Bacillus subtillis with activity of inhibiting citrus canker pathogen and application of bacillus subtillis

Abstract

The invention discloses a bacillus subtillis capable of inhibiting the activity of citrus canker pathogen and application of the bacillus subtillis, the bacillus subtillis NG4 is an endophytic bacterium separated from citrus leaves by using a germ enrichment method, can efficiently antagonize the growth of the citrus canker pathogen, and has a good control effect on canker on the citrus leaves. The genome of the NG4 encodes a plurality of antibacterial metabolites to synthesize a gene cluster, and the fermentation supernatant of the NG4 can also inhibit the growth and pathogenicity of the citrus canker pathogen.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an endophytic Bacillus subtilis NG4 of citrus that can inhibit citrus canker and an application thereof. Background Art

[0002] Citrus canker is a bacterial disease caused by Xanthomonas axonopodispv. citri (Xac). Xac mainly harms the leaves, branches, fruits and sepals of citrus, which can cause huge yield losses. The prevention and control of citrus canker currently relies mainly on chemical agents based on copper preparations, but long-term use of chemical agents has problems such as drug resistance and environmental pollution. As an environmentally friendly, safe and effective means of plant disease prevention and control, biological control has an increasingly important position in the prevention and control of crop diseases. However, there are currently few types of biocontrol bacteria used in the prevention and control of citrus canker, and the prevention effect is unstable. An important reason for this phenomenon is that most of the biocontrol bacteria are isolated and cultured from the soil. These exogenous biocontrol bacteria cannot adapt well to the ecological environment of local plants, resulting in a decrease or even loss of their field prevention effect. It is of great significance to discover new biocontrol bacteria with inhibitory activity against citrus canker. Summary of the invention

[0003] The present invention aims to provide a strain of Bacillus subtilis NG4 having good biocontrol effect on citrus canker and application thereof.

[0004] The purpose of the present invention can be achieved by the following solutions:

[0005] The present invention provides a Bacillus subtilis NG4, which is classified and named Bacillus subtilis (Bacillus subtilis). The strain was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Committee on August 26, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCC No. 31745.

[0006] The Bacillus NG4 described in the present invention is isolated from citrus leaves and has the ability to inhibit the growth and pathogenicity of citrus canker bacteria.

[0007] The present invention also provides a bacterial liquid containing the Bacillus subtilis NG4.

[0008] The bacterial solution of the present invention can be prepared according to conventional methods in the art, such as fermentation broth of Bacillus subtilis NG4, or a bacterial solution formed by separating bacterial cells from fermentation broth and resuspending them in culture medium; in a specific embodiment, the bacterial cell concentration of the bacterial solution is 1*10 7cfu / mL~1*10 10 cfu / mL.

[0009] The present invention also provides the fermentation supernatant of Bacillus subtilis NG4. The fermentation broth is the supernatant retained after the fermentation broth of Bacillus subtilis NG4 is filtered with a bacterial filter to remove bacterial cells.

[0010] The present invention also provides use of the Bacillus subtilis NG4, bacterial liquid or fermentation liquid of the present invention in treating or preventing citrus canker.

[0011] The present invention also provides the use of the Bacillus subtilis NG4, bacterial liquid or fermentation liquid of the present invention in inhibiting citrus canker pathogen. In a specific example, the citrus canker pathogen is Xanthomonas axonopodis pv. citri (Xac).

[0012] The application of the present invention is specifically to apply the bacterial liquid or fermentation supernatant of Bacillus NG4 directly to the location of the pathogen, so that Bacillus NG4 can inhibit the infection of citrus canker bacteria on citrus. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Use the "pathogen enrichment" method to isolate biocontrol bacteria with bactericidal activity;

[0014] Figure 2 The NG4 strain has a strong antagonistic Xac activity;

[0015] Figure 3 Morphological and molecular biological identification showed that NG4 belonged to Bacillus subtilis;

[0016] Figure 4 NG4 can inhibit the pathogenicity of X. citri var.

[0017] Figure 5 The fermentation supernatant of NG4 can inhibit the growth and pathogenicity of citrus canker bacteria. DETAILED DESCRIPTION

[0018] In order to make the principle and method of the present invention more clearly understood, the present invention is further described below with specific examples, but is not limited to these examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0019] Unless otherwise specified, Xac refers to Xanthomonas axonopodis pv. citri (Xac), the specific strain is Xac003, which is preserved in this research group and the applicant promises to make it permanently open to the public.

[0020] Unless otherwise specified, the formula of NG4 fermentation medium is: 9g beef extract, 15g starch, dilute to 1L with distilled water, adjust pH to 6.0 with HCl. Fermentation conditions are: pick a single colony and inoculate it in LB medium, culture at 28℃, 220rpm until the concentration is 1*10 9 cfu / mL, which is the fermentation seed liquid. The seed liquid was inoculated into the fermentation medium at a rate of 3%, and cultured at 28°C, 220rpm, for 72 hours to obtain the NG4 fermentation liquid.

[0021] Example 1: Isolation of citrus endophytic bacteria with inhibitory activity against ulcer pathogens using the “pathogen enrichment” method

[0022] The endophytic microbiome of plants contains a large number of bacteria that share the same ecological niche as plant pathogenic bacteria, and is an important resource for the isolation and identification of biocontrol bacteria. Our laboratory previously developed a "pathogen enrichment" biocontrol bacteria screening platform based on the hypothesis that "biocontrol bacteria and pathogenic bacteria have the same ecology and co-evolution." The present invention is further optimized on this basis. First, the diseased leaves of citrus canker (the pathogen is Xac) are disinfected with alcohol and sodium hypochlorite, and then ground into plant tissue fluid with a juicer. GFP-labeled Xac (Xac003-GFP) was cultured overnight in LB, centrifuged to remove the culture medium, and the cells were resuspended in sterile water to OD 600 =1.0. 20 μL of Xac cell suspension was added to 20 mL of plant tissue solution and cultured at 28°C and 150 rpm for 2 days. The culture was then spread on a PVDF membrane with a pore size of 0.22 μm to simulate the contact between bacteria on citrus leaves and Xac. After the PVDF membrane was cultured on a water agar plate for 2 days, the bacteria on the filter membrane were washed with sterile water and streaked on LB. Xac has a GFP fluorescent marker, and the non-fluorescent colonies around the fluorescent colonies were picked as potential biocontrol bacteria for purification ( Figure 1 The activity of endophytic bacteria against Xac was further analyzed by plate antagonism. Xac was cultured in LB medium until OD 600 = 1.0 and then mixed with LB medium that had been melted and cooled to about 45°C at a ratio of 1:100, and the mixture was poured into a plate and cooled to solidify. 600 =1.0, 5 μL was inoculated on the plate, and after 2 days of culture, the presence of an inhibition zone on the Xac plate was observed. Through screening, a strain NG4 was found to have a strong antagonistic activity against Xac ( Figure 2 ).

[0023] Example 2 Morphological and molecular biological identification of NG4 belonging to Bacillus subtilis

[0024] NG4 was inoculated on LB plates by streaking culture and incubated at 28°C for 1-2 days to observe the morphological characteristics of its colonies. It was found that the colonies on the LB plates were irregular in shape, mainly yellow-white, wrinkled, and the surface of the colonies was not smooth ( Figure 3 A). Colony PCR was performed using 16S rDNA primers, and the 16S rDNA fragment of NG4 was sequenced. Gene sequence comparison was performed using NCBI to determine the classification status of the strain. The 16S rDNA of strains with high homology to NG4 was downloaded, and the phylogenetic tree was constructed using MEGA software to verify the strain classification. It was found that NG4 was most closely related to the Bacillus subtilis strain ( Figure 3 B), it is speculated that NG4 is also Bacillus subtilis.

[0025] Table 1 16S rDNA amplification primers

[0026]

[0027] Example 3 Biocontrol bacteria NG4 can inhibit the infection of citrus canker bacteria

[0028] The biocontrol bacteria to be tested and Xac were cultured in LB to a concentration of 1*10 9 cfu / mL, and then NG4 and Xac were mixed in a volume ratio of 1:1, with LB / Xac mixture as negative control. Take fresh citrus leaves, wipe the citrus leaves with alcohol cotton first, put the wiped leaves into the culture dish, and poke 6-7 small holes on the leaves with the needle hole of a disposable sterile syringe. Then use a pinhole syringe (without the pinhole) to extract 500μL of the mixture, press the leaves to ensure that the mixture enters the leaves and there is residue at the wound of the small holes, put the leaves injected with the mixture on the moist gauze, put it in the culture dish, put the culture dish in a tissue culture box with soaked paper to keep it moist, and observe the disease state of the citrus leaves after 7 days. The results show that NG4 treatment can significantly inhibit the infection of Xac to citrus leaves ( Figure 4 ).

[0029] Example 4: The fermentation supernatant of NG4 can inhibit the growth of Xac and its infection of citrus leaves

[0030] This example further analyzes whether the fermentation supernatant of NG4 has the activity of inhibiting citrus canker bacteria. The cultured NG4 fermentation liquid is filtered with a bacterial filter to remove the bacterial cells and the supernatant is retained. 1 mL of pathogen Xac is applied to the surface of solid LB medium and blown dry. A 1 cm punch is used to punch holes in the medium. 50 μL of NG4 fermentation supernatant is inoculated into the holes on the medium. After 24 hours of observation in a 28°C constant temperature incubator, it is found that the supernatant of NG4 has the activity of antagonizing Xac ( Figure 5 A). Referring to the method of Example 3, the supernatant of NG4 was mixed with Xac and inoculated into citrus leaves. The results showed that the supernatant of NG4 could inhibit the occurrence of citrus canker ( Figure 5 B), indicating that NG4 can secrete anti-ulcer bacteria metabolites into the culture medium, thereby achieving antagonism against citrus canker bacteria.

Claims

1. A strain of Bacillus subtilis NG4, classified and named Bacillus subtilis, with a strain collection number of CGMCC No.31745.

2. A bacterial solution comprising the Bacillus subtilis NG4 according to claim 1.

3. The bacterial solution according to claim 2, characterized in that: The bacterial concentration of the bacterial solution is 1*10 7 cfu / mL~1*10 10 cfu / mL.

4. The bacterial solution according to claim 2, characterized in that: The bacterial liquid is the fermentation liquid of Bacillus subtilis NG4, or the bacterial liquid formed by separating bacterial bodies from the fermentation liquid and resuspending them in culture liquid.

5. The fermentation supernatant of Bacillus subtilis NG4 according to claim 1.

6. The fermentation supernatant according to claim 5, characterized in that The fermentation liquid is the supernatant obtained by removing bacterial cells from the fermentation liquid of Bacillus subtilis NG4.

7. Use of the Bacillus subtilis NG4 according to claim 1, the bacterial liquid according to any one of claims 2 to 4, or the fermentation supernatant according to claim 5 or 6 in treating or preventing citrus canker.

8. Use of the Bacillus subtilis NG4 according to claim 1, the bacterial solution according to any one of claims 2 to 4, or the fermentation supernatant according to claim 5 or 6 in inhibiting citrus canker bacteria.

9. The use according to claim 8, characterized in that: The pathogen causing citrus canker is Xanthomonas axonopodis pv. citri (Xac).

10. The use according to any one of claims 7 to 9, characterized in that: Specifically, Bacillus NG4 or bacterial liquid or fermentation supernatant is directly applied to the location of the pathogen.

Images