Citrus huanglongbing polypeptide strain as well as preparation method and application thereof

Abstract

The invention discloses a Citrus Huanglonglong polypeptide strain as well as a preparation method and application thereof, the strain is Bacillus licheniformis, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO: 31400. The bacillus licheniformis fermentation liquor preparation is prepared in an industrialized mode, and the citrus huanglongbing can be effectively prevented and treated.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of plant diseases, in particular to a citrus Huanglong polypeptide strain and a preparation method and application thereof. Background Art

[0002] Citrus Huanglongbing is a devastating citrus disease caused by bacteria of the genus Phloemella. The pathogen primarily spreads through infected citrus seedlings, infected scion grafts, or infected citrus psyllids, which then spread widely during the growth phase of citrus branches, causing significant damage to citrus production. Citrus Huanglongbing has become a major disease hindering the sustainable and healthy development of my country's citrus industry. Currently, the main prevention and control measures for citrus Huanglongbing are: first, strict control of the disease-transmitting insect psyllid; second, cultivating disease-free seedlings; and third, felling severely infected plants. Psyllids are biological insects that can migrate, making it extremely difficult to completely eliminate them from orchards; the cycle from planting to maturity of virus-free citrus seedlings is long, and if they are replanted in infected soil, they will continue to be infected; citrus Huanglongbing is caused by Gram-negative bacteria of the genus Phloemella, and cutting down diseased plants can only reduce the source of infection. The pathogens remaining in the surrounding environment and on the surface of normal plants will continue to multiply and infect, and cannot be completely eradicated. Therefore, the development and use of an anti-citrus Huanglongbing agent is a low-cost and optimal way to eradicate the disease. Summary of the Invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a citrus Huanglong polypeptide strain and a preparation method and application thereof.

[0004] In order to solve the above technical problems, the present invention discloses a citrus Huanglong polypeptide strain, which is Bacillus licheniformis, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC NO: 31400.

[0005] The invention also discloses a citrus Huanglong polypeptide bacterial liquid, which is prepared by fermenting the Bacillus licheniformis.

[0006] The present invention also discloses a method for preparing a citrus Huanglong polypeptide bacterial liquid, comprising the following steps: S1. Culture the strain in a seed stock shake flask at 37°C and 200 rpm for 16-24 hours. S2. Prepare the nutrient solution, sterilize at 121-123°C for 40-45 minutes, then cool to 37°C and transfer the seed stock to a shake flask. Cultivate at 37°C, 200 rpm, and a 1 VVM air volume for 12-16 hours. Once the seeds are mature, transfer them to a fermentation tank. S3. Prepare nutrients, sterilize at 121-123°C for 40-45 minutes, then cool to 37°C and add seeds to the seed tank. Cultivate at 37°C, 200 rpm, and 1 VVM air volume for 18-24 hours to obtain the fermentation solution.

[0007] Furthermore, the nutrients include corn flour, soybean cake powder, inorganic salts and water.

[0008] The invention also discloses a biological preparation. The obtained biological preparation is directly filled with the Bacillus licheniformis fermentation liquid or spray-dried into a solid for use.

[0009] Preferably, the spray drying air inlet temperature is controlled at 140-160°C, the air outlet temperature is controlled at 80-90°C, the feed amount and the dry powder moisture are controlled to be below 5%.

[0010] The biological preparation prepared from the Bacillus licheniformis fermentation liquid can be used to prevent and treat citrus Huanglongbing disease.

[0011] The present invention has the following advantages: the Bacillus licheniformis provided by the present invention can produce a variety of polypeptide antibacterial substances during the fermentation process, and the antibacterial substances can inhibit the reproduction and multiplication of Gram-negative bacteria in the roots and leaves of diseased citrus plants. By industrializing its fermentation liquid into a product preparation and spraying it on the roots to treat citrus, the purpose of preventing and controlling citrus Huanglongbing can be achieved. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the specific experimental process and experimental data of the present invention.

[0013] The strain used to prevent and control citrus Huanglongbing is Bacillus licheniformis, which was deposited at the General Microbiology Center of China Culture Collection Administration on July 22, 2024. The address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 31400.

[0014] This strain was first isolated from the root soil of tangerine trees in an orchard in Luo Han Guo Town, Yongfu County, Guilin City, Guangxi Zhuang Autonomous Region. The specific steps are as follows: 1. Collect bacterial samples: remove the topsoil, take soil samples and place them in sterilized bags and record them; 2. Preparation of Soil Dilution 3. Coating separation First, dilute the soil solution appropriately using the 10-fold dilution method. Take 0.1 ml of each of the last three dilutions and place them on LB culture plates, averaging two plates for each dilution. Then, use a sterilized spreader to evenly spread the bacterial solution on the LB culture plate and invert it in a 37°C incubator for 24 hours. 4. Colony identification and selection: Gram stain the strains and observe them under an oil immersion microscope. Identify the strains based on their cell morphology and colony characteristics. Select colonies with typical colony morphology, inoculate them onto plates, and incubate them at 37°C for 24 hours before use. 5. Purification: The selected strain was purified on LB medium plates using plate streak separation technique.

[0015] A citrus Huanglong polypeptide bacterial liquid is prepared by fermenting the Bacillus licheniformis. The specific preparation method is as follows: S1. Culture the strain in a seed flask at 37°C and 200 rpm for 16-24 hours. S2. Corn flour, bean cake powder, inorganic salts, defoamer, water, etc. are mixed according to a certain formula: corn flour 1%, bean cake powder 1%, (NH4)2SO4 0.5%, KH2PO4 0.01%, silicone defoamer 0.25%, and the balance is water. The final pH value is 7.1-7.5. Sterilize at 121-123 ° C for 40-45min, then cool to 37 ° C and access the mother shake flask. Incubate at 37 ° C, 200 r / min, and air volume of 1VVM for 12-16 hours. After the seeds mature, transfer them to the fermentation tank; S3. Mix corn flour, soybean meal, inorganic salt, defoamer, and water according to a specific formula: 1% corn flour, 1% soybean meal, 0.5% (NH4)2SO4, 0.01% KH2PO4, 0.25% silicone defoamer, and the balance is water. The final pH value is 7.1-7.5. Sterilize at 121-123°C for 40-45 minutes, then cool to 37°C, add seeds to the seed tank, and cultivate at 37°C, 200 r / min, and an air volume of 1VVM for 18-24 hours to obtain the fermentation solution.

[0016] After the obtained fermentation liquid is placed in a mixing tank, maltodextrin protective agent, silicon dioxide anticoagulant, light calcium carbonate carrier, etc. are added and stirred to mix evenly before entering the spray drying process. The spray drying air inlet temperature is controlled at 140-160℃, and the air outlet temperature is controlled at 80-90℃. The feed amount and dry powder moisture content are controlled below 5%. The dry powder collected by spray drying is a semi-finished product. The semi-finished product is adjusted to product specifications by adding carriers, promoters, etc. according to product needs, and is evenly mixed in a mixing tank and packaged into a solid product preparation.

[0017] The obtained fermentation liquid is placed in a formula tank, and guar gum, maltodextrin protective agent, glacial acetic acid preservative, etc. are added and stirred to mix evenly, and then the mixture is sent to the liquid filling workshop, and the liquid preparation is prepared after liquid metering and filling.

[0018] Application method: Dilute the fermentation liquid of Bacillus licheniformis 1000-10000 times, spray it on both sides of the sugar orange leaves in the evening or before 8 am. Spray the whole tree once every 4-7 days, and irrigate the roots once every 7 days during this period. Continue spraying and irrigating the roots for 3 months.

[0019] The effectiveness of Bacillus licheniformis fermentation liquid against Huanglongbing was tested on 150 citrus trees, which were divided into five groups, with 20 diseased plants and 10 normal plants in each group. The prevention and treatment tests were performed separately. The specific results are shown in the following table: Group Number of survivors Survival rate Number of infections infection rate Group 1 (dilute 1000 times, spray on the liquid surface) 4 20% 2 20% Group 2 (diluted 1000 times, root irrigation) 14 70% 0 0% Group 3 (diluted 1000 times, sprayed + root irrigated) 20 100% 0% 0% Group 4 (diluted 10,000 times, spray + root irrigation) 20 100% 0 0% Control group (clear water) 0 0% 10 100% As can be seen from the above table, the use of Bacillus licheniformis fermentation liquid has a very excellent effect on the prevention and treatment of citrus Huanglongbing, especially when the Bacillus licheniformis fermentation liquid is simultaneously sprayed on the leaves and irrigated with the roots, the prevention and control effect is the best.

[0020] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A citrus Huanglong polypeptide strain, characterized in that: The strain is Bacillus licheniformis, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO: 31400.

2. A citrus Huanglong polypeptide bacterial liquid, characterized in that: The bacterial liquid is prepared by fermenting the Bacillus licheniformis according to claim 1.

3. The method for preparing a citrus Huanglong polypeptide bacterial liquid according to claim 2, characterized in that: The steps include: S1. Culture the strain in a seed flask at 37°C and 200 rpm for 16-24 hours. S2. Prepare the nutrient solution, sterilize at 121-123°C for 40-45 minutes, then cool to 37°C and transfer the seed stock to a shake flask. Cultivate at 37°C, 200 rpm, and a 1 VVM air volume for 12-16 hours. Once the seeds are mature, transfer them to a fermentation tank. S3. Prepare nutrients, sterilize at 121-123°C for 40-45 minutes, then cool to 37°C and add seeds to the seed tank. Cultivate at 37°C, 200 rpm, and 1 VVM air volume for 18-24 hours to obtain the fermentation solution.

4. The method for preparing a citrus Huanglong polypeptide bacterial liquid according to claim 3, characterized in that: The nutrients include corn flour, soybean meal, inorganic salt and water.

5. A biological agent, characterized in that The fermentation broth obtained according to any one of claims 2 to 4 is directly filled or spray-dried into a solid for use.

6. A biological preparation according to claim 5, characterized in that: The spray drying process includes controlling the air inlet temperature at 140-160°C, the air outlet temperature at 80-90°C, and controlling the feed amount and dry powder moisture content below 5%.

7. The use of a biological agent according to any one of claims 5 or 6, characterized in that: The biological preparation is used for preventing and controlling citrus Huanglongbing disease.