Development and Validation of a Multiplex TaqMan Probe qPCR Assay Specific for Three Rust Fungi Infecting Meliosma myriantha.

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Rust fungi are the largest group of obligate plant pathogens and cause severe damage to global forests and agricultural security. Meliosma myriantha, a tree species native to East Asia (China, Japan, and Korea), is vulnerable to three rust species: Neophysopella meliosmae, N. meliosmae-myrianthae, and N. vitis. The early symptoms of infection are indistinguishable between these species, making an accurate and rapid diagnosis challenging. The urediniospores of N. meliosmae-myrianthae and N. vitis are also known to infect economically relevant grapevines (Vitis spp.) and ivies (Parthenocissus spp.), respectively, rendering early detection and identification even more important. To address this issue, we developed a multiplex quantitative polymerase chain reaction assay equipped with TaqMan probes targeting the internal transcribed spacer rDNA sequences specific to the three rust pathogens. This assay successfully detected minute quantities (5 fg for N. meliosmae-myrianthae and 50 fg for N. meliosmae and N. vitis) of DNA from the three Neophysopella species and demonstrated consistent reliability when applied to fresh and herbarium samples collected from M. myriantha, grapevines, and ivies. In conclusion, this novel assay is a rapid and robust diagnostic tool for the three rust pathogens, N. meliosmae, N. meliosmae-myrianthae, and N. vitis, and offers the potential to identify and detect their global movement and spread to grapevines, ivies, and trees.