Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging
Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging
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DOI：
10.1094/mpmi.2004.17.11.1175
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发表时间：
2004-11-01
影响因子：
3.5
通讯作者：
Schulz, A
中科院分区：
文献类型：
--
作者：
Christensen, NM;Nicolaisen, M;Schulz, A
植原体是无细胞壁的细菌，栖息在韧皮部并利用它进行传播。受感染的植物通常表现出生长模式的变化和作物产量的降低。建立了一种实时定量聚合酶链反应（Q-PCR）和生物成像方法，用于植原体的原位定量和定位。根据Q-PCR检测，植原体在一品红源叶中积累不成比例，在较小程度上，在源叶叶柄和茎中积累。然而，植原体积累是小的或检测不到的库器官（根和库叶）。对于生物成像，感染的植物组织用活体荧光染料染色，并使用共聚焦激光扫描显微镜检查。用DNA敏感的染料，病原体只在韧皮部中检测到，在那里它们在长春花的筛管中形成密集的团块。用苯胺蓝复染胼胝质和多光子激发来鉴定筛管。用电位染料，并不是所有的DNA阳性材料染色，这表明染料染色代谢活性植原体。一些高度感染的筛管含有植原体，这些植原体在染色时要么是无活性的，要么是死亡的。
Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.