Optimization of Sample Size and DNA Extraction Methods to Improve PCR Detection of Different Propagules of...

The plant pathogen Phytophthora infestans causes a destructive blight of potato tubers and foliage. A rapid polymerase chain reaction (PCR) assay has been developed for detection of P. infestans in potato tubers. In this study, the effect of method of DNA extraction on different propagule types and the minimal number of propagules of P. infestans detectable by PCR were assessed using the PINF and internal transcribed spacer (ITS)5 primers. Sensitivity of the primers for PCR was high, and DNA was detectable at concentrations as low as 10 pg/ml. Zoospores and oospores responded differently to different extraction methods, whereas all extraction methods worked equally well for sporangia. Freeze-thaw DNA lysis, in which propagules were frozen at -80°C and thawed at 65°C three times for 15 min each, or direct PCR, in which propagules were placed directly in the reaction mix, were effective methods for PCR detection of sporangia or zoospores but were not effective methods for PCR detection of DNA in oospores of P. infestans. DNA from a single sporangium or oospore could be amplified by PCR after hexadecyltrimethyl-ammonium bromide (CTAB) or NaOH lysis extraction methods, whereas DNA from a single zoospore could be amplified by CTAB or direct PCR methods. "IsoCode" Stixs, used in forensic applications, were used to collect the pathogen from leaf and tuber lesions and provided another simple method to extract template DNA. PCR detection of the pathogen in infected tubers using PINF and ITS5 primers was compared to tissue isolation or visual observation. The probability of detection of P. infestans in infected tubers at 7 days post inoculation using the PCR assay, tissue isolation, or visual observation was 0.90, 0.80, and 0.75, respectively. The PINF and ITS5 primers provide a powerful tool for rapid and sensitive detection of zoospores, sporangia, and oospores of P. infestans when used with appropriate extraction methods, and could easily be deployed to reduce spread of the pathogen in potato tubers.