Grapevine trunk diseases (GTDs) represent an increasing problem in viticulture worldwide; however, their etiology in Montenegro has so far remained largely unexplored, and symptoms have traditionally been attributed solely to esca. During surveys conducted in 15 vineyards comprising multiple grapevine cultivars, typical esca symptoms as well as other GTDs-associated symptoms were recorded. A total of 319 fungal isolates were obtained and grouped into 13 morphotypes, from which 20 representative isolates were selected for preliminary molecular identification.The isolates belonged to 15 genera commonly associated with GTDs. Nine isolates were identified to the species level based on morphological characteristics and multilocus phylogenetic analyses (ITS, LSU, act, tub2, tef1a, rpb2, cal, and his). For the first time in Montenegro, the association of Cadophora luteo-olivacea, Diaporthe eres, Seimatosporium vitis viniferae, and four Botryosphaeriaceae species (Botryosphaeria sp., Diplodia mutila, Diplodia seriata, and Neofusicoccum parvum species complex) with grapevine trunk diseases is reported. In addition, Biscogniauxia mediterranea is reported for the first time in Europe in association with grapevine disease symptoms. Pathogenicity tests confirmed the ability of selected isolates to induce necrotic lesions, with varying levels of aggressiveness. The frequent presence of multiple pathogens within the same trunk indicates possible co-infections contributing to symptom expression. This study represents the first comprehensive overview of GTDs fungi in Montenegro and expands existing knowledge of this disease complex in southeastern Europe.
