Measuring quantitative virulence in the wheat pathogen Zymoseptoria tritici using high-throughput automated image analysis.

Zymoseptoria tritici, causal agent of Septoria tritici blotch on wheat, produces pycnidia in chlorotic and necrotic lesions on infected leaves. A high-throughput phenotyping method was developed based on automated digital image analysis that accurately measures the percentage of leaf area covered by lesions (PLACL) as well as pycnidia size and number. A seedling inoculation assay was conducted using 361 Z. tritici isolates originating from a controlled cross and two different winter wheat cultivars. Pycnidia size and density were found to be quantitative traits that showed a continuous distribution in the progeny. There was a weak correlation between pycnidia density and size (r = -0.27) and between pycnidia density and PLACL (r = 0.37). There were significant differences in PLACL and pycnidia density on resistant and susceptible cultivars. In all, >20% of the offspring exhibited significantly different pycnidia density on the two cultivars, consistent with host specialization. Automated image analysis provided greater accuracy and precision compared with traditional visual estimates of virulence. These results show that digital image analysis provides a powerful tool for measuring differences in quantitative virulence among strains of Z. tritici.