Measurement of virulence in Zymoseptoria tritici through low inoculum-density assays

Hitherto, pathogenicity assays with mutants or wildtype variants of Zymoseptoria tritici have been based on pycnidial counts, following inoculation of host leaves with high density inoculum. Here, we present data which suggest that high inoculum densities may mask deficiencies in virulence due to symptom saturation. We describe a low inoculum-density method which obviates this problem. This method can also be used to (i) interrogate the process of lesion formation in Z. tritici (ii) determine whether individuals of the same or different genotypes co-operate or compete during the establishment of apoplastic infections (iii) dissect the determinants of virulence, by assessing a given strain's stomatal penetration efficiency (SPE), its ability to spread within the apoplast and its pycnidiation efficiency. Such methodology can thus be used to investigate the reasons underpinning attenuated virulence in mutant or avirulent wildtype strains.