Comparative metagenomic analysis of the taxonomic composition and function of the oak microbiome in healthy tissue (from trees without AOD) and diseased tissue (from trees with AOD). (
a) The plot depicts the genera represented in all metagenomes based on One Codex binning of raw reads, demonstrating a clear shift in the microbial community between healthy and diseased trees. ( b) Brenneria and Gibbsiella are statistically correlated to diseased tissue, as shown by a principal coordinate ordination analysis based on statistics calculated by Primer v7 and PERMANOVA+ using One Codex binning data. The first two axes depict the plotted community composition. Correlation vectors in the graph are significant using an R 2>0.05. A Welch’s t-test was performed to test significance of differences between key taxa (identified above) and between healthy and diseased trees (pooled abundances for each factor). Resultant P-values from Welch’s t-test are overlaid on the correlation biplot. ( c) Gene groups involved in bacterial phytopathogenic activity are significantly increased in AOD diseased trees, as shown by comparative functional analysis of SEED subsystem categories as annotated by MG-RAST on assembled metagenome contigs. The analysis of SEED subsystem metagenome data was performed using Stamp. Statistically significant functional differences between diseased and healthy communities were calculated using G-test with Yates’ correction. The Newcombe–Wilson test was performed to calculate confidence intervals between two binomial population proportions. ( d) The genomes of 17 species were found to be common across all AOD metagenomes. Visualization of Kraken metagenome analysis of stem samples demonstrates the shifts in bacterial microbiome compositions. Bubble sizes are categorized based on the relative percentage frequencies of Kraken species-level alignment of raw metagenome sequencing reads and are depicted in the figure as 1 (read frequency <0.01), 2 (read frequency 0.01–0.1), 3 (read frequency 0.1–1), 4 (read frequency 1–10) and 5 (read frequency >10). Red bubbles signify samples from diseased trees, while blue bubbles signify samples from healthy trees. The 17 common species were determined based on common occurrence across all diseased tissue samples. Additionally, six species more abundant among the healthy trees were included to provide a contrasting shift.