Yvon Michel, Thébaud Gaël, Alary Rémi, Labonne Gérard
Institut National de la Recherche Agronomique (INRA), UMR BGPI, CIRAD TA A-54/K, Campus international de Baillarguet, Montpellier, France.
Mol Cell Probes. 2009 Oct;23(5):227-34. doi: 10.1016/j.mcp.2009.04.005. Epub 2009 May 4.
'Candidatus Phytoplasma prunorum' is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of 'Ca. P. prunorum' is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of 'Ca. P. prunorum' in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma 'Ca. P. prunorum', 'Ca. P. pyri' (pear decline agent) and 'Ca. P. mali' (apple proliferation agent), as well as for representative strains of the 'Ca. Phytoplasma' genus.
“暂定李属植原体”是一种无细胞壁细菌，与欧洲核果黄化病（ESFY）有关，该病是李属植物（主要是杏树和日本李树）的一种严重病害。它可通过一种昆虫介体李短尾蚜传播，也可通过感染材料的贸易传播。基于PCR的方法能够灵敏且特异检测“暂定李属植原体”，这对该植原体而言至关重要，因为目前它无法培养，且不能通过血清学方法检测。我们开发了一种PCR检测方法，与现有的检测工具相比，该方法能对植物和昆虫中的“暂定李属植原体”进行快速、特异且灵敏的检测。对于需要对植原体滴度进行绝对定量的研究，使用相同引物开发了一种实时PCR检测方法，包括李短尾蚜的标准品。通过系列稀释比较了这些分子工具的灵敏度，并针对密切相关的植原体“暂定李属植原体”、“暂定梨植原体”（梨衰退病原体）、“暂定苹果植原体”（苹果增生病原体）的参考菌株和田间样本，以及“暂定植原体”属的代表性菌株，在计算机模拟和实验中评估了它们的特异性。
