Quantification of 'Verticillium dahliae' in soil
Verticillium wilt in strawberries is an economically important disease caused by the soil-borne fungus Verticillium dahliae. A rapid kit-based extraction method and two quantitative PCR assays using specific primers for the quantification of V. dahliae microsclerotia in soils were developed. For the competitive PCR assay, a heterogeneous competitor DNA fragment was developed as an internal control and found to provide accurate quantification of V. dahliae DNA in the range of 2-200 pg and a detection limit of approximately 60 genomes in soil extract. SYBR Green I PCR had sensitivity down to one genome of V. dahliae DNA and has detected as little as 0.1 colony forming unit per gram of soil. Both PCR methods were tested on 13 naturally infested soil samples and compared with the current wet plating method. In order to relate PCR-based results to infectivity, an approximate estimate of the total number of genomes in microsclerotia was made, based on fresh microsclerotia produced in vitro in four different size ranges, using SYBT Green I PCR and found to be in range of 100 to 1000 genomes per microsclerotia. The variability of wet plating results using a common set of soil samples has highlighted the inaccuracy of this method and made it difficult to achieve a reasonable comparison with the quantitative PCR method. Possible reasons for the differences are discussed. In general, molecular quantification using SYBR Green I PCR offers faster and more efficient identification of the pathogen in soil and is suitable for routine diagnosis and epidemiological studies of Verticillium wilt. Attempts were made to study the effect of Coniothryium minitans mycoparasitism on V. dahliae. The results indicated that C. minitans was a poor biocontrol agent for V. dahliae.