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

Plant Disease
T Wangsomboondee, J B Ristaino

Abstract

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 o...Continue Reading

References

Jul 1, 1994·Applied and Environmental Microbiology·T ErsekJ T English
Aug 25, 1993·Nucleic Acids Research·H WangA J Cutler
Nov 1, 1995·Microbiological Research·F Niepold, B Schöber-Butin
Mar 21, 1998·Applied and Environmental Microbiology·J B RistainoG Parra
Jan 1, 1993·Annual Review of Phytopathology·J M Henson, R French

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Citations

Jul 25, 2009·Critical Reviews in Microbiology·Philip A O'BrienGiles E StJ Hardy
Jun 1, 2011·Plant Disease·Monica Blanco-Meneses, Jean Beagle Ristaino
Nov 3, 2010·Letters in Applied Microbiology·B LlorenteG D Alonso
Oct 6, 2018·The New Phytologist·Gaetan J A ThilliezIngo Hein

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