Besides their symbiotic association with tree rootlets, ectomycorrhizal (EM) fungi have been commonly detected in nature in deadwood and plant debris of various tree species. However, their potential dual roles as symbiotrophs and saprotrophs are still debated. Here, we provide evidence from a series of experiments on the plasticity of symbiotrophic-saprotrophic lifestyles of the ectomycorrhizal fungus Piloderma croceum associated with Quercus robur L. Specifically, we find that P. croceum efficiently colonizes deadwood of oak in an experimental system without living oak. Results based on the productions of hydrolytic enzymes and corticrocin as well as the 14C content in deadwood and mycelium of P. croceum demonstrate its capability of wood decomposition and assimilation of C from the decomposing wood. Our results also show that in presence of wood pieces colonized by saprotrophic mycelium of P. croceum, the roots of oak plants develop true EM symbiosis with Hartig net formation. Collectively, our results indicate a role for mycelium growing in deadwood as an underestimated EM fungus propagule bank, suggesting that deadwood and other decomposing plant material may indirectly influence the productivity of forest ecosystems by contributing to the recruitment of mycorrhizal fungi, thereby enhancing plant nutrient acquisition.
Ectomycorrhizal fungi (EM fungi) play a central role in providing trees with important nutrients especially in boreal and temperate but also in some subtropical and tropical forest ecosystems. The existence and functioning of EM fungal mycelia determine plant growth and productivity in the forest ecosystems and contribute to the large global pool of carbon in soils. The mycelia of EM fungi extend from different individual host tree species and form common mycorrhizal networks in soils, which influence plant nutrient and carbon acquisition at ecosystem level. These mycelia can act as propagules for EM fungi to colonize seedlings and stimulate forest regeneration. In areas where mycorrhizal networks are not available or poorly formed, EM fungal spores in soils can be considered an important source of propagules. EM fungal spores may persist in the soil for decades before colonizing plant roots. Extramatrical mycelium and spores in soils are thus considered as the major propagule banks of EM fungi, but it is currently unknown whether EM fungi colonized plant debris could also serve as propagule banks.
Recent experimental evidence, based on high-throughput sequencing, has demonstrated that deadwood harbors diverse EM fungal communities. EM fungi colonize deadwood and use this niche, among others (litter and living roots). EM fungi have evolved on repeated independent occasions among clades of initially humus and wood saprotrophic fungi, so that EM fungal taxa are spread across the classification of Asco- and Basidiomycota, where they still neighbor some saprotrophic sister taxa within clades. EM fungi can compete with saprotrophs in deadwood. Their competitiveness depends on priority effects (or community assembly history), depending on their early arrival to the substrate or the wood decay stage. Although EM fungi can also be detected at early wood decay stages in some temperate and subtropical tree species, they dominate during the late decomposition stages. Some EM fungi have been suggested to have proteolytic activity via the secretion of relevant enzymes that support the decomposition of dead biomass. Several studies have demonstrated the potential of EM fungi to degrade complex plant polymers, including cellulose, hemicellulose, pectin, and lignin. However, the enzymatic machinery for the decomposition of these polymers is strongly reduced in EM fungi. Some EM fungi can oxidize organic matter, either by Fenton chemistry, such as Paxillus involutus and Piloderma croceum, or by using peroxidases, such as Cortinarius spp.. It has been suggested that these activities serve to obtain nitrogen from organic sources. The conservation level of enzymes for plant polymer decomposition is particularly high in P. croceum, whose genome contains 52, 18, and 38 genes related to cellulose/hemicellulose, pectin, and lignin decomposition, respectively. Such carbon degrading enzyme activities can serve to release glucose and other small carbon compounds and molecules from complex organic sources which can be utilized by P. croceum. Therefore, decomposing plant materials may promote ectomycorrhizal establishment and propagation of P croceum in temperate forest ecosystems. It has been hypothesized to be a potential ectomycorrhizal propagule bank in forest ecosystems. Although some EM fungi might have evolved from either saprotrophic or endophytic fungi, their evolution via saprotrophic fungi has been more common. Decomposing plant materials including, deadwood and leaf litter are present together or in the vicinity with fine roots in the detritusphere and topsoil. Closely related fungi even within the same genera have evolved differently, by specializing in these substrates. While saprotroph fungi have evolved toward litter and deadwood materials with increasing/maintaining of plant cell wall degrading enzymes related genes (especially Carbohydrate-Active EnZymes (CAZymes)), many EM fungi displayed reduction of such genes, while maintaining and increasing symbiosis related genes such as sugar transporters, transposable elements (TEs) and small secreted proteins (SSPs). However, some EM fungi have retained substantial numbers of plant cell-wall degrading genes and have been found to have the ability to decompose plant organic matter. Thus, deadwood and leaf litter can be considered as alternative substrates for such EM fungi, in life phases or compartments where they cannot establish ectomycorrhiza. In forest ecosystem, it is also common that fine root patches of diverse trees colonize decomposing deadwood and leaf litter, from which they obtain nutrients via their EM fungal partners. When EM fungi can survive as saprotroph on decomposing plant materials (especially deadwood), they can increase their potential to find right host plants and to increase the chance of successful colonization.
The plasticity of symbiotroph-saprotroph EM fungal lifestyles has been hypothesized for more than a decade. A study also demonstrated that many saprotrophic fungi are able to colonize fine roots of ectomycorrhizal trees and form either incomplete or complete ectomycorrhizas, indicating the potential for such plasticity. However, thus far there is no substantial evidence that demonstrates the efficient colonization of deadwood and its components by EM fungi and whether EM fungi-colonizing deadwood can change their lifestyles from saprotrophic to ectomycorrhizal programs. The dual role of EM fungi as a symbiotroph and a saprotroph therefore still under debate.
In this study, Piloderma croceum associated with Quercus robur L. was used to demonstrate the plasticity of symbiotroph-saprotroph lifestyles of EM fungi. The saprotroph lifestyle can be demonstrated by the ability of Piloderma croceum to colonize and decompose dead organic matter from wood. The decomposition products (especially glucose) and nutrients released from dead organic matter have to be incorporated into the mycelium of the fungus. To demonstrate the switch from the saprotroph to the symbiotroph- lifestyle, P. croceum living in the saprotroph stage on dead wood, had to be able to colonize the living roots of Q. robur plants and exhibit a clear symbiotic apposition structure (i.e. Hartig net), as well as a benefits of EM formation, i.e. tree growth stimulation. Therefore, in this current work nine experiments were carried out to test whether (1) P. croceum can colonize a decomposing plant material, deadwood (bark, outer sapwood, inner sapwood, and the complete deadwood) of Quercus robur in the absence of living oak plants (experiments 1 to 4) and (2) such colonized decaying wood can serve as a propagule bank for forming new mycorrhizal symbiosis when faced with roots of living oaks (experiments 5 to 9). Specifically, we aimed to answer four questions. First, could P. croceum grow and persist on bark and wood substrates (experiment 1)? This would demonstrate the capacity to live or persist when not in symbiosis with roots (but not yet to establish a saprotrophic lifestyle). Second, what was the fungal enzymatic activity in wood colonized by P. croceum (experiment 2)? This would demonstrate that the fungus can release enzymes (as expected) that are capable of decomposing polymers of wood fibers, i.e., that it is truly saprotrophic. Third, was any wood carbon or related products, as measured via all-E-tetradeca-2, 4, 6, 8, 10, 12-hexaene-l,14-dioic acid (corticrocin) (experiment 3) and carbon isotope (C) (experiment 4), detectable in vegetative tissue of the fungus? This would further demonstrate that the fungus is capable of decomposition and assimilating carbon and glucose from wood fibers and polymers, i.e., saprotrophic. Fourth, can deadwood be a source of dispersal for P. croceum to establish ectomycorrhizal roots (experiments 5 to 9)? This would show that wood can be a source of inoculants for the fungus, a key tenant of the research.
Based on plasticity of symbiotroph-saprotroph lifestyles of EM fungi, we hypothesized that P. croceum is able to grow and persist on bark and wood substrates (hypothesis 1). We hypothesized that based on its gene repertoire, P. croceum can secrete both hydrolytic (β-glucosidase) and oxidative enzymes (laccase and peroxidases) that are important for cellulose/hemicellulose and lignin degradation during an exclusive saprotrophic life phase (hypothesis 2). Furthermore, we expected that P. croceum would produce N-acetyl glucosaminidases and acid phosphatases to acquire N from chitin and organic P. Because of its ability to produce enzymes for C acquisition and lignin degradation/modification, we also hypothesized that the C released from deadwood could be transferred to and assimilated by P. croceum mycelium (hypothesis 3). We expected that Q. robur plants associated with P. croceum originating from deadwood would show the benefit of EM formation and their plasticity to outsource C with higher principal root, lateral root, and total root biomass, and stimulate plant growth with higher stem, shoot, and total plant biomass, compared with non-inoculated plants (hypothesis 4).