CN-115651867-B - Method for improving biocontrol bacteria and preventing and controlling soil-borne bacterial wilt by utilizing obligate phage

Abstract

The invention discloses bacterial wilt biocontrol bacteria YL-Ste-01evo, the preservation number is CCTCC NO: M2022939, bacterial wilt biocontrol bacteria YL-Ste-01, the preservation number is CCTCCNO: M2022324, and a lytic phage YL-Ste-01P, the preservation number is CCTCC NO: M2022460. The invention discloses application of biocontrol bacteria YL-Ste-01evo or biocontrol bacteria YL-Ste-01evo synergistic phage YL-Ste-01P in prevention and control of tomato soil-borne bacterial wilt. The biocontrol strain YL-Ste-01 can remarkably inhibit the growth of bacterial wilt and reduce the incidence rate of bacterial wilt of tomato plants, and the biocontrol strain YL-Ste-01evo with stronger phage resistance and better antibacterial effect is obtained through the evolution of phage stress YL-Ste-01.

Inventors

• WEI ZHONG
• WANG XIAOFANG
• Hou Rujiao
• YANG KEMING
• XU YANGCHUN
• SHEN QIRONG

Assignees

• 南京农业大学

Dates

Publication Date

20260505

Application Date

20221101

Claims (8)

1. 1. A bacterial wilt biocontrol strain YL-Ste-01evo, classified and named as stenotrophomonas maltophilia (Stenotrophomonas maltophilia), is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022939 at the month of 2022 and 21.
2. 2. A microbial inoculum comprising the biocontrol fungus YL-Ste-01evo of claim 1 at a concentration of 1 x10 8 ～5×10 8 CFU/mL.
3. 3. A lytic phage YL-Ste-01P, classified as a stenotrophomonas maltophilia phage (Stenotrophomonas maltophilia phage), is preserved in China center for type culture Collection (CCTCC NO: M2022460) at 4 months 22 of 2022.
4. 4. Use of the biocontrol fungus YL-Ste-01evo of claim 1 or the biocontrol fungus YL-Ste-01evo of claim 1 in cooperation with the phage YL-Ste-01P of claim 3 for controlling tomato soil borne bacterial wilt.
5. 5. A method for preventing and controlling tomato soil-borne bacterial wilt is characterized by comprising the steps of transplanting tomato seedlings for 6-7 days, and adding the biocontrol fungus YL-Ste-01evo microbial inoculum of claim 2 into soil by a root irrigation method.
6. 6. The method for controlling tomato soil-borne bacterial wilt according to claim 5, wherein the final concentration of biocontrol bacteria YL-Ste-01evo is 1X 10 8 ～1×10 9 CFU/plant.
7. 7. A method for improving biocontrol bacteria and preventing soil-borne bacterial wilt by utilizing obligate phage is characterized in that after tomato seedlings are transplanted for 6-7 days, the biocontrol bacteria YL-Ste-01evo microbial inoculum of claim 2 is added into soil by a root irrigation method, and phage YL-Ste-01P is inoculated after 2-3 days.
8. 8. The method for controlling soil-borne bacterial wilt by using obligate phage to promote biocontrol bacteria according to claim 7, wherein the final concentration of biocontrol bacteria YL-Ste-01evo is 1X 10 8 ～1×10 9 CFU/plant and the final titer of phage YL-Ste-01P is 1X 10 8 ～1×10 9 PFU/plant.

Description

Method for improving biocontrol bacteria and preventing and controlling soil-borne bacterial wilt by utilizing obligate phage Technical Field The invention belongs to the technical field of microorganisms, and relates to application of biocontrol bacteria and soil-borne bacterial wilt prevention and control by utilizing specific phage, in particular to application of biocontrol bacteria YL-Ste-01, a specific target host bacteria YL-Ste-01 lytic phage YL-Ste-01P, an evolved biocontrol bacteria YL-Ste-01evo, biocontrol bacteria YL-Ste-01 or biocontrol bacteria YL-Ste-01evo collaborative phage YL-Ste-01P in prevention and control of tomato soil-borne bacterial wilt. Background Soil-borne bacterial wilt caused by Lawsonia solanaceae (Ralstonia solanacearum, abbreviated as bacterial wilt) severely restricts sustainable development of agriculture, and causes serious loss for farmers throughout the year. The physical and chemical prevention and control modes have quick response, but are easy to induce the drug resistance of pathogenic bacteria, and the bacterial wilt is killed and the microbial community is destroyed due to weak targeting in the use process. Soil-borne diseases are prevented and controlled by rhizosphere microorganisms, and the rhizosphere microorganisms become novel environment-friendly biocontrol means [1] such as probiotics, bacteriophage, protozoa and the like. Among them, the phage is a virus that infects bacteria specializedly, and researches show that the specializedly phage of the bacterial wilt plays an important role in the process of preventing and controlling soil-borne bacterial wilt. In addition to pathogenic bacterial obligate phages, there are also a large number of biocontrol phages in the rhizosphere. However, the research on the reduction efficiency of the biocontrol bacteria by the obligate phage of the rhizosphere biocontrol bacteria is relatively less. As a bacterial specificity 'killer', the phage can effectively regulate and control population density, physiology and behavior of rhizosphere biocontrol bacteria, and simultaneously as an important driving force for bacterial evolution, the phage can also change other life history characteristics of host bacteria, such as [2] of growth, movement, metabolism and the like of the biocontrol bacteria while inducing the resistance evolution of the host bacteria, thereby affecting rhizosphere colonization and competition capability of the biocontrol bacteria. Whether the biocontrol bacteria can be directionally domesticated by utilizing the obligate phage of the biocontrol bacteria or not, the efficiency of enhancing the biocontrol bacteria to reduce the bacterial wilt in soil is unclear, and the further research is needed. Disclosure of Invention The invention aims to obtain a biocontrol strain YL-Ste-01 capable of inhibiting bacterial wilt from rhizosphere of tomato plants, and screen and obtain a phage YL-Ste-01P of the biocontrol strain YL-Ste-01, and the inventor finds that under the stress of the phage, the biocontrol strain YL-Ste-01 evolves, and the bacterial wilt inhibition capability and the resistance to the phage are enhanced, so that the biocontrol strain YL-Ste-01evo capable of inhibiting the bacterial wilt is further screened and obtained, and the greenhouse potting experiment shows that the effect of preventing and controlling the bacterial wilt of the tomato can be obviously improved by applying the evolved strain YL-Ste-01 evo. The invention aims at realizing the following technical scheme: A bacterial wilt biocontrol strain YL-Ste-01evo, classified and named as stenotrophomonas maltophilia (Stenotrophomonas maltophilia), is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022939 at the month of 2022 and 21. A bacterial wilt biocontrol strain YL-Ste-01, classified and named as stenotrophomonas maltophilia (Stenotrophomonas maltophilia), is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022324 in the year 2022 and the month 28. A lytic phage YL-Ste-01P belongs to the order of end phages, the family of end phages, and is classified and named as stenotrophomonas maltophilia phage (Stenotrophomonas maltophilia phage), and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2022460 at 4 months of 2022. The specific lytic phage YL-Ste-01P can stress the biocontrol fungus YL-Ste-01 to evolve to obtain the biocontrol fungus YL-Ste-01evo, and compared with the biocontrol fungus YL-Ste-01, the antipathogenic fungus capability and phage resistance of the biocontrol fungus YL-Ste-01evo are obviously enhanced. A method for preparing bacterial wilt biocontrol bacteria YL-Ste-01 by means of phage YL-Ste-01P stress comprises the steps of culturing with NA liquid culture medium to obtain YL-Ste-01 bacterial solution in logarithmic phase, adjusting the OD 600 value of the bacterial solution to