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A PCR‐based ‘molecular tool box’ for in planta differential detection of Verticillium dahliae vegetative compatibility groups infecting artichoke
Author(s) -
ColladoRomero M.,
Berbegal M.,
JiménezDíaz R. M.,
Armengol J.,
MercadoBlanco J.
Publication year - 2009
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/j.1365-3059.2008.01981.x
Subject(s) - verticillium dahliae , biology , verticillium wilt , multiplex polymerase chain reaction , multiplex , polymerase chain reaction , dna extraction , molecular marker , botany , microbiology and biotechnology , genetics , gene
A multiplex‐nested‐PCR procedure was developed for in planta detection of Verticillium dahliae isolates infecting artichoke and assessment of their vegetative compatibility groups (VCGs). PCR markers were identified and assigned to V. dahliae VCGs, including: i) a 334 bp marker amplified from VCG1A or VCG2B 334 isolates; ii) a 688 bp marker amplified from VCG2A or VCG4B isolates; and iii) a 688 bp and a 964 bp PCR marker amplified from VCG2B 824 isolates. The infecting V. dahliae VCGs were identified in artichoke tissues according to specific patterns of amplified markers after two rounds of PCR. The PCR‐based ‘molecular tool box’ was first optimized using DNA extracted from artichoke plants artificially inoculated with isolates representative of known VCGs. Thereafter, the efficiency of the molecular procedure was tested using DNA extracted from naturally‐infected artichoke plants showing a range of symptom severity as well as from symptomless plants. The novel multiplex‐nested‐PCR assay was clearly superior in detecting the pathogen compared to conventional isolation procedures, and in addition was informative about the VCGs. Moreover, the PCR method allowed the detection and VCG identification of V. dahliae infections in symptomless but infected plants, which had yielded false negatives when checked by microbiological isolation procedures. This ‘molecular tool box’ has uncovered the presence of several V. dahliae VCGs infecting the same artichoke plants in the Comunidad Valenciana Region. In addition, it is useful for genetic and pathogenicity diversity studies of V. dahliae populations infecting artichoke, and may help in predicting the severity of verticillium wilt epidemics.