How does the primary mycelium in basidiomycota differ from that of the secondary mycelium?

3.1 Substrate mycelia

Substrate mycelium is known as primary mycelium or vegetative mycelium and grows in both solid-grown and submerged cultures. The substrate mycelium inevitably develops from the germinating spores but never produces spores itself. Many differentiate to form aerial hyphae on solid surfaces. The substrate mycelium of actinobacteria differs in size, shape, color, and thickness as you can see in Fig. 21.2 (Li et al., 2016). The substrate mycelium, which forms branches, is often monopodial, but in some rare cases exhibit dichotomous branching, such as in Thermoactinomyces. The main role of substrate mycelia is to absorb nutrients from the media for the growth of actinobacteria. The release of soluble pigments from some of the mycelia is responsible for the color of the substrate mycelia which can provide an important instance in the determination of new species. The pigment produced may be water-soluble (make the medium with the corresponding color) or fat-soluble (make the colony with the corresponding color). Its color may vary from white or colorless to brown, black, red, pink, yellow, orange, green, and purple (Conn and Conn, 1941). The substrate mycelia appear to be transparent, slender, phase-dark, and more branched than aerial hyphae under the microscope (Li et al., 2016).

Aerial Mycelium

The aerial mycelium bearing the characteristic pigment may be abundantly present in some species and scant in others. The aerial mycelium is more closely packed than the substrate mycelium. In those species that do not form conidia, the aerial mycelium may be altogether absent; Streptomyces verne usually does not possess aerial mycelium. Many species form spirals at the ends of aerial mycelium; these include S. coelicolor, S. albus, S. longisporus and S. diastaticus. Others, such as S. globisporus, S. anulatus, S. vinaceus and S. cinnamonensis, are not known to produce spirals. Aerial mycelium may be profuse in some species, for example S. albus. It may be produced in concentric zones as in S. anulatus, in tufts as in S. viridoflavus, or in whorls as in S. reticuli, S. verticillatus and S. netropsis. Morphogenetic mutants of S. coelicolor such as bld lack aerial mycelium. The colour of the aerial mycelium may vary from species to species:

grey – S. clavulogenes, S. violaceoruber

white – S. somaliensis

red – S. erythrogriseus

yellow – S. fradiae

blue – S. ipomoeae

violet – S. mauvecolor.

The genus can be divided into five main groups based on the structure of sporulating hyphae:

straight, sporulating hyphae without spirals

straight, spore-bearing hyphae arranged in clusters

spiral formation in aerial mycelium with long, open spirals

spiral formation in aerial mycelium with short, compact spirals

spore-bearing hyphae arranged on mycelium in whorls or tufts.

Family Dipodascaceae

Mycelium well-developed, lacking a polysaccharide sheath, septa with clusters of minute pores, fragmenting to form thallic conidia. Asci form by fusion of gametangia from adjacent cells or separate mycelia, usually elongated, more or less persistent, single- to multispored. Ascospores usually ellipsoid, rarely ornamented, usually with a mucous sheath, not blueing in iodine.

Galactomyces (anamorph Geotrichum) – supporting hyphae of gametangia profusely septate. Gametangia on opposite sides of hyphal septa, globose to clavate, fusing at the apices to form the ascus. Asci subhyaline, subglobose to broadly ellipsoid, with one or two ascospores. Ascospores broadly ellipsoid, pale yellow-brown, with an echinate inner wall and an irregular exosporium wall, often with a hyaline equatorial furrow. Geotrichum conidia formed in white, smooth, often butyrous colonies from aerial, erect or decumbent hyaline mycelium functioning conidiogenously. Mycelium dichotomously branched at advancing edge. Conidiogenesis thallic. Conidia hyaline, aseptate, smooth, cylindrical, doliiform, or ellipsoid. Distributed worldwide, from soil, water, air, cereals, grapes, citrus, bananas, tomatoes, cucumber, frozen fruit cakes, milk and milk products; also used with bacteria in fermentation of manioc to produce gari in West Africa.

Pure Mycelium Materials

Pure mycelium is obtained by growing a fungus on a solid or liquid substrate under static growth conditions (Lugones et al., 2004; Haneef et al., 2017; Appels et al., 2018) or as a liquid shaken culture (Appels, 2020). When grown in liquid cultures, the total mycelium can be harvested and used to make mycelium material. In contrast, only the fungal skin can be used when grown on a solid substrate. This skin covers the substrate and contacts the air and is composed of densely packed hyphae. While only part of the mycelium can be harvested in the case of solid substrates, growth of static liquid cultures is labor intensive and challenging to upscale, while growth as liquid shaken cultures such as in bioreactors needs high financial investments. The way industries will grow the fungi in the future to produce pure mycelium materials will depend on both cost of production and material qualities.

The properties of pure mycelium materials not only depend on culture conditions, but are also the result of the type of substrate, the fungus, other environmental growth conditions and post-processing (Haneef et al., 2017; Appels et al., 2018; Appels, 2020). The latter can consist of physical, chemical, and/or biological treatments. Heat pressing and adding plasticizing agents are examples of physical and chemical treatments, respectively, while selective microbial degradation of components of a mycelium material is an example of a biological treatment.

A few reported examples show the impact of the substrate and the type of fungus on material properties of pure mycelium. The mycelium of the basidiomycetes Pleurotus ostreatus and Ganoderma lucidum is more elastic when these fungi are grown on a cellulose-based substrate supplemented with dextrose when compared to growth on cellulose alone. In both cases, the latter fungus forms a stiffer mycelium material when compared to the former fungus (Haneef et al., 2017). Inactivation of a single gene can also impact material properties illustrated by the finding that the mycelium of a strain in which the hydrophobin gene sc3 is inactivated (Δsc3 strain) is stronger when compared to the wild-type strain of Schizophyllum commune (Appels et al., 2018) (see below). SC3 is an amphipathic protein and one of the most abundant proteins in cell walls of aerial hyphae of S. commune, while it also occurs at lower levels in cell walls of hyphae that grow within the substrate. SC3 attaches hyphae to hydrophobic surfaces (Wösten et al., 1994a), mediates escape of hyphae from the aqueous environment into the air (Wösten et al., 1999), and makes aerial structures hydrophobic (Wösten et al., 1993, 1994b). The latter is illustrated by the 115° water contact angle of a wild-type mycelium with aerial hyphae (which is similar to the hydrophobic surface of Teflon), while water immediately soaks into the mycelium of the Δsc3 strain (Wösten et al., 1999).

Pure mycelium materials of S. commune have been studied best. Various examples illustrate the impact of growth conditions on the properties of pure mycelium of this basidiomycete. Dried mycelium films produced with static and liquid shaken cultures show mechanical material properties (i.e., Young׳s modulus, ultimate tensile strength and elongation at breaking; see Table 1 for explanation) similar to materials that are classified in the group of natural materials (Appels et al., 2018; Appels, 2020) (Fig. 1). The macrostructure of pure mycelium material produced with static and liquid cultures is different. The mycelium of static liquid cultures consists partly of submerged and partly of aerial mycelium. The submerged mycelium dries up very dense having almost no air voids with an appearance resembling that of paper. On the other hand, the aerial mycelium is less dense and has a cotton like appearance. Mycelium obtained from liquid shaking cultures has a more homogeneous appearance after drying, being most similar to the submerged mycelium from liquid static cultures. Still, some air voids are observed after drying mycelium from liquid shaking cultures. These air voids can be removed by vacuum drying, which is expected to increase strength of the mycelium material.

Stronger mycelium films of wild-type and Δsc3 strains of S. commune are obtained when dark-grown static liquid cultures are grown at ambient CO2 (400 ppm) when compared to high CO2 levels (i.e., 70,000 ppm). The opposite is observed when the strains are grown in the light. These results reflect events observed under natural growth conditions. Hyphae that grow within a solid substrate are exposed to darkness and high CO2, while aerial hyphae will be exposed to light and ambient CO2. Mycelium growing under the former condition have a higher maximal strength (6.5 MPa) when compared to mycelium grown under the latter condition (5.1 MPa). Intuitively, these results make sense since hyphae growing in wood will experience a higher resistance from the substrate. Yet, mycelium of S. commune is even more strong when grown in the light at high CO2 (9.5 MPa) or grown in the dark under low CO2 (9.6 MPa). Together, it is not yet clear how to relate the mechanical properties of mycelium with the environment of the fungus.

Deletion of sc3 results in a >2-fold increase in material rigidity compared to wild-type S. commune, irrespective of the environmental growth conditions (Appels et al., 2018). Chemical analysis by ATR-FTIR did not show large differences in macromolecular composition of the material of the wild-type and the Δsc3 strain. On the other hand, scanning electron microscopy revealed that the Δsc3 material is more homogeneous and densely packed compared to the wild-type material. Indeed, density of the material is higher when produced with the deletion strain. This density increase correlates strongly with increased rigidity and ultimate tensile strength of the mycelium-based material. Together, Δsc3 material properties are similar to those of polymers, while properties of the wild-type are similar to those of natural materials (Fig. 1).

Post-processing of pure mycelium material can have a high impact on material properties. Plasticizers and crosslinking agents are commonly used chemicals to alter these properties. Plasticizers increase plasticity of a material by lowering the glass transition temperature (Cowie and Caleria, 2007). The plasticizer acts like a lubricant by improving the capacity of polymers to move. Plasticizers such as glycerol and water do so by filling the space between polymer chains, thereby increasing their distance (Cowie and Caleria, 2007). Indeed, elasticity of S. commune pure mycelium material is increased by treatment with glycerol and also reduces ultimate tensile strength and increases elongation at breaking (Appels, 2020). This shift in material properties from being brittle to ductile is reflected by a shift from being a natural-like material to a polymer-like material such as plastic (concentrations of glycerol up to 8%) or even an elastomer-like material like rubber (concentration of glycerol ≥16%) (Fig. 1). Crosslinkers are the opposite of plasticizers and reduce mobility of a polymer structure. Crosslinking enhances ultimate tensile strength and reduces elasticity. Ionic and hydrophobic interactions are examples of non-covalent crosslinks, while chemical crosslinking involves formation of covalent bonds. For instance, dialdehydes such as glutaraldehyde and glyoxal react with amino or hydroxyl groups in polysaccharides (Crini, 2005). Chemical crosslinks can also be the result of for instance gamma- or photo-irradiation or sulfur vulcanization (Bhattacharya et al., 2009). Properties of biological materials, such as fungal mycelium, can be changed by introducing both non-covalent and covalent crosslinks with proteins and polysaccharides (Yang et al., 2005). For instance, pure mycelium materials of S. commune become stronger and more brittle after cross-linking with glutaraldehyde (our unpublished results).

Physiological Traits

Mycelia do not only access and transport nutrients and water but are, at the same time, chemical factories. They produce an abundance of compounds, mainly enzymes and secondary metabolites, such as extrahyphal enzymes to cleave, e.g., cellulose, hormones, toxins, thermal protectors, and fungivore deterrents.

Many mushrooms have mycelia resistant to microbial and predator attacks (e.g., Collembola). Melanins (dark pigment) are well known for their protective qualities (Bell and Wheeler, 1986) (see Box 1). For instance, Bjorbaekmo et al. (2010) reported that many fungi associated with Dryas, an alpine/arctic shrub, show melanized mycelia. Moreover, some volatile organic compounds (VOC) and other secondary metabolites show repelling qualities (Moore et al., 2014; Spiteller, 2015). Especially melanins are costly to synthesize, which probably leads to a trade-off with, e.g., mycelial growth (Koide et al., 2014; Siletti et al., 2017). Conversely, Crowther et al. (2014) found that strong competitors cannot tolerate desiccation well. On the other hand, some species may not only tolerate a specific stressor such as heat but a combination of stressors (Treseder and Lennon, 2015) by employing heat shock proteins and antifreeze compounds (mainly sugars and some lipids). Apart from house-keeping enzymes and hormones, compounds that protect against biotic and abiotic hazards are crucial for survival and fruiting competence.

Box 1

Functional properties of fungal melanins

If not growing in patches not yet colonized by other fungi, many mycelial hyphae show the ability to fend off hyphae of other fungi (competitive exclusion or growth deadlock: Cairney, 2005). Ectomycorrhizal fungi often outcompete saprotrophic taxa (“Gadgil effect”, Cairney, 2005; Gadgil and Gadgil, 1975), though this effect varies with soil horizons and season (Peršoh et al., 2018). The antagonistic mycelial interaction between different taxa is often controlled by excreting secondary metabolites that suppress the growth of foreign hyphae (Kües et al., 2018) or even kill them (Silar, 2012). Not only chemical but also physical defense mechanisms can be observed, e.g., blocking foreign mycelia by hyphal proliferation at the point of contact (Widden, 1997). Ectomycorrhizal taxa and lichens are renowned for their ability to dissolve mineral matter, among others, to enable the uptake of mineral phosphorus, which is not readily soluble (Gadd, 2017).

The degree of hydrophobicity of, e.g., ectomycorrhizal mycelia, seems connected to substrate water content (Smits et al., 2003) and nutrient distribution (Unestam and Sun, 1995). Hydrophilic mycelia are more water stress-tolerant, hydrophobic mycelial strands transport nutrients from more distant patches. Hydrophobicity may dramatically increase with age (Smits et al., 2003). The ecological implications of this phenomenon are unknown.

Isolation/Purification

The mycelium was harvested after 4-7 days growth on a low salts medium. The pigment was obtained by chloroform-methanol (1:1, v/v) extraction of the mycelium in a Soxhlet apparatus, followed by solvent partition with hexane-90% methanol. The 90% methanol layer contained versicolorin A and was separated by chromatography on silica gel H under pressure (1 kg/cm2). The column was developed with chloroform-methanol (97:3, v/v). Crystallization from chloroform-methanol gave pure versicolorin A.

Isolation/Purification

Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was dissolved and chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2% methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 8-ketocalonectrin were combined and obtained pure by preparative TLC.

The mycelium

The mycelium of Aspergillus spp. is similar to that of most other fungi. It is well developed, branching, hyaline and septate. The mycelium can produce copious amounts of enzymes, and some produce mycotoxins. The mycelial phase of Aspergillus spp. is characterized by vigorous growth and an abundant production of conidia.

The architecture of the Aspergillus hyphal wall may have a considerable influence on the response of the fungus to antifungal drugs.11 Caspofungin is an inhibitor of 1,3-β-d glucan synthesis (GS) that produces dramatic morphologic changes in actively growing hyphae. Despite the apparent fungistatic in vitro activity against Aspergillus species, compounds in this class have strong efficacy in vivo.

Isolation/Purification

The mycelium from the fermentation of T. roseum was suspended in warm acetone for 3 hours. After filtration, the mycelium was resuspended in acetone and allowed to stand overnight at room temperature and then filtered. The combined acetone extracts were concentrated to ca.⅓ volume, to which water was added. Extraction with ethyl acetate followed by successive washing with water, drying, and removal of solvent afforded a brown residue, which was saponified with 10% KOH at room temperature to obtain the nonsaponifiable material. Column chromatographic separation was first afforded using ether-benzene. The first fraction (100% benzene) was further chromatographed using a AgNO3 impregnated silica gel column (hexane elution) to obtain pure trichodiene.

ECM in Ecosytems

Mycelium of both ECM and plant litter saprotrophs mingles in the organic soil horizons of forests, and their interactions with each other and with other soil biota are of major significance in ecosystem nutrient dynamics. Basidiomycete mycelium dominates the microbiota of forest soils, and can amount to several tonnes per hectare. Living mycelium accumulates, stores, and redistributes carbon, nitrogen, phosphorus, and other nutrients. Mycorrhizal fungi not only cycle soil nutrients, but may deposit large amounts of recently fixed carbon in soils, building large pools of carbon in the form of complex molecules that contribute to long-term ecosystem carbon sequestration. Until recently it was assumed that soil carbon came mainly from plant remains accumulated aboveground and gradually incorporated into the upper soil horizons as litter fragments and humus. However, recent evidence suggests that organic soil layers may also grow from below, through continuous additions of carbon compounds from roots and their mycorrhiza. A chronosequence of soils under boreal forest that had developed over periods between centuries and millennia was investigated on a cluster of Scandinavian lake islands. The smaller the island, the older the soil carbon, because smaller islands are less frequently burned from lightning strikes, being statistically less likely to be struck. On these small islands, unburned for over 2000 years, the deeper soil layers harbouring predominantly mycorrhizal fungi and roots had accumulated proportionately larger amounts of persistent organic compounds of root and fungal origin, which was associated with tightly-bound nitrogen, leaving little available nitrogen to support plant growth. In other studies, forest fungi have been found to have a range of enzymes able to liberate nitrogen and phosphorus from such complexes. However, because of the variety of chemical bonds, they cannot be easily targeted by most soil microbes and so decompose only slowly.

Extraradical mycelium facilitates acquisition of nutrients from poorly-soluble minerals. Organic acid secretion and hyphal intrusion into rock enables ectomycorrhizal trees growing on nutrient-poor rocky ground to acquire mineral nutrients via mycelium, from underlying rock and from insoluble mineral particles in the soil, as described in Chapter 5, p.181. This is a key process in the establishment and maintenance of boreal forest, since ultimately these inorganic materials are the only source of mineral nutrients for the entire overground biota. Several common ectomycorrhizal species of Suillus, Lactarius, and Piloderma penetrate at least half a metre down into mineral horizons, with some species present exclusively in these layers. Typically the acid, nutrient-poor soils of these forests are strongly layered podsols, with a dark organic horizon overlying a pale, highly-leached eluvial horizon containing mineral particles such as the phosphorus-containing mineral apatite. Other minerals utilised by mycorrhiza include silicates containing calcium, magnesium, and potassium. Hyphae have been found growing into apatite particles, using both hyphal pressure and exudation of citric and oxalate acids, which act both by protonation and chelation to release soluble phosphate. In this way, ECM roots have direct access to phosphorus, bypassing competing biota in the soil and avoiding toxic metal ions such as aluminium that are often present in the acid soil solution of podsols. Other essential cations acquired by ECM from rock include iron, captured by exuded iron-chelating siderophore molecules (Chapter 5, p.159) such as ferricrocin, secreted by the common symbionts Coenococcum geophilum and Hebeloma crustuliniforme. Mineral solubilisation is not confined to mineral particles in soil. Rock surfaces can also be dissolved by fungal bioweathering, and the ability of fungal root symbionts to utilise solid rock as a nutrient source presumably underlies the ability of some pine trees to root directly into the bedrock.

The processes by which a diverse ECM community becomes established is of interest because of the interaction between plant community diversity and that of mycorrhizal fungi. A fungal foray through woodland will typically find scores of sporophores of species belonging to mycorrhizal genera such as Amanita, Boletus, Laccaria, Lactarius, Cortinarius, Tricholoma, and more. Excavating tree rootlets will reveal a similar number of ectomycorrhizal morphotypes, while molecular sampling will reveal the presence of even more species. Most of the diversity can be ascribed to a few common taxa, while intensive sampling will reveal an almost inexhaustible tally of rarities. How do these fungi arrive?

In temperate climates Laccaria amethystina, with distinctive purple sporophores and purple-tipped pale ectomycorrhizal roots, are among the first colonisers of new tree seedlings. They occur on a variety of host trees and colonise readily from easily-germinated spores, so behaving as widespread ruderal members of the ECM community. Others, including, for example, the Cortinarius species typical of ancient woodland, are later colonists of roots. They colonise roots more readily by mycelial growth from already-established ECM, and their spores are slow to germinate.

Arrival and colonisation is limited by the dispersal capacity of fungal species. While airborne spores are produced in staggering abundance by many ECM species, their concentration falls away exponentially with distance from the source, and root colonisation is dose-dependent. Analysis of species and infraspecies diversity across landscape and geographical scales shows that ECM communities are dispersal-limited. It is not true of mycorrhizal fungi that ‘everything is everywhere’ and ECM fungi do not show cosmopolitan distributions. Large geographical areas such as neotropical and palaeotropical rainforest have largely endemic ECM populations. Within a species range, airborne spores serve to facilitate gene flow throughout the population.