Real-Time PCR-Based Monitoring of DNA Pools in the Tri-Trophic Interaction Between Norway Spruce, the Rust Thekopsora areolata, and an Opportunistic Ascomycetous Phomopsis sp.

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nursery-grown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.