Fungal pathogens can be detrimental to forest ecosystems by affecting tree mortality. Other fungal species may be able to colonize niches to the extent of excluding pathogens. However little is known about how tree species diversity affects the fungal community. Are the fungal communities the same in monocultures of a tree species in comparison with those communities that found in more mixed forest stands? To that end, we aim to determine the distribution of fungal species along a tree diversity gradient in one tree species within the experiment at Satakunta, Finland. Birch (Betula pendula) leaves were collected in August 2011 from trees grown in mixture with up to four other tree species. To obtain information about the fungi found in these leaves, a massively parallel high throughput sequencing approach will be taken.

One of the objectives in task 4.6 was achieved with the sampling of fresh leaves from target trees, which was to collect leaves for molecular analysis of the fungal community. Trees were sampled only in Area 1 of Satakunta. Five trees per species were randomly pre-selected for sampling. Birch leaves were sampled from trees along a tree species diversity gradient. The sampling scheme included two plots from the birch monoculture plots, one plot from each two and three mixture containing birch, and two plots from the five species mixture. Four branches were cut per tree: two from the upper canopy facing the North and South, and two from the lower canopy facing the North and South. Leaves were selected randomly from each branch for a total of 20 leaves per tree: five leaves from the upper canopy and five leaves from the lower part of the canopy, and for each branch, the north and south facing expositions. Leaves were dried in paper bags for three days at 60 deg C, and subsequently stored at room temperature.

Plantation: Satakunta, Finland

Between August 12, 2011 and August 15, 2011

Leaves from Betula pendula

DNA was extracted from 10 birch leaves per tree (5 from each of the two canopy levels). Fungal community was amplified with fungal-specific primers fITS7 and barcoded-ITS4, the latter of which would aid in disentangling sequences from different samples. Pooled amplicons were sent to Eurofins MWG Operon for 454 pyrosequencing using GS Junior. Raw sequences were demultiplexed in the SCATA pipeline (http://scata.mykopat.slu.se/) (run number: scata1897) using default parameters and clustering at 1.5% dissimilarity. There were nearly 30,000 sequence reads that were clustered into 163 Operational taxonomic units (OTUs), which are approximately equivalent to species. Singletons were excluded. Non-metric multidimensional scaling ordination will be performed on the data and significance of the ordination can be confirmed by methods such as non-parametric analysis of variance (NPMANOVA) and ANOSIM. Comparison of single species in different treatments will be conducted as well. Raw sequence read counts are reported. It is advisable to convert to each operational taxonomic unit (OUT) per sample into frequencies before any analysis is conducted.