ISHS Acta Horticulturae 730: VI International Symposium on Artichoke, Cardoon and Their Wild Relatives
DETECTION OF VERTICILLIUM DAHLIAE ISOLATES DIFFERING IN VEGETATIVE COMPATIBILITY IN INFECTED ARTICHOKE PLANTS BY MULTIPLEX, NESTED PCR |
| Authors: | M. Collado-Romero, M. Berbegal, J. Mercado-Blanco, J. Armengol, J. García-Jiménez, R.M. Jiménez-Díaz |
| Keywords: | in-planta detection, differential virulence, PCR markers, vegetative compatibility groups |
| Abstract:
A multiplex, nested-PCR (polymerase chain reaction) protocol has been developed and optimized for the detection of Verticillium dahliae in infected artichoke plants, based on the amplification of specific DNA sequences of the pathogen using total DNA extracted from target tissues. Primer pairs for the procedure were designed based on DNA sequences associated with the vegetative compatibility grouping (VCG) of V. dahliae isolates infecting artichoke, which allowed to identify the belonging of isolates to VCGs according to the PCR marker amplicon produced in the second round of the nested-PCR assay. Thus, amplicons were associated with VCG1A and VCG2B334 (334 bp), VCG2A, VCG2B824 and VCG4B (688 bp), or only VCG2B824 (964 bp). That differential detection is of significance because VCG of isolates was shown to correlate with their virulence to artichoke cultivars in previous work. Artichoke plants artificially infected with V. dahliae as well as naturally-infected ones sampled from commercial fields in the Comunidad Valenciana region at eastern-central Spain were used to validate the molecular detection procedure and for comparisons with conventional detection by isolating the fungus from plant tissues in culture media. The molecular detection procedure was always more efficient than the conventional detection. In all cases, results from the multiplex, nested-PCR assays using artificially-infected plants were predictive of the VCG of the infecting V. dahliae isolates. Also, results using plants sampled from commercial fields showed that genetic/molecular diversity exists among V. dahliae isolates infecting artichoke in Valencia and Castellón provinces. Moreover double infection of a plant by different V. dahliae isolates was demonstrated. | |
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