First report of <i>Phytophthora ilicis</i> causing leaf spot, shoot blight and bleeding canker on <i>Ilex aquifolium</i> in Slovenia

English holly (Ilex aquifolium) is a small, evergreen tree that grows naturally in temperate and mediterranean mountain forests in Europe, and is grown throughout the world for its ornamental characteristics. Ecologically, holly is a shade-tolerant and very slow-growing plant that populates the undergrowth in temperate deciduous forests and woodlands dominated by oak or beech (Guerrero Hue et al., 2016). In Slovenia I. aquifolium shows an irregular distribution in wet and shady places with quite mild climates such as the Vipava and Osilniška valleys. Due to illegal removal from the wild, holly has been protected by national legislation in Slovenia. In October 2023, moderate defoliation was found on several young English holly trees located in a mixed forest near Nova Gorica in western Slovenia (Gozd Panovec, 45°56'47.7“N 13°40'06.0”E). Diseased plants exhibited leaf necrosis, shoot blight, defoliation and occasionally stem bleeding cankers and inner bark discolouration (Fig. 1). Field surveys conducted in spring 2024 revealed a disease incidence of 60% along two linear transects of 25 m. Fifteen necrotic leaves and three inner bark (stem) samples were collected from six diseased plants chosen at random in the study area. Samples were processed as described by Bregant et al. (2023) and cultured on carrot agar at 18°C in the dark. A typical slow-growing homothallic Phytophthora species was isolated consistently from the diseased samples (Fig. 2). Isolates produced semi-papillate mostly caducous sporangia (mean size 41.8 ± 6.6 × 26.4 ± 3.6 µm, n = 25) with long pedicels (mean length 17.3 ± 5.4 µm, n = 25) after 36–48 hours on carrot agar flooded in non-sterile pond water (Fig. 2). Based on these morphological data, 14 isolates appeared compatible with the species Phytophthora ilicis Buddenh. & Roy A. Young. The morphological identification was confirmed by molecular analysis based on the sequence of the ITS and cox1 regions obtained following the methodology reported in Bregant et al. (2023). Phylogenetic reconstructions for cox1 sequences of two Slovenian isolates of P. ilicis and nine other isolates together with the other species belonging to ITS clade 3 were performed with MEGA-X 10.1.8, including all gaps in the analyses and choosing the best model determined automatically by the software (Kumar et al., 2018). Maximum likelihood analysis was performed with a neighbour-joining starting tree generated by the software (Fig. 3). Two additional P. ilicis strains isolated in the Marche region (Castelfidardo, Italy) were included in the phylogenetic analysis and a sequence of P. alpina (CBS 146801) was used as an outgroup. The sequences of P. ilicis generated in this study were deposited in GenBank (isolate SLO35 (Slovenia): GenBank Accession Nos.: PQ452714 & PQ472567; isolate SLO43 (Slovenia): PQ452715 & PQ472568; isolate CB1100 (Italy): PQ452716 & PQ472569; isolate CB1101 (Italy): PQ452717 & PQ472570). Mitochondrial cox1 phylogeny shows that the Slovenian population of P. ilicis is identical to those in other parts of the Mediterranean and in North America (Fig. 3). The pathogenicity of Phytophthora ilicis was verified by inoculating asymptomatic young leaves of Ilex aquifolium with a representative isolate (SLO35). The upper surface of the leaf was disinfected with 70% ethanol and inoculated (without wounding) by an agar-mycelium plug (3 mm Ø) cut from the margin of a four-day-old colony on potato dextrose agar (PDA). Eight leaves were inoculated with the pathogen in the trial and another five were inoculated with a sterile PDA plug as a control. Inoculated leaves were maintained in humid chamber at 18 ± 1°C for 72 hours. Phytophthora ilicis proved to be pathogenic on holly leaves, causing extensive necrosis (mean lesion length 710 ±313 mm2) (Fig. 4). Control leaves remained asymptomatic. Phytophthora ilicis was successfully re-isolated from all inoculated leaves, thus fulfilling Koch's postulates. Phytophthora. ilicis is one of the few known host-specific Phytophthora species. It was initially described in the western United States and is also recorded on ornamental holly trees in Canada, UK, the Netherlands, and Spain (Buddenhagen & Young, 1957, Pintos et al., 2012). In 2014 P. ilicis was reported for the first time in natural holly stands in Sardinia and Corsica, where it probably represents an endemic species (Scanu et al., 2014). To our knowledge, this is the first report of P. ilicis on I. aquifolium in Slovenia. A study is currently underway to study the distribution and populations of this species in the rare and fragmented formations of I. aquifolium in continental Italy and Slovenia. This research was funded by the project TESAF1DOR-00414, Public Forest Service funded by Ministry of Agriculture, Forestry and Food (Slovenia), and co-funded by Slovenian Research and Innovation Agency (Research Program P4-0107 Forest Biology, Ecology and Technology).

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