Pantoea capable of efficiently preventing and treating citrus canker and application of pantoea

Abstract

The invention discloses pantoea for efficiently preventing and treating citrus canker and application of the pantoea. The pantoea is named as Pantoea cypripedii Q210, the preservation number is CGMCC NO.36920, the preservation date is December 8, 2025, and the preservation site is the China General Microbiological Culture Collection Center. The bacterial strain is separated and screened from soil of a Gannan navel orange orchard, and it is found that the bacterial strain has the capacity of inhibiting growth of citrus canker pathogen Xcc, has an obvious prevention and treatment effect on citrus canker and can provide a new biological prevention and treatment means for comprehensive prevention and treatment of citrus canker.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a highly effective pan-bacterium for the prevention and control of citrus canker and its application. Background Technology

[0002] Citrus canker is a major quarantine disease threatening the global citrus industry, caused by Xanthomonas citrus subsp. citrus (… Xanthomonas citri subsp. citri, (Xcc) Citrus canker is caused by infection, leading to widespread leaf drop, branch dieback, general tree decline, fruit blemishes, and premature fruit drop, resulting in significant economic losses. Currently, the main methods of control both domestically and internationally include copper-based fungicides and agricultural antibiotics, with copper-based fungicides being the primary chemical agents. However, long-term use of copper-based fungicides not only easily induces an increase in citrus red spider mites and rust ticks but also causes serious environmental pollution and bacterial resistance, hindering the high-quality development of the citrus industry nationwide and globally.

[0003] Biological control, as an environmentally friendly, safe, and effective method for controlling plant diseases, plays an increasingly important role in plant disease management. Therefore, developing a biological control method that can effectively control citrus canker is of great significance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a highly effective pan-bacterium for the prevention and control of citrus canker and its application.

[0005] To achieve the objectives of this invention, the following technical solutions are provided: A highly effective fungus for controlling citrus canker, the fungus being named... Pantoea cypripedii Q210, accession number: CGMCC NO.36920, accession date: December 8, 2025, accession location: China General Microbiological Culture Collection Center.

[0006] The colonies of the aforementioned pantothecin are yellow, moist, rod-shaped under an optical microscope, and Gram-negative.

[0007] On the other hand, the present invention also provides the application of the above-mentioned pantothecin in the prevention and control of citrus canker.

[0008] Furthermore, the application of the aforementioned pantothecin in the prevention and control of citrus canker, wherein the pantothecin includes species containing pantothecin. Pantoea cypripedii Q210 microbial inoculants, Pantothecin Pantoea cypripedii The fermentation supernatant of Q210, wherein the dosage form of the microbial agent includes, but is not limited to, powder, suspension, and microcapsule dosage forms.

[0009] Furthermore, the above-mentioned Pantothecin can be prepared into a bacterial solution or biological agent and then directly applied or sprayed onto plants suffering from citrus canker.

[0010] The present invention also provides a product containing the aforementioned pantothecin. Pantoea cypripedii Preparation method of Q210 bacterial culture: Use the strain with preservation number CGMCC No. 36920... Pantoea cypripedii After overnight activation with Q210, the cells were inoculated into the culture medium at a 1% inoculum and cultured. The cells were then separated by centrifugation, washed with sterile MgCl2 solution, and finally resuspended to OD600 = 0.5 to obtain the bacterial suspension. This invention also provides a solution containing the aforementioned pantothecin. Pantoea cypripedii Preparation method of fermentation supernatant of Q210: The strain *Pantheraea* with preservation number CGMCC No. 36920 was used... Pantoea cypripedii After overnight activation with Q210, inoculate the culture medium at a 1% inoculum rate. After incubation, centrifuge and collect the supernatant. This supernatant is the pantothecin. Pantoea cypripedii Fermentation supernatant of Q210.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolates and screens pan-bacteria from the soil of navel orange orchards in southern Jiangxi. Pantoea cypripedii Q210 has inhibitory effects on citrus canker bacteria. Xcc The fungus can inhibit the growth of bacteria on lemon leaves. Xcc Compared to chemical agents for controlling citrus canker, the pan-fungi strain used in this invention... Pantoea cypripedii Q210 has a short growth cycle, strong antibacterial activity, and is environmentally friendly, providing new resources and ideas for the green prevention and control of citrus canker, and has broad application prospects. Attached Figure Description

[0012] The disclosure of this invention will become more readily understood with reference to the accompanying drawings. It will be readily understood by those skilled in the art that these drawings are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention.

[0013] Figure 1 The colony morphology characteristics and Gram staining images of pan-bacterium Q210 provided by this invention.

[0014] Figure 2 The phylogenetic tree of pan-bacterium Q210 provided by the present invention.

[0015] Figure 3 The pan-bacterium Q210 cell inhibitor provided by the present invention Xcc The effect of growth.

[0016] Figure 4The pantothecin Q210 fermentation broth provided by this invention inhibits... Xcc The effect of growth.

[0017] Figure 5 The diagram shows the effect of the Pan-Bacterium Q210 bacterial solution provided by this invention on the prevention and control of citrus canker on lemon leaves. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are purchased from conventional biochemical reagent stores. The separating agent and usage method provided by this invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0019] The pan-bacterium described in this invention is named *Pan-bacterium pubescens* root pan-bacterium. Pantoea cypripedii Q210, accession number: CGMCC NO.36920, accession date: December 8, 2025, accession location: China General Microbiological Culture Collection Center.

[0020] Example 1: Isolation and Identification of Pantotheca Strain Q210 (1) Isolation and purification of strains The strain Q210 of this invention was isolated and screened from the orchard soil of Gannan navel oranges in Ganzhou City. The isolation medium was TSB medium. The screening conditions were as follows: after activating the purified strain in TSB medium, the bacterial suspension was added dropwise to a plate coated with... Xcc In NA medium, bacteria that exhibit a clear inhibition zone around the bacterial suspension after overnight incubation are considered biocontrol bacteria against citrus canker. The TSB medium formula consists of 17 g tryptone, 3 g soybean papain hydrolysate, 5 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 1 L deionized water, and 20 g agar. The NA medium formula consists of 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L deionized water, and 20 g agar.

[0021] The specific separation and screening steps are as follows: Weigh 10.0 g of soil from the Gannan navel orange orchard in Ganzhou City and place it in an Erlenmeyer flask containing 90 mL of sterile water and sterile glass beads. Shake for 15 min to obtain a soil suspension. Transfer 1 mL of the soil suspension to a large test tube containing 9 mL of sterile water and mix thoroughly. Dilute the soil suspension 10 times to obtain a 10-fold diluted soil suspension. Then, take 1 mL of the 10-fold diluted soil suspension and transfer it to a large test tube containing 9 mL of sterile water and mix thoroughly to obtain a 100-fold diluted soil suspension. Repeat this process to obtain a 100-fold diluted soil suspension. 3 Soil suspension diluted 10 times 4 A soil suspension diluted 10 times. 3 Soil suspension diluted 10 times 4 200 μL of soil suspension (diluted with TSB) was spread onto TSB medium. The TSB plates were then incubated at 30°C for 48 h. Single colonies were picked, streaked again, and purified. The purified single colonies were then transferred to TSB medium and incubated overnight. 5 μL of the TSB-cultured bacterial solution was added dropwise to the pre-spread TSB medium. Xcc On a NA plate, add the bacterial solution at three points, air dry in a clean bench, mark the spots, and then incubate the NA plate at 30°C for 48 hours. The bacteria that appear with a transparent inhibition zone around the added bacterial solution are the *Pantheraea* species described in this invention. Pantoea cypripedii Q210.

[0022] (2) Strain identification 1. The selected antibacterial strain, *Pantotheca acuminata* Q210, was inoculated onto TSB medium and incubated at 30°C for 24 h. Colony characteristics were observed, and Gram staining was performed. *Pantotheca acuminata* Q210 grows rapidly on TSB medium, producing yellow, moist colonies that are easy to pick. Figure 1 As shown in a. It appears pink after Gram staining, indicating a Gram-negative stain, as... Figure 1 As shown in b.

[0023] 2. Genomic DNA was extracted from pantothenic acid Q210 using a bacterial genome extraction kit, and PCR amplification was performed using universal primers for the bacterial 16S sequence. The amplified products were detected by electrophoresis and DNA fragment sequencing was performed.

[0024] The universal primer for the bacterial 16S sequence is: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'; The sequencing results were compared with existing 16S gene sequences in the NCBI database using BLAST analysis. Strains with similar homology were selected, and a phylogenetic tree was constructed using MEGA 7.0 software.

[0025] The 16S sequencing results of *Panthera philoxeroides* Q210 were compared with those of *Panthera philoxeroides* in the NCBI database using BLAST. The results showed that *Panthera philoxeroides* Q210 was similar to *Panthera philoxeroides* root *Panthera philoxeroides*. Pantoea cypripedii The similarity to (Genbank accession number: MW922729) was 99.9%. Combined with the Gram staining results of Pantotheca Q210, Q210 was preliminarily identified as a member of the genus Pantotheca. Pantoea cypripedii Q210). Figure 2 Phylogenetic tree of Pantotheca Q210 strain.

[0026] Example 2 Pantotheca Q210 cells and Xcc Flat-panel standoff The *Pantheraea peruvianum* Q210 colonies from the TSB plate in Example 1 were transferred to TSB medium and cultured in a shaker at 30 °C and 200 rpm. Simultaneously, Xcc The sample was transferred to NB medium (formulation: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L deionized water) and cultured under the same conditions for 16 h. Then, 100 μL of the solution was collected. Xcc The bacterial suspension was spread onto NA plates, and three points were marked at equal intervals in the middle of the bottom of the plate. 5 μL of the cultured Pantotheca Q210 bacterial suspension was added to the marked NA plates. After air-drying in a clean bench, the plates were placed in an inverted incubator at 30℃ and incubated for 48 h.

[0027] Figure 3 Inhibition of pan-bacterial Q210 cells Xcc Images showing the effect of growth. (From...) Figure 3 As can be seen, a transparent inhibition zone forms around the addition of Pantotheca Q210 strain, indicating that Pantotheca Q210 cells can significantly inhibit bacterial growth. Xcc The growth of.

[0028] Example 3: Pantothenic Acid Q210 Fermentation Broth and Xcc Flat-panel standoff Fermentation broth preparation: After activating the *Pantotheca acuminata* Q210 strain in TSB medium, the culture was transferred to Landy medium at a 1% inoculum size. After culturing for 72 h, the supernatant was collected by centrifugation. An equal volume of ethyl acetate was added to the supernatant for extraction. The mixture was separated, and the ethyl acetate phase was collected. After rotary evaporation, the ethyl acetate phase was redissolved in 50% methanol-dichloromethane and the solvent was evaporated at room temperature to obtain the crude fermentation product. 10 μL of the crude fermentation product was dissolved in 990 μL of 20% DMSO aqueous solution to prepare a 10 μL / mL fermentation broth. The Landy medium formulation was as follows: 0.5 g magnesium sulfate, 1 g potassium dihydrogen phosphate, 10 mg manganese sulfate, 0.2 g potassium chloride, 5 mg ferrous sulfate hexahydrate, 0.2 mg copper sulfate pentahydrate, 1 g yeast extract, 2 mg L-phenylalanine, 5 g L-glutamic acid, 20 g glucose, 1 L deionized water, pH 7.5.

[0029] Standoff: Xcc Transferred to NB medium and incubated at 30℃ and 200 rpm for 16 h with shaking. Then, 100 μL of the culture medium was collected. Xcc The bacterial culture was spread onto NA plates, and three sterile Oxford cups were placed at equal intervals in the middle of the plates. 200 μL of the above-mentioned 10 μL / mL pantothenic acid Q210 fermentation broth was added to two of the Oxford cups, and an equal volume of 20% DMSO aqueous solution was added to the third cup. The plates were placed in a constant temperature incubator at 30℃ and incubated upright for 48 h.

[0030] Figure 4 Inhibition of Pantothenic Acid Q210 fermentation broth Xcc Images showing the effect of growth. (From...) Figure 4 It is evident that a transparent inhibition zone, 1.5 cm in diameter, existed around the Oxford cup containing Q210 fermentation broth, while no such zone was observed in the Oxford cup containing 20% ​​DMSO aqueous solution. This indicates that the Q210 fermentation broth has a significant effect on the inhibition of bacteria. Xcc It has a significant inhibitory effect on growth.

[0031] Example 4: Application of Pantothecin Q210 in the prevention and control of citrus canker. Preparation of Pantotheca Q210 bacterial suspension: After overnight activation, Pantotheca Q210 strain was transferred to TSB medium at a 1% inoculum and cultured to the logarithmic phase. The bacterial cells were then collected by centrifugation at 8000 rpm for 2 min and washed twice with 10 mmol / L sterile MgCl2 solution. Finally, the suspension was resuspended to OD600=0.5 to obtain Pantotheca Q210 bacterial suspension.

[0032] Xcc Preparation of bacterial solution: XccAfter overnight activation, the strain was transferred to NB medium at a 1% inoculum. When the strain reached OD600 = 1.0, it was centrifuged at 8000 rpm for 2 min to collect the cells. The cells were washed twice with 10 mmol / L sterile MgCl2 solution and finally resuspended to OD600 = 0.1 to obtain the desired bacterial culture. Xcc Bacterial solution.

[0033] Control of Citrus Canker on Lemon Leaves: Select several young lemon leaves grown under natural conditions (around 20 days after leaf expansion). Using a sterile syringe needle, gently puncture a small hole on each side of the leaf vein (avoiding the large veins). Then, inject a resuspended Panadol Q210 bacterial solution into the leaf using a sterile syringe. Eight hours later, inject a resuspended... Xcc Simultaneously, inject the bacterial solution into both symmetrical sides of the same leaf. Xcc Five leaves were treated with bacterial suspension and 10 mmol / L sterile MgCl2 solution. The leaves continued to grow in situ, and the incidence of citrus canker was observed on the leaves after 5 days.

[0034] Figure 5 The image shows the effect of Pan-Bacterium Q210 bacterial solution on the control of citrus canker on lemon leaves. Figure 5 In the middle section, resuspended Pantotheca Q210 bacterial solution was injected at point A, and resuspended Pantotheca Q210 bacterial solution and resuspended bacteria were injected at point B. Xcc Bacterial solution, injected at point C Xcc Bacterial suspension, inject sterile MgCl2 solution at point D. (From...) Figure 5 It is evident that inoculating leaves with Q210 bacterial solution alone has no significant effect on leaf growth. Xcc The bacterial solution significantly induced citrus canker in lemon leaves, while pre-inoculation with Q210 bacterial solution followed by inoculation... Xcc The symptoms of citrus canker were not obvious in the leaf areas treated with the bacterial solution, indicating that strain Q210 can significantly inhibit the disease. Xcc The pathogenicity of lemon leaves has a preventive effect against citrus canker.

[0035] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. A highly effective pan-fungi strain for controlling citrus canker, characterized in that, The pantothecin was named Pantoea cypripedii Q210, accession number: CGMCC NO.36920, accession date: December 8, 2025, accession location: China General Microbiological Culture Collection Center.

2. The pantothecin strain for controlling citrus canker as described in claim 1 Pantoea cypripedii Application of Q210 in the prevention and control of citrus canker.

3. The application according to claim 2, characterized in that, The citrus species mentioned include any one of mandarin orange, orange, and lemon.

4. The application according to claim 2, characterized in that, The pantothecin Pantoea cypripedii Q210 includes... Pantoea cypripedii Q210 microbial inoculants, Pantothecin Pantoea cypripedii The fermentation supernatant of Q210, wherein the dosage form of the microbial agent includes, but is not limited to, powder, suspension, and microcapsule dosage forms.

5. The application according to claim 2, characterized in that, Pantothecin Pantoea cypripedii After the Q210 strain is prepared into a bacterial solution or biological agent, it can be directly applied or sprayed onto plants suffering from citrus canker.

6. A method for preparing Panax notoginseng bacterial suspension for the prevention and control of citrus canker, characterized in that, The pantothecin described in claim 1 Pantoea cypripedii After overnight activation, Q210 was inoculated into the culture medium at a 1% inoculum and cultured. The cells were then separated by centrifugation, washed with sterile MgCl2 solution, and finally resuspended to OD600=0.5 to obtain the bacterial culture.

7. A method for preparing pancreatic bacteria fermentation supernatant for the prevention and treatment of citrus canker, characterized in that, The pantothecin described in claim 1 Pantoea cypripedii After overnight activation, Q210 was inoculated into the culture medium at a 1% inoculum rate and cultured. After culture, the supernatant was collected by centrifugation.

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