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Title: Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab

Author
item QU, XINSHUN - PENN STATE UNIV, PA
item Wanner, Leslie
item CHRIST, BARBARA - PENN STATE UNIV, PA

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2010
Publication Date: 1/21/2011
Citation: Qu, X., Wanner, L.A., Christ, B. 2011. Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab. Journal of Applied Microbiology. 110:769-777.

Interpretive Summary: Potato skin defects and diseases cause significant economic losses, and can be caused by numerous different pathogens. The symptoms of powdery scab and common scab, two important skin diseases, can be very similar, but management of the two diseases differs. Determination of the cause of disease is the essential first step in disease management. We designed a sensitive quantitative PCR (polymerase chain reaction) assay for simultaneous detection and quantification of the powdery scab and common scab-causing organisms, and demonstrated their utility, specificity and sensitivity in soil and potato tuber samples. This assay can be used to determine the cause of potato scab diseases, and will be useful for researchers, extension personnel and seed producers in determining the level of these pathogens in soil and on seed potatoes.

Technical Abstract: Powdery scab caused by the protist Spongospora subterranea and common scab caused by pathogenic bacteria in the genus Streptomyces are two important potato diseases worldwide. The symptoms of the two diseases are often similar, and diagnosis of the two diseases is therefore challenging for potato researchers and for potato growers. A multiplex real-time PCR assay using TaqMan probes was developed for the simultaneous detection and discrimination of the two pathogens. Real-time PCR primers and probe for S. subterranea were designed based on DNA sequence of the ribosomal RNA ITS2 region. Primers and probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens, and linear responses and high correlation coefficients between the amount of DNA and Ct values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant [or soil] extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, powdery scab and common scab pathogens were detected in some tubers with no visible symptoms and were discriminated in field tubers with confusing scab symptoms. Mixed infection of common scab and powdery scab on single tubers were also revealed. The multiplex real-time PCR assay developed in this study is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens in infected potato tubers or in soil.