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

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 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.

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

“…1997; Judelson and Tooley 2000; Jyan et al. 2002; Wangsomboondee and Ristaino 2002; Hussain et al. 2005) and quantitative real‐time PCR (Böhm et al.…”

Induction of defense-related proteins and growth promotion in tomato by mixture of Trichoderma harzianum OTPB3 and Bacillus subtilis OTPB1 and Pseudomonas putida OPf1 against Phytophthora infestans

“…Thus, development of seed coating formulation with the microbial consortium of OTPB3 and OTPB1 is crucial to raise healthy tomato seedlings as P.infestans is a soil/seed borne pathogen (Wangsomboondee and Ristaino, 2002). In addition to seed and soil borne inoculums, airborne inoculum is also vital to late blight outbreaks under congenial tropical and subtropical conditions.…”

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

“…DNA was extracted using the hexadecyltritnethylamnionium bromide (CTAB) procedure with some modifications in all of the experiments (38,41 ). DNA was extracted using the hexadecyltritnethylamnionium bromide (CTAB) procedure with some modifications in all of the experiments (38,41 ).…”