Comparative studies targeting the drivers of endophytic fungal biodiversity are rare and identified multiple effectors, such as plant chemistry, climate and seasonal attributes. Our project aimed to study the pattern of the leaf-associated mycobiomes of European beech (Fagus sylvatica) at different altitudes to reveal diversity, composition and seasonal dynamics of fungal endophytes by a combination of metabarcoding, cultivation and subsequent ecological analyses. An experimental field site consisting of 100 (2-years old beech) trees was established called ‘beech phytometer system’ at two altitudes (517 and 975 m a.s.l.) in a German mountain forest. Metabarcoding resulted in a total of 15,703,599 demultiplexed and quality filtered ITS1 reads from 165 samples. Clustering at 97% similarity resulted in 1199 OTUs. In addition, 438 isolates from an autumn sampling event were cultivated via the dilution-to-extinction method; endophytes were identified via barcoding based on ribosomal ITS (internal transcribed spacer) markers and Sanger sequencing.
Results/Conclusions
A significant correlation of community composition with elevation was observed. The mycobiome was little affected by the physiological state of the leaves; only a partial shift of taxonomic composition was observed from vital towards senescent leaves. Mycobiome diversity and composition correlated significantly with the origin of the trees, pointing to local habitat condition as the main driver. Under natural conditions, the mycobiome was more diverse at lower elevation. Additionally, leaf chlorophyll and flavonoid contents showed negative correlations with fungal richness in natural stands. Metabarcoding and cultivation approaches resulted in non-overlapping community compositions and pronounced differences in taxonomic classification and trophic stages. However, both methods revealed similar correlations of the fungal communities with local environmental conditions. Our results indicate undeniable advantages of metabarcoding over cultivation in terms of representation of the major functional guilds, rare taxa and diversity signals of leaf-inhabiting fungi. We observed a strong seasonal turnover in phyllosphere fungi in both habitats over the two years of investigation, suggesting that the plant-fungal system not only responds to cyclic climatic conditions but depends as well on various parameters, e.g., geographic position, substrates age and surrounding vegetation. In general, the altitudinal difference is the most important explaining factor for community differences, which shapes many dependent abiotic and biotic habitat factors. Regarding cost and time per sequence, metabarcoding is superior to cultivation approaches and offers surprisingly profound insights by yielding much more data, allowing to test at once multiple hypotheses in fungal ecology.