Use of compound questiomycin a in preventing and treating plant diseases

Abstract

The application relates to application of a compound Questiomycin A in prevention and treatment of plant diseases. The compound not only effectively antagonizes Agrobacterium tumefaciens, but also has high efficient antagonistic activity on botrytis, brown rot and gomme.

Description

Technical Field

[0001] This application relates to the application of compound Questionomycin A in the prevention and control of plant diseases, and belongs to the fields of biotechnology and pesticide technology. Background Technology

[0002] Plant diseases are a significant factor affecting normal plant development. For example, crown gall is a widespread soil-borne bacterial disease in stone fruit cultivation, caused by *Agrobacterium tumefaciens*. Other examples include gray mold caused by *Botrytis cinerea*, brown rot caused by *Monilinia yunnanensis*, and gummosis caused by *Botryosphaeria dothidea*.

[0003] Questionomycin A (hereinafter referred to as QA) possesses antibacterial activity, exhibiting antibacterial activity against Bacillus subtilis, Pseudomonas alkaligenes, and Staphylococcus aureus. Since Questionomycin A is isolated from some phenazine-producing bacteria, it is a natural antibacterial agent and holds promise as a novel biopesticide. However, several factors limit the use of Questionomycin A: on the one hand, the types of pathogens antagonized by Questionomycin A are far from fully understood; on the other hand, there are few known superior strains that produce Questionomycin A. Therefore, discovering more pathogenic microorganisms antagonized by Questionomycin A would expand its application scope; discovering new strains that produce Questionomycin A would provide new methods for its preparation, thus offering new directions for novel bio-based fungicides. Summary of the Invention

[0004] To address the aforementioned technical requirements, this application provides the following technical solution:

[0005] First, the use of Questionomycin A in antagonizing or inhibiting plant pathogens, namely Agrobacterium tumefaciens, Botrytis cinerea, Monilinia yunnanensis, and / or Botryosphaeria dothidea, is provided.

[0006] The use of Questionomycin A in the suppression of plant diseases, including crown gall, gray mold, brown rot, and / or gummosis, is also provided.

[0007] It also provides the use of *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of CM-9 containing Questionomycin A, and / or one or more mixtures of these substances in the inhibition of *Agrobacterium tumefaciens*, *Monilinia yunnanensis*, and / or *Botryosphaeria dothidea*, wherein the CM-9 is registered with the China General Microbiological Culture Collection Center under the number CGMCC No. 18905.

[0008] It also provides the use of *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of CM-9 containing Questionomycin A, and / or one or more mixtures of these substances in the inhibition of crown gall, brown rot, and / or gummosis, wherein the CM-9 is registered with the China General Microbiological Culture Collection Center under the number CGMCC No. 18905.

[0009] It also provides the use of Pseudomonas citrinum CM-9, its fermentation broth, the filtrate of its culture or its complete metabolites in the production of Questionomycin A, wherein the CM-9 is registered with the China General Microbiological Culture Collection Center under the number CGMCC No. 18905.

[0010] A method for preparing Questionomycin A is also provided, comprising the step of isolating Questionomycin A from Pseudomonas citrinum CM-9, its fermentation broth, the filtrate of its culture or its intact metabolites, wherein the CM-9 is registered with the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 18905.

[0011] A method for inhibiting plant pathogens is also provided, comprising applying to plant tissues or cells one or more of the following: *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of *Pseudomonas citrinum* CM-9 containing Questionomycin A, and / or a mixture thereof, wherein the pathogen is *Agrobacterium tumefaciens*, *Monilinia yunnanensis*, and / or *Botryosphaeria dothidea*; wherein the CM-9 is registered with the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 18905.

[0012] A method for inhibiting plant pathogens is also provided, comprising applying a preparation containing Questionomycin A to plant tissues or cells, wherein the pathogens are Agrobacterium tumefaciens, Botrytiscinerea, Monilinia yunnanensis and / or Botryosphaeria dothidea.

[0013] A method for suppressing plant diseases is also provided, comprising applying to plant tissues or cells one or more of the following: *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of *P. citrinum* CM-9 containing Questionomycin A, and / or mixtures thereof, wherein the plant disease is crown gall, brown rot, and / or gummosis; wherein the strain number of *P. citrinum* is CM-9, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 18905.

[0014] And a method for suppressing plant diseases by applying a preparation containing Questionomycin A to plant tissues or cells, said plant diseases being crown gall, gray mold, brown rot, and / or gummosis.

[0015] Advantages of this application:

[0016] 1. This is the first time that *Pseudomonas citrinum* CM9 has been proven to be a strain that produces Questionomycin A. Since *Pseudomonas citrinum* CM9 is a naturally occurring microorganism, Questionomycin A can be produced in large quantities from it, and Questionomycin A can degrade naturally in the environment, avoiding the potential risks to the environment and human health posed by drug residues.

[0017] 2. Expanded the antibacterial spectrum of Questionomycin A. For the first time, Questionomycin A was found to have highly effective antagonistic effects against Agrobacterium tumefaciens, Monilinia yunnanensis, Botryosphaeria dothidea, and Botrytis cinerea.

[0018] 3. Questionomycin A has a long-lasting antibacterial effect. Attached Figure Description

[0019] Figure 1 Research roadmap for CM9 antagonistic metabolites.

[0020] Figure 2 Plate confrontation experiment between CM9 and Agrobacterium tumefaciens.

[0021] Figure 3-1 Phenotypes of CM9 and Agrobacterium tumefaciens infecting plants.

[0022] Figure 3-2 : The area of ​​crown galls in phenotypic photographs of plants infected with CM9 and Agrobacterium tumefaciens.

[0023] Figure 4 : Using non-targeted metabolomics to identify key metabolites.

[0024] Figure 5 Differences in the expression levels of Questionomycin A among different treatment groups.

[0025] Figure 6 Differences in absorbance of biofilms at OD600.

[0026] Figure 7 : Coating experiment of different concentrations of Questionomycin A with Agrobacterium tumefaciens.

[0027] Figure 8-1 Phenotypes of plants infected with different concentrations of Questionomycin A and Agrobacterium tumefaciens.

[0028] Figure 8-2 Phenotypic images of plants infected with different concentrations of Questionomycin A and Agrobacterium tumefaciens show the area of ​​crown galls.

[0029] Figure 9 : Plate confrontation experiment of different concentrations of Questionomycin A with α. Detailed Implementation

[0030] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For example, the *Agrobacterium tumefaciens*, *Botrytis cinerea*, *Monilinia yunnanensis*, and *Botryosphaeria dothidea* used to demonstrate the antibacterial properties of Questionomycin A are well-known species in the art and can be obtained through normal commercial channels, as well as through donations or self-isolation. The experimental effects of the same species are generally similar.

[0032] This application focuses on a plate confrontation model to systematically screen and verify the control effects of compounds. Specifically: ① A plate confrontation system was constructed, and a symbiotic plate culture environment for biocontrol strains and pathogens was prepared to lay the foundation for subsequent experiments. ② The inhibition rate of the biocontrol bacteria was measured, and non-target metabolomics analysis was performed on the metabolites in the inhibition zone. Candidate substances were then introduced into the subsequent plate confrontation system, and the inhibition rate was calculated using pathogen growth indicators to preliminarily screen potential metabolites. ③ Subsequently, the antibacterial function of the compounds was verified. Multiple parallel experiments were designed to replicate the plate confrontation and plant inoculation experiments to verify the stability of the inhibitory effect. Different concentration gradients were set to analyze the dose-response relationship and clarify the effective concentration range. ④ The optimal concentration for inhibitory effect was identified, and the concentration gradient was refined for precise testing. The optimal concentration was determined by combining the growth inhibition rate and physiological indicators of the target organism (see...). Figure 1 ).

[0033] Example 1: Plate confrontation between *Pseudomonas citrus* CM-9 and *Agrobacterium*

[0034] Sample source: *Pseudomonas aurantiaca* CM-9 (hereinafter referred to as CM9), isolated and preserved by our laboratory, with accession number CGMCC No. 18905 at the China General Microbiological Culture Collection Center (see ZL202010990366.3). *Agrobacterium tumefaciens* G13 (hereinafter referred to as Agrobacterium), kindly provided by the team of Li Shifang, Chinese Academy of Agricultural Sciences, with accession number ACCC23308 at the China Agricultural Microbiological Culture Collection Center, was used.

[0035] LB medium (solid): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, autoclaved at 121℃ for 21 min.

[0036] LB medium (liquid): tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, autoclaved at 121℃ for 21 min.

[0037] YEB medium (solid): tryptone 5 g / L, yeast extract 1 g / L, beef meal 5 g / L, sucrose 5 g / L, anhydrous MgSO4 0.24 g / L, agar 15 g / L, pH adjusted to 7.0. Autoclave at 121℃ for 21 min.

[0038] YEB medium (liquid): tryptone 5 g / L, yeast extract 1 g / L, beef meal 5 g / L, sucrose 5 g / L, anhydrous MgSO4 0.24 g / L, agar 15 g / L, pH adjusted to 7.0. Autoclave at 121℃ for 21 min.

[0039] Implementation steps:

[0040] (1) Activation and purification of strains

[0041] CM9 was inoculated onto LB solid medium, and Agrobacterium was inoculated onto YEB solid medium. Streaking was performed using a sterile inoculation loop, and the plates were incubated at 28°C in the dark until single colonies appeared. Single colonies were then picked up using a sterile inoculation loop and inoculated into LB and YEB liquid media, respectively, and incubated overnight at 28°C and 220 rpm in the dark.

[0042] (2) Flat plate confrontation method

[0043] The antibacterial activity of CM9 against Agrobacterium was evaluated using the plate confrontation method. After the YEB solid medium cooled to approximately 55°C, activated Agrobacterium was added to a concentration of 10. 6CFU / mL, shake well and quickly pour into plates until solidified. Inoculate the center of each YEB plate with 3 μL of CM9 strain (concentration 10). 8 (CFU / mL). Incubate at 28℃ for 2-3 days. Five replicates per treatment.

[0044] (3) Calculation of inhibition rate

[0045] When the Agrobacterium in the blank control fully covered the culture dish, the growth area of ​​Agrobacterium in the control group (A1), the area of ​​the inhibition zone in the treatment group (A2), and the growth area of ​​CM9 strain (A3) were measured. The antibacterial activity was expressed as the inhibition rate, which was calculated as follows: Inhibition rate = (A2-A3) / A1 × 100%.

[0046] The results showed that CM9 could significantly inhibit Agrobacterium tumefaciens (see...). Figure 2 ).

[0047] Example 2 Plant infection by *Pseudomonas citrus* CM9 and *Agrobacterium*

[0048] Peach trees, such as Prunus davidiana seedlings, were used as plant material. After soaking and germinating the peach seeds, uniformly sized seedlings were selected and planted in pots, one seedling per pot. These were then placed in a light-dark culture room at 25°C with alternating 16-hour light and 8-hour dark cycles. After 14 days of pre-culture, a 0.5-1 cm incision was made at the rootstock junction of uniformly growing peach seedlings using a sterile scalpel. 50 μL of a 10% concentration was then inoculated into the incision using a sterile syringe. 9 CFU / mL CM9 strain or an equal volume of sterile water. Next, inoculate 50 μL of a 10 CFU / mL solution. 9 Agrobacterium tumefaciens at CFU / mL was used to bandage wounds with sterile, moistened cotton, which was removed after one week. Five biological replicates were set up for each treatment. Plants were cultured in a light-dark chamber at 25°C with alternating 16-h light and 8-h dark cycles. Six weeks after inoculation, the diseased parts of the plants (infected stems) were photographed using a stereomicroscope, and the area of ​​crown galls in each photograph was measured using ImageJ software. The size of crown galls at the diseased sites was statistically analyzed. Phenotypic analysis showed that *Pseudomonas citrinum* CM9 significantly inhibited the area of ​​crown galls in crown gall disease (see...). Figure 3-1 and Figure 3-2 ).

[0049] Example 3: Non-targeted metabolomics analysis to identify key metabolites and validate their functions

[0050] Questiomycin A is a known compound, and isolating and identifying it from the metabolites of questiomycin A-producing strains is a routine technique. The inventors performed non-target metabolomics analysis on the inhibition zone of CM9 antagonistic Agrobacterium and the corresponding range of culture media for each individual culture using ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (LC-MS / MS). The sample was dissolved in 100 μl of 80% methanol and chromatographically separated using a Vanquish UHPLC system (Thermo Fisher, Germany) with a Hypesil Gold column (100 × 2.1 mm, 1.9 μm) C18 column at a flow rate of 0.2 mL / min and a column temperature of 40°C. Mobile phase A is 0.1% formic acid, mobile phase B is methanol, and the gradient elution program is as follows: 0-1.5 min: 2% B, 1.5-3 min: 2% B, 3-10 min: 85% B, 10-10.1 min: 100% B, 10.1-11 min: 2% B, 11-12 min: 2% B.

[0051] During chromatographic separation, different compounds elute at specific retention times according to their characteristics, forming chromatographic peaks. Mass spectrometry analysis was performed using a Q Exactive™ HF / Q Exactive™ HF-X mass spectrometer (Thermo Fisher, Germany). The scan range was 100–1500 m / z. The ESI source settings were as follows: spray voltage: 3.5 kV; sheath gas flow rate: 35 psi; auxiliary gas flow rate: 10 l / min; ion transfer tube temperature: 320 °C; ionization RF level: 60; auxiliary gas heater temperature: 350 °C; polarity: positive, negative; MS / MS secondary scan was a data-dependent scan.

[0052] The results showed that Questionomycin A was significantly enriched in the inhibition zones of *Pseudomonas citrus* CM9 and *Agrobacterium tumefaciens* (see [link to study]). Figures 4-5 ).

[0053] Example 4: Determination of the effect of Questionomycin A on Agrobacterium biofilm using crystal violet staining method

[0054] Implementation steps: Add 191 μL of sterile YEB medium and 5 μL of Agrobacterium tumefaciens bacterial suspension (concentration of 1×10⁻⁶). 6Add 2 μL of different concentrations of the compound (CFU / mL) and a blank control (DMSO) to a sterile 96-well plate, and incubate at 37°C for 48 h. Aspirate the bacterial culture from the wells and gently wash once with PBS buffer (pH 7.2) to remove airborne bacteria. Add 200 μL of 0.1% crystal violet staining solution to each well, stain at room temperature for 20 min, discard the staining solution, and add anhydrous ethanol to dissolve the adsorbed cell membranes. Shake for 20 min until the cell membranes on the well walls dissolve. Measure the absorbance of each well at OD600 using a microplate reader; a higher OD value indicates a greater biofilm content. Repeat three times.

[0055] The results showed that both 10 μg / mL and 80 μg / mL of Questionomycin A significantly inhibited biofilm formation in Agrobacterium tumefaciens (see [link to study]). Figure 6 ).

[0056] Example 5: Plate confrontation between Questionomycin A and Agrobacterium.

[0057] The antibacterial activity of Questionomycin A against Agrobacterium was evaluated using the plate confrontation method. After the YEB solid medium cooled to approximately 55°C, different concentrations of Questionomycin A were added to the medium, and the plates were then poured to prepare metabolite plates. 200 μL of the pathogenic bacterial (G13) culture was used as a 10⁻¹⁰ plate. 6 CFU / mL. Spread the bacterial suspension evenly onto the culture medium. After incubating at 28°C for 96 h, count the number of colonies to determine the inhibitory rate of the compound on the bacteria.

[0058] When the Agrobacterium in the blank control fully covered the culture dish, the number of Agrobacterium growth in the control group (N1) and the number of Agrobacterium growth in the treatment group (N2) were measured. The antibacterial activity was expressed as the inhibition rate, which was calculated as follows: Inhibition rate = N1-N2 / N1×100%.

[0059] Table 1. Results of plate confrontation experiments between different concentrations of QA and Agrobacterium tumefaciens.

[0060]

[0061] The results showed that application of 10 μg / mL and 80 μg / mL of Questiomycin A significantly inhibited the growth of Agrobacterium tumefaciens (see [link to study]). Figure 7 ).

[0062] Example 6: Plant infection by Questiomycin A and Agrobacterium tumefaciens

[0063] Prunus fuciformis seedlings were used as plant material. After 14 days of pre-culture, at the rootstock junction of uniformly growing Prunus fuciformis seedlings, incisions of approximately 0.5 cm–1 cm were made using a sterile scalpel. Using a sterile syringe, 50 μL of 10 μg / mL or 80 μg / mL Questionomycin A or an equivalent volume of 1% DMSO were inoculated at the wound site. Next, 50 μL of 10 μg / mL Questionomycin A was inoculated. 9 Agrobacterium at CFU / mL was used to bandage the wounds with sterile, moist cotton, which was removed after one week. Five biological replicates were set up for each group. The plants were cultured in a light-dark chamber at 25°C with alternating 16-hour light and 8-hour dark cycles. Six weeks after inoculation, the number of diseased plants was counted. Antibacterial activity was expressed as incidence rate and disease index. The incidence rate was calculated as: Incidence rate = (Number of diseased plants / Total number of plants) × 100%. Subsequently, the diseased areas of the plants were photographed using a stereomicroscope, and the size of the crown gall at the diseased areas was determined using ImageJ software.

[0064] The results showed that application of Questionomycin A at concentrations of 10 μg / mL and 80 μg / mL significantly inhibited the growth of crown goiter (see [link to study]). Figure 8-1 and Figure 8-2 ).

[0065] Example 7: Plate confrontation between Questionomycin A and different types of pathogens

[0066] (1) Activation and purification of strains

[0067] After the PDB solid medium cooled to approximately 55°C, different concentrations of Questionomycin A were added to the medium, and the plates were then poured to prepare metabolite plates. Subsequently, the pathogens were inoculated onto the PDB solid medium, and 0.5 cm × 0.5 cm bacterial blocks were transferred using a sterile scalpel to a medium with a 0.5 cm × 0.5 cm core in the center. The plates were then incubated at 28°C in the dark until the pathogen colonies of the blank control (DMSO) filled the entire culture dish. Each treatment was replicated 5 times.

[0068] (2) Flat plate confrontation method

[0069] When the pathogens in the blank control group had covered the entire culture dish, the growth area of ​​the pathogens in the control group (A1) and the growth area of ​​the pathogens in the treatment group (A2) were measured. The antibacterial activity was expressed as the inhibition rate, which was calculated as: inhibition rate = (A1-A2) / A1 × 100%. The results are shown in Tables 2-4.

[0070] Table 2. Results of plate confrontation experiments between different concentrations of QA and *Streptococcus yunnanensis*.

[0071]

[0072] Table 3. Results of plate confrontation experiments between different concentrations of QA and *Botrytis cinerea*.

[0073]

[0074] Table 4. Results of plate confrontation experiments between different concentrations of QA and Staphylococcus aureus.

[0075]

[0076] The results showed that 10 μg / mL and 80 μg / mL of Questionomycin A had significant inhibitory effects on *Streptococcus yunnanensis*, *Botrytis cinerea*, and *Staphylococcus aureus* (see [link to study]). Figure 9 ).

[0077] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The use of Questionomycin A in antagonizing or inhibiting plant pathogens, characterized in that, The plant pathogens are Agrobacterium tumefaciens, Botrytis cinerea, Monilinia yunnanensis, and / or Botryosphaeria dothidea.

2. The use of Questionomycin A in suppressing plant diseases, characterized in that, The plant diseases mentioned are crown gall, gray mold, brown rot, and / or gummosis.

3. The use of *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of CM-9 containing Questionomycin A, and / or one or more mixtures of these substances in the inhibition of *Agrobacterium tumefaciens*, *Monilinia yunnanensis*, and / or *Botryosphaeria dothidea*, wherein the CM-9 is registered with the China General Microbiological Culture Collection Center under the number CGMCC No. 18905.

4. The use of *Pseudomonas citrus* CM-9, its fermentation broth, filtrate of its culture, metabolites of CM-9 containing Questionomycin A, and / or one or more mixtures of these substances in the inhibition of crown gall, brown rot, and / or gummosis, wherein the CM-9 is registered with the China General Microbiological Culture Collection Center under the number CGMCC No. 18905.

5. The use of *Pseudomonas citrinum* CM-9, its fermentation broth, the filtrate of its culture, or its intact metabolites in the production of Questionomycin A, wherein the registration number of CM-9 at the China General Microbiological Culture Collection Center is CGMCC No. 18905.

6. A method for preparing Questionomycin A, characterized in that, The method includes the step of isolating Questionomycin A from Pseudomonas citrinum CM-9, its fermentation broth, the filtrate of its culture or its intact metabolites, wherein CM-9 is registered with the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 18905.

7. A method for inhibiting plant pathogens, characterized in that, Applying *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of *Pseudomonas citrinum* CM-9 containing Questionomycin A, and / or one or more mixtures of these substances to plant tissues or cells, wherein the pathogen is *Agrobacterium tumefaciens*, *Monilinia yunnanensis*, and / or *Botryosphaeria dothidea*; wherein the CM-9 is registered with the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 18905.

8. A method for inhibiting plant pathogens, characterized in that, Applying a preparation containing Questionomycin A to plant tissues or cells, wherein the pathogen is Agrobacterium tumefaciens, Botrytis cinerea, Monilinia yunnanensis and / or Botryosphaeria dothidea.

9. A method for suppressing plant diseases, characterized in that, Applying *Pseudomonas citrinum* CM-9, its fermentation broth, filtrate of its culture, metabolites of *P. citrinum* CM-9 containing Questionomycin A, and / or one or more mixtures of these substances to plant tissues or cells, wherein the plant disease is crown gall, brown rot, and / or gummosis; wherein the strain number of *P. citrinum* is CM-9, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 18905.

10. A method for suppressing plant diseases, characterized in that, Applying a preparation containing Questionomycin A to the tissues or cells of a plant, wherein the plant disease is crown gall, gray mold, brown rot, and / or gummosis.

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