A strain of Bacillus Velezii SF248 for preventing and treating citrus canker and its application

Abstract

The present invention relates to a strain of Bacillus Velez SF248 for preventing and treating citrus canker and its application. The Bacillus Velez SF248 strain has been deposited in the China Center for Type Culture Collection (CCTCC) on June 24, 2024, with a deposit number of CCTCC NO: M 20241355. The strain was isolated from field soil in Minzhu Township, suburbs of Harbin City, Heilongjiang Province. The Bacillus Velez SF248 of the present invention has a significant inhibitory effect on citrus canker pathogens, and at the same time shows a good preventive and therapeutic effect on citrus canker; it can produce auxins and siderophores to promote plant growth and iron absorption. The results of the present invention show that Bacillus Velez SF248 has great application potential in preventing and treating citrus canker and promoting plant iron absorption and growth.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology application, and in particular relates to a Velezella SF248 for preventing and treating citrus canker and an application thereof. Background Art

[0002] Citrus (Citrus spp.) is the largest fruit in my country. It belongs to the genus Citrus of the Rutaceae family and is an economic crop widely distributed in subtropical and tropical regions. According to the 2021 China Rural Statistical Yearbook, China's citrus planting area has reached 2.922 million hectares and its output has reached 55.956 million tons, accounting for 25.81% of the country's total fruit output.

[0003] Citrus canker is a worldwide disease caused by Xanthomonas citri subsp. citri (Xcc). The disease is common in major citrus growing areas in my country, causing serious economic losses. Citrus canker mainly attacks the leaves, fruits and young branches of citrus plants, causing ulcer spots to form on the surface of the fruit. In severe cases, it causes fruit drop and yield reduction, causing serious economic losses to the citrus planting industry (Duan Jiao, Liu Yang, Feng Guangda, et al. Research Progress on Citrus Canker and Its Microbial Control [J]. Journal of Microbiology, 2023, 63(05): 1944-1958. DOI: 10.13343 / j.cnki.wsxb.20230142.). Citrus canker has a strong ability to spread and can spread through wind, rain, insects, agricultural tools, etc. It can overwinter on diseased fruits and leaves, causing continuous disease.

[0004] At present, the methods for preventing and controlling citrus canker mainly rely on chemical pesticides, especially copper and zinc preparations. However, the long-term and large-scale use of chemical pesticides will lead to problems such as pesticide residues, environmental pollution and bacterial resistance. In order to reduce the use of chemical pesticides, researchers have gradually begun to explore biological control methods, using antagonistic microorganisms to inhibit the growth and spread of pathogens. These biological control agents are not only green and environmentally friendly, but also show good effects in improving crop disease resistance and promoting plant growth (Li Qicong, Jin Qiuzhu, Luo Haiyu, et al. Research Progress on Biocontrol Microorganisms for Citrus Canker [J]. Journal of Guangxi Normal University (Natural Science Edition), 2022, 40(05): 388-397. DOI: 10.16088 / j.issn.1001-6600.2022022801.).

[0005] In recent years, with the development of microbial technology, more and more studies have focused on the screening and application of efficient biocontrol strains to control citrus canker. Some studies have isolated endophytic strains with good antagonistic activity from healthy citrus leaves, and verified their control effects through in vitro leaf experiments; for example, Qian Jialin et al. found that the strain Bacillus amyloliquefaciens QC-Y from navel orange leaves can significantly reduce the incidence of citrus canker and the area of ​​leaf lesions (Qian, Jialin, et al. "Biocontrol of Citrus Canker with Endophyte Bacillus Amyloliquefaciens QC-Y." [J]. Plant Protection Science, 2021, 57(01): 1–13.). In addition, some studies have found that certain microorganisms such as Bacillus and Pseudomonas can effectively inhibit the growth of citrus canker pathogens by producing antibiotics and secreting enzyme substances. For example, Chen Jiaomei et al. screened out three Bacillus strains with good protective effects against citrus canker, all of which had strong protease activity (Chen Jiaomei, Cai Xueqing, Qiu Sixin, et al. Isolation and identification of biocontrol bacteria against citrus canker [J]. Journal of Tropical Crops, 2014, 35(07): 1398-1403).

[0006] Patent CN117946926A discloses a Bacillus Velez QK-1, with a deposit number of CGMCCNO.27570 and a deposit date of June 7, 2023. Bacillus Velez QK-1 can have an inhibitory effect on a variety of plant diseases, and can effectively inhibit Rhizoctonia solani, Phytophthora, Black Cordyceps, Anthracnose, Botrytis cinerea, Fusarium oxysporum and Citrus canker pathogen Xanthomonas citrus subspecies, with inhibition rates greater than 35%. In actual production, Bacillus Velez bacteria agents can be widely used in the prevention and treatment of a variety of plant fungal and bacterial diseases. The Bacillus Velez bacteria agent of this scheme emphasizes that it has a broad-spectrum inhibitory effect on a variety of plant diseases, while the inhibition rate for Citrus canker pathogen Xanthomonas is only 34.26%.

[0007] Patent CN114107124A discloses a strain of Bacillus velezensis D-1 and its preparation and application. The name of Bacillus velezensis D-1 is Bacillus velezensis D-1, which was deposited in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures on October 12, 2021, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.23579. The application discloses that the strain has significant prevention and control effects on three types of Fusarium that cause taro dry rot, and also has good inhibitory effects on tea Dichaete spore leaf spot pathogen, kiwi black spot pathogen, kiwi coryneformis leaf spot pathogen, kiwi fruit rot pathogen, kiwi canker pathogen, pepper bacterial spot pathogen, rice white leaf blight pathogen and citrus canker pathogen, showing a broad-spectrum antibacterial activity. Fungi and pathogenic bacteria. The Bacillus Velez bacteria agent in this program also emphasizes its broad-spectrum inhibitory effect on a variety of plant diseases, and the inhibition zone for the citrus canker pathogen Xanthomonas is only 15.05mm.

[0008] At present, there is still a lack of effective and stable biological control products on the market, especially in terms of effectiveness and ease of operation in practical applications. Therefore, it is urgent to screen and obtain more biological resources from the natural environment to provide more effective solutions for the prevention and control of citrus canker. Summary of the invention

[0009] Based on the current situation that the prior art lacks a biocontrol strain with outstanding antibacterial effect on citrus canker pathogen Xanthomonas sp., the present invention provides a strain of Bacillus Velezii SF248 for preventing and treating citrus canker and application thereof.

[0010] The Velez Bacillus SF248 of the present invention has a significant antagonistic effect on citrus canker bacteria and can promote plant growth and iron absorption.

[0011] The Bacillus Velezii SF248 of the present invention can be used as an antagonist of citrus canker bacteria, and provides technical support for the prevention and treatment of citrus canker bacteria diseases.

[0012] The purpose of the present invention can be achieved by the following technical solutions:

[0013] The first aspect of the present invention is to provide a strain of Bacillus velezensis SF248, which was isolated from field soil in Minzhu Township, suburb of Harbin City, Heilongjiang Province on October 4, 2018, and named Bacillus velezensis SF248. The Bacillus velezensis SF248 was deposited in China Center for Type Culture Collection (CCTCC) on June 24, 2024, with a deposit number of CCTCC NO: M 20241355. The deposit address is: Wuhan University, Wuhan, China.

[0014] Through solid culture medium plate culture, it was observed that the colony color of the Bacillus Velez SF248 was white, the edges were not smooth and regular, the surface was rough and opaque.

[0015] The Bacillus Velez SF248 has an antagonistic effect on Xanthomonas citri.

[0016] The plate confrontation culture method was used to detect that Bacillus Velezii SF248 showed significant antagonistic activity against Xanthomonas citri.

[0017] The second aspect of the present invention is to provide a method for isolating the Velez Bacillus SF248: using a gradient dilution plating method, adding sterile water to a soil sample to dissolve and shake for 20 minutes, diluting it in turn into soil bacteria suspensions of different gradients, and respectively spreading them on a NA plate inoculated with citrus canker bacteria, observing the formation of an inhibition zone, streaking the colonies that can produce obvious inhibition zones on the NA plate to obtain single colonies, performing PCR amplification using the bacterial 16S rRNA gene, and constructing a phylogenetic tree to clarify the taxonomic status of the strain.

[0018] The 16S rRNA gene of Bacillus velez SF248 is shown as SEQ ID NO.1.

[0019] The invention uses 16S rRNA gene to construct a phylogenetic tree, performs comparative analysis of kinship, and proves that the strain is Bacillus velezensis.

[0020] The third aspect of the present invention is to provide the 16S rRNA gene of the Bacillus Velezii SF248, as shown in SEQ ID NO.1.

[0021] The fourth aspect of the present invention is to provide an application of the Bacillus Velezii SF248, selected from one or more of the following applications:

[0022] Application as an antagonist of citrus canker bacteria;

[0023] Application as a control agent for citrus canker;

[0024] Application as plant growth promoter;

[0025] Application as an agent to promote plant iron absorption.

[0026] In a specific embodiment of the present invention, the Bacillus Velezii SF248 has a significant antagonistic effect on citrus canker pathogen, and the citrus canker pathogen is Xanthomonas citri Xcc29-1.

[0027] In a specific embodiment of the present invention, the Bacillus Velezii SF248 has a significant preventive and therapeutic effect on citrus canker, and the citrus canker pathogen is Xanthomonas citri Xcc29-1.

[0028] In a specific embodiment of the present invention, the Bacillus Velezii SF248 can produce indoleacetic acid (IAA) to promote plant growth.

[0029] In a specific embodiment of the present invention, the Bacillus Velezii SF248 can produce siderophores to promote iron absorption in plants.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] The Velez Bacillus SF248 provided by the present invention has a significant antagonistic effect on citrus canker pathogens, and at the same time, it is verified by a living biocontrol experiment that it has a certain preventive and therapeutic effect on citrus canker diseases. In addition, the strain also has the ability to produce indoleacetic acid (IAA) and siderophores, which can promote plant growth and iron absorption. The results of the present invention show that the strain has good application potential in preventing and treating citrus canker, and its biocontrol potential needs to be further developed, which will lay a foundation for the research and development of multifunctional biocontrol agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 .Colony morphology of Bacillus velez SF248 on NA medium.

[0033] Figure 2 .16S rRNA gene gel electrophoresis results. A represents the marker; B represents the product of the 16S rRNA gene.

[0034] Figure 3 .Neighbor-Joining phylogenetic tree of Bacillus velez SF248 based on 16S rRNA gene.

[0035] Figure 4 .Antagonistic effect of Bacillus Velez SF248 on Xanthomonas citri Xcc29-1.

[0036] Figure 5 .Antagonistic effect of supernatant of Bacillus velez SF248 on Xanthomonas citri Xcc29-1.

[0037] Figure 6 . Actual picture of the evaluation of the control effect of Bacillus Velez SF248 on living leaves; Figure 6 In the figure, A is control, B is prevention, and C is treatment.

[0038] Figure 7.Test on indoleacetic acid production by Bacillus velez SF248.

[0039] Figure 8 .The effect of Bacillus Velez SF248 producing siderophore on CAS medium. DETAILED DESCRIPTION

[0040] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0041] The bacterial culture medium used in the following examples is as follows:

[0042] NA solid medium (g / L): 3 g beef extract, 5 g polypeptone, 10 g sucrose, 1 g yeast powder, 15 g agar powder, dissolve in water and make up to 1000 mL, adjust pH to 7.0-7.2, and sterilize by high pressure (121°C, 20 min).

[0043] NB liquid culture medium (g / L): 3 g beef extract, 5 g polypeptone, 10 g sucrose, 1 g yeast powder, dissolve in water and make up to 1000 mL, adjust pH to 7.0-7.2, and sterilize by high pressure (121°C, 20 min).

[0044] LB liquid culture medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, dissolve in water to 1 L, pH 7.0-7.2, and sterilize by high pressure (121°C, 20 min).

[0045] Example 1. Acquisition of Bacillus Velez SF248

[0046] 1. Soil source

[0047] Soil collected from the field in Minzhu Township, Harbin, Heilongjiang Province on October 4, 2018

[0048] 2. Screening of strains

[0049] (1) Soil sample collection

[0050] Soil samples of about 10 cm from the surface of the winter wheat field were collected, and 200 g of soil samples were collected at each point. Three soil samples were collected from each plot of land, and the time, location and type of sampling were recorded. The collected soil samples were stored at room temperature for bacterial isolation.

[0051] (2) Isolation of bacteria

[0052] Plate dilution method: weigh 10g soil sample into a conical flask, add 90mL sterile water, and then shake in a shaker at 200rpm and 28℃. Take it out after 20min and let it stand at room temperature for 10min to prepare the soil bacteria suspension stock solution. The stock solution was diluted stepwise to obtain 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , a total of 6 gradient dilutions. Take 200 μL of each bacterial suspension dilution and spread it evenly on the rice leaf streak pathogen RS105 (OD 600 =2.0) on NA plates, with 3 replicates for each gradient. The plates were placed in a 28°C incubator for 48 hours and observed.

[0053] (3) Bacterial purification

[0054] Observe and pick out single colonies with obvious inhibition zones, perform streak purification on NA plates, culture them upside down in a 28°C incubator, pick out single colonies after 12 hours, and number them in sequence.

[0055] (4) Preservation of bacteria

[0056] A single colony of the strain was inoculated into NB liquid culture medium, and after culturing in a shaker at 28°C and 180 rpm for 12 h, 1 mL of the bacterial solution was aspirated with 1 mL of 50% sterile glycerol, and the mixture was gently shaken and stored at -80°C for a long term.

[0057] The colony morphology of Bacillus velez SF248 is as follows Figure 1 shown.

[0058] Example 2 Identification of 16S rRNA Gene of Bacillus Velezii SF248

[0059] The gene was amplified using colony PCR technique.

[0060] Using bacterial 16S rRNA gene primers: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGGCTACCTTGTTACGACTT-3', a small amount of fresh colonies were picked up with a pipette tip as a template for PCR amplification to obtain the target fragment. The PCR reaction system is:

[0061] Table 1 Ex-Taq polymerase chain reaction system

[0062]

[0063] The basic conditions of PCR reaction are: 95℃ pre-denaturation for 3min, 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 60s, 72℃ pre-extension for 10min, 4℃ storage, a total of 30 cycles. After the reaction, the PCR products were tested by 1% agarose gel electrophoresis, and the results were detected and recorded using a gel imager. The gel electrophoresis results of the 16S rRNA gene are shown in Figure 2 As shown, Figure 2 A represents marker; B represents the product of 16S rRNA gene. The PCR stock solution was sent to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared by BLAST on the NCBI website, and the phylogenetic tree was constructed by MEGA 11 software to determine the species of closely related bacterial strains.

[0064] The 16SrRNA gene of Bacillus Velezii SF248 is shown in SEQ ID NO.1, specifically:

[0065]

[0066] The Neighbor-Joining phylogenetic tree constructed by comparing the 16S rRNA gene sequences of Bacillus velez SF248 is shown in Figure 2. Figure 3 As shown, the results showed that the 16SrRNA gene of SF248 had a similarity of 99.58% with Bacillus velezensis strain FZB42 and was in the same evolutionary tree branch as Bacillus velezensis, so F248 should belong to Bacillus velezensis.

[0067] Example 3 Determination of the antagonistic activity of Bacillus Velezii SF248 and its supernatant against Xanthomonas citri

[0068] A single colony of Xanthomonas citri Xcc29-1 was placed in NB medium, and a single colony of Bacillus velez SF248 was placed in LB medium. After culturing for 12 h in a shaking incubator at 28°C and 180 rpm, the bacterial solution concentration was adjusted to OD 600 =2.0. Pipette 200 μL of Xcc29-1 bacterial solution and mix thoroughly with about 25 mL of NA solid medium, then pour the plate, and then place an Oxford cup with a diameter of 8 mm in the center of the NA plate. Each Oxford cup is filled with 50 μL of SF248 bacterial solution. Repeat 3 times and place in a 28°C incubator. After 48 hours of incubation, observe the presence of inhibition zones, record the size of the inhibition zones, and take photos.

[0069] A single colony of Xanthomonas citri Xcc29-1 was picked and cultured in NB medium at 28°C and 180 rpm for 12 h. After that, the concentration of the bacterial solution was adjusted to OD 600 =2.0. Pick a single colony of Bacillus SF248 in LB medium, culture it in a shaker at 28℃ and 180rpm for 48h, centrifuge it at 12000rpm for 5min, aspirate the supernatant, and filter it through a bacterial filter with a diameter of 0.22μm to obtain a sterile supernatant. Pipette 200μL of the bacterial solution of Xcc29-1 and mix it thoroughly with about 25mL of NA solid medium, pour the plate, and then place an Oxford cup with a diameter of 8mm in the center of the NA plate. Each Oxford cup is filled with 200μL of SF248 sterile supernatant. Repeat 3 times and place it in a 28℃ incubator. After culturing for 48h, observe the presence of the inhibition zone, record the size of the inhibition zone, and take pictures.

[0070] The antagonistic effect of Bacillus Velez SF248 on Xanthomonas citri Xcc29-1 is shown in the figure Figure 4 shown.

[0071] The antagonistic effect of the sterile supernatant of Bacillus Velez SF248 on Xanthomonas citri Xcc29-1 is shown in the figure Figure 5 shown.

[0072] The results showed that the diameter of the inhibition zone of Bacillus Velez SF248 against Xanthomonas citri Xcc29-1 was 2.23±0.1247cm, and the inhibition rate was 64.07%±0.43%. The diameter of the inhibition zone of the sterile supernatant of Bacillus Velez SF248 against Xanthomonas citri Xcc29-1 was 2.97±0.0471cm, and the inhibition rate was 73.03%±1.97%. This shows that SF248 has a significant antagonistic effect on citrus canker bacteria, and its antibacterial active substances can be secreted outside the cells and play a role in the supernatant.

[0073] Example 4 Determination of the control effect of Bacillus Velezii SF248 on citrus canker

[0074] A single colony of Xanthomonas citri Xcc29-1 was placed in NB medium, and a single colony of Bacillus velez SF248 was placed in LB medium. After culturing in a shaker at 28°C and 180 rpm for 24 h, the concentration of the Xanthomonas citri strain was adjusted to OD 600 =0.6, the concentration of Bacillus Velez SF248 solution is OD 600 =2.0.

[0075] Control group: select leaves of living plants with the same growth status, prick them with a needle, spray inoculate with Xanthomonas citri strain, repeat 3 times, put them in bags, observe the disease conditions of the leaves (ulcer spots, yellow halos) after 14 days of growth, take pictures and record them.

[0076] Prevention group: Select leaves of living plants with the same growth status, prick wounds with a needle, spray inoculate with Bacillus Velez SF248, repeat 3 times, bag overnight, and spray inoculate with Xanthomonas citri after 24 hours. After 14 days of growth, observe the disease conditions of the leaves (ulcer spots, yellow halos), take pictures and record them.

[0077] Treatment group: Select leaves of living plants with the same growth status, prick wounds with a needle, spray inoculate with Xanthomonas citri solution, repeat 3 times, bag overnight, and spray inoculate with Bacillus Velez SF248 after 24 hours. After 13 days of growth, observe the disease conditions of the leaves (ulcer spots, yellow halos), take pictures and record them.

[0078] Schematic diagram of the control effect of Bacillus Velez SF248 on citrus canker Figure 6 shown.

[0079] The results showed that Bacillus Velezii SF248 had a certain preventive and therapeutic effect on citrus canker, with a therapeutic effect of 57.78±4.16% and a preventive effect of 96.30%±3.46%.

[0080] Example 5 Determination of the ability of Bacillus Velezii SF248 to produce indoleacetic acid

[0081] Pick a single colony of Bacillus Velezii SF248 in LB medium and culture it in a shaker at 28°C and 180 rpm for 24 h. Adjust the bacterial solution concentration to OD 600 =2.0, take 0.2mL and inoculate it into 4mL LB medium containing 3mmol / L tryptophan, and culture it in a shaking incubator at 28℃ and 220r / min for 48h. Take the fermentation broth and centrifuge it at 10000r / min for 5min, take an equal amount of supernatant and react with PC colorimetric solution, protect from light for 30min, and use a microplate reader to measure OD 530 nm value.

[0082] Preparation of IAA standard curve: Prepare standard solution of IAA with concentration gradient of 1, 2, 5, 10, 20, 50, 100 mg / L in LB medium, take equal amount of standard solution and PC colorimetric solution for color reaction, protect from light for 30 minutes, and use microplate reader to measure OD 530 nm value, make a standard curve, y = 0.0157x + 0.1137; y = OD 530 , x = IAA concentration.

[0083] Preparation of PC colorimetric solution: weigh 12g of FeCl3 and dissolve it in 300mL of distilled water, then slowly add 429.7mL of 98% H2SO4 and adjust the volume to 1L after cooling (prepare and use immediately).

[0084] The test results of indoleacetic acid production by Bacillus Velez SF248 are as follows Figure 7 shown.

[0085] The results showed that an obvious color change occurred after the color development reaction between equal amounts of supernatant and PC colorimetric solution, indicating that Bacillus Velezii SF248 can produce indoleacetic acid to promote plant growth at a concentration of 3.31 mg / L.

[0086] Example 6 Identification of siderophore in Bacillus velez SF248

[0087] Pick a single colony of Bacillus Velezii SF248 in LB medium and culture it in a shaker at 28°C and 180 rpm for 24 h. Adjust the bacterial solution concentration to OD 600 =2.0. Place a filter paper in the center of the CAS test medium, drop 5 μl of bacterial solution on the filter paper, and culture at 28°C for 48 hours.

[0088] The test results of siderophore production by Bacillus Velez SF248 are as follows Figure 8 shown.

[0089] The results showed that in the CAS test medium, a clearly visible transparent iron carrier secretion circle was formed around Bacillus Velez SF248, which indicated that Bacillus Velez SF248 could secrete iron carriers, efficiently bind iron in the surrounding environment, and promote iron absorption in plants.

[0090] Therefore, the Velez subtilis SF248 provided by the present invention has a significant antagonistic effect on citrus canker pathogens, has a significant control effect on citrus canker diseases, and provides a new resource for biological control of citrus canker diseases. At the same time, Velez subtilis SF248 can also produce indoleacetic acid and siderophores, so it has great application potential in promoting crop growth and iron absorption.

[0091] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A strain of Bacillus Velezii SF248, characterized in that: It was deposited in the China Center for Type Culture Collection (CCTCC) on June 24, 2024, with the deposit number CCTCC NO: M 20241355.

2. The strain of Bacillus Velezii SF248 according to claim 1, characterized in that: The 16S rRNA gene sequence of the Bacillus Velezii SF248 is shown in SEQ ID NO.

1.

3. The use of Bacillus Velezii SF248 as claimed in claim 1, characterized in that: The application of the Velez Bacillus SF248 in the preparation of a citrus canker disease prevention and control agent.

4. The use of Bacillus Velezii SF248 as claimed in claim 1, characterized in that: The Velez Bacillus SF248 is used as an antagonistic strain against citrus canker bacteria.

5. The use of the strain Bacillus Velezii SF248 according to claim 4, characterized in that: The citrus canker pathogen is Xanthomonas citri subspecies 29-1.

6. Use of the Bacillus Velezii SF248 described in claim 1 in the production of indoleacetic acid.

7. Use of the Bacillus Velezii SF248 according to claim 1 in producing siderophores.

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