Development of Experimental Techniques to Study Blueberry Rust (Pucciniastrum minimum) Urediniospore Survival

Blueberry rust disease is caused by the fungal pathogen Pucciniastrum minimum (syn. Thekopsora minima). Despite its importance as a plant pathogen, there are relatively few published studies on P. minimum. This study investigated and refined methodologies to cultivate and study this obligate parasite. P. minimum was successfully cultivated on detached blueberry leaves by misting leaves with water, followed by dusting with dry urediniospores. In vitro germination of urediniospores on water agar was achieved using a spore dusting technique, and germination rates were 70% higher compared to a spore suspension. Time after leaf detachment affected urediniospore germination and highlighted the importance of the processing time for replicability between experiments. Urediniospore viability could be evaluated by co-staining with fluorescein diacetate and propidium iodide, and the assessed viability was significantly higher than germination rates achieved in vitro. In detached leaf inoculations, leaves sourced from inside the glasshouse developed more rust than those from outside; this is discussed in the context of knowledge gaps on the infection process of P. minimum. This study resolves some key methodological issues involved with studying P. minimum rust urediniospores, and the general protocols we developed can be applied to other rust species for biological survival research.