A Competitive Polymerase Chain Reaction to Quantify DNA ofLeptosphaeria maculansDuring Blackleg Development in Oilseed Rape

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Abstract
An assay based on the competitive polymerase chain reaction technique was developed to quantify Leptosphaeria maculans during blackleg disease development in oilseed rape leaves. By means of primers specific to the highly virulent type of L. maculans, a heterologous internal control template was prepared by amplifying and cloning DNA from Leptosphaeria korrae under low-stringency annealing conditions. Coamplification of L. maculans with the internal control DNA provided accurate quantification of 1 to 10(9) copies of target DNA. The assay was applied to a comparative study of L. maculans colonization of resistant and susceptible rape cultivars. The assay revealed that lesion size was associated with the quantity of L. maculans DNA during the first 12 days after inoculation of the susceptible cultivar Westar and the moderately resistant cultivar Legend. In these cultivars, the quantity of DNA per lesion increased during the first 12 days after inoculation and then declined. This decline in detectable fungal DNA coincided with abundant sporulation, rapid necrosis, and the onset of leaf senescence. Trace amounts of L. maculans DNA were detected in the resistant cultivar Glacier, in which lesion size was similar to that in the wounded, uninoculated check. The assay is rapid, accurate, and very sensitive and can be incorporated into conventional disease screening programs.

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