PCR-Based Detection for the Quarantine Fungus Colletotrichum kahawae, a Biosecurity Threat to the Coffee (Coffea arabica) Industry Worldwide

Abstract

Coffee berry disease is caused by Colletotrichum kahawae, a quarantine fungus still absent from most coffee-producing countries. Given the potential adverse effects on coffee berry production, it is a severe worldwide threat to farmers and industry. Current biosecurity management focuses on exclusion by applying quarantine measures, including the certification of coffee plants and their products. However, methods for detecting C. kahawae by National Plant Protection Organization (NPPO) laboratories still need approval. This research aims to functionally demonstrate, standardize, and validate a method for detecting and discriminating C. kahawae from other Colletotrichum species that may be present in coffee plant samples. The method proposes to use an end-point PCR marker for the mating type gene (MAT1-2-1) and a confirmatory test with a real-time quantitative PCR (qPCR) marker developed on the glutamine synthetase gene. The C. kahawae amplicons for the Cen-CkM10 qPCR marker exhibited specific melting temperature values and high-resolution melt profiles that could be readily differentiated from other tested species, including their relatives. Given the fungus’s quarantine status, specificity was tested using artificial mixtures of DNA of C. kahawae with other Colletotrichum species and coffee plant DNA. The described method will enable NPPOs in coffee-producing and exporting countries, especially Colombia, to prevent this pathogen's entry, establishment, and spread. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .