A Two-Step Molecular Detection Method for Pyrenophora tritici-repentis Isolates Insensitive to Qol Fungicides

作者：Patel Jaimin S; Meinhardt Steven W; Sierotzki Helge; Stammler Gerd; Gudmestad Neil C; Adhikari Tika B^*

来源：Plant Disease, 2011, 95(12): 1558-1564.

DOI：10.1094/PDIS-05-11-0413

Patel, J. S., Meinhardt, S. W., Sierotzki, H., Stammler, G., Gudmestad, N. C., and Adhikari, T. B. 2011. A two-step molecular detection method for Pyrenophora tritici-repentis isolates insensitive to QoI fungicides. Plant Dis. 95:1558-1564.
Tan spot, caused by Pyrenophora tritici-repentis, is an important disease of wheat worldwide. To manage tan spot, quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin have been applied in many countries. QoI fungicides target the cytochrome b (cyt b) site in complex 111 of mitochondria and, thus, pose a serious risk for resistance development. The resistance mechanism to QoI fungicides is mainly due to point mutations in the cyt b gene. The objective of this study was to develop a molecular detection method for the four currently known mutations responsible for shifts in sensitivity toward QoI fungicides in P tritici-repentis. Twelve specific primers were designed based on sequences from the National Center for Biotechnology Information accessions AAX101000704 and DQ919068 and used to generate a fragment of the cyt b gene which possesses four known single-nucleotide polymorphisms (SNPs). These mutant clones served as positive controls because QoI-insensitive and -reduced-sensitive isolates of P tritici-repent is have not yet been reported in the United States. The partial cyt b gene clones were sequenced to identify the SNPs at sites G143A and F129L. Genomic DNA of the mutated partial cyt b gene clones and the European QoI-insensitive and -reduced-sensitive isolates of P tritici-repentis possessing G143A (GCT) and F129L (TTA, TFG, and CTC) mutations were amplified by polymerase chain reaction (PCR) using two specific primer pairs and were further digested with three specific restriction enzymes (BsaJI, Fnu4HI, and Mn/I). The results of the digested PCR product from genomic DNA of known QoI-insensitive and -reduced-sensitive isolates of P tritici-repentis had DNA bands consistent with the mutation GCT at G143A and the mutations TTA, TTG, and CTC at F129L. The amplified region at the F129 site also had 99% sequence similarity with P teres, the net blotch pathogen of barley. To validate mutations, we further tested two isolates of P teres known to have reduced sensitivity to QoI fungicides possessing the mutations TTA and CTC at F129L. After PCR amplification and restriction digestion, DNA bands identical to those observed for the partial cyt b mutant clones were detected. These results suggest that this newly developed two-step molecular detection method is rapid, robust, and specific to monitor QoI-insensitive and -reduced-sensitive isolates of P tritici-repentis.

• 出版日期2011-12