Rural Development Administration, National Academy of Agricultural Science, Suwon 441-707, South Korea.
Mol Biotechnol. 2013 Sep;55(1):43-8. doi: 10.1007/s12033-012-9632-6.
Rice blast caused by Magnaporthe oryzae is a major disease in the paddy field and also a representative model system in the investigation of plant-microbe interactions. This study was undertaken to provide the quantitative evaluation method that specifically determines the amount of M. oryzae proliferation in planta. Real-time PCR was used as the detection strategy in combination with the primer pair and Taqman probe specific to MHP1, a unigene encoding HYDROPHOBIN that is indispensable for normal virulence expression. Based on the crossing point values from the PCR reactions containing a series of increasing concentration of cloned amplicon or fungal genomic DNA, correlation among the template's copy number or its amount and amplification pattern was calculated. Reliability of this equation was further confirmed using the DNA samples from the rice leaves infected with compatible or incompatible strains of M. oryzae. The primer pair used in the Taqman real-time PCR reaction can recognize the existence of fungal DNA as low as 1 pg. In sum, our quantitative evaluation system is applicable and reliable in the blast diagnosis and also in the estimation of objective blast disease progression.
稻瘟病由稻瘟病菌引起，是稻田中的主要病害，也是研究植物-微生物相互作用的代表性模式系统。本研究旨在提供一种定量评估方法，专门测定植物体内稻瘟病菌增殖的数量。实时 PCR 被用作检测策略，结合针对 MHP1 的引物对和 Taqman 探针，MHP1 是编码 HYDROPHOBIN 的单基因，对于正常的毒力表达是必不可少的。基于包含一系列递增浓度克隆扩增子或真菌基因组 DNA 的 PCR 反应的交点值，计算了模板拷贝数与其扩增模式之间的相关性。使用感染了稻瘟病菌亲和或不亲和菌株的水稻叶片的 DNA 样本进一步证实了该方程的可靠性。Taqman 实时 PCR 反应中使用的引物对可以识别低至 1pg 的真菌 DNA 的存在。总之，我们的定量评估系统在稻瘟病诊断和客观稻瘟病进展评估中是适用和可靠的。
