Antibacterial peptide and application thereof in prevention and treatment of citrus bacterial diseases

Abstract

The application discloses an antibacterial peptide and application of the antibacterial peptide to prevention and treatment of citrus bacterial diseases. The application breaks the traditional idea of searching for antibacterial peptides from soil microorganisms, and innovatively searches for possible antibacterial peptides from intermediate insects. Through a series of preliminary researches, six antibacterial peptide sequences are finally determined. It is verified that the natural source antibacterial peptides provided by the application have significant prevention and treatment effects on the citrus bacterial diseases, and the application provides a new pesticide for prevention and treatment of the citrus bacterial diseases, and the pesticide is harmless to the environment and has very obvious effects.

Description

Technical Field

[0001] This invention relates to the field of plant disease control, specifically to an antimicrobial peptide and its application in controlling bacterial diseases of citrus. Background Technology

[0002] Citrus is one of the world's most important fruit trees, widely cultivated globally. Bacterial diseases of citrus are common and difficult to control in orchards. In my country, the most significant bacterial diseases affecting citrus are citrus canker and citrus Huanglongbing (HLB). Citrus canker is caused by a bacterium of the genus Xanthomonas. Canker is a serious bacterial disease affecting citrus, damaging leaves, shoots, and fruit. Seedlings and young trees are particularly severely affected, causing leaf drop, shoot dieback, weakened tree vigor, and even death in young trees. Fruit on fruit-bearing trees is also affected, leading to pre-harvest fruit drop, diseased fruit with lesions, reduced quality, and increased susceptibility to rotting during storage, significantly reducing the fruit's commercial value. HLB is a devastating disease in citrus production, currently prevalent in over 50 countries and regions, including China, the United States, and Brazil. Citrus canker pathogens are bacteria belonging to the family Pseudomonasceae in the phylum Thin-walled Bacteria. There are different pathogenic, serotypes, and genotypes of citrus canker pathogens, generally classified into the following strains: Strain A (CBCD-A, Asian canker or true canker type), distributed in most citrus-growing regions except the Mediterranean Basin and the United States; Strain B (CBCD-B, pseudo-canker type), distributed in Argentina and Uruguay; Strain C (CBCD-C, Mexican lemon type), distributed in Brazil and Paraguay; Strain D (CBCD-D), distributed in Mexico; and Strain E (CBDB-E, citrus bacterial leaf spot), distributed in Florida, USA.

[0003] The pathogen of citrus Huanglongbing (HLB) is a Gram-negative, refractory bacterium belonging to the genus *Candidatus Liberibacter asiaticus* (CLAs), which is divided into three species: Asian (*Candidatus Liberibacter asiaticus*, CLas), American (*Candidatus Liberibacter eramericanus*, CLam), and African (*Candidatus Liberibacter africanus*, CLaf). In China, the pathogen causing HLB is *Candidatus Liberibacter asiaticus*. HLB can cause phloem necrosis, sieve tube blockage, or root rot in citrus, leading to reduced yield, the production of small, sour fruits such as green or red-nosed fruits, and ultimately, the death of citrus plants, seriously threatening the safety of the citrus industry. Due to the lack of effective treatments, HLB is also known as the "cancer" of citrus. Studies have shown that antibiotics can control bacterial pathogens infecting citrus to some extent, but antibiotic use carries significant risks in terms of sustainability and biosafety. With in-depth research into the interaction mechanisms between citrus bacterial pathogens, host plants, and vector insects, an increasing number of natural antimicrobial peptides (AMPs) have been reported as candidate agents for controlling citrus bacterial diseases. Antimicrobial peptides are a class of small molecule polypeptides with antimicrobial activity, widely distributed in organisms, and possess advantages such as broad antimicrobial spectrum, strong antimicrobial activity, and low likelihood of developing resistance. Antimicrobial peptides used for plant disease control are usually plant-derived, but reports on effective antimicrobial peptides targeting citrus bacterial diseases are currently scarce. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing an antimicrobial peptide and its application in preventing and controlling bacterial diseases of citrus.

[0005] To achieve its objective, the present invention employs the following technical solution: A first aspect of the present invention provides an antimicrobial peptide, said antimicrobial peptide being any of the following polypeptides: The amino acid sequence of the antimicrobial peptide DcAMPL1 is shown in SEQ ID NO.19; The amino acid sequence of the antimicrobial peptide DcAMPL2 is shown in SEQ ID NO.20; The amino acid sequence of the antimicrobial peptide DcAMPL3 is shown in SEQ ID NO.21; The amino acid sequence of the antimicrobial peptide DcAMPL4 is shown in SEQ ID NO.22; The amino acid sequence of the antimicrobial peptide DcAMPL5 is shown in SEQ ID NO.23; The amino acid sequence of the antimicrobial peptide DcAMPL6 is shown in SEQ ID NO.24.

[0006] A second aspect of the present invention provides a biomaterial related to the above-described antimicrobial peptide, said biomaterial being any of the following: (1) The nucleic acid molecule encoding the above-mentioned antimicrobial peptide; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1); (4) A recombinant vector containing the expression cassette described in (2); (5) Recombinant microorganisms containing the nucleic acid molecules described in (1); (6) Recombinant microorganisms containing the expression cassette described in (2); (7) Recombinant microorganisms containing the recombinant vector described in (3); (8) Recombinant microorganisms containing the recombinant vector described in (4).

[0007] Among them, (1) the nucleic acid molecule encoding the above-mentioned antimicrobial peptide, and the nucleotide sequences of the nucleic acid molecule encoding the antimicrobial peptide shown in SEQ ID NO.19~24 are shown in SEQ ID NO.13~18 respectively.

[0008] A third aspect of the present invention provides a product for preventing and controlling bacterial diseases of citrus or inhibiting pathogenic bacteria of citrus, wherein the active ingredient is the aforementioned antimicrobial peptide or the aforementioned biological material.

[0009] In the product technical solution described above, the bacterial diseases of citrus include citrus canker and citrus Huanglongbing (HLB).

[0010] In the aforementioned product technical solution, the citrus pathogenic bacteria include citrus canker pathogens and citrus Huanglongbing pathogens; the citrus Huanglongbing pathogens include C Las、 C Lam, C Laf.

[0011] The fourth aspect of the present invention provides the use of the above-described antimicrobial peptide, or the above-described biomaterial, or any of the products described above in any of the following: (1) Inhibits pathogenic bacteria in citrus fruits; (2) Prepare products that inhibit pathogenic bacteria in citrus fruits; (3) Control of bacterial diseases in citrus; (4) Prepare products for the prevention and control of bacterial diseases of citrus.

[0012] In the aforementioned application technical solution, the citrus pathogenic bacteria include citrus canker pathogens and citrus Huanglongbing pathogens; the citrus Huanglongbing pathogens include C Las、 C Lam, C Laf; The bacterial diseases of citrus include citrus canker and citrus Huanglongbing.

[0013] In the aforementioned application technology solution, the product is applied to the diseased plant by injection.

[0014] In the application technical solution described above, the product is an antimicrobial peptide solution with a concentration of 0.5~2 mg / ml; preferably, the concentration is 0.5~1.5 mg / ml.

[0015] The beneficial effects of this invention are as follows: It breaks away from the traditional approach of studying antimicrobial peptides from citrus plant sources or citrus soil microorganisms, and innovatively seeks potential antimicrobial peptides for Huanglongbing (HLB) from the citrus psyllid, the insect vector of HLB. After a series of preliminary studies, six antimicrobial peptide sequences were finally identified. Verification showed that the natural antimicrobial peptides provided by this invention have significant control effects on bacterial diseases of citrus, and have significant control effects on HLB. This provides a new pesticide for the control of bacterial diseases of citrus (especially HLB, which is currently very difficult to control), is environmentally friendly, and has a very obvious effect. Attached Figure Description

[0016] Figure 1 This is a diagram showing the results of an inhibition zone experiment on antimicrobial peptides against Rhizobium fischeri and Citrus canker.

[0017] Figure 2 The image shows the use of an auto-injector to inject antimicrobial peptides into sweet orange seedlings.

[0018] Figure 3 This study uses real-time fluorescence quantitative PCR to detect changes in the content of Huanglongbing bacteria in sweet orange seedlings before and after injection of PBS and antimicrobial peptides. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0020] Example 1: Screening of antimicrobial peptides against citrus canker and citrus Huanglongbing – Inhibition zone experiment Our research team previously used an antimicrobial peptide database to screen a batch of potential insect antimicrobial peptide gene sequences from the genomes of insects that act as vectors of citrus diseases using bioinformatics. Based on the analysis, we selected six of these sequences for further validation studies.

[0021] I. Amplification of antimicrobial peptide genes in target species The target species is the citrus psyllid ( Diaphorina citri Amplify the antimicrobial peptide genes that were initially screened.

[0022] (1) PCR amplification Design PCR primers to amplify the full-length gene: Design amplification primers based on the screened antimicrobial peptide gene sequence.

[0023] Total RNA was extracted from citrus psyllids and reverse transcribed into cDNA. Amplification system: 2 μL cDNA, 1 μL 10 μM upstream primer, 1 μL 10 μM downstream primer, 25 μL 2*Primer STAR Mix, 21 μL ddH2O, total volume 50 μL. Amplification program: 98℃ for 5 min; 98℃ for 20 s, 60℃ for 30 s, 72℃ for 24 s, 36 cycles; 72℃ for 10 min, storage at 12℃. Primer sequences corresponding to the antimicrobial peptide gene are shown in Table 1.

[0024] Table 1

[0025] The amplification products were recovered using a gel extraction kit and ligated into the pET-28a vector: the amplification products and the pET-28a prokaryotic expression vector plasmid were digested with EcoRI and HindIII and then ligated with T4 DNA ligase to obtain the recombinant vector, which was then verified by sequencing.

[0026] The full-length sequence of the antimicrobial peptide gene was obtained by sequencing. This gene was compared with the gene in the target species database. The signal peptide fragment in the antimicrobial peptide gene was removed and used to design specific amplification primers.

[0027] The nucleotide sequences of each antimicrobial peptide gene are shown in Table 2: Table 2

[0028]

[0029] II. Prokaryotic Expression and Purification of Antimicrobial Peptides The nucleotide sequences in Table 2 were synthesized and constructed into the pET-28a vector using a double digestion method (EcoRI and HindIII). Sequencing was then performed for verification. The verified pET-28a-AMP vector was transformed into BL21 *E. coli*. Single colonies were picked from the transformed plates and transferred to kanamycin liquid LB medium. The mixture was shaken at 220 rpm and 37 °C until the OD600 value reached approximately 0.6-0.8. The mixture was then incubated on ice for two minutes, and IPTG inducer was added at a 1:200 ratio. The mixture was shaken at 220 rpm and 37 °C for 4-5 h. The medium was removed by centrifugation, and the cells were resuspended in 1×PBS. The resuspended bacterial culture was then lysed with a protease inhibitor (PMSF) and homogenized until clear. Coomassie brilliant blue staining and Western blot analysis were performed. Successful protein induction was indicated by single and congruent bands in both Coomassie brilliant blue and Western blot analyses. After large-scale induction to express the target protein, *E. coli* were collected by centrifugation and resuspended in PBS in 50 ml centrifuge tubes. Dissolve inclusion bodies with binding wash buffer and incubate at room temperature for 30–60 min to allow the precipitate to dissolve completely. Remove the antibody purification column, remove the bottom cap, and remove excess liquid. Equilibrate the column with twice the volume of binding wash buffer (equilibration column packing volume) at a flow rate of 0.5–1 ml / min. Add the bounding-wash buffer-treated bacterial culture from the top of the column. Wash the resin with twice the volume of binding wash buffer and collect the eluent. Repeat this step until the absorbance of the eluent is at the baseline of 280 nm. Elute the His-tagged protein from the resin with twice the volume of Elution buffer (equilibration volume), repeating this step twice. The purified antimicrobial peptides are obtained; the amino acid sequences of each antimicrobial peptide are shown in Table 3.

[0030] Table 3 Antimicrobial peptide sequences

[0031] III. Antimicrobial zone detection to assess the antimicrobial peptide's antimicrobial ability Currently, in this technical field, citrus bacterial diseases mainly include *Citrus canker* and *Huanglongbing*. *Citrus canker* can be cultured in vitro and therefore used directly. However, since *Huanglongbing* has not yet been successfully isolated artificially, it cannot be directly used for inhibition zone experiments. Instead, its close relative, *Rhizobium fischeri*, is used. Sinorhizobium fredii The antibacterial effect was tested.

[0032] Preparation of antimicrobial peptide solution: Take the purified antimicrobial peptide obtained above and add it to 1× PBS solution to prepare an antimicrobial peptide solution with a concentration of 2-3 mg / ml.

[0033] Prepare *Rhizobium fischeri* and *Citrus canker* ( Xanthomonas axonopodis pv . citri Place 50ml centrifuge tubes, filter paper discs, and LB solid medium into an autoclave and sterilize at 120℃ for 20 min. Prepare 3 groups of petri dishes, with 6 petri dishes in each group. Prepare *Rhizobium fischeri* and *Citrus canker* separately in advance using antibiotic-free LB liquid medium to adjust the OD values ​​of the UV spectrophotometer. 600 At approximately 0.5, add 50 ml of LB medium + 500 μL of bacterial suspension (OD) to each 50 ml centrifuge tube. 600 Approximately 0.5). The antibacterial test employed the pre-added bacterial suspension pour plate method. Bacterial suspensions of *Rhizobium fischeri* or *Citrus canker* were injected into a culture medium cooled to approximately 50°C. The bacterial suspension and culture medium were mixed thoroughly, poured onto plates, and allowed to solidify horizontally. Filter paper discs were sterilized, dried, and immersed in a purified antimicrobial peptide solution for 5 seconds before being placed in the center of the test plate. Figure 1 The small white disc in the center of the plate is the filter paper. The plate was incubated at 28℃ for 24 h, and the size of the inhibition zone was measured. The diameter of the inhibition zone was measured by taking the average value using a ruler using the cross-hatching method, and the diameter was used to represent the size of the inhibition zone.

[0034] The results are as follows Figure 1 As shown in Table 4, all antimicrobial peptides have significant antibacterial effects against citrus canker pathogens.

[0035] Table 4 Results of inhibition zones

[0036] Example 2: Antimicrobial peptide injection experiment in citrus plants Preparation of antimicrobial peptide injection: Take the purified antimicrobial peptide obtained from prokaryotic expression in Example 1 and add it to 1× PBS to prepare an antimicrobial peptide injection with a concentration of 1 mg / ml.

[0037] Injecting antimicrobial peptide solution into sweet orange seedlings that were found to be infected with citrus Huanglongbing pathogen (C. HLB). Figure 2 Using an auto-injector, 10 ml of antimicrobial peptide injection solution was injected into the phloem of the tree trunk at a rate of 0.1 ml / h. Diseased trees injected with PBS buffer served as a control. Injection was completed after 96 hours. Five days were observed, and the bacterial load on the leaves of diseased plants was measured before and five days after injection. DNA was extracted from the main veins of different leaves taken from the same branch, and real-time quantitative PCR was used to detect Huanglongbing (HLB). C Ct value of Las (in sweet orange) 18S rRNA (as an internal reference), primer sequences are shown in Table 5: Table 5 Primers for Real-Time Quantitative PCR

[0038] The relative content of Huanglongbing bacteria was calculated using a formula, and the changes in the Ct value of Huanglongbing bacteria titer expressed in plants of the antimicrobial peptide injection treatment group and the control group were detected at the nucleic acid level.

[0039] Excel software was used for data statistics, and GraphPad Prism 8.0 software was used for data statistical analysis and chart generation.

[0040] The results are as follows Figure 3 As shown: The content of Huanglongbing (HLB) in sweet oranges was detected before and after AMP / PBS injection. The results showed that the content of HLB in the control group injected with PBS buffer increased, while the content of HLB in the treatment group injected with antimicrobial peptides decreased significantly compared with that before the injection due to the inhibition of antimicrobial peptides. All six antimicrobial peptides in Table 3 had a significant inhibitory effect on HLB.

[0041] The above experiments show that the symptoms of Huanglongbing (HLB) in citrus plants infected with HLB pathogens were significantly suppressed after injection of antimicrobial peptides, and the plants recovered significantly.

Claims

1. An antimicrobial peptide, said antimicrobial peptide being any of the following polypeptides: The amino acid sequence of the antimicrobial peptide DcAMPL1 is shown in SEQ ID NO.19; The amino acid sequence of the antimicrobial peptide DcAMPL2 is shown in SEQ ID NO.20; The amino acid sequence of the antimicrobial peptide DcAMPL3 is shown in SEQ ID NO.21; The amino acid sequence of the antimicrobial peptide DcAMPL4 is shown in SEQ ID NO.22; The amino acid sequence of the antimicrobial peptide DcAMPL5 is shown in SEQ ID NO.23; The amino acid sequence of the antimicrobial peptide DcAMPL6 is shown in SEQ ID NO.

24.

2. A biomaterial related to the antimicrobial peptide of claim 1, wherein the biomaterial is any one of the following: (1) A nucleic acid molecule encoding the antimicrobial peptide of claim 1; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1); (4) A recombinant vector containing the expression cassette described in (2); (5) Recombinant microorganisms containing the nucleic acid molecules described in (1); (6) Recombinant microorganisms containing the expression cassette described in (2); (7) Recombinant microorganisms containing the recombinant vector described in (3); (8) Recombinant microorganisms containing the recombinant vector described in (4).

3. The biomaterial according to claim 2, characterized in that: in, (1) The nucleic acid molecule encoding the antimicrobial peptide of claim 1, wherein the nucleotide sequences of the nucleic acid molecule encoding the antimicrobial peptide shown in SEQ ID NO.19~24 are as shown in SEQ ID NO.13~18 in sequence.

4. A product for preventing and controlling bacterial diseases of citrus or inhibiting pathogenic bacteria of citrus, wherein the active ingredient is the antimicrobial peptide as described in claim 1 or the biomaterial as described in claim 2.

5. The product according to claim 4, characterized in that: The bacterial diseases of citrus include citrus canker and citrus Huanglongbing.

6. The product according to claim 4, characterized in that: The citrus pathogenic bacteria include citrus canker pathogens and citrus Huanglongbing pathogens; the citrus Huanglongbing pathogens include C Las、 C Lam, C Laf.

7. The use of the antimicrobial peptide of claim 1, the biomaterial of claim 2, or the product of any one of claims 4 to 6 in any of the following: (1) Inhibits pathogenic bacteria in citrus fruits; (2) Prepare products that inhibit pathogenic bacteria in citrus fruits; (3) Control of bacterial diseases in citrus; (4) Prepare products for the prevention and control of bacterial diseases of citrus.

8. The application according to claim 7, characterized in that: The citrus pathogenic bacteria include citrus canker pathogens and citrus Huanglongbing pathogens; the citrus Huanglongbing pathogens include C Las、 C Lam, C Laf; The bacterial diseases of citrus include citrus canker and citrus Huanglongbing.

9. The application according to claim 8, characterized in that: The product is applied to the diseased plants by injection.

10. The application according to claim 9, characterized in that: The product is an antimicrobial peptide solution with a concentration of 0.5~2 mg / ml; preferably, the concentration is 0.5~1.5 mg / ml.

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