# Methods for Studying Bacterial-Fungal Interactions in the Microenvironments of Soil
## 2 Fungi, Bacteria and Their Microenvironment
### Bacterial-Fungal Interactions: A Harsh Existence
Fungi actively explore the soil pore space through hyphal spread, and cope well with heterogeneous distribution of nutrients [46]. They can cross air-water interfaces and nutrient-depleted spots to gain access to distant nutrient resources. The extensive mycelial architecture enables fungi to easily and efficiently re-allocate useful compounds to substrate-limited regions, to the benefit of exploratory colonisation of more unfavourable habitats [47]. A relevant feature of microbial interaction related to mycelial growth is that hyphae serve as dispersal vectors for motile bacteria (i.e., fungal highways) [48] and, hence, allow for bacterial colonization of new micro-habitats [49; 50]. Moreover, the mycelia of fungi and oomycetes enhance bacterial activity by nutrient and water transfer (excretion) from the hyphae to the bacterial cells, thus enabling bacterial growth in otherwise too oligotrophic habitats [51], or enhancing microbial activity in dry soils [52]. An important example of such a symbiotic interaction is extraradical mycelia of arbuscular mycorrhizal and other mycorrhizal fungi in the rhizosphere of plants, where a large array of bacteria can directly consume fungal exudates released into the environment [34]. The mutualistic relationship can also be reciprocal: fungi take advantage of their bacterial partners to improve their carbon source pool in a mechanism named bacterial farming [53]. Fungal hyphae can also become an ideal hotspot for horizontal gene transfer [54; 55; 56] or bacterial prey populations [57], as they facilitate dispersal and preferential contact of bacteria in the hyphae. In this regard, it should be mentioned that, apart from fungal hyphae, plant roots and dead organisms also behave as hot spots for highly interactive (e.g., competition, pathogeny, mutualism, predation) microbial communities. Nonetheless, these many physical interactions between bacteria and hyphae-forming fungi may represent short-lived associations, as microscale communities frequently assemble and disassemble by migration, attachment, and detachment from surfaces and cells. In this quite lonesome and unforgiving scenario, physical contact between cells is often not realizable. Hence, molecular compounds are employed and secreted in the soil solution to sense potential partners in their surroundings. Importantly, the release of molecular signals represent a cost, both energetic and elemental, and hence are regulated to maximize the fitness of the organism [27]. As molecules diffuse freely from any pore and are easily removed from the microenvironments, molecular interactions can only occur at relatively short distances. On leaf surfaces, for example, interactions among bacteria have been found to occur principally in the 5 to 20 \(\upmu\)m range [58], whereas in soil, where local patches of cells may reside in a pocket of soil solution, diffusible metabolites can reach neighbouring cells up to 100 \(\upmu\)m away [44; 59]. The interactive dynamics are very different in soil crusts, biofilms, or mats where cells are well physically constrained and in direct contact with each other (e.g., [60]). Sensing and recognition of fungi and bacteria includes also solution-independent ways. Volatile organic compounds (VOCs) are one important quorum sending vehicle-enabling communication over longer distances, especially in vision of the spatial separation between soil particles often occurring in unsaturated water conditions [61; 62]. Finally, it is worth mentioning that bacteria and fungi can also indirectly interact by modifying their microenvironment in ways that can positively or negatively affect their partners (i.e., niche modulation) [34]. For example, the acidification or the neutralization of pH values influences the solubility of soil nutrients (e.g., phosphorus [24]), thus inhibiting or stimulating overall bacterial growth and metabolism [63; 64; 65]. Nutrient depletion is one example of the many ways of indirect interaction.