A novel Sphingomonas bacterium and its application in preventing and controlling citrus canker

Abstract

The present invention discloses a novel Sphingomonas sp. and its application in preventing and controlling citrus canker. Sphingomonas sp. A58, with the preservation number: GDMCC No: 64393, was preserved at the Guangdong Microbial Culture Collection Center on March 7, 2024. This strain is a new species of the genus Sphingomonas. Spraying the bacterial liquid of the novel Sphingomonas sp. A58 of the present invention on citrus leaves can significantly reduce its disease symptoms and disease index; and can efficiently produce siderophores and IAA. Therefore, the novel Sphingomonas sp. A58 of the present invention has great application potential in aspects such as preventing and controlling citrus canker, promoting iron absorption and growth of crops, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a novel Sphingomonas strain and its application in controlling citrus canker. Background Art

[0002] As a major cash crop in southern China, citrus canker is an important quarantine disease and one of the important diseases in citrus production areas in China. Citrus canker is mainly caused by Xanthomonas citri subsp. citri, which mainly damages citrus branches, leaves and fruits, seriously affecting the quality of citrus. At present, the main method for controlling citrus canker is through chemical agents, but the improper use of chemical agents makes the pathogen produce drug resistance and pollutes the environment, which does not conform to the current purpose of food security. Therefore, it is very necessary to study a safe and efficient control method. Biological control is favored by the public because of its safety, high efficiency, less pollution and low cost. In recent years, more and more scholars have studied biological control, and using biological control methods to deal with citrus canker has great potential.

[0003] Sphingomonas sp. is a genus of Gram-negative bacteria belonging to the phylum Bacteroidetes and is widely distributed in soil, water and the rhizosphere of plants. Sphingomonas sp. may have the following effects on plants: it can produce antibiotics or compounds that inhibit the growth of plant pathogens, helping to protect plants from rhizosphere diseases; Sphingomonas sp. is known for its excellent degradation ability, and it can decompose a variety of organic compounds, including polycyclic aromatic hydrocarbons, pesticides, organic wastes, etc., which helps to improve soil texture and provide nutrients required by plants; it can also produce plant growth hormones, such as indole acetic acid, which helps to promote the growth and development of plants; it can participate in the nitrification and denitrification processes of nitrogen in the soil, thus helping to provide the nitrogen source required by plants; it has antioxidant properties, which can help plants fight oxidative stress and improve their resistance, especially under adverse conditions.

[0004] Iron is one of the essential micronutrients for plant growth and development. In plants, iron plays many important physiological and metabolic roles, especially crucial for chlorophyll synthesis and respiration processes. The presence of siderophores can enhance the plant growth-promoting performance, provide the iron element required by the plant, and enhance the growth and development of the plant by promoting chlorophyll synthesis and photosynthesis. IAA can alleviate the damage of biological and abiotic stresses to plants by reducing membrane lipid peroxidation, improve the adaptability of plants in stress environments, and at the same time can promote plant growth and increase yield. The synergistic effect of siderophores and IAA can better enhance plant stress resistance and contribute to the healthy growth of plants. Therefore, screening siderophore-producing strains that synergistically act with plant secretions has great application value.

[0005] In summary, Sphingomonas, as an important microbial resource, can be effectively applied to the development of microbial fertilizers and inoculants by enhancing the diversity of microbial resources, exploring and identifying their potential functions. Such applications can not only promote the ecological balance of agriculture but also serve as an important means to drive the development of sustainable and green agriculture. Summary of the Invention

[0006] The first object of the present invention is to provide a strain of Sphingomonas sp. A58, with a preservation number of: GDMCC No: 64393.

[0007] A novel strain of Sphingomonas provided by the present invention is a potential new species among Sphingomonas. It not only has a significant control effect on citrus canker but also has the functions of producing siderophores and indole-3-acetic acid (IAA), showing great potential for promoting plant stress resistance and growth.

[0008] The second object of the present invention is to provide the application of the above-mentioned Sphingomonas A58 in the control of citrus canker.

[0009] Preferably, the pathogen of citrus canker is Xanthomonas citri subsp. citri (Xcc).

[0010] The third object of the present invention is to provide the application of the above-mentioned Sphingomonas A58 in the production of siderophores and IAA.

[0011] The fourth object of the present invention is to provide the application of the above-mentioned Sphingomonas A58 in promoting plant growth and iron uptake.

[0012] Preferably, the fermentation broth of Sphingomonas A58 is used to irrigate plants to promote plant iron uptake and growth.

[0013] The fifth object of the present invention is to provide a biological agent using the above-mentioned Sphingomonas A58 or its fermentation broth as an active ingredient.

[0014] Preferably, the biological agent is a microbial inoculant, microbial fertilizer, microbial pesticide, or microbial additive.

[0015] The sixth object of the present invention is to provide a method for controlling citrus canker, which includes the following step: applying the above-mentioned biological agent to citrus leaves.

[0016] Preferably, the pathogen of citrus canker is Xanthomonas citri subsp. citri (Xcc), and the Sphingomonas A58 in the biological agent has an optical density (OD) 600 = 0.6 - 0.8.

[0017] The present invention has the following advantages and effects compared with the prior art:

[0018] The present invention discovered for the first time a novel Sphingomonas sp. A58, which is a new species of Sphingomonas sp. and enriches my country's beneficial microbial resources.

[0019] This study, published in the journal Nature Communications, discovered that the novel Sphingomonas strain A58 significantly reduces citrus canker symptoms and disease index, while also producing siderophores and IAA, demonstrating multiple beneficial functions, including disease resistance and growth promotion. Therefore, the discovery and identification of the novel Sphingomonas strain A58 will provide new insights into the development of novel biological agents and methods for the prevention and treatment of citrus canker.

[0020] Sphingomonas sp. A58 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on March 7, 2024, with the address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 64393. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of the novel Sphingomonas A58 on NA medium.

[0022] Figure 2 This is the phylogenetic tree of the novel Sphingomonas A58.

[0023] Figure 3 This is the inhibitory effect of the new Sphingomonas A58 on the citrus canker pathogen Xcc.

[0024] Figure 4 The control effect of the new Sphingomonas A58 on citrus canker on citrus leaves.

[0025] Figure 5 Yellow siderophore secretion circle produced by the novel Sphingomonas sp. A58 on CAS detection medium

[0026] Figure 6 The new type of Sphingomonas A58 reacts with Salkowski colorimetric solution to produce a red color. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions therein, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] Example 1: Isolation of novel Sphingomonas A58

[0029] The leaves used to isolate Sphingomonas sp. nov. A58 were collected from the Jinyou Park in Nanfu Village, Yanyang Town, Meixian District, Meizhou City, Guangdong Province (N24°24′50″, E116°19′54″). The collected leaves were cut into 5 mm × 5 mm squares in a sterile bag and mixed evenly. According to the leaf weight, sterile water was added at a ratio of 1:10 for grinding. The ground leaves were homogenized and diluted 10-fold in gradients. 100 μL of each concentration was taken and spread on NA medium (Guangdong Huankai, product number 022020) for inoculation. After culturing in an incubator at 28 °C for 5 days, colonies were selectively picked with an inoculation loop according to colony characteristics for purification culture. The colony morphology of Sphingomonas sp. nov. A58 after culturing on NA medium for 3 days is as Figure 1 shown. The single colony is circular, with a diameter of 1 - 2 mm, orange-yellow, smooth surface, opaque, and neat edges.

[0030] Example 2: Analysis of the 16S rRNA gene sequence of Sphingomonas sp. nov. A58

[0031] The DNA of Sphingomonas sp. nov. A58 was extracted by the alkaline lysis method: A small amount of bacterial cells was placed in a 200 μL centrifuge tube containing 16.6 μL of alkaline lysis solution, and the bacterial cells were lysed at 95 °C for 30 min in an alkaline environment; after cooling, 16.6 μL of neutralization buffer was added to each well, mixed evenly, and stored in a -20 °C refrigerator. Alkaline lysis solution: 25 mM NaOH and 0.2 mM Na2-EDTA (pH = 12). Neutralization buffer: 40 mM Tris-HCl (pH = 7.5). Then, the 16S rRNA gene sequence of Sphingomonas sp. nov. A58 was amplified using the 16S rRNA specific primers of bacteria 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCA ATCGC-3′). The amplified product was about 1400 bp by electrophoresis analysis. The amplified product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequence obtained by sequencing was spliced by DNA MAN software to obtain the 16S rRNA sequence (SEQ ID NO.1), with a length of 1490 bp. The 16S rRNA gene sequence of Sphingomonas sp. nov. A58 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that the similarity between the 16S rRNA gene sequence of Sphingomonas sp. nov. A58 and the type strain Sphingomonas rubra CGMCC 1.9113 (accession number, jgi.1058074) was the highest, which was 98.30%.

[0032] Example 3: Analysis of the whole gene sequence of Sphingomonas sp. nov. A58

[0033] The bacterial solution of the novel Sphingomonas sp. A58 was sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for genome sequencing. The software QUAST v5.0.2 was used to analyze the genome of the strain Sphingomonas sp. A58. The results showed that: the genomic sequence of the novel Sphingomonas sp. A58 consisted of 19 contigs in total, the total length of the genome was 2,988,474 bp, and the content of genomic DNA G+C was 70.55%. The software UBCG v3.0 was used to construct the genomic phylogenetic tree of the novel Sphingomonas sp. A58 and its closely related reference model strains. The results were as Figure 2 shown. Sphingomonas sp. A58 was most closely related to Sphingomonas abaci, Sphingomonas metalli, Sphingomonas rubra, and Sphingomonas jinjuensis. Moreover, compared with the above three strains with the closest relatedness, the ANI values of Sphingomonas sp. A58 were 77.61 - 85.29%, lower than the critical values proposed for species demarcation of 95 - 96%, and the dDDH values were between 23.50 - 53.3%, lower than the species demarcation threshold of 70% (Table 1). The results of both ANI and dDDH supported that the strain Sphingomonas sp. A58 was a new species of the genus Sphingomonas.

[0034] Therefore, it was named Sphingomonas sp. A58. This strain has been deposited in the Guangdong Provincial Culture Collection of Microorganisms (GDMCC), Address: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Guangdong Province, Zip Code: 510070, Deposit Number: GDMCC No: 64393, Deposit Date: March 7, 2024.

[0035] Table 1 shows the ANI and dDDH values of the novel Sphingomonas sp. A58 and its related strains

[0036]

[0037] Example 4: Antagonistic ability of the novel Sphingomonas sp. A58 against Xanthomonas citri subsp. citri and its control ability against citrus canker

[0038] The antagonistic ability of the novel Sphingomonas sp. A58 against Xcc was determined by the double-layer agar diffusion method. Pour about 1 mm thick NA medium into a petri dish and spread it flat. Place three Oxford cups with a diameter of 6 mm in each petri dish, and then spread 5 mm thick NA medium containing Xcc (Xcc concentration: 0.1% v / v; agar content 1.5% m / v). After cooling, pull out the Oxford cups to form 3 round holes with a diameter of 6 mm. Take 30 μL of the bacterial solution of the novel Sphingomonas sp. A58 (OD 600(= 0.8) was added into the holes and cultured at 28 °C for 24 h, and the diameter of the inhibition zone was measured using a vernier caliper. As Figure 3 shown, after 24 h of adding the bacterial solution of the novel Sphingomonas sp. A58, clear inhibition zones were formed around the round holes, and their diameters were 18.75, 18.95, and 19.11 mm respectively, with an average value of 18.94 mm. The results showed that the novel Sphingomonas sp. A58 had a significant inhibitory effect on the growth of the pathogen Xcc of citrus canker.

[0039] The ability of the novel Sphingomonas sp. A58 to control citrus canker was verified using in vitro Citrus maxima cv. Jinyou leaves. The fully expanded leaves after one week were picked, cleaned, placed in a sterile petri dish, and a layer of sterile wet filter paper was placed at the bottom; after pricking the inoculation points on the back of the leaves with an inoculation needle, the A58 bacterial suspension with OD 600 = 0.6 was sprayed on the leaves, and about 10 8 CFU / mL of the pathogen of citrus canker was sprayed 24 h later. The petiole was moisturized with cotton wrapped with sterile water. The control leaves were only inoculated with the pathogen of citrus canker, and all the experiments were carried out in a sterile operating table. The disease incidence at the pricking points was observed 5 days after inoculation, and the lesion area was measured using ImageJ 2.0 software. The lesion area (R / mm 2 ) was divided into 6 grades. 1: R ≤ 2, 2: 2 < R ≤ 4, 3: 4 < R ≤ 6), 4: 6 < R ≤ 8, 5: 8 < R ≤ 10, 6: R > 10. The disease index = Σ [the number of lesions at each grade × the corresponding grade] / (the total number of lesions × the maximum grade) × 100%. Three replicates were performed. As Figure 4 shown, compared with CK, spraying the A58 bacterial solution on the Citrus maxima cv. Jinyou leaves significantly reduced the lesion area and the disease index by 60.40% and 50.50% respectively.

[0040] Example 5: Analysis of the ability of the novel Sphingomonas sp. A58 to produce siderophores and IAA

[0041] The strain Sphingomonas sp. A58 was activated and cultured on an NA plate at 28 °C for 48 h. A single colony was picked with a toothpick and spot-inoculated on a CAS detection medium plate. After culturing at 28 °C for 3 d, the soluble index of the strain was calculated. A single colony was picked and inoculated into the modified M9 medium, cultured on a shaker at 28 °C and 150 r / min for 2 d. The bacterial suspension was centrifuged at 10000 r / min for 10 min, and the supernatant was mixed with an equal volume of CAS detection solution, allowed to stand for 1 h, and its OD 630Absorbance value (As), using the culture medium without inoculating Sphingomonas sp. strain A58 as a control, measuring its absorbance value (Ar) by the same method, calculating the siderophore activity (su), and the calculation formula is: su = [(Ar - As) / Ar] × 100%. Judging the siderophore-producing ability of the strain by As / Ar, the smaller the ratio, the stronger the siderophore-producing ability. When the As / Ar value is 0 - 0.6, it belongs to high siderophore production; when the As / Ar value is 0.6 - 0.8, it belongs to medium siderophore production; when the As / Ar value is 0.8 - 1.0, it belongs to low siderophore production.

[0042] Modified M9 medium:

[0043] 1. Prepare M9 salt solution, and the components and concentrations of M9 salt solution are: Na2PO4·7H2O (12.8 g / 200 mL), KH2PO4 (0.3 g / 200 mL), NaCl (0.5 g / 200 mL), NH4Cl (1 g / 20 mL), and the rest is water;

[0044] 2. Prepare 0.75 M MgSO4·7H2O solution by adding 18 g of MgSO4·7H2O to 100 mL of double-deionized water;

[0045] 3. Add 14.7 g of CaCl2·2H2O to 100 mL of ddH2O to make 1 M CaCl2·2H2O solution;

[0046] 4. Prepare PIPES buffer by dissolving 6.048 g of PIPES in 156 mL of ddH2O by stirring, and adjust the pH to 6.8 with 5 M NaOH;

[0047] 5. Dissolve 20 g of glucose in 100 mL of ddH2O to prepare 20% glucose solution;

[0048] 6. Prepare the solutions. Except for the glucose solution, the rest of the solutions are sterilized by autoclaving separately, and the glucose solution is used after filtering through a high-pressure sterilization filter (0.22 μm);

[0049] 7. Mix 156 mL of PIPES buffer, 40 mL of M9 salt solution, 20 μL of CaCl2·2H2O solution, 266 μL of MgSO4·7H2O solution and 4 mL of 20% glucose solution in a biosafety cabinet to make the modified M9 culture medium;

[0050] 8. To store the modified M9, seal the container, cover it with aluminum foil to prevent ultraviolet light, and place it at 4°C.

[0051] CAS detection solution:

[0052] Solution A: 1 mM Ferric Chloride Stock Solution: 0.2703 g of ferric chloride hexahydrate is dissolved in 1 L of 10 mM hydrochloric acid;

[0053] Solution B: CAS Stock Solution: 0.2421 g of CAS (Chromeazurol sulphonate) is dissolved in 200 mL of deionized water;

[0054] Solution C: HTDMA Solution (Cetyltrimethylammonium Bromide): Weigh 0.0219 g of HTDMA and dissolve it in 50 mL of deionized water;

[0055] Solution D: Piperazine Buffer Solution: Weigh 4.3079 g of anhydrous piperazine and dissolve it in 30 mL of water, adjust the pH to 5.6 with hydrochloric acid;

[0056] Take 1.5 mL of Solution A, add 7.5 mL of Solution B and mix well. While stirring, add 50 mL of Solution C, then add 30 mL of Solution D, and finally add 11 mL of deionized water to make a final volume of 100 mL of CAS detection solution.

[0057] CAS Detection Medium Plate:

[0058] Prepare PIPES buffer solution: Dissolve 15.12 g of PIPES in 375 mL of ddH2O, stir gently, adjust the pH to 6.8 with 5 M NaOH, add deionized water to make the volume reach 450 mL, and add 5 g of agarose to the solution. Autoclave the PIPES buffer solution and the CAS detection solution at 121 °C for 30 minutes. Carefully add 50 mL of the CAS detection solution to all (450 mL) of the PIPES buffer solution in a biosafety cabinet.

[0059] The ability of Sphingomonas novella A58 to secrete IAA was determined by the Salkowski colorimetric method. The principle is that under the action of perchloric acid, indole-3-acetic acid turns red when it meets FeCl3, and the deeper the color, the more IAA is secreted. The well-activated strain of Sphingomonas novella A58 was inoculated into R2A liquid medium (Qingdao Haibo, product number HB0167-2) containing 200 mg·L -1 L-tryptophan and cultured in a shaker at 28 °C and 200 rpm for 72 h. Pipette 1 mL of the bacterial solution into a 1.5 mL centrifuge tube, centrifuge at 12000×g for 5 min, pipette 100 μL of the supernatant into a 96-well plate, and at the same time add 100 μL of Salkowski reagent. Use the culture solution without inoculating Sphingomonas novella A58 as a control and add an equal volume of Salkowski reagent. Let it stand at room temperature in the dark for 30 min, and then measure the OD 530 absorbance value. The prepared 400 mg·L -1The IAA standard solution was diluted, and the IAA concentration gradients were 0, 25, 50, 100, 200, 400 mg·L -1 , and then mixed with an equal volume of Salkowski reagent and left to stand in the dark at room temperature for 30 min. Then, the absorbance value of OD 530 was measured with an enzyme-labeled instrument, the data were statistically analyzed, the IAA standard curve was plotted, and the concentration of IAA produced by the target strain was calculated.

[0060] After the novel Sphingomonas sp. A58 grew on the CAS detection medium plate for 3 days, an obvious yellow halo was found ( Figure 5 ), indicating that it could produce siderophores; the siderophore activity (su) of the strain was 54.46 ± 2.35, and As / Ar was 0.48 ± 0.01, indicating that the novel Sphingomonas sp. A58 was a high siderophore-producing strain. It can be seen from Figure 6 that the reaction of an equal volume of the supernatant of the novel Sphingomonas sp. A58 with Salkowski reagent made it turn red, and the IAA production was quantitatively calculated to be 21.58 ± 2.71 mg / L (Table 2).

[0061] Table 2 Determination of the siderophore-producing ability and IAA ability of the novel Sphingomonas sp. A58

[0062]

[0063] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be regarded as a limitation of the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, several improvements and refinements can be made without departing from the spirit and scope of the present invention, and these improvements and refinements should also be regarded as the protection scope of the present invention.

[0064] SEQ ID NO.1 (16S rRNA sequence of Sphingomonas sp. A58)

[0065]

Claims

1. A strain of Sphingomonas sp. A58, characterized in that, The preservation number is: GDMCC No: 64393.

2. Use of Sphingomonas sp. A58 according to claim 1 in the prevention and treatment of citrus canker.

3. The application according to claim 2, wherein The pathogen of the citrus canker is Xanthomonas citri subsp. citri.

4. Use of Sphingomonas sp. A58 according to claim 1 in the production of siderophores and IAA.

5. Use of Sphingomonas sp. A58 according to claim 1 in promoting plant growth and iron uptake.

6. The application according to claim 5, wherein It is to irrigate plants with the fermentation broth of Sphingomonas sp. A58 to promote plant iron uptake and growth.

7. A biological agent, characterized in that, Using Sphingomonas sp. A58 according to claim 1 or its fermentation broth as an active ingredient.

8. The biological agent according to claim 7, wherein The biological agent is a microbial fertilizer, a microbial pesticide or a microbial additive.

9. A method for preventing and controlling citrus canker, characterized in that, It includes the following steps: applying the biological agent according to claim 7 or 8 to citrus leaves.

10. The method according to claim 9, wherein The pathogen of citrus canker is Xanthomonas citri subsp. citri, and the OD of Sphingomonas A58 in the biological agent is 600 0.6 - 0.8.

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