Standardizing experimental approaches to investigate interactions between bacteria and ectomycorrhizal fungi

Abstract

Bacteria and ectomycorrhizal fungi (EcMF) represent two of the most dominant plant root-associated microbial groups on Earth, and their interactions continue to gain recognition as significant factors that shape forest health and resilience. Yet, we currently lack a focused review that explains the state of bacteria-EcMF interaction research in the context of experimental approaches and technological advancements. To these ends, we illustrate the utility of studying bacteria-EcMF interactions, detail outstanding questions, outline research priorities in the field, and provide a suite of approaches that can be used to promote experimental reproducibility, field advancement, and collaboration. Though this review centers on the ecology of bacteria, EcMF, and trees, it by default offers experimental and conceptual insights that can be adapted to various subfields of microbiology and microbial ecology.

Keywords: bacteria; bacteria-EcMF-plant interactions; ectomycorrhizal fungi (EcMF).

Figure 1.

Overview of bacteria-EcMF interaction categories. (Left) Interactions between bacteria and ectomycorrhizas may require physical contact, or produced and consumed metabolites may traverse some distance, rendering two broad categories of interactions—contact-dependent and contact-independent. However, these two categories are not necessarily mutually exclusive. Interactions may shift throughout the course of symbiosis, and/or the strength of the interaction may be attenuated or strengthened depending on both the amount of contact and the effect distance of produced/consumed metabolites. Interactions can also be categorized as direct (e.g. consumer-producer exchange of resources) or indirect (e.g. pathogen suppression via antibiotic or siderophore production, resource competition, and spatial residence depletion). (Right) These interaction relationships can be viewed along a tripartite symbiosis spectrum. In this three-dimensional space, benefits derived (e.g. resources, residence, pathogen suppression) from symbiosis may differ among interacting members. For example, the maximal benefits for a given bacterial species may be highest when the plant gains effectively no benefit (i.e. commensalism), whereas another bacterial species may derive maximal benefit only when the plant and or EcMF is also deriving maximal benefit (i.e. tripartite synergistic symbiosis). Since the success of EcMF is largely contingent on the success of the plant host, these relationships tend to be viewed with a higher degree of interdependency. Therefore, as shown, EcMF will likely never gain maximal benefits unless the plant host derives some benefit. It is likely, however, that EcMF species with greater saprotrophic capabilities will be less host dependent. This tripartite symbiosis spectrum nevertheless functions as a conceptual framework to assess these relationships, which may also change along the fourth dimension, time.

Figure 2.

Schematic experimental overview of bacteria-EcMF interaction research. (A) Common inoculation conditions to assess interactions between bacteria, EcMF, and plants. (B) Commonly measured plant responses to bacteria and EcMF inoculations. Note that the duration of experiments post inoculation tends to span anywhere between 1 and 12 months. (C) Plant and microbial responses that have yet to be addressed. Priority effects, or when species arrive, have not been tested variables in bacteria-EcMF research. Instead, experiments simultaneously inoculate plants with combinations of bacteria and EcMF, leaving much uncertainty about the potential priming effects or niche preemption that either bacteria or EcMF may facilitate. Microscale changes, such as localization to plant roots, fungal hyphae, or neighboring soils, have likewise received little attention, despite several reports indicating that significant microbial filtering events occur across these microscale zones. In addition, the molecular changes that are associated with these microscale shifts (e.g. changes to gene expression) remain largely unknown.

Figure 3.

Developmental stages of bacteria-EcMF interactions. (A) Germination of EcMF spores. In nature, spores germinate in the presence of bacterial communities, and the composition of these communities, their metabolic potential, and the consortium of molecules that they produce affect spore germination. (B) EcMF plant root colonization. The mantle that EcMF produce as they colonize plant roots can create a unique niche that selects for bacteria and/or bacterial communities that further facilitate or enhance EcMF colonization and ectomycorrhizal symbiosis. (C) Symbiosis maintenance among bacteria, EcMF, and plants. Here, we show how dead or decaying plant or fungal tissue can select different bacterial communities compared to healthy plant and fungal tissue. The connection between this differential enrichment and plant responses remains unexplored.

Figure 4.

Emerging methods for studying bacteria-EcMF interactions across spatial and informational scales. A holistic understanding of bacteria-EcMF interactions will require an interdisciplinary methodological approach, whereby symbiotic community formation is studied from the chemical (i.e. individual molecules) to the environmental level (i.e. changes to the climatic environment). Summarized are emerging or underutilized experimental approaches (top) that may be applied to study bacteria-EcMF interactions across spatial and informational scales (bottom). Arrows indicate the approximate experimental scale of the system studied, colored by approach. The arrow bar adjacent to the corresponding arrow for Cryo-ET represents metabolomics, MALDI, and mass spectroscopy, which are ideal for this scale range.