Application of tryptophol in prevention and treatment of maize bacterial stalk rot

Abstract

The application relates to the application of chromanol in the prevention and treatment of corn bacterial stalk rot, relates to the technical field of biological prevention and treatment, and the pathogenic bacteria of the corn bacterial stalk rot is Dickeya zeae. It is found for the first time that chromanol can effectively reduce the pathogenicity of corn bacterial stalk rot by inhibiting the motility of Dickeya zeae, reducing the activity of cell wall degrading enzyme and reducing the degree of disease, but does not have adverse effects on the cell activity and morphology of Dickeya zeae, indicating that the effect of chromanol on Dickeya zeae is not achieved by killing the bacteria, but by inhibiting the pathogenicity of the bacteria. Therefore, chromanol is applied to corn bacterial stalk rot, is not easy to produce drug resistance, and is expected to become a substitute for antibiotics and be used for preventing and treating corn bacterial stalk rot. The application can not only effectively control the disease, but also slow down the selective pressure of drug resistance development, and provides a new solution for the sustainable prevention and treatment of corn bacterial stalk rot.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to the application of chromol in the control of bacterial stalk rot in maize. Background Technology

[0002] Bacterial stalk rot of maize is a soil-borne disease caused by *Dickeya zeae*, which can lead to yield reductions of 21% to 98.8% in severe cases. This disease has been reported in the United States, Canada, China, India, and Africa, and poses a significant threat in tropical and subtropical maize-growing areas. Currently, chemical fungicides and resistant varieties are common methods for controlling bacterial plant diseases. However, both methods have limitations. On the one hand, genetic research on maize bacterial diseases is relatively scarce, resulting in slow progress in the breeding of resistant varieties; on the other hand, the extensive use of toxic chemicals such as fungicides not only harms the ecological environment but may also promote drug resistance in pathogens, thus increasing the difficulty of disease control.

[0003] In recent years, biological control has received widespread attention as a green and environmentally friendly strategy for controlling plant diseases. Tryptophol (TOL), scientifically known as indole-3-ethanol, is a natural product widely found in plants and fungi, possessing various biological activities. Patent CN202311129661.X discloses the application of tryptophol in enhancing the efficacy of yeast in controlling postharvest diseases of fruits and vegetables. Exogenous addition of tryptophol can promote the inhibitory effect of a specific yeast (Scheffersomyces spartinae W9) on the spore germination of pathogens such as Botrytis cinerea and Sclerotinia sclerotiorum, enhancing the biocontrol effect of yeast. This demonstrates the significant potential of tryptophol in biological control.

[0004] However, there is currently no research on the use of tryptol for the prevention and control of bacterial stalk rot in maize caused by Dickeya zeae. Summary of the Invention

[0005] The purpose of this invention is to provide the application of chromool in the prevention and control of bacterial stalk rot in maize in order to solve the above-mentioned problems.

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

[0007] This invention provides the application of tryptophan in the prevention and control of bacterial stalk rot in maize.

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

[0009] As a further optimization of the present invention, the chromool has a CAS registration number of 526-55-6 and a chemical formula of C. 10 H 11 No, specifically

[0010] This invention also provides the application of chromol in the preparation of a drug for the prevention and control of bacterial stalk rot in maize, wherein the main component of the drug is chromol.

[0011] As a further optimization of the present invention, the concentration of crcotol in the drug is ≥0.5mM.

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

[0013] This invention is the first to discover that tryptol can effectively reduce the pathogenicity of *Dickeya zeae* by inhibiting its motility, reducing the activity of cell wall degrading enzymes, and mitigating the severity of disease. However, it does not adversely affect the cell activity and morphology of *Dickeya zeae*, indicating that its effect on *Dickeya zeae* is not achieved by killing the bacteria, but by inhibiting its pathogenicity. Therefore, applying tryptol to corn bacterial stalk rot is unlikely to induce drug resistance. Tryptol is expected to become an alternative to antibiotics for the prevention and control of corn bacterial stalk rot. This application can not only effectively control the disease, but also alleviate the selective pressure of drug resistance development, providing a new solution for the sustainable prevention and control of corn bacterial stalk rot. Attached Figure Description

[0014] Figure 1 The figure shows the experimental results of the effect of tryptophan on the motility of Dickeya zeae.

[0015] Figure 2 Figure 1 shows the experimental results of the effect of tryptophan on the activity of cell wall degrading enzymes in Digesterone maize.

[0016] Figure 3 The figure shows the experimental results of the effect of tryptophan on the severity of bacterial stalk rot in maize.

[0017] Figure 4 The figure shows the experimental results of the effect of tryptophan on the cell viability of Digesterone maize.

[0018] Figure 5 The figure shows the experimental results of the effect of croterol on the cell morphology of Diggia maize. Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection 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 culture medium: Add 10g of tryptone, 10g of NaCl and 5g of yeast extract to deionized water, and bring the volume to 1L with deionized water. Adjust the pH to 7.0 and sterilize at 121℃ for 20min.

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

[0023] 3. Protease culture medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1.5g agar, diluted with pure water to 100mL, sterilized at 121℃ for 20min.

[0024] 4. The CAS registry number for chromol is 526-55-6, and its chemical formula is C. 10 H 11 NO, with a molecular weight of 161.20.

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

[0026] II. Methods

[0027] 2.1 Inhibitory effect of tryptol on motility of Digesterone maize

[0028] 1) Dickeya zeae was spread on LB solid medium and incubated at 28°C for 15 hours. Single colonies were then picked and transferred to LB liquid medium and incubated overnight at 28°C and 200 rpm with shaking. The overnight culture was then adjusted to OD200. 600 =1.0, spare;

[0029] 2) Pour 0.25% agar LB medium (add chromol to the medium to a final concentration of 0.5mM; use no chromol as a control) into a 60mm petri dish and let it solidify; take 2μl of the prepared Diggia corniculata suspension and drop it into the center of the medium, and incubate it in a constant temperature incubator at 30℃. Observe and measure the colony diameter after 3 days.

[0030] The results are as follows Figure 1As shown, compared with the control group without added chromol, chromol significantly reduced the motility of Diggia corniculata on agar plates, with a 50% reduction in motility diameter.

[0031] 2.2 Inhibitory effect of tryptophan on the activity of cell wall degrading enzymes in Digesterone maize

[0032] 1) Spread *Digitaria zei* on LB solid medium and incubate at 28°C for 15 hours. Then, pick single colonies and incubate them overnight at 28°C and 200 rpm with shaking. Adjust the overnight culture to OD200. 600 =1.0, spare;

[0033] 2) Add 10g of skim milk powder to 100mL of purified water and sterilize at 115℃ for 15min.

[0034] 3) Transfer the skim milk powder solution from step 2) to protease medium at a transfer rate of 10% (v / v), and add troponin to the medium to a final concentration of 0.5 mM (with no troponin added as a control). Mix thoroughly and pour into a 100×100 mm petri dish. Take 2 μl of the prepared Diggia corniculata suspension and drop it onto the medium. Incubate in a 28℃ constant temperature incubator upside down for 48 h, and then observe and measure the radius of the transparent zone.

[0035] The results are as follows Figure 2 As shown, compared with the control group without added croteric alcohol, croteric alcohol significantly inhibited the protease activity of Diggia corniculata.

[0036] 2.3 Effects of tryptol on the severity of bacterial stalk rot in maize

[0037] 1) Sterilized corn B73 seeds were sown in sterile soil. After 4 weeks of growth, corn plants with uniform growth were selected for stem injection inoculation. Two treatment groups were established in this experiment:

[0038] Treatment group 1 consisted of: 0.5 mM ... 600 =1.0 of the Diggia cornuta bacterial suspension was mixed thoroughly;

[0039] Treatment group 2 consisted of: *Digitaria zeylinum* bacterial suspension (50 μl OD). 600 =1.0 of *Digitaria zebrina* bacterial suspension;

[0040] 2) During injection, insert the syringe needle vertically into the corn stalk core and slowly inject the suspension of the above treatment groups into different corn stalks respectively; after injection, wrap a layer of sterile gauze around the inoculation point; after all treatment groups are cultured at 30℃ for 3 days, observe the disease status of the corn plants; take pictures of the corn plants and observe whether the stalks of the corn plants in different treatment groups have softened; then cut the corn plant stalks in the middle to observe the disease status inside the stalks and take pictures.

[0041] The results are as follows Figure 3 As shown, compared with the control group without added chromol, the corn plants treated with chromol showed a significant reduction in lodging and the disease symptoms were also alleviated. Although brown lesions still formed on the stems of the infected corn plants, the corn plants in the control group without added chromol showed constriction at the inoculation site, while the corn plants in the chromol-treated group did not show this phenomenon.

[0042] 2.4 Effects of tryptophan on the cell viability of Digesterone maize

[0043] 1) Pick single colonies of *Digitaria zebufo* and incubate them overnight at 28°C and 200 rpm. Collect the cells by centrifugation at 6000 rpm for 5 min at room temperature and wash three times with fresh LB medium. Then, transfer *Digitaria zebufo* to fresh LB (negative control) and LB + chromol (final chromol concentration 0.5 mM), and incubate with shaking at 28°C and 200 rpm for 12 h. Collect the cells by centrifugation at 6000 rpm for 5 min at 4°C, wash the precipitate and resuspend it in 0.85% NaCl. Adjust the bacterial concentration to approximately 10% with 0.85% NaCl. 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 bacterial suspension. After thorough mixing, incubate at room temperature in the dark for 15 min. After incubation, take 5-8 μL of bacterial suspension to prepare a slide and observe the results under a laser confocal microscope.

[0045] The results are as follows Figure 4 As shown, in the control group without added croteric alcohol, all Diggia corniculata cells appeared green; in contrast, in the croteric alcohol-treated group, only a few cells showed red, while the vast majority of cells remained green; indicating that croteric alcohol treatment does not affect the cell viability of Diggia corniculata.

[0046] Effects of 2,5-Croterol on Cell Morphology of Diggia maize

[0047] 1) Pick a single colony of Diggia corniculata and incubate it overnight at 28°C and 200 rpm. Collect the bacterial cells by centrifugation at 4000 rpm for 5 min at room temperature and wash them three times with fresh LB medium. Then transfer Diggia corniculata to fresh LB (negative control) and LB + chromol (final concentration of 0.5 mM), and incubate with shaking at 28°C and 200 rpm for 12 h.

[0048] 2) Bacterial treatment and fixation: Take 5 mL of bacterial culture, centrifuge at 4000 rpm for 5 min to collect bacterial cells, and discard the supernatant; wash the precipitate with 0.1 M phosphate buffer, centrifuge at 4000 rpm for 5 min to collect cells, repeat twice; add 2.5% glutaraldehyde, mix by pipetting and vortex to distribute the bacterial cells evenly in the solution, and fix overnight at 4℃ (dark treatment).

[0049] 3) Ethanol gradient dehydration: After fixation in step 2), centrifuge at 4000 rpm for 5 min and discard the supernatant; add 0.1M PBS (pH=7.2), vortex at room temperature, let stand for 20 min, centrifuge at 4000 rpm for 5 min and discard the supernatant, repeat 3 times; use a series of gradient ethanol solutions of 30%, 50%, 70%, 80%, 90%, and 100% for dehydration, incubating at 4℃ for 10 min after each addition; discard the solution, add 100% ethanol, and incubate at 4℃;

[0050] 4) Freeze-drying: Centrifuge the sample containing 100% ethanol at 4000 rpm for 5 min and discard the supernatant. Blow air in a clean bench for 5 min. Freeze the sample at -20℃ for 2 h and dry it in a freeze dryer for 5 h. Then plate it with gold. Observe the sample with a scanning electron microscope.

[0051] The results are as follows Figure 5 As shown, the cells of *Digitalis maize* in the control group without added chromol and the chromol-treated group were intact, with smooth surfaces and typical rod-shaped structures, indicating that chromol treatment does not affect the cell morphology of *Digitalis maize*.

[0052] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of tryptol in the control of bacterial stalk rot in maize, characterized in that, The pathogen causing the bacterial stalk rot of corn is *Digitaria zebrina*. Dickeya zeae .

2. The application of tryptophan in the preparation of a drug for controlling bacterial stalk rot in maize, characterized in that, The main component of the drug is chromol, and the pathogen of the bacterial stalk rot of corn is *Digitaria zeyeris*. Dickeya zeae .

3. The application according to claim 2, characterized in that, The concentration of tryptophan in the drug is ≥0.5 mM.

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