National Center for Citrus Improvement, Hunan Agricultural University, Changsha 410128, Hunan, China.
Curr Microbiol. 2012 Sep;65(3):304-12. doi: 10.1007/s00284-012-0155-y. Epub 2012 Jun 7.
Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.
柑橘溃疡病菌（Xac）是引起柑橘细菌性溃疡病的病原菌，该病是世界柑橘产业的一种重要经济病害。为了监测柑橘溃疡病菌在不同柑橘品种中的侵染过程，构建了增强型绿色荧光蛋白（EGFP）可视化系统，用于观察病原菌在植物体内的繁殖和定位。首先，从感病的‘冰糖橙’甜橙叶片中分离出野生型 Xac，然后通过三亲交配将 EGFP 标记到野生型 Xac 中。通过 PCR 鉴定，比较了转化体与野生型 Xac 的生长动力学和致病性。通过喷雾接种和叶肉浸润将 EGFP 标记的细菌接种到‘冰糖橙’甜橙叶片上。接种后不同时间间隔，在共聚焦激光扫描显微镜下直接观察细菌细胞的增殖和扩散过程。结果表明，荧光显微镜下清晰发出绿色荧光的 EGFP 标记 Xac 显示了感染过程，并且与野生型对柑橘的致病性相同。因此，标记的 Xac 可作为监测病原菌感染的有效工具。
