First Report of Neofusicoccum australe and N. luteum Associated with Canker and Dieback of Quercus robur in Portugal.

Quercus robur L., commonly known as "pedunculate oak," is a long-lived deciduous tree native to most of Europe. It is of great ecological and forestry importance. It is also commonly cultivated as an ornamental tree in parks and gardens. Since 2009 and most likely related to increased drought periods, diseased ornamental trees have been observed in the campus of the University of Aveiro, Portugal. More than 50% of the trees are already damaged by the disease. The symptoms included twig and branch dieback and sunken necrotic bark lesions that could progress to the trunk, resulting in the death of large sections of the tree. Ascomata and conidiomata typical of Botryosphaeriaceae were observed on branches of symptomatic trees. Ascospores were hyaline, aseptate, ovoid to fusoid, and conidia were hyaline, aseptate, smooth, thin-walled, and fusiform with base truncate. Single spore isolates were obtained from samples. In culture, single ascospore isolates produced conidia similar to the ones found on the host. Diseased branch tissues were surface sterilized with 5% NaOCl, plated on potato dextrose agar (PDA), and incubated at 25°C. Fungal isolates recovered produced white aerial mycelium that darkened with age becoming grey to dark grey and conidia that were similar in all aspects to the ones produced by single spore isolates. All isolates produced on PDA a yellow pigment that diffused into the agar and disappeared after 6 to 7 days. Morphological and cultural aspects of the isolates were similar to the species Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips and N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (2). Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR fingerprinting divided the isolates into two groups (1). Two isolates (CAA352 and CAA392), one from each group, were selected for further identification by sequencing of the internal transcribed spacer (ITS) region of the rDNA and part of the elongation factor 1-alpha gene (EF1-α) (Accession No. ITS: JX975212 JX975213; EF1-α: JX975210 JX975211). BLAST analysis showed that ITS and EF1-α sequences from group 1 and 2 had 99 to 100% similarity to reference cultures (including ex-type) of N. luteum and N. australe, respectively. To confirm pathogenicity and fulfill Koch's postulates, six 2-year-old seedlings of Q. robur were artificially infected with isolates CAA352 and CAA392 and kept at approximately 20 to 25°C. A shallow wound was done with a scalpel on the basal part of the stem of each seedling, a bark portion was removed aseptically and a PDA disc (0.5 cm) of an actively growing culture was placed on the wound. Control seedlings received sterile PDA discs. The inoculation site was wrapped in Parafilm to prevent desiccation. Within 8 weeks, infected seedlings developed canker lesions associated with vascular necrosis around the inoculation point. A third of the seedlings died and developed abundant pycnidia on the stem. Control seedlings remained symptomless. Both pathogens were successfully reisolated from the infected tissue. N. luteum and N. australe are increasingly reported as causing diseases to a wide range of woody hosts of economic and forestry importance (3). To our knowledge, this is the first report of both species causing dieback and canker disease on Q. robur. References: (1) A. Alves et al. Res. Microbiol. 158:112, 2007. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) B. Slippers et al. Fungal Biol. Rev. 21:90, 2007.