First Record of Ash Dieback Caused by Hymenoscyphus fraxineus on Fraxinus excelsior in the Apennines (Tuscany, Italy)

Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz & Hosoya represents a relevant fungal pathogen in Europe causing a wide range of symptoms on common ash trees (Fraxinus excelsior) (Kowalski 2006). The infection consists of necrosis on leaflets and rachises, twigs dieback, and finally the death of the tree (Kräutler and Kirisits 2012). In Italy, the disease has been reported on F. excelsior since 2010 in the northern part of the peninsula, close to the Alps (Ogris et al. 2010). Surveys were conducted in recent years by the Italian regional phytosanitary services in the central peninsula, particularly in Tuscany. However, the pathogen was not discovered. In July 2015, symptomatic trees of F. excelsior were observed in Tuscany in central Italy (Montepiano, PO; 44°5′34″ N; 11°09′30″ E; 710 m elevation). Symptoms included blackish discoloration of leaves; necrotic leaf rachises; wilting and leaf dropping; dieback of shoots, twigs, and branches; and necrosis of bark tissue. Isolations were made from symptomatic leaf petioles, rachises, and twigs after surface sterilization with sodium hypochlorite and ethanol according to Kräutler and Kirisits (2012). Fragments of necrotic tissue were placed on 2% malt extract agar (MEA) plates and incubated at 10°C in the dark. After 3 weeks, small, dull white to fulvous brown colonies were observed and subcultured in MEA at 5°C to stimulate the anamorphic stage of the pathogen. After 15 days, subcylindrical to obclavate, 12.6 to 21.6 (16.9) μm long phialides, arising from the surface of the colony, were observed. Venters were short, cylindrical, 6.0 to 13.6 (9.6) μm long, and 3.1 to 4.6 (3.9) μm wide. Collarettes were cylindrical 5.6 to 9.1 (7.3) μm long and 2.0 to 2.7 (2.3) μm wide. Phialoconidia, 2.2 to 4.1 (3.0) μm long and 1.6 to 2.6 (1.9) μm wide, were formed in slimy droplets and sometimes in short chains. The first formed conidia measured 5.0 to 7.4 (6.0) μm long and 1.7 to 2.3 (1.9) μm wide. These morphological characteristics are consistent with those of H. fraxineus reported in the published literature (Kowalski 2006; Ogris et al. 2010). Molecular identification was conducted by amplifying and sequencing the ITS1-5.8S-ITS2 region using universal primers ITS1/ITS4 (White et al. 1990). Consensus sequences were obtained. BLASTn search in GenBank revealed a 100% homology with H. fraxineus species. The ITS sequences (strains CNRHfrax300, CNRHfrax301, CNRHfrax24.4, CNRHfrax51.1) have been deposited in GeneBank under Accession Nos. KT696592, KT696593, KT696594, and KT696595. Based on morphological characteristics and DNA sequencing, the fungus was identified as H. fraxineus. The strains were stored in the culture collection of the Institute for Sustainable Plant Protection (IPSP-CNR, Sesto Fiorentino, Italy). Based on the literature, this is the first report of H. fraxineus in central Italy in the Apennines, an area strongly influenced by Mediterranean climatic conditions. This represents the southernmost limit of the current known distribution of the pathogen in Europe (McKinney et al. 2014).