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

## TL;DR

This paper develops methods to study the survival of blueberry rust spores, which could help in managing the disease.

## Contribution

The study introduces new cultivation and viability assessment techniques for Pucciniastrum minimum urediniospores.

## Key findings

- Urediniospores of P. minimum germinate better when dusted on agar compared to spore suspensions.
- Viability assessment using co-staining showed higher viability than in vitro germination rates.
- Leaves from inside a glasshouse produced more rust than those from outside.

## Abstract

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.