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A PCR-based Assay for Detection of Alternaria radicina on Carrot Seed

January 2001 , Volume 85 , Number  1
Pages  18 - 23

B. M. Pryor and R. L. Gilbertson , Department of Plant Pathology, University of California, Davis 95616

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Accepted for publication 30 August 2000.

A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.

Additional keywords: black rot of carrot, Daucus carota, seedborne pathogen

© 2001 The American Phytopathological Society