巴西果胶杆菌噬菌体的分离纯化及应用

Isolation,purification,and application efficacy of Pectobacterium brasiliense Phage

目的:对巴西果胶杆菌(Pectobacterium brasiliense)的噬菌体进行分离纯化与稳定性分析及生防效果测定,为防治铜陵白姜茎腐病提供科学依据。方法:将从发病姜块中分离到的菌株纯化后,通过测序鉴定获得宿主菌。采集发病大白菜的根际土和养猪场污水,对获得的宿主菌进行噬菌体分离;测定噬菌体的宿主范围、稳定性;利用注射法测定噬菌体对铜陵白姜茎腐病的生防功效。结果:从72株分离菌株中鉴定出22株果胶杆菌,以P.brasiliense KH10作为宿主菌从根际土和污水中共分离出5株噬菌体,挑选3株裂解性较强的噬菌体分别命名为PB-1、PB-3和PB-5;3株噬菌体都具有较强的宿主专一性;均能在4~55℃内保持稳定,但不耐高温;在pH值范围为4~10时均能保持活性,紫外线照射1 h后效价均保持在10^(5)PFU/mL以上。同时,盆栽试验表明,与接种单一噬菌体相比,噬菌体组合处理对铜陵白姜茎腐病的发生具有更好的防控效果。结论:本研究分离得到的3株噬菌体具有良好的应用潜力,为进一步开发噬菌体类新型抗菌剂提供基础,同时为探讨铜陵白姜茎腐病的防治提供重要依据。

Objective:To isolate and purify the phages of Pectobacterium brasiliense,analyze its stability and test its biocontrol effect,and to provide scientific basis for the control of stem rot of Tongling white ginger.Methods:The strains isolated from infected ginger were purified in the laboratory,and the host bacteria were identified by sequencing.The rhizosphere soil of infected Chinese cabbage and the sewage of pig farm were collected for phages isolation.The host range and stability of phages were determined,and the efficacy of phages on stem rot of Tongling white ginger was measured by injection method.Results:Twenty-two strains of Pectobacterium spp.were identified from 72 isolates,and a total of five phages strains were isolated from the rhizosphere soil and effluent by using P.brasiliense KH10 as the host bacterium,and three phages strains with high lysogenicity were selected and named PB-1,PB-3,and PB-5,respectively.All three phages had strong host specificity,and all could maintain stability in the range of 4—55℃,but not resistant to high temperature.The activity could be maintained in the pH range of 4—10,and the titer remained above 10^(5) PFU/mL after ultraviolet irradiation for 1 h.Meanwhile,the pot experiment showed that compared with single phage inoculation,phage combination treatment had better control effect on the occurrence of stem rot of Tongling white ginger.Conclusion:The three phages strains isolated in this study had good potential for application and provided a basis for further development of phage-based novel antimicrobial agents,as well as an important basis for exploring the prevention and control of white ginger stem rot in Tongling.