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Lupins anthracnose is a destructive seed and airborne disease caused by <i>Colletotrichum lupini</i>, affecting stems and pods. Primary seed infections as low as 0.01&#8722;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 <i>C. lupini</i> 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 <i>C. lupini</i> primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of <i>C. lupini</i>, 23 different <i>Colletotrichum</i> species, and 21 different organisms isolated from seeds of <i>Lupinus albus</i> cv. Multitalia, <i>L. luteus</i> cv. Mister, and <i>L. angustifolius</i> cv. Tango. The protocol described here enabled the detection of <i>C. lupini</i> in samples artificially infected with less than 1/10,000 infected seed.