Fungal Diversity in the Phyllosphere of Pinus heldreichii H. Christ-An Endemic and High-Altitude Pine of the Mediterranean Region

Pinus heldreichii is a high-altitude coniferous tree species naturaly occurring in small and disjuncted populations in the Balkans and southern Italy. The aim of this study was to assess diversity and composition of fungal communities in living needles of P. heldreichii specifically focusing on fungal pathogens. Sampling was carried out at six different sites in Montenegro, where 2-4 year-old living needles of P. heldreichii were collected. Following DNA isolation, it was amplified using ITS2 rDNA as a marker and subjected to high-throughput sequencing. Sequencing resulted in 31,831 high quality reads, which after assembly were found to represent 375 fungal taxa. The detected fungi were 295 (78.7%) Ascomycota, 79 (21.0%) Basidiomycota and 1 (0.2%) Mortierellomycotina. The most common fungi were Lophodermium pinastri (12.5% of all high-quality sequences), L. conigenum (10.9%), Sydowia polyspora (8.8%), Cyclaneusma niveum (5.5%), Unidentified sp. 2814_1 (5.4%) and Phaeosphaeria punctiformis (4.4%). The community composition varied among different sites, but in this respect two sites at higher altitudes (harsh growing conditions) were separated from three sites at lower altitudes (milder growing conditions), suggesting that environmental conditions were among major determinants of fungal communities associated with needles of P. heldreichii. Trees on one study site were attacked by bark beetles, leading to discolouration and frequent dieback of needles, thereby strongly affecting the fungal community structure. Among all functional groups of fungi, pathogens appeared to be an important component of fungal communities in the phyllosphere of P. heldreichii, especially in those trees under strong abiotic and biotic stress.