| Summary: | Two hundred and twenty-five <i>Diaporthe</i> isolates were collected from 2005 to 2019 in almond orchards showing twig cankers and shoot blight symptoms in five different regions across Spain. Multilocus DNA sequence analysis with five loci (ITS, <i>tub</i>, <i>tef-1α</i>, <i>cal</i> and <i>his</i>), allowed the identification of four known <i>Diaporthe</i> species, namely: <i>D. amygdali</i>, <i>D. eres</i>, <i>D. foeniculina</i> and <i>D. phaseolorum</i>. Moreover, a novel phylogenetic species, <i>D. mediterranea</i>, was described. <i>Diaporthe</i> <i>amygdali</i> was the most prevalent species, due to the largest number of isolates (85.3%) obtained from all sampled regions. The second most frequent species was <i>D. foeniculina</i> (10.2%), followed by <i>D. mediterranea</i> (3.6%), <i>D.</i> <i>eres</i> and <i>D. phaseolorum</i>, each with only one isolate. Pathogenicity tests were performed using one-year-old almond twigs cv. Vayro and representative isolates of the different species. Except for <i>D. foeniculina</i> and <i>D. phaseolorum</i>, all <i>Diaporthe</i> species were able to cause lesions significantly different from those developed on the uninoculated controls. <i>Diaporthe mediterranea</i> caused the most severe symptoms. These results confirm <i>D. amygdali</i> as a key pathogen of almonds in Spain. Moreover, the new species, <i>D. mediterranea</i>, should also be considered as a potential important causal agent of twig cankers and shoot blight on this crop.
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