Terrestrial plants including forest trees are generally known to live in close association with microbial organisms. The inherent features of this close association can be commensalism, parasitism or mutualism. The term “microbiota” has been used to describe this ecological community of plant-associated pathogenic, mutualistic, endophytic and commensal microorganisms. Many of these microbiota inhabiting forest trees could have a potential impact on the health of, and disease progression in, forest biomes. Comparatively, studies on forest tree microbiomes and their roles in mutualism and disease lag far behind parallel work on crop and human microbiome projects. Very recently, our understanding of plant and tree microbiomes has been enriched due to novel technological advances using metabarcoding, metagenomics, metatranscriptomics and metaproteomics approaches. In addition, the availability of massive DNA databases (e.g., NCBI (USA), EMBL (Europe), DDBJ (Japan), UNITE (Estonia)) as well as powerful computational and bioinformatics tools has helped to facilitate data mining by researchers across diverse disciplines. Available data demonstrate that plant phyllosphere bacterial communities are dominated by members of only a few phyla (Proteobacteria, Actinobacteria, Bacteroidetes). In bulk forest soil, the dominant fungal group is Basidiomycota, whereas Ascomycota is the most prevalent group within plant tissues. The current challenge, however, is how to harness and link the acquired knowledge on microbiomes for translational forest management. Among tree-associated microorganisms, endophytic fungal biota are attracting a lot of attention for their beneficial health- and growth-promoting effects, and were preferentially discussed in this review.

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.

The opportunistic pathogen Sphaeropsis sapinea (≡Diplodia sapinea) is one of the most severe pathogens in Scots pine, causing the disease Diplodia tip blight on coniferous tree species. Disease symptoms become visible when trees are weakened by stress. Sphaeropsis sapinea has an endophytic mode in its lifecycle, making it difficult to detect before disease outbreaks. This study aims to record how S. sapinea accumulates in trees of different health status and, simultaneously, monitor seasonal and age-related fluctuations in the mycobiome. We compared the mycobiome of healthy and diseased Scots pines. Twigs were sampled in June and September 2018, and filamentous fungi were isolated. The mycobiome was analyzed by high-throughput sequencing (HTS) of the ITS2 region. A PERMANOVA analysis confirmed that the mycobiome community composition significantly differed between growth years (p < 0.001) and sampling time (p < 0.001) but not between healthy and diseased trees. Sphaeropsis sapinea was the most common endophyte isolated and the second most common in the HTS data. The fungus was highly abundant in symptomless (healthy) trees, presenting in its endophytic mode. Our results highlight the ability of S. sapinea to accumulate unnoticed as an endophyte in healthy trees before the disease breaks out, representing a sudden threat to Scots pines in the future, especially with increasing drought conditions experienced by pines.

There is an increasing need to calibrate microbial community profiles obtained through next generation sequencing (NGS) with relevant taxonomic identities of the microbes, and to further associate these identities with phenotypic attributes. Phenotype Microarray (PM) techniques provide a semi-high throughput assay for characterization and monitoring the microbial cellular phenotypes. Here, we present detailed descriptions of two different PM protocols used in our recent studies on fungal endophytes of forest trees, and highlight the benefits and limitations of this technique. We found that the PM approach enables effective screening of substrate utilization by endophytes. However, the technical limitations are multifaceted and the interpretation of the PM data challenging. For the best result, we recommend that the growth conditions for the fungi are carefully standardized. In addition, rigorous replication and control strategies should be employed whether using pre-configured, commercial microwell-plates or in-house designed PM plates for targeted substrate analyses. With these precautions, the PM technique is a valuable tool to characterize the metabolic capabilities of individual endophyte isolates, or successional endophyte communities identified by NGS, allowing a functional interpretation of the taxonomic data. Thus, PM approaches can provide valuable complementary information for NGS studies of fungal endophytes in forest trees.