Phytopathology. 2002 Nov;92(11):1227-35. doi: 10.1094/PHYTO.2002.92.11.1227.
ABSTRACT Peronospora tabacina is an obligately parasitic oomycete that causes blue mold, a devastating disease of tobacco. Genetic studies of this pathogen have been hampered by the lack of molecular markers. We generated a set of molecular markers for P. tabacina by collecting sporangiospores from infected tobacco leaves, extracting spore DNA, and cloning it in a plasmid vector. The resulting clones were then used to probe DNA from a collection of P. tabacina isolates to survey for polymorphisms. Most probes gave unexpected hybridization patterns with signal intensities that varied significantly from one DNA sample to another or between different DNA preparations of the same isolate. These results indicated that certain DNA preparations contained DNA from a source other than P. tabacina, which in turn suggested that some probes might have been derived from contaminating organisms present in the spore suspensions. Therefore, we characterized the inserts of several recombinant plasmids to determine their origins. Sequence analysis revealed that several of the inserts encoded peptides with similarity to bacterial proteins, suggesting that they were derived from bacterial contaminants. Of the remaining clones, five exhibited similarity to retroelements, one resembled eukaryotic helicase genes, and nine had no similarity to sequences in the databases. These were postulated to be true P. tabacina DNA clones. Verification of the origin of each probe was achieved by filtering a spore suspension, extracting DNA from the retentate and filtrate, and probing Southern blots of these DNA samples. These experiments confirmed the probe origins predicted by sequence analysis, resulting in the generation of 20 different restriction fragment length polymorphism probes that are specific for P. tabacina DNA. These probes should enable identification of reliable genetic markers for population studies of the blue mold organism.
摘要 烟草霜霉病菌（Peronospora tabacina）是一种专性寄生卵菌，可引起烟草霜霉病，这是一种毁灭性的烟草病害。由于缺乏分子标记，该病原体的遗传研究受到了阻碍。我们通过从感染的烟草叶片中收集游动孢子，提取孢子 DNA 并将其克隆到质粒载体中，为烟草霜霉病菌生成了一组分子标记。然后，使用这些克隆探针来探测来自一系列烟草霜霉病菌分离株的 DNA，以调查多态性。大多数探针与 DNA 样品之间的信号强度存在差异，这表明某些探针可能来自游动孢子悬浮液中存在的污染生物。因此，我们对几个重组质粒的插入物进行了特征分析，以确定其来源。序列分析表明，几个插入物编码的肽与细菌蛋白具有相似性，这表明它们来自细菌污染物。在剩余的克隆中，有五个与逆转录元件相似，一个与真核解旋酶基因相似，还有九个与数据库中的序列没有相似性。这些被推测为真正的烟草霜霉病菌 DNA 克隆。通过过滤游动孢子悬浮液、从截留物和滤液中提取 DNA 并探测这些 DNA 样本的 Southern 印迹，证实了每个探针的来源。这些实验证实了序列分析预测的探针来源，从而生成了 20 个不同的限制片段长度多态性探针，这些探针特异性针对烟草霜霉病菌 DNA。这些探针应该能够为该蓝霉菌种的群体研究提供可靠的遗传标记。
