Quantification of disease progression of Alternaria spp. on potato using real-time PCR

Potato early blight and brown spot are important fungal diseases responsible for premature defoliation and yield loss of potato. Pathogens considered to be involved in leaf necrosis are Alternaria solani and A. alternata, respectively. Both diseases are commonly characterized by the visualization of leaf lesions. Current detection and identification methods for Alternaria species rely primarily on cultural and morphological characteristics, the assessment of which is time-consuming and not always suitable. Sensitive, reliable methods for estimating infection severity are therefore desirable. In this study, an Alternaria-specific real-time PCR assay was developed using primers based on internal transcribed spacers (ITS) 1 and 2. The assays facilitated species detection and clearly discriminated between A. solani and A. alternata. The use of real-time PCR allowed quantitative estimation of fungal biomass in plant tissues. Detection sensitivities were in the range of >100 fg. Real-time PCR applications used to accurately assess the extent of colonization by Alternaria spp. during disease development are reported here for the first time. Additionally, Alternaria genomic DNA levels were verified not only in potato leaves showing different levels of disease progress, but also in symptomless leaves. This assay provides a useful tool to quantify pathogen levels during initial latent stages of infection and will thus help in the early detection and quantification of Alternaria spp..