The term necrobiome was originally used to describe “the community of species (e.g., prokaryotic and eukaryotic) associated with decomposing remains of heterotrophic biomass, including animal carrion and human corpses” [21]. This term, initially focusing mainly on vertebrate carrions, was later expanded by Benbow et al. [22] to include other forms of necromass, such as leaves, wood, and dung. Therefore, the term necrobiome can be associated with vegetable and animal necromass decomposition. Some aspects of the decomposing action of the necrobiome are common to all types of necromass, such as tissue disintegration, microbial activity, and the release and recycling of nutrients within the ecosystem. Conversely, other aspects are characteristic of each different form of necromass, such as community dynamics, decomposition rates, and specific decomposing taxa [22].

Deadwood is an important source of organic matter consisting of simple sugars, organic acids, and complex structural biopolymers such as cellulose, hemicellulose, and lignin. It is a crucial factor in nutrient recycling in forest soil [19]. In fact, through decomposition, the structural polymers of wood cell walls are demolished and transferred to the soil, making the nutrients contained therein available for soil microorganisms and absorption by plant roots [14].

The refractoriness of the lignin–cellulosic complexes is the main driver controlling the decay rate of deadwood. Only a limited number of fungi and prokaryotes possess the capability to decompose the lignin–cellulosic complexes, as they have developed the enzymes necessary to break them down [23]. The action of pedofauna, consisting mainly of invertebrates such as xylophagous insects, is fundamental in exposing a larger surface of the wood to microorganism attack. Through the chewing apparatus, the pedofauna fragment deadwood, favoring the mixing of organic molecules and soil mineral components [24].

Unicellular protozoa can also significantly contribute to mineralization processes and the recycling of the nutrients contained in deadwood [25].

The deadwood necrobiome is composed of organisms belonging to different kingdoms that have evolved to use decaying organic matter as a nutrient resource or as a habitat, interacting with each other through relationships that can be synergistic, antagonistic, and/or neutral [26]. Through these interactions, the necrobiome influences the deadwood decay rates and plays a crucial role in the balance between organic matter decomposition, carbon sequestration, and gas exchanges with the atmosphere (mainly carbon dioxide; CO₂) [27,28].

The necrobiome colonizes plant tissues immediately after or before their death and may have an internal (endonecrotic community) or external (epinecrotic community) origin [22]. Climate and the quality of the woody substrates (density, pH, moisture content, total lignin, and cellulose contents) are the driving forces of deadwood decomposition, as they strongly influence the structure of the necrobiome and the speed of the decomposition process [29,30]. As the woody material decays, its structure and chemical composition gradually change, inducing a succession of microbial communities, as species are progressively replaced by other species that are better adapted to the new habitat [31,32,33,34,35,36]. The incorporation on the soil surface organic layer of woody material at various stages of decomposition can have consequences on the chemical and biological properties of forest soil [37].

Fungi, in particular Basidiomycetes and Ascomycetes, have always been considered pioneering microorganisms in the wood decomposition process [33]. Thanks to their ability to secrete various enzymes that attack the structural biopolymers of wood, fungi can readily colonize deadwood and modulate the availability of nutritional resources for their growth [23,33]. Furthermore, they promptly contribute to opening the way in the recalcitrant substrate for colonization by other microorganisms, including bacteria [40]. Several authors found that Ascomycetes are more abundant than Basidiomycetes, especially in the early phases of deadwood decomposition [28,33,41,42].

Endophytes are generally the first colonizing fungi [42]. Outside taxa preferentially take over in the later stages [43,44]. Fungi use two main colonization strategies: the dispersion of spores in the atmosphere and the invasion of mycelial filaments from the surrounding soil [45]. Following these stochastic events, the ability of certain fungal groups to degrade different substrates leads to temporary changes in the chemical composition of deadwood. As the state of decay progresses, a greater number of ecological niches become available to support the colonization of a highly diversified microbial community. As the wood decomposes, a greater variety of substrates gradually becomes available, requiring a greater metabolic diversity within the active bacterial and fungal communities [36,42].

Fungal taxa exhibit specific preferences for wood at a definite stage of decay [46]. Based on the different degradation strategies they operate, fungi are commonly classified into soft rot fungi, white rot fungi, and brown rot fungi. The soft rot fungi are mostly Ascomycetes and Deuteromycetes and are among the first colonizers [47,48]. They can degrade cellulose and hemicellulose, creating localized gaps that allow access to the necromass for other decomposing microorganisms. The term soft rot was originally proposed by Savory in 1954 [49] to distinguish the decay caused by these fungi, which attack only the cellulose, making the surface of the wood very soft, from the action of the white and brown rot fungi, which, conversely, destroy the wood [50]. White rot fungi and brown rot fungi are mainly taxonomically classified within the subdivision of Basidiomycetes [51,52]. White rot fungi secrete a plethora of extracellular oxidative enzymes capable of degrading all cell wall biopolymers [14,53]. They significantly reduce the lignin content of deadwood thanks to the action of oxidative metalloenzymes, such as laccase and manganese peroxidase [23,54,55]. Some species preferentially remove the lignin, leaving white degraded areas mainly consisting of cellulose, while other species concurrently degrade lignin and cellulose [56]. After a first phase dominated by soft rot and white rot fungi, the decay process is predominantly driven by brown rot fungi, which can attack hemicellulose and cellulose, causing a relative increase in lignin [48,52]. They are not able to mineralize lignin into CO₂ and do not secrete peroxidase, but they apply a singular mechanism that uses hydroxyl radicals, produced by the non-enzymatic Fenton reaction, as an oxidizing agent [23,52]. Therefore, the lignin is modified while maintaining its polymeric structure [52]. Despite having limited ligninolytic activity, brown rot fungi are good competitors and are abundant in the late stages of deadwood decomposition [31,52,57].

Hypoxylon frangiforme and Lopadostoma turgidum were found almost exclusively on deadwood at early stages of decomposition while Trechispora farinacea and Phanerochaete velutina were more frequently isolated from deadwood at late decay stages [58]. A key player in Norway spruce decomposition is Guehomyces pullulans, which is very efficient in conquering the habitat in the early stages of decomposition and, successively, as the decay progresses, providing its biomass to feed other species [42]. The white rot fungus Phlebia radiata proved to be an efficient early colonizer of deadwood, contributing to wood decomposition by the secretion of oxidative and carbohydrate-active enzymes [23]. In Norway spruce deadwood, these enzyme activities were associated with the emission of volatile organic compounds (VOCs), such as methyl-3-furoate [23]. Fungal VOCs are considered important signaling molecules in hyphal interspecific interactions.

Several species, such as Eutypa spinosa and Fomes fomentarius, have been identified as endophytes latently present in the living sapwood of the European beech [59]. They can utilize the available nutritional resources early, before the entry of secondary and late colonizers who may be stronger competitors [60]. Other examples of late colonizers are Mycena haematopus and Pluteus spp. [57]. The species Resinicium bicolor (Hymenochaetales), Fomitopsis pinicola (Polyporales), and Heterobasidion spp. (Russulales) have been found in the deadwood of conifers (Picea abies) in temperate and boreal forests [61,62,63]. Trametes versicolor (Polyporales) and members of Xylariales have frequently been found in European temperate beech and oak forests. Resinicium spp. have been considered functional and structural key members of the necrobiome and are abundant in deadwood of both deciduous and coniferous species [64].

Bacteria are also involved in the deadwood decomposition process [65]. They colonize deadwood in early decay stages growing on easily degradable substrates such as sugars, organic acids, pectin, and easily accessible cellulose. In recent years, it has been shown that even bacteria can degrade wood’s structural biopolymers, including lignin, and catabolize secondary products deriving from lignin’s incomplete degradation by fungi [66,67]. However, their role in cellulose and lignin decomposition is lesser than that of fungi. Tláskal et al. [65] found that more than 91% of the transcripts involved in the degradation of structural wood biopolymers were of fungal origin, while only 7% were assigned to bacteria. By using a gene-centric approach, selecting 60 enzyme-encoding genes putatively involved in lignin depolymerization and the metabolism of lignin-derived aromatic compounds, Díaz-García et al. [67] predicted that some species within the Pseudomonadaceae family could possess broad and relevant ligninolytic activity.

Based mainly on electron microscope ultrastructural observations, three main types of bacterial decay, clearly different from those operated by fungi, have been recognized: erosion, tunneling, and cavitation [56]. The erosion bacteria degrade the cellulose and hemicellulose of the secondary walls, producing deep channels parallel to the cell-wall microfibrils and leaving lignin residues. The tunneling bacteria produce tiny tunnels in the secondary walls and medium lamellae. The cavitation bacteria form small diamond-shaped or irregular cavities in the secondary wall [56]. While tunneling bacteria appear to require the presence of oxygen for their activity, erosion bacteria can tolerate conditions of extremely low oxygen levels [70].

Most deadwood saproxylic bacteria belong to the Proteobacteria, Actinobacteria, and Acidobacteria phyla [71]. They participate in the whole deadwood decay, being able to use a wide variety of more or less labile substrates and interacting with fungi through complex synergistic and competitive relationships [72,73,74]. Bradyrhizobium and Caulobacter were found to be the most frequent proteobacterial species in deciduous temperate mixed forest ecosystems [28]. The presence of Bradyrhizobium could potentially contribute to N-enrichment, thanks to its nitrogen (N₂)-fixing activity [28].

Deadwood is a favorable environment for the growth of actinobacteria. Many actinobacterial species can secrete cellulases and hydrolytic enzymes that degrade cellulose and hemicellulose [72]. Furthermore, they are presumably also involved in lignin degradation [75], although their role is not yet clear. Lynd et al. [76] retain the actinobacteria as early colonizers of deadwood; thanks to their cellulolytic action, they contribute to increasing the permeability of water and the humidity of the wood, thus favoring fungal colonization [77]. On the other hand, in the more advanced stages of decomposition, a greater metabolic specialization is hypothesized for actinobacteria, thus confirming their limited ability to degrade lignin [78]. Pastorelli et al. [36] found a greater number of actinobacterial taxa in the early stages of deadwood decay than in the more advanced phases. This finding suggests a greater involvement of this bacterial group in the degradation of more labile structural compounds, such as hemicellulose and cellulose, compared to more recalcitrant compounds, such as lignin.

Although the cultivation of the acidobacterial group has proved to be challenging, some members belonging to subdivision 1 have been isolated from deadwood colonized by the white rot fungus Hypholoma fasciculare [73]. Subdivision 1 hosts acidobacteria preferring a moderately acidic pH range [79] which are, therefore, presumably well adapted to the deadwood environment. By using an Illumina MiSeq platform, Lee et al. [28] identified Terriglobus as the most cosmopolitan species in the acidobacterial deadwood community. Acidobacteria have been described as abundant in the forest soil methylotrophic community [80]. However, their physiology and role in deadwood decomposition are still poorly known.

A significant portion of the deadwood prokaryotic necrobiome consists of bacteria capable of using reduced carbon substrates without CC bonding, such as methanol, a by-product of the lignin decomposition operated by fungi [65]. Vorob’ev et al. [81] identified the presence of methylotrophic microorganisms in association with the H. fascicular fungus, in decaying beech wood. The obligate methanotroph Methyloferula sp. was found in association with Amelanchier arborea deadwood [28]. The group of methylotrophic bacteria also includes members with the ability to oxidize methane (CH₄), the so-called methanotrophic bacteria. In a mesocosm experiment performed on Pinus nigra deadwood fragments, methanotrophic bacteria increased with increasing decay class and CH₄ consumption, suggesting relatively greater involvement of this microbial group as decomposition progresses [36]. Mäkipää et al. [82] found methanotrophs as the main group within the N₂-fixing prokaryotic community in spruce deadwood. They assumed the presence of synergistic interactions between methanotrophs and fungi, with the former providing ammonium (NH₄⁺) to the fungi in return for the methanol produced by the latter [83].

Nitrogen-fixing bacteria also significantly increase as decomposition progresses [36,84]. Using a meta-transcriptomic approach to analyze the microbiome associated with European beech deadwood decay, Tláskal et al. [65] found that N₂-fixation is one of the dominant processes of the N cycle occurring in deadwood, second only to the incorporation of NH₄⁺ into organic molecules. On the contrary, the respiratory pathway (denitrification) that reduces nitrates (NO₃⁻) and nitrites (NO₂⁻) seems to be considerably less important in deadwood than in soil, and the transcripts related to the nitrification process were absent. Denitrifying bacteria are abundant and widespread in forest soils [27]. Denitrification is a stepwise process that involves several enzymes (reductases) and results in the conversion of dissolved NO₃⁻ and NO₂⁻ to molecular nitrogen (N₂), passing through the production of nitrous oxide (N₂O) [85]. Not all denitrifying bacteria harbor the complete battery of genes encoding for all the reductases of the denitrification process. Some denitrifying species have a truncated metabolic pathway [85]. Pastorelli et al. [36] quantified two key denitrification genes, the nirK, and nosZ genes, and N₂O potential emission from black pine deadwood. The obtained results suggested that the deadwood necrobiome may host a diversity of species that could drive denitrification towards a complete reduction in nitrate up to the release of N₂, thus lowering N₂O emissions.

Little is known about the metabolic activities of the prokaryotes belonging to the Archaea domain and their involvement in deadwood degradation. Enzymes such as cellulase and xylanase have been discovered in extremophilic archaea [87], but it is not known whether these enzymes may also be present in temperate archaea [88].

Numerous members of this domain can produce CH₄ as a metabolic by-product. The CH₄ emission is the result of the activity of a consortium of microorganisms, where simple C-compounds are produced by the degradative and/or fermentative activity of other microorganisms and used as terminal electron acceptors by methanogenic archaea [89]. The colonization of living tree tissue by methanogenic archaea was documented as early as the 1970s [90]. The early stages of decomposition showed the highest methanogenic activity [91]. This finding suggested that methanogenesis is fueled by non-structural labile C substrates, most abundant in less-decayed wood [91]. Fungi break down cell structural biopolymers, generating by-products that other wood microorganisms may use to produce CO₂ and H₂, primary substrates for the methanogenesis process [92]. However, non-methanogenic archaea have also been found in decaying wood, indicating archaea as integral and dynamic members of the plant necrobiome [88].

To date, Thaumarchaeota have been found as prominent members of the archaea community of forest necromass, highlighting the versatility and cosmopolitan nature of this phylum in the natural environment [88]. However, it is easier to assume that CH₄ evolution occurs mainly as a result of symbiotic interactions between methanogenic archaea and xylophagous insects, protozoa, or fungi inhabiting the deadwood [92,93,94]. In a mesocosm experiment conducted on P. nigra deadwood, Pastorelli et al. [36] found the presence of Methanobrevibacter strongly correlated with the high production of CH₄. Methanobrevibacter is a strictly anaerobic species that generally live as a symbiote of protozoa or are attached to the intestinal epithelium of both lower and upper termites. Furthermore, a great abundance of Methanobrevibacter has also been found within the cells of Spirotrichonympha leidyi, a flagellate of the parabasalid group that lives in the termite intestine [94].

Ciliophora is one of the most abundant phyla of the protozoan community in soil, but ciliates can also inhabit mosses, lichens, litter, and deadwood [95,96]. Ciliates are important members of the ecosystem trophic network since they prey on microorganisms and are preyed upon by other protozoa and metazoans, thus playing a very central role in nutrient recycling [95]. Understanding ciliate functions and diversity is essential to extending the knowledge about the nutrient cycle in forest ecosystems.

Different ciliate species have specific food preferences and tolerate specific microclimates and abiotic conditions [96]. Ciliates are highly adaptable to environmental changes and, thanks to their ability to develop inactive forms (cysts), they can survive adverse conditions [97], such as wet–dry alternations. Pastorelli et al. [98] showed that, like the other members of the necrobiome, the composition of the ciliate community varies as deadwood decays, becoming more and more homogeneous. The early stages of deadwood decomposition are characterized by great variability in deadwood quality, probably due to stochastic events that led to colonization by bacterial and fungal taxa with different degrading capacities [45] and ciliate taxa with different food preferences. As the decomposition progresses, the bacterial, fungal, and ciliate taxa are mainly selected according to deterministic mechanisms [99] controlled by the metabolic processes involved in the degradation of complex wood residues and by the palatability of the degrading microorganisms [19].

Ciliates are ubiquitous and can be easily dispersed in the air [97]. They are susceptible to a wide range of environmental factors, such as humidity, temperature, pH, and food abundance, which can induce changes in their community composition [100,101]. Since ciliates need water to be active, the daily fluctuations of temperature and humidity can induce cycles of encystment and excystment so that, due to the different tolerance to abiotic conditions and food preferences, at any moment the habitat is occupied by active and inactive individuals [96].

The ciliate trophic groups in deadwood are very similar to those in soil with the prevalence of bacterivores and predators in the most decomposed deadwood [102]. Through the predation and secretion of metabolites, ciliates regulate the size and composition of bacterial communities and influence C and N cycling [19]. The nutrients temporarily immobilized in the bacterial biomass are released by the ciliate predatory action. Overall, deadwood ciliates affect the rates of nutrients released into the soil and atmosphere and significantly contribute to improving plant growth [100]. Part of the ingested C is used for new ciliated biomass production while the rest is returned to the atmosphere as CO₂. Organic molecules not used by any species of fungi, bacteria, or ciliates become a non-recycled end-product and accumulate in the soil, contributing to humus formation [96]. Nitrogen excesses are excreted as NH₄⁺, readily available to other organisms, improving the total N content of deadwood and soil fertility [100]. Consistently, the abundance of ciliates in deadwood was found to be positively correlated with CO₂ production, N content, and bacterial abundance [98].

The ciliate species identified in deadwood generally belong to soil-inhabiting genera within the Colpodea and Spirotrichea classes [98]. Colpodea are abundant in soils, especially in polluted soil [103,104], and are typically bacterivorous [95]. Interestingly, Jia et al. [19] showed a vesicular Colpodea strongly related to catalase and polyphenol oxidase, both enzymes involved in the degradation of refractory C sources, such as lignin. This finding suggested a potential role of Colpodea in deadwood degradation. Spirotrichea are common in soil, freshwater, and marine environments. Bartošová and Tirjaková [102] identified Colpodea, Spirotrichea, and Lithostomes as dominant systematic groups in decaying bark and wood.