A COMPETITIVE POLYMERASE CHAIN-REACTION TO QUANTIFY DNA OF LEPTOSPHAERIA-MACULANS DURING BLACKLEG DEVELOPMENT IN OILSEED RAPE

An assay based on the competitive polymerase chain reaction technique was developed to quantify Leptosphaeria maculans during blackleg disea se development in oilseed rape leaves, By means of primers specific to the highly virulent type of L. maculans, a heterologous internal cont rol template was prepared by amplifying and cloning DNA from Leptospha eria korrae under low-stringency annealing conditions, Coamplification of L. maculans with the internal control DNA provided accurate quanti fication of 1 to 10(9) copies of target DNA, The assay was applied to a comparative study of L. maculans colonization of resistant and susce ptible rape cultivars, The assay revealed that lesion size was associa ted with the quantity of L. maculans DNA during the first 12 days afte r inoculation of the susceptible cultivar Westar and the moderately re sistant cultivar Legend, In these cultivars, the quantity of DNA per l esion increased during the first 12 days after inoculation and then de clined, This decline in detectable fungal DNA coincided with abundant sporulation, rapid necrosis, and the onset of leaf senescence, Trace a mounts of L. maculans DNA were detected in the resistant cultivar Glac ier, in which lesion size was similar to that in the wounded, uninocul ated check, The assay is rapid, accurate, and very sensitive and can b e incorporated into conventional disease screening programs.