An optimized sporulation method for the wheat fungal pathogen Pyrenophora tritici-repentis

Silke Jacques,Kar-Chun Tan,Julie Lawrence,Leon Lenzo,Kofi Stevens

doi:10.1186/s13007-021-00751-4

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 1500 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.

Highlights

The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) causes tan spot of wheat and accounts for significant yield losses worldwide
The routinely used method described by Lamari and Bernier [29] typically yields between a 1000–3000 spores per vegetative plate, which is relatively low compared to most other fungi [13, 31]
To raise sufficient spore inoculum for wheat bio-assays, numerous petridishes are needed for vegetative growth and subsequent conidial generation, thereby significantly increasing space occupancy and labour time

Summary

Introduction

The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) causes tan (syn. yellow) spot of wheat and accounts for significant yield losses worldwide. Yellow) spot (TS) is an economically important disease of wheat and causes significant crop losses worldwide [1]. ToxA induces dark necrotic lesions occasionally associated with a chlorotic halo, whilst ToxB induces chlorosis as does ToxC, with the latter able to spread the chlorosis through the leaf [14, 15]. These disease symptoms will only effectuate on those wheat lines carrying the dominant susceptibility genes which are Tsn, Tsc and Tsc that interact with ToxA, ToxB and ToxC, respectively [16]. The molecular knowledge gained within the Ptr-wheat pathosystem and the establishment of a global race structure allows for the screening of host genetic resistance and the development of more effective and sustainable control measures [18]

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