Molecular Detection of the Seed-Borne Pathogen Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster.

Lupins anthracnose is a destructive seed and airborne disease caused by , affecting stems and pods. Primary seed infections as low as 0.01-0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of , 23 different species, and 21 different organisms isolated from seeds of cv. Multitalia, cv. Mister, and cv. Tango. The protocol described here enabled the detection of in samples artificially infected with less than 1/10,000 infected seed.