Genomic variation within Monilinia laxa, M-fructigena and M-fructicola, and application to species identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. f ructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isola tes collected in France was amplified by Polymerase Chain Reaction (PCR) us ing universal primers and sequenced. Multiple alignment of the ITS sequence s and comparison with published sequences revealed very little intraspecifi c variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356 bp fragment for each of the three species. The specificity of the three primer pairs was s uccessfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countri es, unequivocally confirming the identification of each isolate based on mo rphological and cultural traits. Using stringent PCR conditions, no cross-r eaction was observed with any of the isolates tested. The specificity of th e PCR assays was also successfully confirmed with DNA extracted from differ ent fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doub tful isolates can be directly identified in a single PCR run. Moreover, det ection and identification of the Monilinia species were successfully achiev ed directly on diseased fruits. This simple and rapid method can be particu larly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.