Cork oak decline has been recorded in Portuguese oak forests during recent decades. Several symptoms have been associated with the decline of Quercus suber, namely thinning of the canopy, dry and discolored leaves, dry branch tips, and cankers on the trunk and branches. During a survey to determine the impact of Botryosphaeriaceae species on Q. suber, samples were collected from trees showing cankers and dieback symptoms in Ribatejo and Alentejo stands. Isolations were performed on potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate. Pure cultures were obtained from symptomatic tissues and induced to sporulate by plating on 2% water agar containing sterilized pine needles and incubating at 23°C under 12 h cycles of near UV light, until pycnidia developed. On the basis of micromorphological characters of conidia, the isolates were assigned to genus Diplodia. Isolates were deposited in the Micoteca da Estação Agronómica Nacional (MEAN) at the Instituto Nacional de Investigação Agrária e Veterinária (INIAV) in Portugal. To confirm their identity, the internal transcribed spacer (ITS) of the rDNA and the translation elongation factor 1-alpha (tef-1α) regions were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone et al. 1999). BLAST searches against GenBank showed that Diplodia corticola was the closest match. Isolates MEAN 1016 and 1017 showed 100% sequence similarity with ITS-rDNA (GenBank accessions KU311197 and KU311198) and 99% with tef-1α (KU311200 and KU311201) of the ex-holotype isolate of Diplodia quercivora CBS 133852 (Linaldeddu et al. 2013). To verify Koch’s postulates, pathogenicity tests were performed in a greenhouse by inoculating 10 Q. suber seedlings (stem diameters 5 to 7 mm) with isolate MEAN 1016. Small strips of bark (10 × 1 mm) were cut from the stems and pieces of PDA colonized by the fungus were placed in the wounds and covered with Parafilm. Ten seedlings inoculated with sterile PDA served as negative controls. Since D. corticola is considered very aggressive to Q. suber in Portugal (Alves et al. 2004), to compare both species regarding their capacity to cause symptoms in this host, 10 seedlings were inoculated with D. corticola isolate MEAN 1020 (KU891979 for ITS and KU891980 for tef-1α). The first symptoms appeared after 7 and 10 days for D. corticola and D. quercivora, respectively. After one month, all plants inoculated with D. corticola and two plants inoculated with D. quercivora were dead. No external symptoms were observed on 50% of the plants inoculated with D. quercivora and also on control plants. The species inoculated were reisolated from all symptomatic plants. Based on a scale of external symptoms comprising five levels (healthy, <25% affected leaves, 50% affected leaves, >75% affected leaves, and all plant affected), Kruskal-Wallis test shows that D. quercivora tends to be less aggressive to Q. suber than D. corticola (P < 0.001), but further experiments are necessary to confirm this. D. quercivora was first described as causing shoot dieback and cankers on Q. canariensis in Tunisia (Linaldeddu et al. 2013) and on Q. virginiana in the United States (Dreaden et al. 2014). To our knowledge, this is the first report of D. quercivora on Q. suber and in Europe. More studies are needed to assess the risk this pathogen poses to cork oak stands.
