Blueberry rust caused by the fungal pathogen Pucciniastrum minimum (previously Thekopsora minima) is a significant disease of blueberry. Severe infections result in defoliation and yield losses. The complete lifecycle of P. minimum requires two plant hosts an Ericaceae, such as blueberry, and a Tsuga species (hemlock). However, in Australia, P. minimum persists in the blueberry plantations in the absence of Tsuga where it seems to continuously reinfect through urediniospore production. There are few publications available on the biological mechanisms of this infection process and whether there are dormant phases of the uredinia.
My presentation will focus on techniques we have developed to maintain a working collection of P. minimum in planta and to reliably germinate urediniospores in vitro, to enable further studies on pathogen biology. As an obligate parasite, only growing on a host, establishing and maintaining a biotrophic culture has been difficult with replication of published methodologies being largely unsuccessful. Similarly getting reliable germination of P. minimum urediniospores in vitro has been a challenge, despite availability of published methods.
Techniques tested for maintaining P. minimum in planta included inoculation of potted blueberry plants in a controlled-environment greenhouse, compared with those maintained outdoors, and a detached leaf culture. We found that the controlled environment of our greenhouse was unsuitable for P. minimum, with poor inoculation success. Plants outside were successfully inoculated and the disease spread readily. Detached leaf biotrophic cultures were also successful in maintaining P. minimum.
Methods investigated to germinate P. minimum urediniospores in vitro included: various formulations of media, using agar media in Petri dishes versus on microscope slides, and water suspensions of urediniospores versus a dusting technique. The germination rate of urediniospores over time after leaf detachment was also measured. The method of urediniospore transfer was the largest contributor to inoculation and in vitro germination success. Unlike other rusts, using a water suspension of P. minimum urediniospores often resulted in germination failure. Dusting of dry urediniospores onto leaves and plants, following by misting, gave greater inoculation success in planta. Similarly, dry dusting onto agar media resulted in much higher germination rates in vitro. Germination rate also changed significantly over time after leaf collection, highlighting the importance of running assays at the same intervals to get consistent results.
Maintaining collections of obligate parasite pathogens like rust is notoriously difficult. The methodologies presented in this study provide a foundation for more reliable maintenance of working collections of rust. Thereby increasing pathological research capacity on rust biology and supporting the collection and preservation of single-pustule derived cultures for genomic research.
Whilst there are many publications reporting on germination of urediniospores in vitro for many different rust species, they do not include detail on the intricacies of working with rust pathogens. With this study we hope that a more in-depth exploration of the factors that affect germination rates in P.minimum and the publication of our failures will allow other researchers to refine their own methodology when working with rust species.