Detection and Quantification of Peronospora tabacina Using a Real-Time Polymerase Chain Reaction Assay

Abstract

Peronospora tabacina is an obligate plant pathogen that causes blue mold of tobacco. The disease is difficult to diagnose before the appearance of symptoms and can be easily spread in nonsymptomatic tobacco seedlings. We developed a real-time polymerase chain reaction (PCR) assay for P. tabacina that uses 5′ fluorogenic exonuclease (TaqMan) chemistry to detect and quantify pathogen DNA from diseased tissue. The primers and probe were designed using 5.8S ribosomal DNA sequences from 12 fungal and oomycete tobacco pathogens and 24 Peronospora spp. The PtabBM TaqMan assay was optimized and performed with a final concentration of 450 nM primers and 125 nM probe. The real-time TaqMan assay was assessed for sensitivity and the lower detection limit was 1 fg of DNA. The assay was specific for P. tabacina. None of the DNA from other tobacco pathogens, nonpathogens, or the host were amplified. The PtabBM TaqMan assay was useful for detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings. The assay was used to quantify host resistance and it was possible to detect the pathogen 4 days postinoculation in both medium-resistant and susceptible tobacco cultivars. The real-time PCR assay for P. tabacina will be a valuable tool for the detection of the pathogen and of use to regulatory agencies interested in preventing the spread of blue mold.