Bacillus amyloliquefaciens jdf1 and application thereof in preventing and treating citrus canker

Abstract

The application discloses a high-temperature-resistant Bacillus amyloliquefaciens JDF1 and application thereof in preventing and treating citrus canker. The application experiment shows that low-concentration JDF1 strains can efficiently prevent and treat citrus canker, and the supernatant of the bacterial liquid can also effectively inhibit the occurrence of the citrus canker; the JDF1 strains treated at 50 DEG C can significantly reduce the harm of Xanthomonas campestris to citrus, and have strong high-temperature resistance, thus providing a new microbial strain and a prevention and treatment method strategy for the frequently-occurring citrus canker in a high-temperature and high-humidity environment, and the application of the strain has good biological prevention and treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticide technology, specifically relating to a thermotolerant Bacillus amyloliquefaciens strain JDF1 and its application in the prevention and control of citrus canker. Background Technology

[0002] Citrus canker is caused by Xanthomonas citrus (… Xanthomonas citri subsp. citri, Xcc Citrus canker is a global plant disease that seriously threatens the sustainable development of the citrus industry. The pathogen invades the host through stomata or wounds, causing typical canker spots on leaves and fruits, leading to leaf and fruit drop and reduced quality. According to the Food and Agriculture Organization of the United Nations (FAO), the annual loss rate of citrus canker in major producing areas such as Asia and the Americas can reach 20%-30%, and there is a lack of effective treatments.

[0003] Traditional control methods mainly rely on copper-based fungicides (such as Bordeaux mixture) and antibiotics (such as streptomycin), but long-term use leads to multiple problems. Studies show that... Xcc Copper-resistant strains have emerged widely in Brazil, China, and other regions, and the overuse of antibiotics may induce cross-resistance in human pathogens. Furthermore, the strict limits on copper residues in agricultural products (≤5 mg / kg) imposed by EU regulations (EC No 396 / 2005) further restrict the application of chemical control.

[0004] Biological control has become an alternative due to its environmentally friendly characteristics, but field application requires high concentrations of bacterial solutions, which is costly and can easily trigger phytotoxic reactions. The poor thermal stability of bacterial agents and the easy loss of activity during formulation processing and application are also significant issues. Bacillus strains have attracted considerable attention due to their sporulation capacity and broad-spectrum antibacterial activity. Similarly, Bacillus subtilis QST713 (trade name Serenade®) is registered in the United States for the control of citrus diseases, but the recommended field concentration is 1×10⁻⁶. 8 CFU / mL, high doses may cause slight scorching of plant leaf margins. Bacillus licheniformis ENV123 at 1×10⁻⁶... 7 The efficacy against peptic ulcer disease was 65% at a concentration of CFU / mL, but this was reduced to 1×10⁻⁶. 6 At CFU / mL, the efficacy drops sharply to 38%, and at high concentrations, necrotic spots appear on 5%-10% of the leaves. The lipopeptides (such as surfactantin) secreted by *Bacillus amyloliquefaciens* FZB42 show a decrease in antibacterial activity of over 60% after treatment at 50°C for 2 hours, leading to significant loss of activity during high-temperature processing (such as spray drying) or storage. Similar problems exist in *Bacillus subtilis* GB03, whose antibacterial proteins are completely inactivated after treatment at 55°C for 1 hour. Summary of the Invention

[0005] To overcome the above-mentioned shortcomings of the prior art, the present invention provides a thermoresistant Bacillus amyloliquefaciens strain JDF1 ( Bacillus amyloliquefaciens JDF1 (accession number CCTCC NO.M2025865, accession date: April 23, 2025, accession institution code: CCTCC-China Center for Type Culture Collection) and its application in the control of citrus canker. This invention, through antagonistic activity assays on the pathogen *Xanthomonas citrus*, shows that JDF1 can significantly inhibit the growth of citrus canker, forming an inhibition zone, and effectively inhibit the infection of citrus canker by the pathogen. Appropriate concentrations of *Bacillus amyloliquefaciens* JDF1 bacterial suspension and supernatant can effectively control the occurrence of citrus canker. Even after treatment with a 50℃ high-temperature water bath for 6 hours, the bacterium still has a high control effect on citrus canker and does not cause necrotic lesions at wound sites.

[0006] The first objective of this invention is to provide Bacillus amyloliquefaciens JDF1 ( Bacillus amyloliquefaciens (JDF1), its accession number is CCTCC NO.M2025865.

[0007] A second objective of this invention is to provide a fermented product, which is the fermentation broth or fermentation broth filtrate of the aforementioned Bacillus amyloliquefaciens JDF1.

[0008] A third objective of this invention is to provide a biocontrol agent containing the aforementioned Bacillus amyloliquefaciens JDF1 and / or the aforementioned fermentation product as active ingredients.

[0009] Preferably, the biocontrol agent is an agent used to control citrus canker.

[0010] Preferably, the biocontrol agent is the fermentation broth of Bacillus amyloliquefaciens JDF1, and the OD of the fermentation broth is 0.2 to 1.0.

[0011] Preferably, the fermentation broth of Bacillus amyloliquefaciens JDF1 is prepared by inoculating Bacillus amyloliquefaciens JDF1 into LB liquid medium and culturing it at 28°C and 200 rpm for 24–36 h to obtain the fermentation broth of Bacillus amyloliquefaciens JDF1.

[0012] Preferably, the biocontrol agent is the fermentation broth filtrate of Bacillus amyloliquefaciens JDF1, which is prepared by the following method: Bacillus amyloliquefaciens JDF1 is inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 24–36 h to obtain the fermentation broth, which is then filtered through a 0.22 µm sterile filter membrane, and the filtrate is the fermentation broth filtrate of Bacillus amyloliquefaciens JDF1.

[0013] A fourth objective of this invention is to provide the application of the aforementioned Bacillus amyloliquefaciens JDF1, the aforementioned fermented product, or the aforementioned biocontrol agent in the prevention and control of citrus canker.

[0014] Preferably, the citrus canker is caused by Xanthomonas citrus subsp. citrus (…). Xanthomonas citri Citrus canker caused by subsp. citri.

[0015] The fifth objective of this invention is to provide a method for preventing and controlling citrus canker, which includes applying the Bacillus amyloliquefaciens JDF1, the fermented product, or the biocontrol agent to the surface of citrus plants or injecting them into citrus leaves or fruits.

[0016] The present invention has the following beneficial effects:

[0017] This invention obtained Bacillus amyloliquefaciens JDF1 through screening. The results of source, morphology and 16S rDNA sequence comparison show that it is a new Bacillus amyloliquefaciens resistant to citrus canker.

[0018] The JDF1 strain provided by this invention can effectively prevent and control citrus canker, and has a high control effect at low concentrations. In vivo experiments show that a low concentration of JDF1 bacterial solution with OD=0.2 can effectively prevent and control citrus canker, and the control effect is comparable to that of high concentrations, effectively reducing the amount used and saving production costs significantly. Low-concentration application can avoid leaf wound necrosis caused by high concentrations, and significantly improve the safety of field application.

[0019] In addition, citrus canker generally occurs in high temperature and high humidity environments. The high concentration of JDF1 bacterial solution with OD=1.0 still has a strong control effect after being treated at 50℃, and its virulence is reduced, which can avoid the problem of poor thermal stability in the field and further improve the safety of field application.

[0020] Both live JDF1 bacteria and the supernatant from the bacterial solution independently inhibited citrus canker, indicating that JDF1 possesses the dual functions of colonizing live bacteria and secreting antimicrobial metabolites. This characteristic ensures the stability of the fungicide's efficacy under different environmental conditions and avoids the risk of failure due to a single mechanism of action. Therefore, this strain can be used as an environmentally friendly green agent to replace chemical pesticides in the control of citrus canker.

[0021] The antibacterial efficiency of JDF1 bacterial agent at both high and low concentrations can reach 99.99%, and the antibacterial efficiency after high-temperature treatment is still above 99%. The unique low-concentration high efficiency, high-temperature resistance, and dual-pathway antibacterial function of JDF1 strain provide an innovative solution to overcome the bottlenecks of existing technologies. This invention provides a new microbial strain and control method strategy with excellent biological control effects.

[0022] Preservation Instructions

[0023] The present invention Bacillus amyloliquefaciens JDF1 (Bacillus amyloliquefaciens JDF1) was deposited at the China Center for Type Culture Collection (CCTCC) on April 23, 2025, with accession number CCTCC NO.M2025865, depositary code CCTCC-China Center for Type Culture Collection, and address of the depositary at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0024] Figure 1 These are the bacteria selected through screening that exhibit anti-ulcer activity on agar plates. Xcc Pathogenic bacteria with significant antibacterial effects.

[0025] Figure 2 The results show the colony morphology of strain JDF1 and its antagonistic effect against citrus canker pathogens; where A represents the single colony morphology of JDF1; B represents the plate inhibition of citrus canker pathogens; C represents the comparison of JDF1 with the morphology of antagonistic bacteria against citrus canker pathogens in published patents and papers; and D represents the 16S rDNA homology comparison results.

[0026] Figure 3 The graph shows the inhibitory effect of JDF1 bacterial suspensions with OD=0.2 and 1.0 on citrus canker after inoculation of young citrus leaves.

[0027] Figure 4 This is a graph showing the inhibitory effect of JDF1 bacterial supernatant on citrus canker after inoculation of young citrus leaves.

[0028] Figure 5 The inhibitory effect of JDF1 bacterial solution with OD=1.0 on citrus canker after being treated at 50℃ and then inoculated onto young citrus leaves (A) is compared with the inhibitory effect of JDF1 bacterial solution without high temperature treatment (B).

[0029] Figure 6 It uses citrus leaves Xcc The antibacterial effect of JDF1 was evaluated by pathogen plate counting. Detailed Implementation

[0030] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0031] The culture medium used in the following examples:

[0032] LB solid medium: 5.0 g yeast extract, 10.0 g peptone, 5.0 g NaCl, 8.0 g agar, distilled water to a final volume of 1000 mL, sterilize at 121℃ for 20 min, pour into plates for later use.

[0033] LB liquid medium: 5.0 g yeast extract, 10.0 g peptone, 5.0 g NaCl, distilled water to a final volume of 1000 mL, sterilize at 121℃ for 20 min, and set aside.

[0034] Example 1: Screening and isolation of strains

[0035] Wash the leaves of *Veronica persica* with clean water, immerse them in 75% alcohol for 30 seconds in a clean bench, and then rinse them three times with sterile water. Then cut off pieces approximately 1 cm in size. 2 Three to five leaf tissue samples were placed in 1.5 mL centrifuge tubes, 1 mL of sterile water was added, and the samples were ground thoroughly. After standing for 30 min, 100 μL of the suspension was pipetted and evenly spread onto LB solid medium. This process was repeated three times, and the samples were incubated at 28 °C for 1 day. The results were observed and recorded. Based on the color and morphology of the colonies, different bacterial strains were activated in LB liquid medium, and the resulting strains were stored at -80 °C for later use.

[0036] Example 2: Screening and Identification of Antagonistic Bacteria

[0037] In a clean bench, 100 μL of activated 1.0 × 10⁻⁶ μL was aspirated. 6 cfu / mL Xcc The bacteria were evenly spread on LB solid medium, dried, and then a nine-square grid was drawn on the bottom of the plate. Approximately 2 μL of the 1000 strains isolated in Example 1 were spotted into each square. After drying, the plates were incubated at 28°C for 1-2 days. Nine antagonistic bacteria with inhibition zones were screened. 16S rDNA sequencing results showed that these included two strains of *Bacillus amyloliquefaciens* and seven strains of *Pseudomonas*. Inoculation of citrus leaves showed that the seven *Pseudomonas* strains were highly pathogenic to citrus leaves and therefore could not be used for the control of citrus canker. The two *Bacillus amyloliquefaciens* strains effectively prevented the occurrence of citrus canker. Furthermore, strain JDF1 amplified rapidly and was easily scraped off the viscous material from the plate; therefore, it was selected for further analysis and verification. Figure 1 ).

[0038] Morphological identification: The JDF1 strains obtained from the above screening were placed separately in a 1.0×10⁻⁶ saturated tank. 6 cfu / mL Xcc When evenly spread on LB agar plates and incubated at 28°C, a distinct inhibition zone is observed. The growth morphology of colonies on LB agar plates is as follows: Figure 2 As shown in A: Single colonies are white with neat edges, semi-transparent and mucous-like. Colonies grown by spot inoculation show obvious layers and concentric rings from the inside out. Figure 2 A and B in the text). A significant morphological difference was found when compared with the morphology of antagonistic bacteria against citrus canker in published patents and papers. Figure 2The C in the sample has only 85.12% similarity to the 16S rDNA of F9, which is the closest in morphology.

[0039] 16S rDNA identification: JDF1 bacterial culture was used as a template for 16S rDNA amplification. PCR amplification was performed using universal 16S rDNA primers (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.3: 5'-TACGGCTACCTTGTTACGACTT-3'). The PCR reaction system (total 50 μL) contained 25 μL ddH2O, 19 μL Mix, 2 μL each of forward and reverse primers, and 2 μL of bacterial culture template. The reaction conditions were 98℃ for 3 min; 98℃ for 10 s, 55℃ for 15 s, and 72℃ for 20 s, for 45 cycles. After PCR amplification, 1.0% agarose gel electrophoresis was performed at 120V for 25 min. The amplified fragment length was approximately 1500 bp. The PCR product of the target band was sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing. The obtained sequence was 1438 bp in length. According to the 16S sequencing results, the nucleotide sequence of the strain's 16S rDNA is shown in SEQ ID NO.1. Homology comparison analysis was performed between the obtained strain sequence and nucleic acid sequence libraries registered on NCBI. The results showed that the 16S rDNA sequences of this strain were mostly from the genus *Bacillus* (…). Bacillus sp.) - Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Its sequence also differs from the most similar Bacillus amyloliquefaciens by 6 bases, with the highest similarity being 99%. Figure 2 (D in the text). These results demonstrate that this JDF1 strain, resistant to citrus canker, is a novel Bacillus amyloliquefaciens.

[0040] Based on the above morphological identification, 16S rDNA, and homology comparison results, strain JDF1 was named Bacillus amyloliquefaciens JDF1 (Bacillus amyloliquefaciens JDF1).

[0041] Example 3: Application of JDF1 in the prevention and control of citrus canker

[0042] 1. Control of citrus canker using JDF1 bacterial solution

[0043] To test the control efficacy of strain JDF1 in live outdoor plants, a live inoculation experiment was conducted on potted citrus (Eureka lemon) seedlings. Inoculation was mainly performed by injecting the bacterial solution into the leaves. Prepare 1.0 × 10⁻⁶ seedlings. 6 cfu / mL Xcc The bacterial suspensions and JDF1 bacterial suspensions with OD=0.2 and 1.0 were respectively sampled from the above... XccEqual volumes of bacterial suspension and JDF1 bacterial suspension were mixed to form the treatment group, with sterile water replacing JDF1 bacterial suspension. Xcc A control group was prepared by mixing bacterial suspensions. Young citrus leaves sown in seed trays were selected, and needles were used to prick both sides of the leaves for easy injection. During inoculation, 100 μL of bacterial suspension from both the treatment and control groups was drawn up with a 1 mL syringe and injected into both sides of the leaves. Five leaves were prepared for each treatment as replicates. The incubators were covered and placed in a constant temperature incubator with humidity, in the dark for 1-2 days, followed by normal light for 10-15 days. The incubation period was 24℃, 75% relative humidity, 10000 lux light intensity, and a 16-hour light-8-hour dark cycle. Disease incidence was observed daily, and the control effect was assessed after approximately 15 days. In the control group, a yellow halo or callus protrusion appeared around the leaf, indicating green fluorescence. Xcc The abundant proliferation in the leaves of the control group indicates that in this experiment... Xcc Xanthomonas citrus subsp. citrus can successfully infect citrus leaves; however, leaves injected with a bacterial solution containing JDF1 strain did not show yellow halos or callus protrusions, and the leaf tissue did not show obvious symptoms of canker. However, high concentrations (OD=1.0) of JDF1 bacterial solution caused hypersensitive necrosis at the wound site, while low concentrations (OD=0.2) of JDF1 bacterial solution did not cause hypersensitive necrosis at the wound site, indicating that this JDF1 bacterial solution has a low-concentration, high-efficiency control effect. Figure 3 The results showed that the bacterial solution containing Bacillus amyloliquefaciens strain JDF1 could effectively inhibit the infection of citrus canker by Bacillus citrus.

[0044] 2. Control of citrus canker using JDF1 bacterial supernatant

[0045] To test the control effect of JDF1 bacterial supernatant on live plants outdoors, a live inoculation experiment was also conducted on potted citrus (Eureka lemon) seedlings. Inoculation was mainly carried out by injecting the bacterial supernatant into the leaves. The bacterial supernatant was JDF1 bacterial suspension with OD=1.0. The suspension was centrifuged at 12000 rpm, the supernatant was discarded, and an equal volume of sterile water was added. The suspension was shaken well and incubated at 28℃ for 12 h. The supernatant was then collected by centrifugation at 12000 rpm and filtered through a 0.22 µm sterile filter membrane to obtain the JDF1 bacterial supernatant. 1.0 × 10⁻⁶ cells were prepared. 6 cfu / mL Xcc Bacterial suspension. Take samples of the above... Xcc Equal volumes of bacterial suspension and JDF1 bacterial suspension supernatant were mixed to form the treatment group. Sterile water was used to replace the JDF1 bacterial suspension supernatant. XccA control group was prepared by mixing bacterial suspensions. Young citrus leaves sown in seed trays were selected, and needles were used to prick both sides of the leaves for easy injection. During inoculation, 100 μL of bacterial suspension from both the treatment and control groups was drawn up with a 1 mL syringe and injected into both sides of the leaves. Five leaves were prepared for each treatment as replicates. The incubators were covered and placed in a constant temperature incubator with humidity and darkness for 1-2 days, followed by continued incubation under normal light for 10-15 days. The incubation period was 24℃, 75% relative humidity, 10000 lux light intensity, and a 16-hour light-8-hour dark cycle. Disease incidence was observed daily, and the control effect was assessed after approximately 15 days. In the control group, a yellow halo or callus protrusion appeared around the leaf, indicating green fluorescence. Xcc The abundant proliferation in the leaves of the control group indicates that in this experiment... Xcc Xanthomonas citrus subsp. citrus can successfully infect citrus leaves; however, leaves injected with supernatant containing JDF1 bacterial solution do not show a yellow halo, but exhibit mild canker symptoms. This does not lead to hypersensitive necrosis at the wound site, and the green fluorescence indicates that the JDF1 bacterial solution supernatant can effectively inhibit the reproduction of canker pathogens. Figure 4 The results showed that the supernatant of the bacterial solution containing Bacillus amyloliquefaciens JDF1 could effectively prevent the infection of citrus canker by Bacillus amyloliquefaciens.

[0046] 3. High-temperature activity test of JDF1 bacterial culture

[0047] To test the heat resistance of JDF1 bacterial solution, a high-concentration Bacillus amyloliquefaciens JDF1 bacterial solution with an OD of 1.0 was treated with a 50°C water bath for 4 hours as the treatment bacteria. The experimental procedure was the same as the JDF1 bacterial solution control experiment for citrus canker in this example. The control effect was observed after about 15 days. In the control group, yellow halos or callus protrusions appeared around the same leaf, while the leaves injected with JDF1 bacterial solution treated at 50°C only showed mild citrus canker symptoms, but did not cause hypersensitive necrosis at the wound site. Green fluorescence indicated that JDF1 bacterial solution treated at 50°C could also effectively inhibit the reproduction of canker bacteria. Figure 5 (A) We also compared them on the same leaf. Xcc The efficacy of JDF1 bacterial suspension with OD=1.0, JDF1 bacterial suspension with OD=0.2, and JDF1 bacterial suspension treated with a 50℃ high-temperature water bath in preventing and treating peptic ulcer disease was compared. Symptom observation and fluorescence detection results showed that the efficacy of JDF1 bacterial suspension with OD=1.0 was higher than that with JDF1 bacterial suspension with OD=0.2, and the efficacy of JDF1 bacterial suspension treated with a 50℃ high-temperature water bath for 6 hours was higher than that with JDF1 bacterial suspension treated with a 50℃ high-temperature water bath for 6 hours. Xcc control group ( Figure 5 (B in the middle).

[0048] Pathogens XccContent determination: Samples were taken from the inoculation site of the leaves using a punch, and placed into 2 mL centrifuge tubes. The samples were disinfected in a clean bench with 75% alcohol for 3-5 min, rinsed 3-4 times with sterile water, dried, and then ground with an appropriate amount of sterile water. The mixture was diluted tenfold, and an equal volume of the homogenate was spread onto LB solid medium and incubated upside down at 28℃ for 36-48 h. Once single colonies appeared on the plates, the number of pathogens was counted, and the bacterial content was determined. Plate counting results showed that the antibacterial efficiency of JDF1 bacterial suspensions with OD=1.0 and 0.2 was 99.99%, and the antibacterial efficiency of JDF1 bacterial suspension with OD=1.0 after heat treatment was approximately 99.00%. Figure 6 The results showed that the bacterial solution containing Bacillus amyloliquefaciens JDF1 had sustained high-temperature resistance.

Claims

1. Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens JDF1, characterized in that, The accession number is CCTCC NO.M2025865.

2. A fermented product, characterized in that, The fermentation broth or fermentation broth filtrate of Bacillus amyloliquefaciens JDF1 as described in claim 1.

3. A biocontrol agent, characterized in that, The active ingredients include Bacillus amyloliquefaciens JDF1 as described in claim 1 and / or the fermentation product as described in claim 2.

4. The biocontrol agent according to claim 3, characterized in that, The aforementioned biocontrol agent is used to control citrus canker.

5. The biocontrol agent according to claim 3, characterized in that, The biocontrol agent is the fermentation broth of Bacillus amyloliquefaciens JDF1, and the OD of the fermentation broth is 0.2 to 1.

0.

6. The biocontrol agent according to claim 5, characterized in that, The preparation method of the fermentation broth of Bacillus amyloliquefaciens JDF1 is as follows: Bacillus amyloliquefaciens JDF1 is inoculated into LB liquid medium and cultured at 28℃ and 200 rpm for 24-36 h to obtain the fermentation broth of Bacillus amyloliquefaciens JDF1.

7. The biocontrol agent according to claim 3, characterized in that, The biocontrol agent is the fermentation broth filtrate of Bacillus amyloliquefaciens JDF1, which is prepared by the following method: Bacillus amyloliquefaciens JDF1 is inoculated into LB liquid medium and cultured at 28℃ and 200 rpm for 24-36 h to obtain the fermentation broth. The broth is then filtered through a 0.22 µm sterile filter membrane, and the filtrate is the fermentation broth filtrate of Bacillus amyloliquefaciens JDF1.

8. The use of Bacillus amyloliquefaciens JDF1 as described in claim 1, the fermented product as described in claim 2, or the biocontrol agent as described in any one of claims 3-7 in the prevention and control of citrus canker.

9. The application according to claim 8, characterized in that, The citrus canker disease mentioned above is caused by Xanthomonas citrus subsp. citrus.

10. A method for preventing and controlling citrus canker, characterized in that, The method includes the steps of applying the Bacillus amyloliquefaciens JDF1 of claim 1, the fermented product of claim 2, or the biocontrol agent of any one of claims 3-7 to the surface of citrus plants or injecting it into citrus leaves or fruits.

Images