qPCR assays for sensitive and rapid detection of Quambalaria species from plant tissues.

Several species from the genus Quambalaria (order Microstromatales) cause diseases on eucalypts (Eucalyptus and related genera) both in plantations and natural ecosystems. We developed real-time qPCR assays to rapidly detect and distinguish five Quambalaria species.The design of the species-specific qPCR assay for each species, Q. pitereka (PIT), Q. coyrecup (COR), Q. cyanescens (CYN), Q. pusilla (PUS) and Q. eucalypti (EUC) was based on the ITS1 region, and was evaluated for specificity and sensitivity. The CYN qPCR assay could amplify as little as 1fg µl-1 from pure culture, the PIT and COR qPCR assays could amplify 10fg µl-1, while PUS and EUC qPCR assays could amplify 100 fg µl-1 of their target species. The PIT, COR and CYN qPCR assays were further validated using artificially and naturally infected samples of their plant host Corymbia calophylla. These assays will be used for rapid diagnostics and for future experiments on the infection process.