Identification of the tobacco blue mold pathogen, Peronospora tabacina, by polymerase chain reaction

Tobacco blue mold, caused by the oomycete pathogen Peronospora tabacina, is a highly destructive pathogen of tobacco (Nicotiana tabacum) seed beds, transplants, and production fields in the United States. The pathogen also causes systemic infection in transplants. We used polymerase chain reaction (PCR) with the primers ITS4 and ITS5, sequencing, and restriction digestion to differentiate P. tabacina from other important tobacco pathogens, including Alternaria alternata, Cercospora nicotianae, Phytophthora glovera, R parasitica, Pythium aphanidermatum, P. dissotocum, R myriotylum, P. ultimum, Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, Thielaviopsis basicola, and related Peronospora spp. A specific PCR primer, called PTAB, was developed and used with ITS4 to amplify a 764-bp region of DNA that was diagnostic for P. tabacina. The PTAB/ITS4 primers did not amplify host DNA or the other tobacco pathogens and were specific for P. tabacina on tobacco. DNA was detected to levels of 0.0125 ng. The PTAB primer was useful for detection of the pathogen in fresh, air-dried, and cured tobacco leaves. This primer will be useful for disease diagnosis, epidemiology, and regulatory work to reduce disease spread among fields.