Quantification of Verticillium biomass in wilt disease development

A polymerase chain reaction (PCR) assay has been adapted for the quantification of Verticillium biomass in wilt disease development. Using previously designed V. albo-atrum or V. dahliae specific primers, homologous and heterologous internal control templates were prepared which could be amplified with experimental samples to permit an accurate quantification of the unknown products and to detect inhibitory substances in the plant extracts. For homologous templates, a partial deletion in the target templates resulted in shorter products which could readily be differentiated by gel electrophoresis. For the heterologous templates, unrelated sequences were amplified from Fusarium genomic DNA under relatively non-specific conditions and a shorter fragment was again selected. Subsequent assays with both types of control template resulted in accurate calibration curves but since calculations with homologous templates were complicated by additional products of in vitro recombination, the heterologous templates were adapted for the standard assay. The standard test was applied to a comparative study of V. albo-atrum colonization in alfalfa or V. dahliae in sunflower. Inhibitory substances, which were particularly significant in sunflower, were readily detected using the internal control templates and appropriate dilutions were used to eliminate this artefact. The results accurately revealed substantial differences between the two species and in the alternate tissues and were similar to those obtained by conventional cytological or maceration and plating techniques, but the PCR-based assay was faster, more sensitive and significantly more accurate.