Novel amplified fragment length polymorphism (AFLP) markers for typing medically relevant Fusarium and allied fusarioid genera

Abstract

Fusariosis is an emerging mycosis caused by diverse Fusarium and allied fusarioid genera that are characterized by spindle-shaped macroconidia. These fungi possess a broad ecological distribution, causing infections in a wide diversity of hosts, spanning the animal and plant kingdoms. The spectrum of human fusariosis encompasses superficial lesions like keratitis and onychomycosis to invasive fungal diseases. Notable genera within the medically relevant fusarioid group include Neocosmospora, Fusarium s. str., and Bisifusarium. While Neocosmospora species (formerly F. solani species complex) are primary causative agents of human fusariosis, instances involving Fusarium s. str. and Bisifusarium (formerly F. dimerum species complex) have been reported. There is an urgent need for DNA-based markers to explore the epidemiology of these emerging fusarioid pathogens using molecular methods. We took advantage of fusarioid genomes available in NCBI (n = 20) to optimize the development of novel amplified fragment length polymorphism (AFLP) markers by conducting in-depth in silico analyses to refine their applicability for studies on these pathogens’ genetic epidemiology. In-silico screening highlighted eight primer pair combinations (C1–C8) to be tested in vitro. The AFLP protocol was used for genotyping 40 medically relevant fusarioid fungi. Based on the overall scored AFLP markers (77–93 fragments), the values of polymorphism information content (PIC = 0.3474–0.3725), marker index (MI = 0.0038–0.0056), effective multiplex ratio (E = 26.3750–40.4750), resolving power (Rp = 40.1500–54.6000), discriminating power (D = 0.7978–0.8857), expected heterozygosity (H = 0.4476–0.4949), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Neocosmospora, Fusarium s. str., and Bisifusarium species. Of relevance, some AFLP panels were better than others at studying genetic trends in Neocosmospora (#2 EcoRI-AT/MseI-TA, #3 EcoRI-AA/MseI-TT, and #5 EcoRI-AT/MseI-AG) or Fusarium s. str. (mainly #2 EcoRI-AT/MseI-TA and #6 EcoRI-GA/MseI-TT) and Bisifusarium (#1 EcoRI-GA/MseI-AG and #6 EcoRI-GA/MseI-TT), and these combinations will better resolve disease transmission routes. Our DNA fingerprint assay has proven effective by exhibiting rapidity, reproducibility, and high discriminatory capabilities, which represents a valuable asset in the ongoing efforts to combat fusariosis and enhance our scientific understanding of medically relevant Fusarium and allied fusarioid genera.