Development of Pathotype-Specific SCAR Markers for Detection of Verticillium albo-atrum Isolates from Hop

Abstract

Rapid polymerase chain reaction (PCR) assays were developed for the identification and detection of Verticillium albo-atrum hop pathotypes PG1 and PG2 from Slovenia. Of 17 pathotype-linked amplified fragment length polymorphism (AFLP) markers, 11 were cloned successfully and sequenced. To convert polymorphic AFLP markers into pathotype-specific sequence-characterized amplified region (SCAR) markers, 22 PG2- and 10 PG1-specific primer pairs were designed from 16 sequences. When primer specificity was tested on a wide range of Verticillium isolates, 10 PG2- and 6 PG1-specific primer pairs retained amplification specificity for V. albo-atrum Slovene hop isolates, but also amplified sequences in V. albo-atrum and V. dahliae hop isolates from different hop production areas in Europe, as well as in some isolates from other hosts. Primer combinations obtained from the AFLP-9-1 marker were specific only for V. albo-atrum PG2 isolates. The highly specific primers were used in multiplex PCR and a nested PCR to detect the V. albo-atrum PG2 pathotype in xylem tissue of hop plants. These new SCAR markers provide a valuable tool for rapid identification of V. albo-atrum PG1 and PG2 hop pathotypes.