|Detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (P. schachtii) using spore traps and quantitative PCR assays|
S. J. KLOSTERMAN (1), A. Anchieta (2), N. McRoberts (3), S. Koike (4), K. V. Subbarao (5), H. Voglmayr (6), Y. J. Choi (7), M. Thines (8), F. N. Martin (9). (1) USDA ARS, Salinas, CA, U.S.A.; (2) USDA, Salinas, CA, U.S.A.; (3) University of California, Davis, CA, U.S.A.; (4) University of California Cooperative Extension - Monterey County, Salinas, CA, U.S.A.; (5) Univ of California, Salinas, CA, U.S.A.; (6)
Downy mildew of spinach, caused by <i>Peronospora effusa, </i>is a disease constraint on spinach production worldwide. The aim of this study was to develop a real-time quantitative PCR assay for detection of airborne inoculum of <i>P. effusa</i> in California. This type of assay may, in combination with disease-conducive weather forecasting, be helpful to time fungicide applications for disease management. Among oomycete ribosomal DNA (rDNA) sequences examined, the highest nucleotide sequence identity was observed between rDNA sequences of <i>P. effusa </i>and <i>Peronospora schachtii, </i>the cause of downy mildew on <i>Beta vulgaris</i> (beet and Swiss chard). Single nucleotide polymorphisms (SNPs) were detected between <i>P. effusa</i> and <i>P. schachtii </i>18S rDNA regions for design of <i>P. effusa-</i> and <i>P. schachtii-</i>specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both <i>P. effusa</i> and <i>P. schachtii</i> rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of<i> P. effusa</i> from impaction spore trap samples. The rDNA copy numbers of <i>P. effusa</i> were on average 3400-fold higher from trap samples collected near an infected spinach field than at sites remote from such fields.