An Optimized Sporulation Method for the Wheat Fungal Pathogen Pyrenophora Tritici-Repentis

Abstract

BackgroundThe necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) causes tan (syn. yellow) spot of wheat and accounts for significant yield losses worldwide. Understanding the molecular mechanisms of this economically important crop disease is crucial to counteract the yield and quality losses of wheat globally. Substantial progress has been made to comprehend the race structure of this phytopathogen based on its production of necrotrophic effectors and genomic resources of Ptr. However, one limitation for studying Ptr in a laboratory environment is the difficulty to isolate high spore numbers from vegetative growth with mycelial contamination common. These limitations reduce the experimental tractability of Ptr. ResultsHere, we optimized a multitude of parameters and report a sporulation method for Ptr that yields robust, high quality and pure spores. Our methodology encompasses simple and reproducible plugging and harvesting techniques, resulting in spore yields up to 1,500 fold more than the current sporulation methods and was tested on multiple isolates and races of Ptr as well as an additional seven modern Australian Ptr isolates. Moreover, this method also increased purity and spore harvest numbers for two closely related fungal pathogens (Pyrenophora teres f. maculata and f. teres) that cause net blotch diseases in barley (Hordeum vulgare), highlighting the usability of this optimized sporulation protocol for the wider research community. ConclusionsLarge-scale spore infection and virulence assays are essential for the screening of wheat and barley cultivars and combined with the genetic mapping of these populations allows pinpointing and exploiting sources of host genetic resistance. We anticipate that improvements in spore numbers and purity will further advance research to increase our understanding of the pathogenicity mechanisms of these important fungal pathogens.

Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat

“…An attached leaf assay with modifications was used to carry out multi-strain coinfection (85). Briefly, the first leaves of two-week old wheat seedlings were inoculated with a P. nodorum spore suspension [1 x 10 6 spores/ml in 0.02 % (v/v) Tween 20] using an airbrush kit (6ml/min, Ozito, Perth, Australia).…”

“…The wheat lines were H213, H324 (snn1, snn3b1, insensitive to Tox2A) and H086, H336, H091 (Snn1, snn3b1, insensitive to Tox2A). Leaves were allowed to dry and incubated in a controlled growth chamber as described previously (85). Two and four days post-inoculation, infected leaves were harvested with a scalpel and snap-frozen in liquid nitrogen (three biological repeats per time point per wheat line per treatment).…”