21st Century Guidebook to Fungi, SECOND EDITION, by David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 11: Exploiting fungi for food
2. Chapter 12: Development and morphogenesis
   1. Development and morphogenesis
   2. The formal terminology of developmental biology
   3. The observational and experimental basis of fungal developmental biology
   4. Ten ways to make a mushroom
   5. Competence and regional patterning
   6. The Coprinopsis fruit body: making hymenia
   7. Coprinopsis and Volvariella making gills (not forgetting how polypores make tubes)
   8. The Coprinopsis fruit body: making stems
   9. Co-ordination of cell inflation throughout the maturing fruit body
   10. Mushroom mechanics
   11. Metabolic regulation in relation to morphogenesis
   12. Developmental commitment
   13. Comparisons with other tissues and other organisms
   14. Genetic approaches to study development: through the classic to genomic systems analysis
   15. Senescence and death
   16. Basic principles of fungal developmental biology
   17. Relevance to commercially cultivated fungi
   18. Chapter 12 References and further reading
   19. Buy a PDF of Chapter 12
3. Chapter 13: Ecosystem mycology: saprotrophs, and mutualisms between plants and fungi

12.1 Development and morphogenesis

In a great many fungi hyphae differentiate from the vegetative form that ordinarily composes a mycelium and aggregate to form tissues of multihyphal structures. These may be linear organs (that emphasise parallel arrangements of hyphae), such as strands, rhizomorphs and fruit body stems (CLICK HERE to view the page in Chapter 9); or globose masses (that emphasise interweaving of hyphae), such as sclerotia, fruit bodies and other sporulating structures of the larger Ascomycota and Basidiomycota (CLICK HERE to view the page in Chapter 9).

Development of any of these fungal multicellular structures requires that hyphal growth takes on a particular ‘pattern’. A ‘pattern’ that, time after time, produces the same species-specific structure and morphology of that structure, a process that demands precise control and regulation. Formation of a multicellular structure begins with a localised association of aerial hyphae into a hyphal tuft (also called a hyphal knot), which gradually enlarges and differentiates into a primordium of the fruit body (or other structure, according to circumstances) from which the fruit body (etc.) finally emerges.

The differential growth represented in this morphogenesis, and which gives rise to the development of the variety of tissues that make up a fungal multicellular structure involves detailed control and regulation of wall synthesis. Most of the descriptions of wall formation we have given so far have concentrated on hyphal tip (apical) growth, but in development of multicellular structures, 'mature' hyphal wall distant from its hyphal apex can restart wall formation to remodel and reshape the cell. In addition, two adjacent hyphal branches can be joined together by synthesis of a joint wall, which can be stronger than the original (see Section 12.7). Also, there are many observations of hyphal walls being thickened internally by synthesis of a secondary wall, mostly made up of thick fibrils, which are probably constructed of glucans accumulated as a nutritional reserve. Fungal wall synthesis, resynthesis and secondary wall formation are topics worthy of separate treatment and we have dealt with them in detail in Chapter 6.

Differentiation events can be limited to particular hyphae within the structure (indeed, to particular cells in individual hyphae), and differential growth of tissues can generate mechanical forces that change the macroscopic shape of the whole structure. In developmental terminology this is pattern formation (creation of a particular spatial arrangement of tissues that will generate the final morphology of the structure or organ) caused by regional patterning (regional specification) of the differentiation pathways followed by the cells within those patterns. That these processes take place can be deduced from relatively simple experiments and observations even though it is not yet known how the hyphae that will differentiate are specified. Pattern formation and regional specification are examples of development-specific terminology, most of which has been coined in conjunction with research on animals, so it is appropriate to consider how far it can be applied to fungi.

We have already used some development-specific terminology, namely pattern formation and regional specification. We have also mentioned some important, indeed crucial, features that uniquely characterise fungal development. This is the dependence of fungal multicellular development on control and adaptation of the normal growth and branching of vegetative hyphae, and in particular the fact that formation of any multicellular structure in fungi requires reversal of the outward, exploratory growth habit that characterises vegetative hyphae, specifically: altered autotropisms. To contribute to the aggregations that become multicellular structures the hyphae concerned must convert the negative autotropism that ensures outward growth of hyphal tips in mycelia into a positive autotropism (see the discussion in Section 4.10) that permits the tips of branches to approach each other and other hyphae and create the hyphal tuft that is the initiation point for the multicellular structure.

In the next section we will illustrate how the formal principles of fungal developmental biology have been established by experiment and observation of the patterns of hyphal growth, branching and interactions that achieve the tissue patterns represented in the diverse morphologies of fungal sporophores and similar multicellular structures; and then theorise about how these patterns are achieved. But first we have a few words about the terminology that is employed.

But first we want to make a point about ‘real world’ mushroom development and differentiation as it occurs on a mushroom farm. Once the compost is completely colonised, the challenge is to turn the fungal biomass into mushrooms. In order to expedite mushroom formation, a layer of buffered peat or coir is applied on top of the compost. This casing layer is at field capacity for moisture and is around pH 8, and additionally is low in nutritional value. The mushroom grower will then allow the casing layer to be colonised by mushroom mycelium, and then the room will be ‘flushed’ to reduce the CO₂ level to below 1000 ppm and reduce the air temperature to around 17°C, together with heavy application of water. In other words, reduced temperature and reduced CO₂ concentration are triggers for mushroom formation in Agaricus.

There is another, perhaps more interesting factor occurring in the casing layer on a mushroom farm. Back in the 1970s, it became clear that Pseudomonas bacteria played a role in stimulating pin initiation (Hayes et al., 1969). Later work by Noble et al. (2009) further refined the role of bacteria, with Pseudomonas metabolising Agaricus VOCs (Volatile Organic Compounds). This change in the chemistry of the casing layer stimulates pin formation. Removal of these self-inhibitors of sporophore initiation by other microorganisms in the compost is a critical trigger for Agaricus sporophore development (Baars et al., 2020). Zhang et al. (2019) transformed Agaricus bisporus with a bacterial Pseudomonas gene encoding 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and found that the transformant mycelium made primordia 5 days sooner than the wild-type strain. It appears that the bacterial ACC deaminase reduces ethylene synthesis, eliminating ethylene inhibition of mycelial growth and promoting primordium formation in A. bisporus.

It is unclear if bacteria fulfil similar roles in other mushroom forming fungi in vivo, but the reverse interkingdom interaction has been reported. Specifically, when Streptomyces is grown with yeast, some of the bacteria change their growth pattern into a non-branching vegetative hypha-like exploratory form that can rapidly explore the substratum. These exploratory Streptomyces can then communicate with nearby bacteria with a volatile signal that stimulates them to begin exploring too (Jones et al., 2017).

Mushrooms of the genus Pleurotus are cultivated on a variety of lignocellulosic substrates. Mushroom shelves, suspended systems and plastic bags with perforations are the main substrate containers used for Pleurotus cultivation (Sánchez, 2010). The pasteurized substrate is spawned and placed into the container, which is maintained under the temperature, moisture and other conditions optimal for mycelium growth and fruiting. Fruiting initiation can be triggered by a variety of environmental and physicochemical stimuli. In bags used for cultivation, mushrooms begin to form around the edges of the bag’s perforations, which indicates that higher levels of oxygen are needed for fruiting. The influence of several factors on fruiting and fruit body development, such as humidity, light, oxygen, temperature, pH, and medium composition (among others) have been studied over the years (Chang & Miles, 2004; Upadhyay & Singh, 2010; Sánchez, 2010; Sakamoto et al., 2018; Bellettini et al., 2019). The cellular age is also crucial for fruiting. It was observed that changes during hyphal maturation appear to prepare the mycelia for the formation of sporophore initials in Pleurotus pulmonarius. In mature hyphae, glucan content and protease activity are increased, whereas protein content is reduced, and laccases and β-1,3-glucanases showed less activity (Sánchez et al., 2004).

In addition, the effect of some substances of natural or synthetic origin has been studied in sporophore formation. Berne et al. (2007) studied the effect of ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of P. ostreatus, which slightly inhibited the mycelial growth but strongly induced primordium formation in agar plates. Ostreolysin also stimulated the subsequent development of primordia into sporophores.

In the natural environment, Volvariella volvacea, the Chinese straw mushroom, is found in tropical and subtropical grasslands. The vegetative mycelium grows optimally at 32 to 35° C but cannot tolerate temperatures below 10° C (Li et al., 1992). V. volvacea has been widely cultivated on a commercial scale in many parts of Asia; indeed, it may have been in cultivation longer than any other mushroom (Singer, 1961) and in many markets this is the preferred fresh culinary mushroom owing to its unique flavour (Mau et al., 1997).

Traditional cultivation mainly uses rice straw (hence its alternative common name of paddy straw mushroom), although several other agricultural wastes (and industrial materials like cotton wastes) make suitable substrates. Traditional preparation of the substrate is limited to tying the straw into bundles before soaking in water for 24 to 48 h. The soaked straw is piled into heaps about 1 m high which are inoculated with spent straw from a previous crop. In less than one month, a synchronised flush of egg-like sporophores appears. These immature sporophores (in which the universal veil is intact and completely encloses the immature mushroom are sold for consumption just like the young sporophores (‘baby buttons’) of Agaricus (though oyster and shiitake mushrooms are sold mature).

Improvements in V. volvacea cultivation have been limited due to its low biological efficiency (that is, low rate of conversion of growth substrate to mushrooms), although improved fruiting was observed on cotton waste supplemented with sodium acetate. It being suggested that treatment of the cultivation substrate with this chemical improved the availability of nutrients for vegetative hyphal growth, particularly the proliferation of hyphal branching (Hou et al., 2017) of the sort that promotes sporulation in saprotrophs (Moore et al. 2008).

Other issues that need to be addressed in Volvariella volvacea cultivation are its sensitivity to low temperatures, which makes it difficult to maintain a good quality in post-harvest storage. Even in cold storage the crop turns brown and autolyses within 2-3 days. Most important of all, though, is the ambiguous sexuality pattern of V. volvacea that has restricted the breeding of improved strains. This latter point is explored further in Section 12.17, below.

Lentinula edodes was traditionally grown on wood logs (Chang & Hayes, 1978; Chang & Miles, 1989). This method has been replaced by artificial log cultivation that uses heat-treated substrates based on sawdust enclosed in plastic bags (‘bag-log cultivation’). The main advantages of this method are the short time to complete a crop cycle and the higher yields (Sánchez, 2004). However, in an attempt to increase mushroom yields in log wood and/or sawdust-based substrates, other fruiting stimuli have been studied.

A pulsed high-voltage stimulation technique has been applied as an electrical stimulation to accelerate fruiting and increase yield in woody substrates used for mushroom cultivation (Ohga et al., 2004; Takaki et al., 2009). Takaki et al. (2014) applied electrical stimulation to a sawdust-based substrate of Lyophyllum decastes and natural logs, in which Lentinula edodes, Pholiota nameko, and Naematoloma sublateritium were grown, and observed sporophore induction and hydrophobin release from the vegetative hyphae (in Lentinula edodes) one day after the stimulation. It was suggested that the pulsed electric field affects the hyphae through an electrostatic force, increasing the hypha activity (Takaki et al., 2014).

Current methods used in commercial production of Ganoderma, include wood log, tree stump, sawdust bag, short blocks of basswood (from the tree genus Tilia, known as Lime or Linden in Europe) and bottle procedures (Hapuarachchi et al., 2018). Mycelial growth, fruiting and development of sporophores require specific nutritional factors and environmental conditions (Zhou, 2017). In particular, Ganoderma is very sensitive to light/dark conditions; light inhibits mycelial growth, but low light levels can promote fruiting (Zhou et al., 2012, Zhou, 2017; Montoya et al., 2018).

Updated October, 2020