一组靶向烟草青枯病的噬菌体鸡尾酒筛选与防控效果

doi:10.13560/j.cnki.biotech.bull.1985.2024-1000

摘要/Abstract

摘要：

目的从江西抚州烟区分离并系统解析针对烟草青枯病菌的专性噬菌体，筛选互补性强的株系并调制“噬菌体鸡尾酒”，为本地化噬菌体资源库建设和烟草青枯病生物防治提供技术支撑。方法针对抚州烟区的青枯菌，从发病区的原位根际土壤中筛选青枯菌专性噬菌体，探究其基本生物学特性和抑菌效果，进一步组合成噬菌体鸡尾酒，通过室内孔板试验和盆栽试验，探究其对烟草青枯病的防控效果。结果从抚州市的烟草根际土中共分离出22株青枯菌专性噬菌体，其中噬菌体GC3和LC5是肌尾噬菌体，拥有正二十面体头部和10 nm左右的尾巴，基因组中存在大量与尾管和鞘相关的基因，可能与自身病毒体结构相关，YH1和YH3是短尾噬菌体，拥有正二十面体头部和10 nm左右的尾丝，携带溶菌素、几丁质酶等基因，可能与侵染过程或细菌的裂解相关。室内孔板和盆栽试验证明，青枯菌专性噬菌体鸡尾酒能够提高对青枯菌的抑菌效果，降低青枯病的发生。进一步研究发现随噬菌体鸡尾酒接种浓度的增加，防控效果逐渐增强，高达65.59%。结论本研究从原位分离青枯菌专性噬菌体，系统地研究了其基础生物学特性，通过构建噬菌体鸡尾酒，防控效果提高至65.59%，为建立青枯菌专性噬菌体资源库和烟草青枯病的噬菌体疗法提供技术支撑。

关键词: 噬菌体鸡尾酒, 青枯病, 烟草, 根际土

Abstract:

Objective This study aims to isolate and systematically characterize specific bacteriophages targeting Ralstonia solanacearum, the causal agent of tobacco bacterial wilt, from the Fuzhou tobacco region of Jiangxi province. Strains with strong complementarity were screened and phage cocktail was modulated, providing technical support for the construction of a localized phage resource library and the biocontrol of tobacco bacterial wilt. Method We screened P. chrysogenum-specific phages from the in situ inter-root soil of the incidence area for P. chrysogenum in Fuzhou, explored their basic biological properties and bacteriostatic effects, and then further combined them into phage cocktails to investigate their preventive and control effects against tobacco green blight through indoor hole-plate and pot planting experiments. Result A total of 22 strains of P. chrysogenum-specific phages were isolated from the tobacco inter-root soil in Fuzhou city, among which phages GC3 and LC5 are mycotail phages, possessing an ortho icosahedral head and a tail of about 100 nm, with a large number of genes in the genome related to the tail tubes and sheaths, which may be related to their own virulence structure. Phage YH1 and YH3 are short-tailed phages, possessing an ortho icosahedral head and a 10 nm or so YH1 and YH3 are short-tailed phages with ortho icosahedral heads and tail filaments carrying genes such as lysozyme and chitinase, which may be related to the process of infestation or lysis of bacteria. Indoor hole plate and pot experiment proved that the Cyanobacteria-specific phage cocktail improved the inhibitory effect on Cyanobacteria and reduced the occurrence of Cyanobacteria. Further studies revealed that the control effect was gradually enhanced with increasing phage cocktail inoculation concentration up to 65.59%. Conclusion In this study, we isolated P. chrysogenum-specific phage from in situ, systematically studied its basic biological properties, and improved the prevention and control effect to 65.59% by constructing phage cocktail, which provided technical support for the establishment of P. chrysogenum-specific phage resource library and phage therapy for tobacco blight.

Key words: phage cocktail, bacterial wilt, tobacco, rhizosphere soil

冯小虎, 张文梅, 徐婧, 熊书斌, 王利兵, 宋文静. 一组靶向烟草青枯病的噬菌体鸡尾酒筛选与防控效果[J]. 生物技术通报, 2025, 41(11): 311-318.

FENG Xiao-hu, ZHANG Wen-mei, XU Jing, XIONG Shu-bin, WANG Li-bing, SONG Wen-jing. Screening and Biocontrol Efficiency of Phage Cocktail Targeted on a Group of Tobacco Bacterial Wilt[J]. Biotechnology Bulletin, 2025, 41(11): 311-318.

图/表 6

表1 噬菌体的筛选

Table 1 Screening of Bacteriophage

噬菌体编号 Phage number	平均噬菌斑直径 Mean phage diameter （mm）	地点 Location	噬菌体编号 Phage number	平均噬菌斑直径 Mean phage diameter （mm）	地点 Location
YH1	1.313±0.149	宜黄	LC1	2.080±0.097	黎川
YH2	1.064±0.156	宜黄	LC2	2.125±0.110	黎川
YH3	0.819±0.074	宜黄	LC3	2.218±0.131	黎川
YH4	1.679±0.181	宜黄	LC4	1.265±0.137	黎川
YH5	1.655±0.142	宜黄	LC5	2.458±0.055	黎川
YH6	1.643±0.142	宜黄	GC1	2.213±0.083	广昌
YH7	2.374±0.238	宜黄	GC2	1.858±0.160	广昌
YH8	1.125±0.170	宜黄	GC3	2.013±0.061	广昌
YH9	0.857±0.117	宜黄	GC4	1.605±0.032	广昌
YH10	0.868±0.192	宜黄	GC5	1.743±0.072	广昌
YH11	1.063±0.146	宜黄	GC6	2.098±0.149	广昌

图1 青枯菌（A）及其专性噬菌体（B）的平板形态及青枯菌专性噬菌体的电镜形态（C-F）

Fig. 1 Plate morphology of R. solanacearum (A) and its obligate bacteriophage (B), electron microscopic morphology of 4 strains of obligate bacteriophage of R. solanacearum (C-F)

图2 青枯菌专性噬菌体的系统发育距离

Fig. 2 Phylogenetic distance of an obligate phage of R. solanacearum

图3 4株青枯菌专性噬菌体的基因图谱

Fig. 3 Gene map of four phages specific to R. solanacearum

图4 噬菌体鸡尾酒与单一噬菌体对青枯菌的抑制效果（48 h）不同小写字母表示差异的显著性P<0.05，下同

Fig. 4 Inhibitory effects of phage cocktail and single phage on R. solanacearum (48 h)Different lowercase letters indicate the significance of differences P<0.05. The same below

图5 不同噬菌体鸡尾酒接种浓度对烟草青枯病发生的影响A：青枯病发生过程；B：病情严重程度；C：盆栽结束时的病情指数

Fig. 5 Effects of different phage cocktail inoculation concentrations on the occurrence of tobacco bacterial wiltA: Occurring process of bacterial wilt. B: Severity of disease. C: Disease index at the end of potted plant

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