Novel sphingomonas and application thereof in prevention and treatment of citrus canker

Abstract

The invention discloses a novel sphingomonas strain and application thereof in prevention and treatment of citrus canker. The invention discloses sphingomonas sp. A58, the preservation number of the sphingomonas sp. A58 is GDMCC No: 64393, and the sphingomonas sp. A58 is preserved in Guangdong Microbial Culture Collection Center on March 7, 2024. The strain is a new species of sphingomonas. The novel sphingomonas A58 bacterial liquid disclosed by the invention is sprayed on citrus leaves, so that the disease and disease index of the citrus leaves can be obviously reduced; and moreover, siderophores and IAA can be efficiently generated. Therefore, the novel sphingomonas A58 has great application potential in the aspects of preventing and treating citrus canker, promoting iron absorption and growth of crops and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a novel sphingomonas strain and application thereof in preventing and treating citrus canker. Background Art

[0002] Citrus is the main economic crop in southern my country. Citrus canker is an important quarantine disease and one of the important diseases in China's citrus producing areas. Citrus canker is mainly caused by Xanthomonas citrus subspecies citrus, which mainly causes damage to citrus branches, leaves and fruits, seriously affecting the quality of citrus. At present, the method of preventing and controlling citrus canker is mainly through chemical agents. However, the improper use of chemical agents makes the pathogens resistant and pollutes the environment, which is not in line with the current purpose of food security. Therefore, it is very necessary to study a safe and efficient prevention and control method. Biological control is popular among the public because of its safety, high efficiency, low pollution and low cost. In recent years, more and more scholars have studied biological control, and there is great potential to use biological control methods to deal with citrus canker.

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

[0004] Iron is one of the essential trace nutrients for plant growth and development. In plants, iron plays many important physiological and metabolic roles, especially in the synthesis of chlorophyll and the respiratory process. The presence of siderophores can enhance the growth-promoting properties of plants, provide the iron required by plants, and enhance the growth and development of plants by promoting chlorophyll synthesis and photosynthesis. IAA can alleviate the damage of biotic and abiotic stresses to plants by reducing membrane lipid peroxidation, improve the adaptability of plants under stress environments, and promote plant growth and increase yield. The synergistic effect of siderophores and IAA can better enhance the stress resistance of plants and help plants grow healthily. Therefore, screening siderophore-producing strains that synergize with plant secretions has great application value.

[0005] In summary, Sphingomonas, as an important microbial resource, can be effectively used in the development of microbial fertilizers and agents by enhancing the diversity of microbial resources and exploring and identifying their potential functions. This application can not only promote the ecological balance of agriculture, but also is an important means to promote the development of sustainable agriculture and green agriculture. Summary of the invention

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

[0007] The novel Sphingomonas provided by the present invention is a potential new species among Sphingomonas, which not only has a significant preventive and therapeutic effect on citrus canker, but also has the functions of producing siderophores and indoleacetic acid (IAA), and has great development potential in plant stress resistance and growth promotion.

[0008] The second object of the present invention is to provide the use of the above-mentioned Sphingomonas A58 in preventing and treating citrus canker.

[0009] Preferably, the pathogen of citrus canker is X. citrus canker Xcc.

[0010] The third object of the present invention is to provide the use 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 use of the above-mentioned Sphingomonas A58 in promoting plant growth and iron absorption.

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

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

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

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

[0016] Preferably, the pathogen of citrus canker is citrus canker Xcc, and the OD of Sphingomonas A58 in the biological preparation is 600 =0.6-0.8.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

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

[0019] The present invention first discovered that the novel Sphingomonas A58 can significantly reduce the symptoms and disease index of citrus canker, and can also produce siderophores and IAA, and has multiple beneficial functions of disease resistance and growth promotion. Therefore, the discovery and identification of the novel Sphingomonas A58 will provide new ideas for the development of new biological agents and means for the prevention and treatment of citrus canker.

[0020] Sphingomonas sp. A58 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on March 7, 2024, with the address at 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 pathogen of citrus canker 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 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 the new Sphingomonas A58 were collected from Jinyou Park, Nanfu Village, Yanyang Town, Meixian District, Meizhou City, Guangdong Province (N24°24′50″, E116°19′54″). The collected leaves were cut into 5mm×5mm cubes in a sterile bag and mixed evenly. Sterile water was added at a ratio of 1:10 according to the weight of the leaves, and the ground leaves were diluted 10 times according to the gradient homogenate. 100μL of each concentration was applied to NA culture medium (Guangdong Huankai, product number 022020) for inoculation. After culturing in a 28°C incubator for 5 days, the colonies were selectively picked with an inoculation loop according to the characteristics of the colonies and purified and cultured. The colony morphology of the new Sphingomonas A58 after 3 days of culture on NA culture medium is as follows Figure 1 As shown, a single colony is round, 1-2 mm in diameter, orange-yellow in color, with a smooth, opaque surface and neat edges.

[0030] Example 2: 16S rRNA gene sequence analysis of novel Sphingomonas sp. A58

[0031] The DNA of the novel Sphingomonas A58 was extracted by alkaline lysis method: a small amount of bacteria was placed in a 200 μL centrifuge tube with 16.6 μL alkaline lysis solution, and the bacteria were lysed at 95°C for 30 minutes in an alkaline environment; after cooling, 16.6 μL of neutralization buffer was added to each well, mixed, 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). The 16S rRNA gene sequence of the novel sphingomonas A58 was amplified using the bacterial 16S rRNA specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCA ATCGC-3′). The amplified product was about 1400 bp after electrophoresis analysis, and the amplified product was sent to Beijing Qingke 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), which was 1490 bp in length. The 16S rRNA gene sequence of sphingomonas A58 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that the 16S rRNA gene sequence of Sphingomonas rubra CGMCC 1.9113 (accession number, jgi.1058074) had the highest similarity of 98.30% between Sphingomonas A58 and the model strain Sphingomonas rubra CGMCC 1.9113.

[0032] Example 3: Full gene sequence analysis of novel Sphingomonas A58

[0033] The bacterial culture of the new Sphingomonas A58 was sent to Shanghai Meiji Biotechnology Co., Ltd. for genome sequencing. The genome of the strain Sphingomonas A58 was analyzed using the software QUASTv5.0.2. The results showed that the genome sequence of the new Sphingomonas A58 contained 19 contigs, the total genome length was 2988474bp, and the G+C content of the genomic DNA was 70.55%. The software UBCGv3.0 was used to construct a phylogenetic tree of the genome of the new Sphingomonas A58 and closely related reference model strains. The results are as follows Figure 2 As shown, Sphingomonas A58 is most closely related to Sphingomonas abaci, Sphingomonasmetalli, Sphingomonas rubra, and Sphingomonasjinjuensis. Compared with the three most closely related strains, the ANI value of Sphingomonas A58 is 77.61-85.29%, which is lower than the critical value of 95-96% proposed for species delimitation, and the dDDH value is between 23.50-53.3%, which is lower than the species delimitation threshold of 70% (Table 1). The results of ANI and dDDH support that the strain Sphingomonas A58 is a new species of the genus Sphingomonas.

[0034] Therefore, it was named Sphingomonas sp. A58. The strain has been deposited in Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit number is GDMCC No: 64393, and the deposit date is March 7, 2024.

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

[0036]

[0037] Example 4: Antagonism of the novel Sphingomonas sp. A58 against citrus canker pathogen Xcc and its ability to prevent and treat canker

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

[0039] The ability of the new Sphingomonas A58 to prevent and treat citrus canker was verified by using isolated pomelo leaves. The leaves were fully expanded for one week, cleaned, and placed in a sterile culture dish with a layer of sterile wet filter paper at the bottom. After pricking the inoculation point on the back of the leaf with an inoculation needle, the OD 600 = 0.6 A58 bacterial suspension was sprayed on the leaves. After 24 hours, the spraying concentration was about 10 8 CFU / mL of citrus canker. The petioles were wrapped with cotton containing sterile water to keep them moist. The control leaves were only inoculated with citrus canker. The experiments were performed in a sterile operating table. The incidence of the puncture points was observed 5 days after inoculation, and the lesion area was measured using ImageJ 2.0 software. The lesion area (R / mm 2 ) is divided into 6 levels. 1: R≤2, 2:2 <R≤4,3:4<R≤6),4:6<R≤8,5:8<R≤10,6:R> 10. Disease index = Σ[number of lesions at each level × corresponding level] / (total number of lesions × maximum level) × 100%. Repeat 3 times. Figure 4 As shown, compared with CK, spraying A58 bacterial solution on pomelo leaves significantly reduced the lesion area and disease index by 60.40% and 50.50%, respectively.

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

[0041] The strain Sphingomonas A58 was activated and cultured on NA plates at 28°C for 48 hours. A single colony was picked with a toothpick and spotted on a CAS test medium plate. After culturing at 28°C for 3 days, the solubility index of the strain was calculated. A single colony was picked and inoculated into a modified M9 medium, and cultured on a shaking table at 28°C and 150 r / min for 2 days. The bacterial suspension was centrifuged at 10000 r / min for 10 minutes, and the supernatant was mixed with an equal volume of CAS test solution, left to stand for 1 hour, and its OD was measured. 630The absorbance value (As) was measured by the same method with the culture fluid of the non-inoculated Sphingomonas A58 strain as the control (Ar), and the siderophore activity (su) was calculated using the following formula: su = [(Ar-As) / Ar] × 100%. The siderophore production capacity of the strain was determined by As / Ar. The smaller the ratio, the stronger the siderophore production capacity. As / Ar values ​​of 0-0.6 were high-yield siderophores; As / Ar values ​​of 0.6-0.8 were medium-yield siderophores; and As / Ar values ​​of 0.8-1.0 were low-yield siderophores.

[0042] Modified M9 medium:

[0043] 1. Prepare M9 salt solution. The composition and concentration 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 balance is water;

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

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

[0046] 4. Prepare PIPES buffer by dissolving 6.048 g PIPES in 156 mL 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 a 20% glucose solution.

[0048] 6. Prepare solutions. Except for the glucose solution, all other solutions are sterilized by autoclave. The glucose solution is filtered through an autoclave filter (0.22 μm) before use.

[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 prepare a modified M9 culture medium;

[0050] 8. To store the modified M9, seal the container, cover with aluminum foil to protect against UV rays, and place at 4°C.

[0051] CAS test fluid:

[0052] Solution A: 1 mM ferric chloride stock solution: 0.2703 g ferric chloride hexahydrate dissolved in 1 L 10 mM hydrochloric acid;

[0053] Solution B: CAS stock solution: 0.2421 g CAS (Chromeazurolsulphonate) dissolved in 200 mL deionized water;

[0054] Solution C: HTDMA solution (hexadecyltrimethylammonium bromide): weigh 0.0219 g HTDMA and dissolve it in 50 mL deionized water;

[0055] Solution D: Piperazine buffer: 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 1.5mL of solution A and add 7.5mL of solution B to mix well. Add 50mL of solution C while stirring, then add 30mL of solution D, and finally add 11mL of deionized water to finally prepare 100mL of CAS detection solution.

[0057] CAS assay medium plates:

[0058] Prepare PIPES buffer solution: dissolve 15.12g PIPES in 375mL ddH2O, stir gently, adjust pH to 6.8 with 5M NaOH, add deionized water to 450mL, add 5g agarose to the solution. Autoclave PIPES buffer solution and CAS detection solution at 121℃ for 30 minutes. Carefully add 50mL CAS detection solution solution to the total (450mL) PIPES buffer solution in the biosafety cabinet.

[0059] The ability of the novel Sphingomonas A58 to secrete IAA was determined by the Salkowski colorimetric method. The principle is that under the action of perchloric acid, indoleacetic acid will turn red when it encounters FeCl3. The darker the color, the more IAA is secreted. The activated Sphingomonas A58 strain was inoculated in a solution containing 200 mg·L -1 L-tryptophan R2A liquid medium (Qingdao Haibo, catalog number HB0167-2), 28°C, 200rpm shaker culture for 72h. Pipette 1mL of bacterial solution into a 1.5mL centrifuge tube, centrifuge at 12000×g for 5min, pipette 100μL of supernatant into a 96-well plate, and add 100μL of Salkowski reagent. Use the culture medium without inoculation of Sphingomonas A58 as a control, and add an equal volume of Salkowski reagent. Stand at room temperature in the dark for 30min, and then measure OD with an enzyme reader 530 The absorbance value of the prepared 400mg·L -1IAA standard solution was diluted to a concentration gradient of 0, 25, 50, 100, 200, and 400 mg·L -1 Then, it was mixed with an equal volume of Salkowski reagent and placed in the dark at room temperature for 30 min, and then the OD was measured with an enzyme-labeled instrument. 530 The absorbance values ​​were statistically analyzed, the IAA standard curve was drawn, and the concentration of IAA produced by the target strain was calculated.

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

[0061] Table 2 Determination of the ability of the new Sphingomonas A58 to produce siderophore and IAA

[0062]

[0063] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

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

[0065]

Claims

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

2. Use of Sphingomonas A58 according to claim 1 in preventing and treating citrus canker.

3. The use according to claim 2, characterized in that: The pathogen of citrus canker is citrus canker pathogen Xcc.

4. Use of Sphingomonas A58 according to claim 1 in producing siderophores and IAA.

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

6. The use according to claim 5, characterized in that: The method is to water plants with the fermentation liquid of Sphingomonas A58 to promote plant iron absorption and growth.

7. A biological agent, characterized in that: The Sphingomonas A58 or its fermentation liquid as claimed in claim 1 is used as an active ingredient.

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

9. A method for preventing and treating citrus canker, characterized in that: The method comprises the following steps: applying the biological agent described in claim 7 or 8 to citrus leaves.

10. The method according to claim 9, characterized in that The pathogen of citrus canker is citrus canker pathogen Xcc, and the OD of Sphingomonas A58 in the biological preparation is 600 =0.6-0.8.

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