A PCR-Based Assay for Early Diagnosis of the Coffee Leaf Rust Pathogen Hemileia vastatrix

Abstract

Early detection and identification of plant pathogens is one of the most important strategies for sustainable plant disease management. Fast, sensitive, and accurate methods that are cost-effective are crucial for plant disease control decision-making processes. Coffee leaf rust (CLR) caused by Hemileia vastatrix is a devastating worldwide fungal disease which causes serious yield losses of coffee, especially relevant for Coffea arabica. A rapid PCR assay for detecting and characterizing H. vastatrix with high specificity, high sensitivity and simple operation has been developed based on specific amplification of the Internal Transcribed Spacer (ITS) region of ribosomal genes. The specificity of the primers was determined using isolates DNA of H. vastatrix, Coleosporium plumeriae, and other fungal species that infect coffee plants and are common in coffee leaves, such as Lecanicillium sp (the H. vastatrix hyperparasite fungi), Cercospora coffeicola, Colletotrichum gloeosporioides, amongst others. Results showed specific amplification of a 396-bp band from H. vastatrix DNA with a detection limit of 10 pg/μl of pure genomic DNA of the pathogen. The PCR assay described in the current chapter allows to detect H. vastatrix rapidly and reliably in naturally infected coffee tissues, vital for the early detection and diagnostics of H. vastatrix and CLR epidemiology.