Radišek Sebastjan, Jakše Jernej, Javornik Branka
Plant Protection Department, Slovenian Institute of Hop Research and Brewing, Cesta Žalskega tabora 2, SI-3310 Žalec, Slovenia.
Centre for Plant Biotechnology and Breeding, Agronomy Department, Biotechnical Faculty, Jamnikarjeva 101, Ljubljana 1000, Slovenia.
Plant Dis. 2004 Oct;88(10):1115-1122. doi: 10.1094/PDIS.2004.88.10.1115.
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.
已开发出快速聚合酶链反应（PCR）检测方法，用于鉴定和检测来自斯洛文尼亚的黑白轮枝菌啤酒花致病型PG1和PG2。在17个与致病型相关的扩增片段长度多态性（AFLP）标记中，成功克隆并测序了11个。为了将多态性AFLP标记转化为致病型特异性序列特征扩增区域（SCAR）标记，从16个序列中设计了22对PG2特异性引物和10对PG1特异性引物。当在广泛的轮枝菌分离株上测试引物特异性时，10对PG2特异性引物和6对PG1特异性引物对斯洛文尼亚啤酒花黑白轮枝菌分离株保持扩增特异性，但也能扩增来自欧洲不同啤酒花产区的黑白轮枝菌和大丽轮枝菌啤酒花分离株以及一些来自其他寄主的分离株中的序列。从AFLP - 9 - 1标记获得的引物组合仅对黑白轮枝菌PG2分离株具有特异性。这些高特异性引物用于多重PCR和巢式PCR，以检测啤酒花植株木质部组织中的黑白轮枝菌PG2致病型。这些新的SCAR标记为快速鉴定黑白轮枝菌PG1和PG2啤酒花致病型提供了有价值的工具。
