Background: Species identification is essential for controlling disease, understanding epidemiology, and to guide the implementation of phytosanitary measures against fungi from the genusDiaporthe. AccurateDiaporthespecies separation requires using multi-loci phylogenies. However, defining the optimal set of loci that can be used for species identification is still an open problem.Methods: Here we addressed that problem by identifying five loci that have been sequenced in 142Diaportheisolates representing 96 species:TEF1,TUB,CAL,HISand ITS. We then used every possible combination of those loci to build, analyse, and compare phylogenetic trees.Results: As expected, species separation is better when all five loci are simultaneously used to build the phylogeny of the isolates. However, removing the ITS locus has little effect on reconstructed phylogenies, identifying theTEF1-TUB-CAL-HIS4-loci tree as almost equivalent to the 5-loci tree. We further identify the best 3-loci, 2-loci, and 1-locus trees that should be used for species separation in the genus.Discussion: Our results question the current use of the ITS locus for DNA barcoding in the genusDiaportheand suggest thatTEF1might be a better choice if one locus barcoding needs to be done.

Blueberry, an increasingly cultivated fruit crop in Portugal, is known to be susceptible to twig blight and dieback caused by species of Diaporthe. The diversity of Diaporthe species associated with symptomatic and asymptomatic Vaccinium corymbosum plants in Portugal was assessed. A multilocus sequence analysis of the rDNA internal transcribed spacer (ITS) region and the translation elongation factor 1-alpha (tef1-α), β-tubulin (tub2), calmodulin (cal), and histone 3 (his3) genes revealed the presence of Diaporthe foeniculina, Diaporthe rudis, and four new species, which are described as Diaporthe crousii, Diaporthe phillipsii, Diaporthe rossmaniae, and Diaporthe vacuae. These new species were characterized in terms of their morphology, mating strategies, and temperature growth requirements. In artificial inoculation trials of V. corymbosum cv. Bluecrop plants, all Diaporthe species caused minor symptoms. Further, no differences in aggressiveness were apparent between species. This study provides the first survey of Diaporthe species associated with blueberry twig blight and dieback in Portugal. It disclosed the occurrence of a diverse assemblage of Diaporthe species, whose status and impact as pathogens of blueberry is not yet fully understood.