Soil Fungi Lecture Notes PDF

Chapter 4 Soil fungi Introduction There is now considerable circumstantial evidence that true fungi (Kingdom Eumycota) were instrumental in both the colonization of land by the ancestors of terrestrial plants and the termination of carbon deposition into geolog...

Chapter 4 Soil fungi Introduction There is now considerable circumstantial evidence that true fungi (Kingdom Eumycota) were instrumental in both the colonization of land by the ancestors of terrestrial plants and the termination of carbon deposition into geological reserves (i.e., fossil fuels, Floudas et al. 2012: see Textbox). The traits that underlie these major evolutionary and ecological transitions illustrate why fungi play such important roles in soils. Most fungi interact intimately with both living and dead organisms, especially plants. The mycorrhizal symbiosis with plant roots is thought to have permitted aquatic plants to transition into the challenging terrestrial habitat. Mycorrhizal and other interactions with living plants (pathogens, endophytes) may be highly specific or generalized with outcomes that vary among taxa, but influence the structure and function of plant communities. Fungi have profound influences on biogeochemical cycles through their growth habits, which include external digestion of food resources using a powerful arsenal of degradative enzymes and secondary metabolism. The filamentous habit common to the majority of soil-dwelling fungi allows them to bridge gaps between pockets of soil water and nutrients; force their way into substrates such as decaying wood; and redistribute C, minerals, and water through the soil. Filamentous growth may underlie the abilities of some fungi to withstand soil water deficits and cold temperatures that are beyond the tolerance of bacteria and archaea. Fungi constitute large fractions of living and dead soil biomass, particularly in forested habitats. Their growth and production of cell wall materials lead to the creation and stabilization of soil aggregates, which are key elements of soil structure. Rates of turnover of fungal biomass have important consequences for C cycling and long-term sequestration in soil. Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period. (Floudas et al. 2012. The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed From 31 Fungal Genomes. Science 336(6089): 1715-9. doi: 10.1126/science.1221748.) 31 1. Taxonomic situation and general characteristics The Fungi (capitalized italic when used as a formal Linnean taxon) are recognized as a Kingdom. Although sometimes loosely referred to as microbes, fungi are eukaryotes and most are multicellular. A superkingdom of eukaryotes that includes fungi and animals is the Opisthokonta. The evolutionary lines that constitute the true fungi, or Eumycota, are all descended from a single common ancestor (i.e., constitute a monophyletic clade) within the Opisthokonta. Various molecular-clock estimates place the origin of the Eumycota from 600 million to>1 billion years before the present, but a robust estimate will require more reliable fossil calibration points. Fungi are characterized by a number of shared, derived traits. Fungi depend on organic compounds for C, energy, and electrons, taking up these resources via osmotrophy. Although many fungi can fix CO2 using enzymes of central anabolic cycles (e.g., pyruvate carboxylase) they are considered heterotrophs in a broad sense. Chitin, a polymer of N-acetylglucosamine, is a feature of the cell wall matrix of most fungi, although a few parasitic lineages have life stages that lack cell walls (e.g., Rozella), and some groups have little or no chitin in their walls (e.g. ascomycete yeasts). Most fungi also use the sugar trehalose as an energy store, display apical growth, and have spindle-pole bodies rather than centrioles (with the exception of ancient, flagellated lineages). In the more ancient fungal groups, the filaments in which the nuclei are housed do not contain cross-walls (septa), whereas in other groups, septa divide hyphal filaments into distinct cells. Although not representing any particular taxon, the features shown in Fig. 4.1 are characteristic of the Dikarya. Cross walls or septa separate individual cells (numbers of nuclei are usually variable in Ascomycota, but are more often fixed in Basidiomycota). As a mycelium grows, hyphae branch at regulated intervals in response to external and internal signals. In many fungi, cytoplasm is retracted from older parts of the mycelium, leaving walled-off empty cells. The newly formed, thin and soft hyphal tip extends due to turgor pressure. The growing tip is the area of most active enzyme secretion and nutrient uptake. In the two most recently evolved phyla, the Ascomycota and Basidiomycota, nuclear division and apportionment to the cells comprising the hyphae is tightly regulated, whereas in groups without septa, nuclei flow freely through the entire mycelium (e.g., in phylum Glomeromycota). Although the evolutionary cohesion of the Eumycota is widely accepted, some uncertainties and surprises with respect to membership have attracted attention in the last decade. Molecular phylogenies confirm the long-held view that the Oomycota, which includes the filamentous plant pathogens Pythium and Phytophthora, belong to the superkingdom Stramenopiles (heterokonts), while the slime molds belong to the superkingdom Amoebozoa, both outside the Opisthokonta. 32  Fig. 3.1 Filamentous fungal growth form. Diagram depicts a mycelium growing from left to right. 2. Phylogeny Molecular systematics of the Eumycota are in the midst of radical revision. About 20 years ago, a system of five fungal phyla achieved universal recognition based, in part, on initial rDNA phylogenies. The five phyla were the Chytridiomycota (water molds), Zygomycota (bread molds), Glomeromycota (arbuscular mycorrhizal fungi), Ascomycota (cup fungi), and Basidiomycota (club fungi). But later multilocus, molecular analyses suggest that neither the Zygomycota nor the Chytridiomycota are monophyletic groups. Several new phyla, subphyla, and unranked higher taxa have been proposed (Fig. 4.2). Even extremely data-rich phylogenomic analyses have yet to resolve with certainty relationships at the base of the fungal tree, so we can expect further rearrangements and optimization of high-order taxonomy in the years to come. It is also for these reasons that we will not delve deeper into these taxonomic issues. Current understandings of the major evolutionary lines of fungi, along with a few exemplar taxa and their trophic niches, are presented in Figs. 3.2 - 3.4. The phylum Glomeromycota encompasses all fungi that form arbuscular mycorrhizae. There is some evidence that the Glomeromycota is basal to the Dikarya, but this is not yet certain. The subkingdom Dikarya is comprised of the most recent and derived “crown” phyla Ascomycota and Basidiomycota. The Ascomycota constitute the most species-rich fungal phylum, accounting for roughly 75% of described fungal species, and falling into three subphyla. The diverse phylum Basidiomycota is also divided into three well-supported subphyla (Fig. ). 33  Fig. 3.2 Overview of fungal phylogeny. Currently recognized phyla and subphyla in the Kingdom Fungi. Exemplar taxa and their ecological roles are provided on the right. Fig. 3.2 Currently recognized subphyla and classes within the phylum Basidiomycota. Several subphyla and classes that contain only a few, rarely encountered species are not shown. Exemplar taxa and their ecological roles are provided on the right. 34 Fig. 3.4. Currently recognized subphyla and classes within the phylum Ascomycota. Several subphyla and classes that contain only a few, rarely encountered species are not shown. Exemplar taxa and their ecological roles are provided on the right. 3. Occurrence Fungi are present and prominent in all soils. At broad phylogenetic scales, beyond soils, they are also found in nearly every other habitat on Earth, including deep sea hydrothermal vents and sediments, subglacial sediments, ancient permafrost, sea ice, hot and cold deserts, salterns, and soils of the Dry Valleys of Antarctica. Many clearly are extremophiles, and although no fungi match the extreme thermotolerance of Archaea and Bacteria from hot springs and hydrothermal vents, thermophilic fungi are among the most heat-tolerant eukaryotes. Furthermore, certain species equal or surpass any prokaryotes in cold tolerance and also occupy extreme habitats with respect to salt, desiccation, hydrostatic pressure, and pH. However, the most luxuriant fungal growth can be seen in moist, aerobic soils with large amounts of complex organic C. Fungi generally dominate microbial biomass and activity (i.e., respiration) in soil organic horizons, particularly in forests. Bacterial-to-fungal ratios tend to be lower in acidic, low- nutrient soils with recalcitrant litter and high C-to-N ratios, whereas bacteria are increasingly prominent in high N+P, saline, alkaline, and anaerobic (waterlogged) soils. 35 Fungal biomass varies widely within and across biomes in relation to litter composition, root density, and nutrient availability. Fungi may comprise up to 20% of the mass of decomposing plant litter. In biomes dominated by EMF plants, extraradical mycelium may comprise 30% of the microbial biomass and 80% of the fungal biomass. Although fungal abundance and ratios of fungal to bacterial biomass tend to increase as soil pH decreases, other studies suggest that fungal distributions are more influenced by N and P availability than pH per se. Estimates of fungal biomass turnover in soils are on the order of months (i.e., 130-150 days). Fungal mycelia generally grow radially as fractal networks in soil, wood, and litter. Fungi alter hyphal development in response to environmental conditions to minimize cost-to-benefit ratios in terms of C or nutrient capture versus expenditure on growth. Very fine “feeder hyphae” are elaborated in resource-rich patches, whereas nutrient-poor areas are less densely colonized by hyphae specialized for efficient searching and nutrient transport. The transport hyphae may aggregate into tightly woven bundles called cords, strands, or rhizomorphs depending on their developmental structure. All these aggregations provide larger diameter transport tubes. However, species vary considerably in hyphal growth patterns, the size and structure of transport networks, and the resulting foraging strategies. 4. Diversity The true diversity of the Eumycota is uncertain and controversial. There are roughly 100,000 described taxa, which is thought to include many synonyms due to both duplicate descriptions and anamorph-teleomorph pairs (i.e., the tradition in mycology of giving separate names to asexual and sexual forms). New species are being described at a rapid rate, and estimates of true fungal diversity come to 5-6 million species! In mainstream ecology, the term community refers to the set of sympatric, metabolically active organisms that interact or can potentially interact. We know little about when, where, and how fungi interact with other organisms in soil, aside from conspicuous manifestations, such as mycorrhizal colonization of plant roots or nematode-trapping fungi. Fungi in soil vary at least four orders of magnitude in size. Single-celled yeasts may be 3-10 μm in diameter. In contrast, a single mycelial individual of the white rot, root pathogen Armillaria mellea, spanned 15 ha, with a predicted mass greater than 10,000 kg. In some areas of mycology, more careful attention has been paid to the spatial definition of community. In particular, researchers studying wood, litter, and dung decay have recognized that fungal species must colonize, grow, and reproduce within the confines of a particular substrate, leading to the designation of “unit communities”. For example, the fungi that occupy a single, isolated leaf might constitute a unit community. The application of the unit community perspective to soil is difficult due to the complex distribution of resources and lack of distinct spatial boundaries. Fungal communities in soil can be extremely species rich and patchy at small spatial scales. For example, high throughput clone sequencing of fungi in cores collected approximately one m apart in a boreal forest site revealed in excess of 300 fungal taxa in 0.25 g soil. Moreover, the dominant taxa in the first core were quite distinct from the dominant taxa in the second core. 36 5. Role in carbon and nutrient dynamics in soil Fungi are crucial players in nearly all processes occurring in soil, including soil weathering and formation, host-pathogen interactions (where fungi can be both pathogen or host, i.e. cause and suppress diseases), shaping the composition of plant communities, aggregation and formation of soil structure, etc. Fungi mediate nearly every aspect of organic matter production, decomposition, and sequestration, with concomitant roles in the mineralization and cycling of N and P, with many links to the other processes mentioned. Filamentous fungi support plant production through mycorrhizal associations that enhance the acquisition of water and nutrients. As agents of organic matter decomposition, fungi mediate the creation and protection of soil organic matter (SOM), as well as its mineralization. Filamentous fungi promote macroaggregate formation by binding soil particles with hypha and producing cell wall materials that act as adhesives. Aggregate formation promotes soil C sequestration by providing physical protection from decomposers and their degradative enzymes. The most commonly measured characteristics of soil fungi are their biomass, elemental stoichiometry, growth rates and efficiencies, and activities of their extracellular enzymes. These parameters define the size of fungal C and nutrient pools and rates of fungal-mediated biogeochemical reactions. Fungi must generate small molecular mass growth substrates by enzymatically degrading complex organic matter outside the cell. However, enzymes released to the environment by intent or as a result of cell lysis are beyond the control of that organism (Fig. 4.1). As a result, a substantial fraction of the enzymatic potential of soils is a legacy of enzymes that are spatially and temporally displaced from their origins. 5.1. Enzymatic activity (see also Chapter 5. Soil enzymes) Fungi are considered the principal degraders of plant cell wall material, especially during the early stages of decomposition when their filamentous growth form and their capacity to secrete a variety of glycosidases and oxidases allows them to ramify through the cellular structure of plant litter. The capacity to at least partially degrade cellulose, especially after it has been decrystallized, is widespread in fungi, including basal lineages. Ascomycota and Basidiomycota have the widest genetic and ecological capacity, which is facilitated by the synergistic expression of a variety of other polysaccharide-degrading enzymes. Production of laccases and other phenol oxidases are also widely distributed among Ascomycota, Basidiomycota, and Glomeromycota. In some organisms, mainly saprotrophs, these enzymes function primarily in the degradation of lignin and other secondary compounds in plant cell walls, often indirectly through the production of small reactive oxidants known as redox mediators. Other saprotrophs oxidatively degrade SOM to obtain chemically protected C, N, and P. Mycorrhizal fungi appear to use the same strategy to mine N and P. In many taxa, a large but indeterminate portion of oxidative activity is related to morphogenesis (e.g., melanin production), detoxification, and oxidative stress, rather than nutrient acquisition. But once released into the environment through biomass turnover, these activities also contribute to the oxidative potential of soils, which catalyzes nonspecific condensation and degradation reactions that contribute to both the creation and loss of SOM. Peroxidases, which have 37 greater oxidative potential than laccases, are produced by some members of the Ascomycota and Basidiomycota. Some Basidiomycota, principally wood-rotting fungi, produce lignin peroxidase, which directly oxidizes aromatic rings. ECM fungi were once thought to have little saprotrophic capability compared to wood and litter decay fungi. Some recent genomic data support this perspective. But there is growing evidence that at least some ECM fungi can attack a wide range of organic polymers, but these activities are likely under different environmental controls than those to which pure decomposer fungi respond. The key issue is that decomposer fungi are usually C-limited, while ECM fungi produce degradative enzymes to access and mobilize N and P. Thus the cost- benefit ratios that drive the adaptive evolution of enzyme activities differ for ECM versus decomposer fungi. 5.2. N cycling The potential to use chitin as an N source is widespread among fungi, largely because fungal cell walls include chitin and related compounds. In some studies, chitinase activity is used as an indicator of fungal biomass and metabolism. However, proteins and their degradation products are the largest source of organic N in soils, and it is likely that most saprotrophic and biotrophic fungi obtain much of their N from degradation of peptides. The role of fungi in the N cycle was once considered primarily assimilatory. That is, fungi assimilated inorganic N and N-containing organic molecules to support the production of new fungal biomass and supply plant hosts. Recent studies have shown that dissimilatory denitrification and codenitrification pathways are widespread among Ascomycota and are responsible for a large fraction of nitrous oxide efflux, especially in arid soils (Spott et al., 2011; Shoun et al., 2012). Anthropogenic N deposition has been shown to decrease fungal diversity, biomass, and respiration. Nitrogen deposition also alters the economics of plant-fungal symbioses, which may also contribute to observed decreases in fungal biomass and activity. Another contributing factor may be the two- to three-fold difference in biomass C-to-N ratio of fungi (10- 20) and bacteria (5-10), which might promote bacterial growth relative to fungi when N availability is high. 5.3. P cycling Many soil microorganisms produce extracellular phosphatases. Consequently, phosphatase activities in soil are generally greater than any other measured enzymatic activity, making it difficult to resolve the contributions of individual taxa to P mineralization. For fungi at least, it appears that most phosphatases released for extracellular P acquisition have acidic pH optima, whereas those intended for intracellular reactions have optima at neutral to alkaline pH. Like all extracellular phosphatases, enzyme expression is induced by P deficiency. Mineral phosphate is also important to fungi and plants. In alkaline soil, calcium phosphates may be abundant, whereas weathered acid soils may have high concentrations of iron and 38 aluminum phosphates. Some fungi, particularly EMF Basidiomycota, solubilize phosphate from mineral sources using low molecular weight organic acids, such as oxalate. 5.4. Bioremediation The filamentous growth habit and enzymatic versatility of fungi can also be adapted to treat waste streams and remediate soils contaminated with organic pollutants or toxic metals. The most effective pollutant remediators belong to the phyla Ascomycota and Basidiomycota. Most of this capacity is related to the production of a broad spectrum of extracellular laccases and peroxidases with varying redox potentials, pH optima, and substrate specificity that oxidatively modify or degrade aliphatic and aromatic pollutants, including halogenated compounds. In addition, some fungi also produce nitroreductases and reductive dehalogenases that further contribute to the degradation of explosive residues and halogenated contaminants. Intracellularly, many fungi also have cytochrome P450 oxidoreductases that can mitigate the toxicity of a broad range of compounds. The toxicity of metal contaminants can be mitigated by translocation and sequestration in chemically inaccessible complexes. In natural systems, improving the bioremediation capabilities of ECM fungi is of particular interest because the C supply from host plants may support fungal growth into contaminated hotspots and stimulate cometabolic reactions. 39