Molecular fingerprinting of antagonist Aureobasidium pullulans isolates by fluorescent amplified fragment length polymorphism

The epiphytic yeast-like fungus Aureobasidium pullulans includes strains showing biocontrol activity on different stored crops against the major postharvest pathogens. Molecular fingerprinting of biocontrol agents is pivotal both for environmental monitoring and registration purposes. Several strains of A. pullulans with different levels of antagonistic activity were analysed by fAFLP (fluorescent Amplified Fragment Length Polymorphism). Total genomic DNA was digested with two restriction endonucleases, and compatible oligonucleotide adapters were ligated to the resulting fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by four selective sets of primers (AC/CC, AT/CG, AC/CA, G/CT) extending beyond the adapter and restriction site sequences. In each set, one of the primers was labelled with a fluorescent dye, thus enabling amplified fragments to be detected and sized with an automated DNA sequencer. This procedure generated 150 to 280 fragments, 35 to 500 bp in size. A dendrogram was obtained by analysing the four fAFLP patterns by using Dice similarity coefficient (SD) and clustering of fingerprints performed with the unweighted pair groups (UPGMA). All isolates fell into four clusters with a level of similarity ranging from 0.02 to 0.98. Only two isolates displayed high similarity under each fAFLP condition. fAFLP appears to be a fast and reproducible molecular tool for characterising intraspecific genetic variability between A. pullulans strains. Further, it could pave the way to the identification of genetic marker(s) to be used in ecological studies for monitoring the environmental fate of these microorganisms when applied for biocontrol purposes.