First Record of Thousand Cankers Disease Geosmithia morbida and Walnut Twig Beetle Pityophthorus juglandis on Juglans nigra in Europe

Thousand cankers disease (TCD) of walnut is responsible for widespread mortality of black walnut (Juglans nigra L.) in the United States since the mid-1990s (2). The disease is caused by the fungus Geosmithia morbida Kolařik (Ascomycota, Hypocreales), vectored by the walnut twig beetle Pityophthorus juglandis Blackman 1928 (Coleoptera, Scolytinae). In September 2013, TDC was observed in northeastern Italy (Bressanvido, Vicenza, 45°39′ N, 11°38′ E) in black walnuts of different ages: ~80-year-old plants growing in a garden and 17-year-old trees belonging to a nearby walnut plantation for timber production. Main symptoms were yellowing, wilting, twig and branch dieback, and a high number of small bark cankers (3). Longitudinal and radial sections collected through the cankers revealed gray to brown discoloration of both phloem and outer bark, and the presence of bark beetle galleries radiating from the mating chamber and developing horizontally (across the wood grain), and vertical (along the grain) larval galleries. Occasionally, discoloration involved the outward xylematic tissues. Fungal fruiting bodies were not found on or near the cankers. Whitish mycelium, sometimes producing verticillate conidiophores, was frequently detected inside galleries. A number of 1- to 3-cm diameter twigs showing cankers up to 2 cm long surrounding bark beetle penetration holes were randomly collected. From samples, emerging beetles were identified as P. juglandis both morphologically (4) and genetically. DNA extraction was carried following a standard salting out protocol. The barcode region of the mitochondrial gene cytochrome oxydase I was then amplified using universal primers (1) and sequenced, obtaining 627 bp. BLAST analysis showed 100% identity to P. juglandis. Sequences were finally deposited in the BoldSystem database (GenBank Accession No. KF725084). From the necrotic margin of six cankers previously surface-sterilized with 3% sodium hypochlorite, two 3-mm-wide chips per canker were placed on potato dextrose agar and incubated at 23 ± 1°C in the dark. Among a variety of microorganisms, slow growing lobate, plane, yellowish-ochre colonies with hyaline mycelium appeared in 6 days. After subculturing to the same medium, growing features, mycelium, conidiophores, and conidia with morphological characteristics matching Kolařik's description of G. morbida (2) were observed. Same result was obtained culturing the mycelium growing inside the galleries. The ITS region of rDNA was amplified using ITS1F and ITS4 primers and sequenced, obtaining 597 bp. BLAST analysis showed 100% identity to G. morbida strain U173 (HF546283.1) for 558 bp. To our knowledge, this is the first record of TCD and P. juglandis to Europe, where walnut species (mainly J. regia, J. nigra, and their hybrids) are intensively cultivated for timber production. This finding is therefore of particular importance. An intensive survey of the disease is suggested, both to assess fungus/beetle presence and to verify possible pathways of introduction, likely associated to importation of infested/infected timber from native Nearctic regions. Voucher specimens are stored in the TeSAF herbarium (fungus) and in the DAFNAE insect collection.