ARS | Publication request: Multiplex PCR for four Sclerotinia species

Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2011
Publication Date: August 8, 2011
Citation: Abd-Elmagid, A.W., Garrido, P., Hunger, R.M., Melouk, H.A., Arif, M., Garzon, C.D. 2011. Multiplex PCR for four Sclerotinia species [abstract]. Phytopathology. 101:S2.

Technical Abstract: Sclerotinia homeocarpa, S. minor, S. sclerotiorum, and S. trifoliorum are common species within the genus Sclerotinia, where the morphological identification is challenging, especially when one crop hosts multiple species. The objective of this study was to design species specific primers compatible with multiplexing, for rapid and accurate species identification. Highly specific primers working under similar PCR conditions were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of, S. trifoliorum, the elongation factor-1 alpha gene of S. homeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of primers were tested individually and in multiplex against isolates of each species and each consistently amplified DNA of their target species only. Four DNA fragments of different sizes were amplified: a 264-bp PCR product for S. minor, a 218-bp product for S. homeocarpa, a 171-bp product for S. sclerotiorum, and a 97-bp product for S. trifoliorum. Primer sets differed in their lower sensitivity limits: SMlac2 = 1pg/microliter; SHelf1 = 0.1pg/microliter; SSaspr, and STCad = 10pg/microliter. These primer sets can be used individually for verifying the identity of isolates of a particular species or as a multiplex assay. This multiplex assay is accurate and rapid tool to differentiate between these plant pathogenic Sclerotinia species in a single PCR reaction.