水华蓝藻噬藻体对不同条件培养的宿主细胞感染性分析

ANALYSIS OF THE CYANOPHAGE (PAV-LD) INFECTION IN HOST CYANOBACTERIA UNDER DIFFERENT CULTURE CONDITIONS

在从武汉东湖水样中培养分离水华蓝藻噬藻体(Planktothrix agardhii Virus from Lake Donghu,PaV-LD)的基础上,对在不同条件培养的宿主蓝藻细胞中,PaV-LD增殖效率及裂解作用进行了测定分析。分别将PaV-LD接种到生长期、半连续培养更新率或光照不同的宿主蓝藻液中,并采用稀释培养计数(Mostprobable number,MPN)方法与电镜观察,测定子代PaV-LD释放量及宿主细胞的裂解作用。结果显示:对数生长期宿主蓝藻单个细胞中子代PaV-LD的平均释放量为350感染单位(Infectious Units,IU/cell),显著高于稳定生长期的平均释放量110 IU/cell。在用新鲜培养基更新率为0%、35%、50%和65%的半连续培养宿主蓝藻中,接种PaV-LD 5d之后,噬藻体的释放量分别约为50 IU/cell、70 IU/cell、220 IU/cell或310 IU/cell,表明子代PaV-LD释放率随培养基更新率的增加而显著提高。在光照条件下感染3—4d后,宿主蓝藻细胞充分裂解,并释放大量子代PaV-LD,滴度可由初始7.00×103IU/mL快速增加到8.56×107IU/mL;但在遮光条件下,同样感染的蓝藻细胞未见裂解,也检测不到释放的子代噬藻体。电镜观察显示,在光照条件下感染的蓝藻细胞类囊体膜结构消失,而大量子代PaV-LD颗粒主要分布在原有类囊体的部位。显然,宿主蓝藻细胞的培养条件和状态可能对获得噬藻体纯培养有决定性影响。

Based on a cyanophage PaV-LD （Planktohtrix agardhii Virus isolated from Lake Donghu） infecting bloom-forming filamentous cyanobacterium Planktothrix agardhii from a typical shallow freshwater lake--Lake Donghu, the impact of host cyanobacterial culture conditions on the cyanophage efficiency and lytic activity of this cyanophage was analyzed in this study. PaV-LD was inoculated into cyanobacterial cultures of host under different states, such as growth phase, renewal rate of semicontinuous cultures and illumination condition, and the burst size of progeny cyanophage and host cell lysis efficiency of host cyanobacteria was determined by the most-probable number （MPN） method and transmission electron microscopy （TEM） observation, respectively. The results showed that the av- erage burst size of infectious progeny PaV-LD released from each infected host cell at exponential phase was 350 infec- tious units （IU）/cell, which was significantly higher than that of each infected host cell at stationary phase （110 IU/cell）; the initial cell densities of host cultures at exponential phase and stationary phase was 1.38×10^6 cells/mL and 1.43× 10^7 cells/mL, and respectively decreased to 1.75× 10^3 cells/mL and 3.0×10^3 cells/mL at 5 days after infection, indicating that no significant difference was found in the sensitivity of host cell at different growth stage to PaV-LD infection. Semi- continuous culture experiment was performed, either 0, 35%, 50% and 65% of the fresh BG11 medium was replaced and 5 days after infection, the burst size of PaV-LD were estimated to be ca. 50 IU/cell, 70 IU/cell, 220 IU/cell and 310 IU/cell. Thus, the burst size of PaV-LD increased with the addition of medium renewal rate in a certain range. Infected host cells were lysed under light 3 or 4 days later. Numerous progeny PaV-LD was observed with TEM. Additionally, the titer aggrandized rapidly from 7.0×10^3 IU/mL to 8.56×10^7 IU/mL during this period. On the contrary, the infected host ceils were not cracked under darkness. Simultaneously, progeny PaV-LD could not be detected. Further, TEM ob- servation showed that thylakoid membranes of infected host cells under light were disappeared, and progeny PaV-LD were mainly distributed in the thylakoid location of the original. The results showed that cyanobacteria cell culture conditions might play a decisive role in obtaining pure cultures of the cyanophage