Montes-Borrego Miguel, Muñoz Ledesma Francisco J, Jiménez-Díaz Rafael M, Landa Blanca B
Institute of Sustainable Agriculture (IAS), Córdoba, Spain.
Phytopathology. 2009 Jan;99(1):73-81. doi: 10.1094/PHYTO-99-1-0073.
A sensitive nested-polymerase chain reaction (PCR) protocol was developed using either of two primer pairs that improves the in planta detection of Peronospora arborescens DNA. The new protocol represented an increase in sensitivity of 100- to 1,000-fold of detection of the oomycete in opium poppy tissue compared with the detection limit of single PCR using the same primer pairs. The new protocol allowed amplification of 5 to 0.5 fg of Peronospora arborescens DNA mixed with Papaver somniferum DNA. The protocol proved useful for amplifying Peronospora arborescens DNA from 96-year-old herbarium specimens of Papaver spp. and to demonstrate that asymptomatic, systemic infections by Peronospora arborescens can occur in wild Papaver spp. as well as in cultivated opium poppy. Also, the increase in sensitivity of the protocol made possible the detection of seedborne Peronospora arborescens in commercial opium poppy seed stocks in Spain with a high frequency, which poses a threat for pathogen spread. Direct sequencing of purified amplicons allowed alignment of a Peronospora arborescens internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequence up to 730-bp long when combining the sequences obtained with the two primer sets. Maximum parsimony analysis of amplified Peronospora arborescens ITS rDNA sequences from specimens of Papaver dubium, P. hybridum, P. rhoeas, and P. somniferum from different countries indicated for the first time that a degree of host specificity may exist within populations of Peronospora arborescens. The reported protocol will be useful for epidemiological and biogeographical studies of downy mildew diseases as well as to unravel misclassification of Peronospora arborescens and Peronospora cristata, the reported causal agents of the opium poppy downy mildew disease.
利用两对引物中的任意一对开发了一种灵敏的巢式聚合酶链反应（PCR）方案，该方案提高了在植物中检测树状霜霉（Peronospora arborescens）DNA的能力。与使用相同引物对的单重PCR检测限相比，新方案在罂粟组织中检测这种卵菌的灵敏度提高了100至1000倍。新方案能够扩增与罂粟（Papaver somniferum）DNA混合的5至0.5飞克树状霜霉DNA。该方案被证明可用于从96年历史的罂粟属植物标本馆标本中扩增树状霜霉DNA，并证明树状霜霉的无症状系统性感染可发生在野生罂粟属植物以及栽培罂粟中。此外，该方案灵敏度的提高使得能够高频检测西班牙商业罂粟种子库存中种传的树状霜霉，这对病原体传播构成了威胁。对纯化扩增子进行直接测序，将用两组引物获得的序列组合后，可比对出长达730 bp的树状霜霉内部转录间隔区（ITS）核糖体DNA（rDNA）序列。对来自不同国家的野罂粟（Papaver dubium）、杂种罂粟（P. hybridum）、虞美人（P. rhoeas）和罂粟的标本中扩增的树状霜霉ITS rDNA序列进行最大简约分析，首次表明树状霜霉种群中可能存在一定程度的寄主特异性。所报道的方案将有助于霜霉病的流行病学和生物地理学研究，以及揭示树状霜霉和冠状霜霉（Peronospora cristata）的错误分类，后者是报道的罂粟霜霉病的致病因子。
