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

Abstract

A quantitative polymerase chain reaction (PCR)-based assay for evaluation of propagules of Verticillium tricorpus (VT) in soils is presented. The assay is based on competitive PCR amplification applied directly to soil extracts. The accuracy of the assay was tested with a known number of VT propagules. The enumeration of propagules can be expressed both as number of microsclerotia or number of spores per gram of soil. Co-amplification of VT DNA with competitor DNA provided accurate quantification in the range of 102 to 106 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 conditions and the number of spores estimated by competitive PCR. Enumeration of propagules on potato field soils is presented for uninoculated and inoculated soils, at different inoculum concentrations The number of propagules detected varied from 0.16 to 19.2 microsclerotia per gram of soil. The number of propagules at harvest time was not correlated with the initial amount of inoculum used at planting time. Virulence of VT on potato plants is discussed in relation to inoculum built up in soils. The use of an accurate and reliable competitive PCR assay in combination with simple and fast methods for extracting DNA from soils should find many applications for such studies as pathogen survival ability in soils, competitiveness with other soil-borne pathogens, and cross-protection to the more pathogenic Verticillium species of potato.