Quantitative monitoring of colonization of olive genotypes by Verticillium dahliae pathotypes with real-time polymerase chain reaction

For Verticillium wilt diseases, quantitative measurement of the pathogen in infected tissues is necessary for characterizing the disease reaction of host genotypes to pathogen strains. The amount of pathogen DNA in the plant should be representative of the number of potential propagules. In this work, we quantified the DNA of highly virulent, defoliating (D) and mildly virulent, nondefoliating (ND) Verticillium dahliae in Verticillium wilt resistant (Acebuche-L), and -susceptible (Arbequina and Picual) olive genotypes by real-time polymerase chain reaction (PCR) assays. We show that the amount of pathogen DNA quantified in each olive genotype correlated with susceptibility to Verticillium wilt, but was influenced less by virulence of the infecting V. dahliae pathotype, suggesting that the extent of pathogen colonization does not clearly determine the virulence phenotype. Maximal pathogen DNA occurred in root and stem tissues before disease had fully developed in the susceptible olive genotypes, with pathogen DNA content in stem tissue being lower than that in root tissues. A boost in V. dahliae propagules in root tissues, as indicated by the amount of D and ND DNA, took place I week after inoculation, followed by a decrease over time. That decrease was sharp in Arbequina and Acebuche-L plants compared with a progressive reduction in Picual. The amount of V. dahliae DNA in roots of Picual plants remained at a high level for a long period of time following inoculation, but it eventually decreased too. Similar changes in pathogen DNA amounts over time occurred also in stem. Verticillium wilt hardly developed in Acebuche-L plants, but the amount of D and ND V. dahliae DNA in roots was comparable to that found in Arbequina plants. However, pathogen DNA could not be quantified in stems of Acebuche-L plants though it was detectable in them by nested-PCR. The results in our study show that real-time PCR is an excellent tool for quantitative diagnosis of olive infection by D and ND V. dahliae; as well as for monitoring pathogen colonization and assessing resistance and tolerance to Verticillium wilt in olive genotypes. This would help in olive breeding programs for resistance to the disease. (C) 2003 Elsevier Ltd. All rights reserved.