Development and use of new sensitive molecular tools for diagnosis and detection of Melampsora on cultivated poplar

Poplar rusts due to Melampsora larici-populina (Mlp), M. allii-populina (Map) and M. medusae f. sp. deltoidae (Mmd) are the most serious disease in Europe on cultivated poplars, that is, Populus 9 euramericana and P. 9 interamericana hybrids. These pathogenic species can be identified by the observation of morphological characteristics of urediniospores but this method is not appropriate for high-throughput analysis and cannot be used on other spore stages, such as aeciospores or teliospores, that are morphologically similar. The aim of this study was to develop a rapid and sensitive molecular method based on PCR amplification that was able to specifically detect these species on various hosts for routine analysis. Three primer pairs ITS-MLP-F/ITS-MLP-R, ITS-MAP-F/ITS-MAP-R and ITS-MMD-F/ITS-MMD-R were designed within the internal transcribed spacer (ITS) sequences of ribosomal DNA to target Mlp, Map and Mmd, respectively, and their specificity were confirmed on a wide range of isolates and species. ITS-MLP-F/ITS-MLP-R and ITS-MAP-F/ITS-MAP-R primers proved to be highly specific to Mlp and Map, respectively, whereas ITS-MMD-F/ITS-MMD-R cross-reacted with DNA from M. larici-tremulae and M. pinitorqua. However, these species are not pathogenic on cultivated poplars that all belong to sections Aigeiros and Tacamahaca of the genus Populus. Specific Mmd primers proved to be very sensitive as a positive signal could be obtained with DNA extracts from 6 target urediniospores mixed with 800 000 urediniospores of Mlp. An internal amplification control (IAC) was included to discriminate false negative results due to the potential presence of inhibitory compounds in DNA extracts. ITS-MMD-F/ITS-MMD-R primers are therefore efficient for the detection of the quarantine pathogen Mmd on samples collected on poplar or larch and are fit for use in official tests. This new PCR assay has been used in routine for ten years, and Mmd has hitherto never been detected in commercial poplar nurseries in France.