Pseudomonas aeruginosa BL11 and application of pseudomonas aeruginosa BL11 in prevention and treatment of tropical crop diseases such as bacterial wilt and areca leaf spot disease
By using the antagonistic function of Pseudomonas aeruginosa BL11, microbial agents used to prevent and treat betel leaf spot disease and the solanaceae crops were developed, and the problems of environmental pollution and consumption risks in the prior art were solved, and these were achieved. Effective green prevention and control of diseases.
Patent Information
- Application Number
- CN202411329611.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art can easily cause environmental pollution and potential consumption risks when preventing and controlling betel leaf spot disease and solanaceae crops, and lack green prevention and control measures.
A strain of Pseudomonas aeruginosa BL11 was used to prevent and control anthrax, stemophobia and cyperus through its significant antagonistic function, and microbial agents were developed for prevention and control.
It significantly reduces the incidence of betel leaf spot disease, provides effective bioprevention effect on betel nut and solanaceae crops, and avoids the environmental pollution and consumption risks of chemical drugs.
Smart Images
Figure CN119193380A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biocontrol bacteria, and specifically relates to a strain of Pseudomonas aeruginosa BL11 and application thereof in preventing and controlling tropical crop diseases such as bacterial wilt and betel nut leaf spot. Background Art
[0002] Betel nut is an important economic crop and medicinal plant in Hainan Province. Betel nut leaf spot disease is serious, among which the betel nut leaf spot disease caused by Colletotrichum is one of the important diseases. In the early stage of the disease, small yellow spots of chlorosis appear on the betel nut leaves. As the lesions grow, they will continue to expand into larger lesions. In the later stage, multiple lesions can be connected into one piece, causing large-scale leaf death, and finally weakening the growth of betel nut trees, affecting the yield of betel nut. The disease occurs in betel nut trees at all growth stages, which has a huge impact on the yield and product quality of betel nut. In addition, the crop bacterial wilt disease caused by Ralstonia solanacearum (commonly known as solanacearum) is a global soil-borne plant disease with a wide host range. It can infect more than 500 crops in 45 families, especially Solanaceae crops, which are extremely harmful and have become the second largest plant bacterial disease in the world. There are reports of bacterial wilt in nearly 30 provinces in my country, especially in South China.
[0003] Nowadays, the main means of treating betel leaf spot disease and solanaceous crop bacterial wilt is chemical drug control, but it is easy to cause environmental pollution and potential food risks. In order to solve the green prevention and control problems of betel leaf spot disease caused by anthracnose and phomopsis, and solanaceous crop bacterial wilt, the present invention proposes a strain of Pseudomonas aeruginosa BL11 that has significant antagonistic functions to pathogenic fungi such as anthracnose and phomopsis, as well as to Ralstonia solani, and the biocontrol effect shows that it can significantly reduce the incidence of betel leaf spot disease, and has a good biocontrol effect. In view of the serious harm of betel leaf spot disease to the betel nut industry, and the serious harm of Ralstonia solani to many crops, the invention has important application value. Summary of the invention
[0004] The present invention provides a Pseudomonas aeruginosa BL11, characterized in that its strain preservation information is as follows: name of the preservation unit: Guangdong Microbial Culture Collection Center; address of the preservation unit: 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences; preservation date: August 27, 2024; preservation number: GDMCC No.65060; classification name: Pseudomonas aeruginosa.
[0005] Another embodiment of the present invention provides the use of the above-mentioned Pseudomonas aeruginosa BL11 in preventing and treating betel leaf spot disease. The betel leaf spot disease is caused by Colletotrichum and / or Phomopsis.
[0006] Another embodiment of the present invention provides the use of the above-mentioned Pseudomonas aeruginosa BL11 in preventing and controlling bacterial wilt of Solanaceae crops.
[0007] Another embodiment of the present invention provides a microbial agent for preventing and controlling betel leaf spot disease, characterized in that the active ingredient of the microbial agent comprises the above-mentioned Pseudomonas aeruginosa BL11.
[0008] Another embodiment of the present invention provides a microbial agent for preventing and controlling bacterial wilt of Solanaceae crops, characterized in that the active ingredient of the microbial agent comprises the above-mentioned Pseudomonas aeruginosa BL11.
[0009] Compared with the prior art, the invention has the following advantages: BL11 is isolated from betel nut leaves with leaf spot disease, and 16S rDNA and r-POD gene sequence comparison shows that BL11 has high homology with Pseudomonas aeruginosa, and is a new strain of Pseudomonas aeruginosa (preservation number: GDMCC No.65060; classification name: Pseudomonas aeruginosa); the invention also conducts antagonistic experiments on Pseudomonas aeruginosa BL11 to anthracnose, phomopsoma pseudostomum and solanacearum, and the results show that Pseudomonas aeruginosa BL11 has significant prevention and control effects on betel nut leaf spot disease and solanacearum wilt of Solanaceae crops, and is expected to be developed and used as a novel biocontrol agent. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 It is an electrophoresis detection diagram, in which the left figure is the PCR amplification product of 16SrDNA of BL11, and the right figure is the PCR amplification product of r-POD gene of BL11.
[0011] Figure 2 This is the result of Pseudomonas aeruginosa BL11 antagonizing Ralstonia solanacearum EP1.
[0012] Figure 3 This is the result of Pseudomonas aeruginosa BL11 antagonizing anthrax CB1. The left picture is the control, which only has anthrax strains, and the right picture is the biocontrol bacteria BL11 added around it, with anthrax in the middle.
[0013] Figure 4 These are the results of Pseudomonas aeruginosa BL11 antagonizing Phomopsis. The left picture is the control, which only has Phomopsis strains, and the right picture shows the biocontrol bacteria BL11 added around it, with Phomopsis in the middle.
[0014] Figure 5 These are the results of the BL11 strain controlling betel leaf spot disease caused by anthracnose under greenhouse conditions, where A is the effect diagram, B is the statistical diagram, and * represents a significant difference in the number of leaf spots compared with the pathogen inoculation group. DETAILED DESCRIPTION
[0015] Example 1 Isolation, Culture and Identification of Pseudomonas aeruginosa BL11
[0016] 1. Isolation and culture of BL11
[0017] 1. Cut the betel nut leaves with disease spots into pieces and put them into a sterilized conical bottle. Add sterile water to cover the leaves and treat with ultrasound for 30 minutes.
[0018] 2. Take out the leaves and grind them thoroughly in a mortar;
[0019] 3. Take 100 μL of grinding solution, apply it to LB medium, and culture and observe at 30°C;
[0020] 4. Pick a single colony from the plate and place it in LB, separate and purify it using the streak method, and culture it at 30°C for observation.
[0021] 2. Identification of BL11
[0022] 1. Activate the isolated BL11 strain on LB plate and culture at 30°C for observation;
[0023] 2. Scrape some BL11 cells into a 2 ml EP tube, extract DNA using a DNA extraction kit (Tian Gen), and perform electrophoresis detection ( Figure 1 );
[0024] 3. The extracted DNA was amplified for 16S rDNA (amplification primers are 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-TACCTTGTTACGACTT-3′) and r-POD sequence (amplification primers are PsEG30F: ATYGAAATCGCCAARCG and PsEG790R: CGGTTGATKTCCTTGA), and the PCR products were sent to a sequencing company for identification.
[0025] In the NCBI database, the 16S rDNA sequence comparison showed that BL11 had the highest homology with Pseudomonas aeruginosa; in order to further clarify its classification, the specific detection primer PsEG790F / R in the Pseudomonas genus was used for amplification, and the results showed that BL11 had the highest sequence consistency with Pseudomonas aeruginosa. The strain preservation information is as follows: Name of the depository: Guangdong Microbiological Culture Collection Center; Address of the depository: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences; Date of deposit: August 27, 2024; Deposit number: GDMCC No.65060; Classification name: Pseudomonas aeruginosa.
[0026] Example 2 Pseudomonas aeruginosa BL11 antagonism experiment
[0027] 1. BL11 antagonizes Ralstonia solanacearum EP1
[0028] 1. Activation of strains: Activate BL11 on LB plates and EP1 on TTC plates.
[0029] (TTC formula: casein acid hydrolyzate 1g; glucose 10g; peptone 10g)
[0030] 2. Shake and transfer: Scrape BL11 from the plate and place it in LB liquid medium, and scrape EP1 from the plate and place it in TTC medium and culture it in a shaking incubator at 30°C overnight.
[0031] 3. Measure the OD of bacterial solution 600 Measure the concentration of EP1 bacterial solution and dilute it to 1.0.
[0032] 4. When the TTC temperature reaches 30°C, add EP1 (TTC: EP1 = 200 μL: 1 μL), mix well and pour into the plate. Add 20 ml of culture medium to each plate. After air-drying, punch three holes on each plate to form an equilateral triangle distribution. Add 40 μL of BL11 bacterial solution to each well. After the liquid in the plate is air-dried, seal the plate and culture at 30°C. Observe and record the diameter of the antagonism zone.
[0033] 5. After the plate was incubated for 48 hours, it can be seen that most of the culture medium in the unpunched area became turbid, and a transparent circle appeared around the hole. The diameter of the transparent circle was measured and the average was 2.2 cm ( Figure 2 ). This indicates that strain BL11 can antagonize the growth of Ralstonia solanacearum.
[0034] 2. BL11 antagonizes anthrax CB1
[0035] 1. Activation of strains BL11 and anthrax CB1 were activated on LB plates.
[0036] 2. Shake and transfer. Scrape BL11 from the plate into LB liquid medium, culture at 30℃ overnight in a shaker, use a pipette to make a stem on the anthrax CB1 plate, transfer to new LB solid medium, and culture at 30℃ overnight.
[0037] 3. When the Anthrax CB1 grows to the size of a one-yuan coin, add 1 μL of BL11 bacterial solution around it and another 1 μL of BL11 bacterial solution in the middle of its stipe. Place it at 30°C for static culture, observe and record the diameter of the Anthrax CB1.
[0038] 4. After 2 days of static culture, observe the size of the fungus, and measure and record its diameter when the size of the fungus no longer changes. It can be observed that with the growth of BL11, the diameter of Anthrax CB1 gradually decreases, the hyphae gradually changes from the initial white to light yellow, and the hyphae become moist, and the entire fungus no longer grows. In this experiment, the average diameter of the fungus added with bacteria for four weeks to Anthrax CB1 was 4.1cm, and the diameter of the anthrax colony without the addition of biocontrol bacteria was 7.8cm. This shows that adding the biocontrol bacteria BL11 for four weeks can significantly inhibit the growth of anthrax ( Figure 3 ).
[0039] 3. BL11 antagonism against Phomopsis
[0040] 1. After the strains are activated, activate BL11 and Phomopsis on LB plates.
[0041] 2. Shake and transfer. Scrape BL11 from the plate into LB liquid medium, culture at 30℃ overnight in a shaker, use a pipette to make a stipe on the Phomopsis plate, transfer to new LB solid medium, and culture at 30℃ overnight.
[0042] 3. When the Phomopsis spp. grows to the size of a one-yuan coin, add 1 μL of BL11 bacterial solution around it and place it at 30°C for static culture. According to observation and statistics, after culturing the strain BL11 around Phomopsis spp., the growth of Phomopsis spp. can be inhibited, showing a certain antagonistic activity ( Figure 4 ).
[0043] Example 3 Experiment on the biocontrol effect of Pseudomonas aeruginosa BL11 on betel leaf spot disease
[0044] In this experiment, 6-month-old betel nut seedlings were selected for biocontrol experiments. All seedlings were cultured in a 28°C greenhouse with 12 hours of darkness and light. The biocontrol strain BL11 was cultured in a shaker at 30°C and 200 rpm for 48 hours to obtain a bacterial suspension of BL11. After centrifugation at 8000 rpm for 10 minutes, it was resuspended with 10 mM MgCl2, then centrifuged and resuspended twice, and finally the bacterial concentration was adjusted to 10 with 10 mM MgCl2.8 CFU / mL.
[0045] The experiment was divided into 3 groups. The first group was the control group, in which only 10mM MgCl2 was sprayed on the leaves; the second group was the pathogen inoculation group, in which the betel nut leaves were sprayed with 10mM MgCl2 and then sprayed with the mycelium suspension of the anthrax strain CB1, as the pathogen inoculation group; the third group was the biological control group, in which 1mL of the supernatant of the strain BL11 was applied on the betel nut leaves, and then the mycelium suspension of the anthrax strain CB1 was sprayed again 2 days later. The leaves were placed under the above-mentioned greenhouse conditions for cultivation and the experimental results were observed and recorded. In each group of experiments, at least 5 replicates were set, and the leaf disease situation was determined by the number of lesions on the leaves.
[0046] According to statistics, in the control group that was not inoculated with the mycelial suspension of anthracnose fungi CB1, all leaves had lesions, while the leaves inoculated with the mycelial suspension had more typical leaf spots. The number of lesions was significantly reduced when the BL11 suspension was used before inoculation with the pathogen. This greenhouse experiment further showed that the BL11 suspension can significantly reduce the leaf spot disease caused by pathogens such as anthracnose fungi ( Figure 5 ).
Claims
1. A Pseudomonas aeruginosa BL11, characterized in that The strain preservation information is as follows: Name of the preservation unit: Guangdong Microbial Culture Collection Center; Address of the preservation unit: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Preservation date: August 27, 2024; Preservation number: GDMCC No.65060; Classification name: Pseudomonas aeruginosa.
2. Use of Pseudomonas aeruginosa BL11 described in claim 1 in preventing and treating betel nut leaf spot disease.
3. The use according to claim 2, characterized in that The betel leaf spot disease is caused by Colletotrichum and / or Phomopsis sp.
4. Use of the Pseudomonas aeruginosa BL11 according to claim 1 in preventing and treating bacterial wilt of Solanaceae crops.
5. A microbial agent for preventing and treating betel leaf spot disease, characterized in that The active ingredient of the microbial agent comprises the Pseudomonas aeruginosa BL11 described in claim 1.
6. A microbial agent for preventing and treating bacterial wilt of Solanaceae crops, characterized in that The active ingredient of the microbial agent comprises the Pseudomonas aeruginosa BL11 described in claim 1.
7. Use of the microbial agent according to any one of claims 5 to 6 in preventing and controlling betel nut leaf spot disease and / or bacterial wilt disease of Solanaceae crops.