DEVELOPMENT OF A PCR-BASED DIAGNOSTIC ASSAY FOR THE DETECTION OF COLLETOTRICHUM LUPINI ON LUPIN SEEDS

Lupins anthracnose is a destructive seed- and airborne disease affecting stems and pods, caused by Colletotrichum lupini. The pathogen is a member of the C. acutatum species complex, and contrasts with other members of the latter by its host specificity and by its apparent low genetic variability. Primary seed infections as low as 0.01-0.1% can cause very severe infections. Under this conditions, one 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 PCRquality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared and a 72 h incubation of the seeds with yeast malt broth amended with ampicillin, streptomycin and lactic acid was the most efficient technique to increase C. lupini biomass before DNA extraction. A species specific C. lupini primer pair was developed, based on rDNA IGS region sequences. The specificity was evaluated against 23 different Colletotrichum species and 21 different non target organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/1,000 infected seed.