drug resistance, thereby increasing the difficulty of disease control.

[0003] In recent years, biological control, as a green and environmentally friendly plant disease control strategy, has received extensive attention. Tryptophol (TOL), scientifically named indole-3-ethanol, is a natural product widely present in plants and fungi and has various biological activities. The invention patent with the patent number CN202311129661.X discloses the application of tryptophol in improving the control efficacy of postharvest diseases of fruits and vegetables by yeast. Exogenous addition of tryptophol can promote the inhibitory effect of a specific yeast (Scheffersomyces spartinae W9) on the spore germination of pathogenic fungi such as Botrytis cinerea and Monilinia fructigena, enhancing the biocontrol effect of yeast. Thus, it shows that tryptophol has great potential in biological control.

[0004] However, there is currently no research on the use of tryptophol to control corn bacterial stalk rot caused by Dickeya zeae. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of tryptophol in controlling corn bacterial stalk rot in order to solve the above problems.

[0006] The present invention achieves the above purpose through the following technical solutions:

[0007] The present invention provides the application of tryptophol in controlling corn bacterial stalk rot.

[0008] As a further optimized scheme of the present invention, the pathogen of the corn bacterial stalk rot is Dickeya zeae.

[0009] As a further optimized solution of the present invention, the CAS registration number of the chromanol is 526-55-6, and the chemical formula is C 10 H 11 NO, specifically

[0010] The present invention also provides the application of chromanol in the preparation of drugs for preventing and treating corn bacterial stalk rot, and the main component of the drug is chromanol.

[0011] As a further optimized solution of the present invention, the concentration of chromanol in the drug is ≥0.5 mM.

[0012] The beneficial effects of the present invention are as follows:

[0013] The present invention discovers for the first time that chromanol can effectively reduce its pathogenicity by inhibiting the motility of Dickeya zeae, reducing the activity of cell wall degrading enzymes and alleviating the disease severity, but has no adverse effects on the cell viability and morphology of Dickeya zeae, indicating that its effect on Dickeya zeae is not achieved by killing bacteria, but by inhibiting the pathogenicity of the bacteria. Therefore, when chromanol acts on corn bacterial stalk rot, it is not easy to produce drug resistance, and chromanol is expected to become a substitute for antibiotics for preventing and treating corn bacterial stalk rot. This application can not only effectively control the disease, but also slow down the selective pressure of the development of drug resistance, providing a new solution for the sustainable prevention and treatment of corn bacterial stalk rot. Description of the Drawings

[0014] Figure 1 It is a graph of the experimental results of the effect of chromanol on the motility of Dickeya zeae;

[0015] Figure 2 It is a graph of the experimental results of the effect of chromanol on the activity of cell wall degrading enzymes of Dickeya zeae;

[0016] Figure 3 It is a graph of the experimental results of the effect of chromanol on the disease severity of corn bacterial stalk rot;

[0017] Figure 4 It is a graph of the experimental results of the effect of chromanol on the cell viability of Dickeya zeae;

[0018] Figure 5 It is a graph of the experimental results of the effect of chromanol on the cell morphology of Dickeya zeae. Detailed Embodiments

[0019] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] I. Materials

[0021] 1. LB liquid medium: Add 10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract to deionized water, and make up the volume to 1 L with deionized water. After adjusting the pH to 7.0, sterilize at 121 °C for 20 min.

[0022] 2. LB solid medium: Add 1.5% (w / v) agar to the LB liquid medium.

[0023] 3. Protease medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1.5 g of agar, make up the volume to 100 mL with pure water, and sterilize at 121 °C for 20 min.

[0024] 4. The CAS registration number of tryptophol is 526-55-6, and its chemical formula is C 10 H 11 NO, and its molecular weight is 161.20.

[0025] Unless otherwise specified, the methods used in this example are conventional methods well known to those skilled in the art. Unless otherwise specified, the reagents and other materials used are commercially available products.

[0026] II. Methods

[0027] 2.1 Inhibitory effect of tryptophol on the motility of Dickeya zeae

[0028] 1). Spread Dickeya zeae on the LB solid medium and culture it at 28 °C for 15 h. Then pick a single colony and inoculate it into the LB liquid medium, and culture it overnight at 28 °C and 200 rpm; adjust the OD 600 of the overnight cultured test bacterial solution to 1.0 for standby;

[0029] 2). Pour 0.25% agar LB medium (add tryptophol to this medium to make the final concentration of tryptophol 0.5 mM; and use the medium without added tryptophol as a control) into a 60 mm petri dish and let it solidify; take 2 μl of the prepared Dickeya zeae bacterial suspension and drop it in the center of the medium, and culture it statically in a constant temperature incubator at 30 °C. After 3 days, observe and measure the colony diameter.

[0030] The results are as Figure 1As shown, compared with the control group without added chromanol, it can be seen that chromanol significantly reduced the motility of Dickeya zeae on the agar plate, and the motility diameter decreased by 50%.

[0031] 2.2 Inhibitory effect of chromanol on the activity of cell wall degrading enzymes of Dickeya zeae

[0032] 1), Spread Dickeya zeae on the LB solid medium. After culturing at 28 °C for 15 h, pick a single colony into the LB liquid medium and culture it overnight at 28 °C and 200 rpm; adjust the OD 600 of the overnight cultured test bacterial solution to 1.0 for standby;

[0033] 2), Dissolve 10 g of skim milk powder in pure water to a volume of 100 mL, sterilize it at 115 °C for 15 min for standby;

[0034] 3), Transfer the skim milk powder solution in step 2) to the protease medium at a transfer volume of 10% (v / v), and add chromanol to this medium to a final concentration of 0.5 mM (using the absence of chromanol as a control). After thorough mixing, pour it into a 100×100 mm petri dish; take 2 μL of the prepared Dickeya zeae bacterial suspension and drop it on the medium, place it in an incubator at 28 °C for inverted culture for 48 h, and then observe and measure the radius of the clear zone.

[0035] The results are as Figure 2 shown. Compared with the control group without added chromanol, it can be seen that chromanol significantly inhibited the protease activity of Dickeya zeae.

[0036] 2.3 Effect of chromanol on the incidence of corn bacterial stalk rot

[0037] 1), Sow the disinfected corn B73 seeds in sterile soil. After growing for 4 weeks, select corn with consistent growth for stem injection inoculation. Two treatment groups were set up in this experiment:

[0038] Treatment group 1 is: chromanol + Dickeya zeae bacterial suspension (mix 50 μL of 0.5 mM chromanol and 50 μL of Dickeya zeae bacterial suspension with OD 600 = 1.0);

[0039] Treatment group 2 is: Dickeya zeae bacterial suspension (50 μL of Dickeya zeae bacterial suspension with OD 600 = 1.0);

[0040] 2) During injection, insert the syringe needle vertically into the core of the corn stalk, and slowly inject the suspension of the above treatment groups into different corn stalks respectively. After injection, wrap a sterile gauze around the inoculation point. After all treatment groups are cultured at 30°C for 3 days, observe the disease incidence of the corn plants, take pictures of the corn plants, and observe whether the stalks of the corn plants in different treatment groups become soft. Then cut the stalks of the corn plants in half to observe the internal disease incidence of the stalks and take pictures.

[0041] The results are as Figure 3 shown. It can be seen from the comparison with the control group without added colored alcohol that for the corn plants treated with chromanol, the lodging situation is significantly reduced, and the disease symptoms are also alleviated. Although brown lesions still form on the stalks of the infected corn plants, constriction appears at the inoculation sites of the corn plants in the control group without added colored alcohol, while this situation does not occur in the corn plants in the chromanol treatment group.

[0042] 2.4 Effect of chromanol on the cell viability of Dickeya zeae

[0043] 1) Pick a single colony of Dickeya zeae and inoculate it into LB liquid medium, and culture it overnight at 28°C and 200 rpm. Centrifuge at 6000 rpm for 5 min at room temperature to collect the bacterial cells, and wash them three times with fresh LB medium. Subsequently, transfer Dickeya zeae to fresh LB (negative control) and LB + chromanol (final concentration of chromanol is 0.5 mM), and culture them with shaking at 28°C and 200 rpm for 12 h. Centrifuge at 6000 rpm for 5 min at 4°C to collect the bacterial cells, wash the precipitate and resuspend it in 0.85% NaCl. Adjust the bacterial cell concentration to about 10 8 CFU / mL;

[0044] 2) Prepare a 2× stock solution of the LIVE / DEAD BacLight staining reagent mixture, mix the 2× stock solution sample with an equal volume of the bacterial suspension, incubate for 15 min in the dark at room temperature after thorough mixing. After the incubation, take 5 - 8 μL of the bacterial suspension to make a slide, and observe the results under a laser confocal microscope.

[0045] The results are as Figure 4 shown. In the control group without added colored alcohol, all the Dickeya zeae cells show green. On the contrary, in the chromanol treatment group, only sporadic cells show red, and the vast majority of cells still show green, indicating that chromanol treatment does not affect the cell viability of Dickeya zeae.

[0046] 2.5 Effect of chromanol on the cell morphology of Dickeya zeae

[0047] 1), Pick a single colony of Dickeya zeae and inoculate it into LB liquid medium, and culture it overnight at 28 °C and 200 rpm; centrifuge at 4000 rpm for 5 min at room temperature to collect the bacteria, and wash them three times with fresh LB medium; then transfer Dickeya zeae to fresh LB (negative control) and LB + tryptophol (the final concentration of tryptophol is 0.5 mM), and culture it with shaking at 28 °C and 200 rpm for 12 h;

[0048] 2), Bacterial treatment and fixation: Take 5 mL of the bacterial solution, centrifuge at 4000 rpm for 5 min to collect the bacteria, and discard the supernatant; wash the precipitate with 0.1 M phosphate buffer, centrifuge at 4000 rpm for 5 min to collect the cells, and repeat twice; add 2.5% glutaraldehyde, pipette and mix well, then vortex to make the bacteria evenly distributed in the solution, and fix overnight at 4 °C (dark treatment);

[0049] 3), Ethanol gradient dehydration: After fixation in step 2), centrifuge at 4000 rpm for 5 min to discard the supernatant; add 0.1 M PBS (pH = 7.2), vortex at room temperature, then let it stand for 20 min, centrifuge at 4000 rpm for 5 min to discard the supernatant, and repeat 3 times; dehydrate with a series of gradient ethanol of 30%, 50%, 70%, 80%, 90%, and 100%, each time add and keep at 4 °C for 10 min; aspirate and discard the solution, add 100% ethanol, 4 °C;

[0050] 4), Freeze-drying: Centrifuge the sample containing 100% ethanol at 4000 rpm for 5 min to discard the supernatant, and blow it in the ultra-clean workbench for 5 min; freeze the sample at -20 °C for 2 h, dry it in a freeze-dryer for 5 h, and coat it with gold; observe the sample with a scanning electron microscope.

[0051] The results are as Figure 5 shown. The cell morphology of Dickeya zeae in the control group without tryptophol addition and the tryptophol-treated group is intact, the surface is smooth, and it shows a typical rod-shaped structure, indicating that tryptophol treatment does not affect the cell morphology of Dickeya zeae.

[0052] The above-described embodiments only represent several implementation manners of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Application of tryptophan in the prevention and treatment of corn bacterial stalk rot.

2. The use according to claim 1, characterized in that: The pathogen of the corn bacterial stalk rot is Dickeya zeae.

3. The use according to claim 1, characterized in that: The CAS registration number of the tryptophan is 526-55-6, and the chemical formula is C 10 H 11 NO, specifically 4. The use of tryptophan in the preparation of a drug for preventing and treating corn bacterial stalk rot, characterized in that: The main component of the medicine is tryptol.

5. The use according to claim 4, characterized in that: The concentration of tryptophan in the drug is ≥0.5 mM.

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