Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia

Sclerotinia sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Eriks, and S. homoeocarpa F.T. Benn are the most relevant plant pathogenic species within the genus Sclerotinia because of their large range of economically important hosts, including tomato, peanut, alfalfa, and turfgrass, among others. Species identification based on morphological characteristics is challenging and time demanding, especially when one crop hosts multiple species. The objective of this study was to design specific primers compatible with multiplexing, for rapid, sensitive and accurate detection and discrimination among four Sclerotinia species. Specific primers were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of S. trifoliorum, the elongation factor-1 alpha gene of S. homoeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of each primer set was tested individually and in multiplex against isolates of each species and validated using genomic DNA from infected plants. Each primer set consistently amplified DNA of its target gene only. DNA fragments of different sizes were amplified: a 264bp PCR product for S. minor, a 218bp product for S. homoeocarpa, a 171bp product for S. sclerotiorum, and a 97bp product for S. trifoliorum. These primer sets can be used individually or in multiplex for identification of Sclerotinia spp. in pure culture or from infected plants. The multiplex assay had a lower sensitivity limit than the simplex assays (0.0001pg/μL DNA of each species). The multiplex assay developed is an accurate and rapid tool to differentiate between the most relevant plant pathogenic Sclerotinia species in a single PCR reaction. ► We designed four Sclerotinia species specific end-point PCR primer sets. ► Our primers are compatible with multiplex-PCR. ► Primer specificity and sensitivity were tested individually and in multiplex. ► Diagnostic assays were validated using genomic DNA from fungi and infected plants. ► Our multiplex-PCR assay accurately discriminates four Sclerotinia species.