Rapid loss of virulence during submergence of Z. tritici asexual spores

Abstract Zymoseptoria tritici , the causal agent of Septoria tritici blotch, is a notable pathogen of temperate-grown wheat. To better understand the mechanisms underpinning pathogenicity, leaf infection assays are commonly used to compare either the virulence of Z. tritici wildtype or mutant strains, or the susceptibility of wheat cultivars. These assays, which control for many biotic, abiotic and experimental variables, involve the application of known spore numbers to leaves. To achieve this, spore numbers are quantified during a period of aqueous suspension. Published methods rarely state the period in which spores are held in suspension, suggesting that this variable may be uncontrolled. Using simple, agar-based plating experiments, this work firstly demonstrates that blastospore culturability (the ability to form a colony when plated on appropriate agar) decreases rapidly over time during maintenance in aqueous suspension. It is subsequently shown that this reduction in culturability correlates to a reduction in the virulence of the blastospore population. This is shown in three wild type Z. tritici strains. From this, it is concluded that suspension time is a variable of major importance in experimental design and one which, if not controlled, may lead to erroneous conclusions from inter-strain comparisons. The conidia of the unrelated fungus Magnaporthe oryzae also rapidly lose culturability when stored in aqueous suspension, whereas the microspores of Fusarium oxysporum f. sp. cubense do not, suggesting that this phenomenon occurs in some but not all other fungi. Finally, a droplet method of inoculations is proposed to decrease the variability in the numbers of spores applied, within and between experiments.