Quantitative and qualitative influence of inoculation methods on in planta growth of rice blast fungus

ABSTRACT Molecular analyses of early disease events require infected plant tissue in which the pathogen is present in high quantities and interacts with the plant in a way found in the field. In this study, a quantitative polymerase chain reaction (Q-PCR) assay was developed to determine an "infection ratio" of fungal to plant cells in infected tissues. This assay was used to evaluate four inoculation methods (spray, mist, dip, and sheath) as well as use of whole plants or excised parts. Fluorescence stereomicroscopy was used to follow individual lesions developing from appressoria to macroscopic symptoms. Disease progression and outcomes were documented from 24 to 96 h postinoculation (hpi), as well as effectiveness of Pi-ta-mediated resistance. Even at 96 hpi, fungus proliferated well ahead of visible plant damage, especially in veins. Developing lesions sometimes were surrounded by greener areas in detached leaves. Spray inoculation was not sufficient for detecting fungal gene expression in planta before 96 h. Alternatively, a leaf sheath assay produced infected tissues containing 10 to 30% fungal DNA by 34 h. Used together, Q-PCR quantification and fluorescence stereomicroscopy will facilitate studies of early plant invasion because infection density and fungal growth stages are directly observed, not assumed from incubation time.