A competitive PCR-based assay to quantify Verticillium tricorpus propagules in soil

A quantitative polymerase chain reaction (PCR)-based assay for evaluation o f propagules of Verticillum tricorpus (VT) in soils is presented. The assay is based on competitive PCR amplification applied directly to soil extract s. The accuracy of the assay was tested with a known number of VT propagule s. The enumeration of propagules can be expressed both as number of microsc lerotia or number of spores per gram of soil. Go-amplification of VT DNA wi th competitor DNA provided accurate quantification in the range of 10(2) to 10(6) spores and 1 to 500 microsclerotia. A strong correlation (r = 0.99) was found between number of spores added to VT-free soil under controlled c onditions and the number of spores estimated by competitive PCR. Enumeratio n of propagules on potato field soils is presented for uninoculated and ino culated soils, at different inoculum concentrations The number of propagule s detected varied from 0.16 to 19.2 microsclerotia per gram of soil. The nu mber of propagules at harvest time was not correlated with the initial amou nt of inoculum used at planting time. Virulence of VT on potato plants is d iscussed in relation to inoculum built up in soils. The use of an accurate and reliable competitive PCR assay in combination with simple and fast meth ods for extracting DNA from soils should find many applications for such st udies as pathogen survival ability in soils, competitiveness with other soi l-borne pathogens, and cross-protection to the more pathogenic Verticillium species of potato.