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. Degeneration, 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

12.17 Relevance to commercially cultivated fungi 

We have focussed on the developmental biology of Coprinopsis cinerea in this Chapter so far, but there are over 1000 fungal genomes listed as complete in at least draft form in the Mycocosm portal [http://jgi.doe.gov/fungi]. We will end by briefly assessing the relevance of our discussion to some commercially farmed fungi, specifically: the button mushroom (Agaricus), shiitake (Lentinula), oyster mushroom (Pleurotus), and the paddy-straw mushroom (Volvariella) (Table 12.2).
We also include Ganoderma, which is unique in being cultivated (and consumed) for its pharmaceutical value rather than as a food. It is cultivated by being inoculated into short segments of wooden logs which are then covered in soil in an enclosure (often a plastic-covered ‘tunnel’) which can be kept moist and warm. The sporophores then emerge in large number quite close together and the conditions encourage the fungus to form the desirable long stemmed fruit body (Moore & Chiu, 2001).

Recent megaphylogeny studies have determined evolutionary relationships between the clades represented by these genera. Varga et al. (2019) used multigene and genome-based data to assemble a 5,284-species phylogenetic tree with which to infer ages and patterns of speciation, extinction and morphological innovation in Agaricomycetes. They suggested that the subclass Agaricomycetidae diverged from other Agaricomycetes about 185 Million years ago (range 192 to174 Mya) and the Agaricales about 173 Mya (range 182 to 160 Mya). The position of the Polyporales is uncertain in this study but the clade may have diverged from the Agaricomycetes shortly before the Agaricomycetidae. He et al. (2019) compared six gene sequences in 430 species from Agaricomycotina (with six outgroup species from Pucciniomycotina) and estimated the divergence of class Agaricomycetes at 298 Mya, the Polyporales 138 Mya and Agaricales 136 Mya. Families within Agaricales were estimated to have diverged 125 Mya (Agaricaceae and Psathyrellaceae), with the Omphalotaceae (Lentinula) diverging as recently as 71 Mya.

In the following paragraphs we mention a few of the more recent ‘omics’ publications that deal with what we believe to be significant themes in current research on the five most commonly farmed Agaricomycetes. We also note a few research themes worthy of closer attention (Loftus et al., 2020).

Agaricus. In the last century, Loftus et al. (1988) demonstrated that restriction fragment length polymorphisms (RFLPs) had great potential in the development of the genetics and breeding of Agaricus bisporus by providing the basis for genetic finger-printing, differentiating between homokaryons and heterokaryons and following gene segregations in crosses.

Earlier this century an international group of scientists from seven countries completed the sequencing and initial analysis of the Agaricus bisporus genome (Morin et al., 2012) [and see the following URL: http://www.emr.ac.uk/projectposts/agaricus-genome-project/]. They reported a genome of around 30 million bases spread across thirteen chromosomes. An alignment with Coprinopsis cinerea showed considerable synteny. Further analyses of the A. bisporus genome and transcriptome revealed the necessary genes for the degradation of polymers found in plant litter (specifying enzymes able to digest lignin, cellulose, hemicellulose and proteins; see Chapter 11). However, Agaricus grows poorly on plant litter unless it is already partially decomposed, which is why in the European tradition mushroom farming has come to mean cultivation of a mushroom crop on composted plant litter (Junior Letti et al., 2018). Production of compost for mushroom farming has become a self-contained industry, and mushroom farms depend on highly selective substrate, with defined levels of ammonia, protein, pH and moisture. Although widely distributed in nature, A. bisporus can be difficult to find. However, one ecological niche, now well known, is the litter of cypress trees (Arora, 1986). Today’s industry can be viewed as a ‘joint evolution’ of substrate and cultivar, resulting in high cropping densities by an otherwise unremarkable and not very abundant mushroom (Buth, 2017; and see Section 12.6).

Transcriptome (Kerrigan et al., 2013) and proteome (Ham et al., 2020) analyses provide a molecular explanation of Agaricus biology compared to other wood- or leaf- degrading fungi. In particular, A. bisporus has a large number of genes specifying enzymes able to metabolise the breakdown products of lignin, and these genes are upregulated in the presence of partially decomposed plant litter. Partially decomposed materials are called humic substances; these being naturally occurring organic compounds resulting from the decomposition and transformation of plant, animal, and microbial residues. They are important components of humus, which is the major organic fraction of soil. The demonstration by the transcriptome analysis of gene composition and expression profiles that A. bisporus is highly specialised to a humic-rich substrate led to the conjecture that the behaviour might be regulated by a new humic response promoter element (Kerrigan et al., 2013).; These authors also proposed the category humicolous as a new eco-nutritional classification that expands lignocellulosic digestion categories beyond white-rot fungi (that break down the lignin in wood, leaving the lighter-coloured cellulose behind, though some digest both lignin and cellulose) and brown-rot fungi (that break down hemicellulose and cellulose, leaving the lignin) (and see Riley et al., 2014).

It is interesting to recall that Wood (1980) demonstrated that during mycelial growth of Agaricus bisporus, a large proportion of the compost lignin was degraded, and correspondingly high activities of laccase were recorded (this one enzyme could amount to 2% of the total fungal protein). Yet, as the culture formed sporophores laccase activity was rapidly lost, initially by inactivation and subsequently by proteolysis; this pattern of behaviour reflecting the changing nutritional demands of fungal mycelia as they process through successive developmental phases and the ability of the mycelium to act on its environment to satisfy those demands.

Agaricus bisporus is globally distributed and a substantial collection of wild-collected strains is available (Sonnenberg et al., 2017). Such collections provide opportunities for directed breeding (O’Connor et al., 2019) of button mushrooms as genomics analysis improves understanding of relationships with disease organisms (Bailey et al., 2013), and potential for improvement of stress resistance (Sun et al., 2019) and other improved agronomic characteristics, and ‘mining’ for single nucleotide polymorphisms (SNPs) to develop cleaved amplified polymorphic sequences (CAPS) (An et al., 2021).

Ganoderma. Under the names lingzhi or reishi, Ganoderma lucidum is a cultivated mushroom which is unique in being consumed for its pharmaceutical value (real or imagined) rather than as a food. Ganoderma is highly regarded as a traditional herbal medicine, though many of the claims made for it are clinically unproven. Several species of the G. lucidum complex provide various commercial brands of nutriceuticals, in the form of health drinks, powders, tablets, capsules and diet supplements. Current research is focussed on purification and characterisation of the bioactive components and determination of clinical value, especially putative anti-tumour and anti-aging properties.

The morphology of Ganoderma sporophores varies greatly and at least some of that variation is likely to be due to misidentifications as the taxonomy of the Ganoderma lucidum complex has been described as ‘chaotic’. Analysis of 32 collections of the complex from Asia, Europe and North America using both morphology and molecular phylogenetics recovered a total of 13 taxonomically distinct species within the complex (Zhou et al., 2015). In sharp contrast, a survey of the molecular phylogenetics of 20 specimens of the related clade, Ganoderma sinense, from China were found to exhibit varied sporophore morphology, even though they possessed identical nucleotide sequences (Hapuarachchi et al., 2019). Evidently, phenotypic plasticity (= varied sporophore morphology) of a specimen or strain of Ganoderma can be influenced greatly by extrinsic factors, such as climate, nutrition, vegetation, and geographical environment rather than being necessarily associated with genotypic variation.

Ganoderma is mainly farmed for use as a traditional Chinese medicine; this is discussed in Section 18.14. Triterpenoids and polysaccharides are the two major categories of pharmacologically active compounds in Ganoderma lucidum. Genome sequence analysis (Chen et al., 2012) has revealed the impressive array of genes encoding cytochromes, transporters and regulatory proteins that cooperate in this secondary metabolism in G. lucidum and prepare the way for the metabolomics that will further exploit it (Bhardwaj et al., 2017; Ma et al., 2018). Transcriptome analysis of a strain of Ganoderma lucidum has correlated lignocellulose degradation with carbohydrate and triterpenoid metabolism (Zhou et al., 2021). Initial genomic studies of the related Ganoderma australe, which is a widespread and common tree pathogen in the British Isles and mainland Europe, also reveal considerable biotechnological potential (Agudelo-Valencia et al., 2020).

Another species of Ganoderma of major global significance is G. boninense, which causes basal stem rot disease of oil palm. The genome sequence of this species has been reported (Utomo et al., 2019) and an environmentally-friendly potential necrotrophic mycoparasite (Scytalidium parasiticum; Ascomycota) has been identified as a potential candidate for biocontrol of this disease in oil palm (Goh et al., 2016; Section 16.15).

Lentinula. Generally known by its common (Japanese) name shiitake, Lentinula edodes is one of the most popular edible mushroom species, second only in production volume to Agaricus. It is widely cultivated in many Asian countries, especially China, Japan and Korea and is increasingly penetrating the markets in Australia, Europe, and the USA. It is a white rot fungus with highly developed lignocellulose degradation ability and is consequently widely distributed as a member of the wood-decay community (Chiu et al., 1999). Shiitake mushrooms are also the source of polysaccharides with verified immunomodulatory activity; Lentinan is a β-1,3-glucan with β-1,6 branching and a molecular weight of 500,000 Da, which has been used in several clinical trials in cancer patients, though well-designed large-scale studies are still lacking [view: https://www.mskcc.org/cancer-care/integrative-medicine/herbs/lentinan].

Chen et al. (2016b) report the genome sequence of Lentinula edodes and initiate the transcriptome analysis of the expression of genes encoding cellulases and transcription factors up-regulated when mycelia were briefly cultivated in cellulose medium versus glucose medium. Their aim is to understand the molecular mechanism of lignocellulose degradation sufficiently to allow partial replacement of wood sawdust with agricultural wastes in L. edodes cultivation.

Transcriptome analysis has also been done to study postharvest problems in L. edodes, such as gill browning, sporophore softening, and lentinan degradation, and including light-induced browning (Sakamoto et al., 2017; Yoo et al., 2019). These analyses revealed a rapid sporophore autolysis system in L. edodes, with many cell wall-degrading enzymes being upregulated after harvest, along with many transcription factor genes. Clusters of genes specific to light-induced browning were associated with photoreceptors and melanogenesis via activation of tyrosinases, as well as cell wall degradation enzyme systems. These studies also found that several cell death related proteins were also upregulated postharvest.

This last observation is interesting because senescence and cell death are important aspects of the biology of fungi just as they are in the other two major eukaryotic kingdoms (Shefferson et al., 2017). There is only one experimental study of the longevity of fungal sporophores; that by Umar & Van Griensven (1997a, 1997b, 1998), which we discuss in Section 12.15. This study found that even in severely senescent sporophores healthy, living cells were found. We suggest that senescence and death of Lentinula sporophores would be a suitable topic for genomic analysis so that the postharvest findings can be understood in their natural biological context (Wiemer et al., 2016). Zhang et al. (2021) have constructed an ultra-high-density genetic map of L. edodes using next generation sequencing of RNA (RNA-Seq) of sporophores from 110 dikaryons. They combined genomic, genetic and phenotypic data to establish the genetic architecture of sporophore development. Twenty-nine quantitative trait loci (QTLs) and three main genomic regions were associated with sporophore development of shiitake. In the mapped QTLs, the expression of 246 genes were found to correlate significantly with phenotypic traits, thirty-three of which could represent candidate genes controlling the shape and size of sporophores.

Another aspect we suggest worthy of transcriptomic/metabolomic analysis is the accumulation in Agaricus bisporus and Lentinula edodes of the sugar alcohol mannitol, which is synthesised to serve an osmoregulatory function. Mannitol can amount to as much as 50% of the total mushroom dry weight in Agaricus bisporus, and 20 - 30% in L. edodes (Section 12.13, above).

Pleurotus. The genus Pleurotus includes several species of commercial value, such as P. ostreatus, P. pulmonarius, P. cornucopiae, P. eryngii, P. ostreatoroseus, and P. sajor caju, among others. Pleurotus mushrooms are distributed globally and have been cultivated in different parts of the world, ranking second in the world mushroom market (Bellettini et al., 2019). These fungi are white-rot fungi that secrete enzymes able to degrade lignin present in lignocellulosic substrates. Pleurotus enzymatic machinery is able to degrade complex compounds, constituting a great tool for the bioremediation of polluted environments. Genome sequencing has recently revealed the wood-degrading machinery of P. ostreatus to be typical of white rot fungi (Riley et al. 2014).

P. ostreatus is the most cultivated species of the genus. This fungus is also known as ‘oyster mushroom’ or ‘grey oyster mushroom’ to distinguish it from other Pleurotus species. In Asian countries, it is also called ‘hiratake’, ‘shimeji’, or ‘houbitake’. Several studies have shown that P. ostreatus has medicinal properties and potential biotechnological applications. It has been shown to produce pleuran, a bioactive glucan which has immunomodulatory and antioxidant properties. Pleuran has β(1,4)- or β(1,6)-branches at every fourth residue in its β(1,3)-glucan backbone (Sánchez, 2017a; 2017b). On the other hand, P. ostreatus is one of the carnivorous mushrooms known to be capable of killing and digesting nematodes as a nitrogen source, which raises interest in them as biocontrol agents as alternatives to conventional nematicides which have a negative impact on the global ecosystem (Thorn & Barron 1984, Thorn et al. 2000; Balaeș & Tănase, 2016).

Chiu et al. (1998) and Sadiq et al. (2019) reported that the spent mushroom compost of P. pulmonarius and P. ostreatus was able to degrade toxic organic pollutants such as pentachlorophenol and endosulfan, pesticides reported to be carcinogenic. Ahuactzin-Pérez et al. (2018) found that P. ostreatus degrades and uses as carbon and energy source high concentration (1000 mg l^-1) of di(2-ethyl hexyl) phthalate, which is a plasticizer that interferes with endocrine systems in mammals and has been detected as contaminant in the environment. The potential for using Pleurotus for pollutant and plastic degradation is discussed in Section 13.6, below, and by Sánchez et al. (2020). Nepomuceno et al. (2021) review the use of various agricultural wastes as resources in agriculture.

The demand for improved Pleurotus strains with high productive capacity has been rising. Currently, new generation approaches such as molecular breeding, genetic transformation and genome editing are used for strain improvement (Barh et al., 2019). So far, seven species of Pleurotus (P. ostreatus, P. platypus, P. citrinopileatus, P. salmoneo-stramineus, P. eryngii, P. tuber-regium and P. tuoliensis) have been sequenced, having genome sizes between 33.5 MB and 49.9 MB (Barh et al., 2019). Comparative genome sequence analysis showed that the sizes of P. eryngii (49.92 MB) and P. tuoliensis (48.23 MB) genome are significantly larger than other Pleurotus species, mainly due to their higher percentage of transposable elements and high long-terminal repeat transposable elements (Zhang et al. 2018). These transposable elements have epigenetic roles and cause silencing in Pleurotus genomes (Borgognone et al. 2018).

Gao et al. (2018) reported the first genetic linkage map integrated with physical mapping of the de novo sequenced genome and the mating type loci of P. tuoliensis. This is an important cultivated mushroom in China, where commercial profits are low, mainly due to a long cultivation period, sensitivity to diseases and a low yield. The de novo sequenced and annotated genome, assembled using a 2b-RAD (Restriction site Associated DNA) generated linkage map, provides a basis for marker-assisted breeding of this mushroom, which is crucial to improve agronomically-important traits. The 2b-RAD method uses type IIB restriction enzymes to generate a restriction-site associated library (Wang et al., 2012). This information and other research findings on genetic improvement are crucial in future breeding programs.

Volvariella. Breeding of improved strains in Volvariella volvacea has been hampered by its ambiguous sexuality pattern so it is in this aspect that genomic analysis could make the greatest contribution. Vegetative hyphal compartments of V. volvacea, and the hyphae of sporophore tissues are multinucleate and their septa lack clamp connections. An apparently normal meiosis occurs with DNA replication taking place at prophase I of meiosis in basidia that form four basidiospores into which the four meiotic nuclei migrate individually. The basidiospores remain uninucleate as there is no post-meiotic mitosis until germination (Chiu, 1993).

As long ago as 1987, Royse et al. demonstrated Mendelian segregation of five allozyme loci in V. volvacea. Further, there seems to be no barrier to cross-fertility, with no evidence for the expression of mating type genes. On the other hand, although the overwhelming majority of the basidiospores are uninucleate, many of these uninucleate basidiospores germinate to form mycelia which are self-fertile and able to form sporophores. In many cases, tests for self-fertility showed a 1:1 ratio of self-fertile to self-sterile among progenies. Even more unusual is that published evidence shows that progenies resulting from selfing in V. volvacea are regularly phenotypically diverse, implying that the selfed diploid that enters meiosis is not homozygous (see discussion and references in Chiu & Moore, 1999).

After considering likely mechanisms for generating genetic variation, Chiu & Moore (1999) suggested that a plausible mechanism, which could generate variation and produce 1:1 ratios from initially heterozygous loci, might be to regulate meiotic recombination mechanisms such that repair of base mismatches in hybrid DNA is reduced or eliminated. The result would be that (a) in every meiosis at least a proportion of the heterozygosity of the diploid nucleus would be preserved in the haploid daughter nuclei in the form of base mismatches in hybrid DNA; (b) at the first postmeiotic mitosis (at germination) these mismatches would be segregated into daughter nuclei and the V. volvacea spore germling would immediately become dikaryotic, even though the basidiospore had a single haploid nucleus; (c) assuming that recombination and mismatch-repair patterns differed between meioses then different spores would produce different dikaryons; (d) if half the base-mismatches at an initially heterozygous hypothetical fertility locus were repaired prior to the post-meiotic mitosis then a 1:1 ratio could result of self-fertile (still heterozygous in the sporeling dikaryon): self-sterile (repaired to homozygosity).

Analyses of the Volvariella genome are beginning to resolve some of these issues. Bao et al. (2013) reported the mating type system of V. volvacea to be similar to the bipolar system in basidiomycetes, suggesting that the organism might be secondarily homothallic. Chen et al. (2016a) studied the life cycle of V. volvacea using whole genome sequencing; demonstrating that the heterothallic life cycle is bipolar, as MAT-A, and not MAT-B, controlled heterokaryotisation. They cloned the MAT loci and found MAT locus polymorphisms in a series of strains. Crosses performed to establish the role(s) of the different MAT loci in mating established the presence of three coexisting, homo- and heterothallic life cycles. This study confirmed all of the peculiarities in segregation patterns previously demonstrated decades before with allozymes, colony morphology markers, restriction fragment length polymorphisms and electrophoretic karyotypes described in the previous paragraph. Also, the demonstration of structural variation (SV) markers relating to both alleles of both parents in single spore isolates that were clearly not heterokaryotic, and doubled SV markers in large numbers of single spore isolates suggested that the DNA structural peculiarities hypothesised by Chiu & Moore (1999) remained plausible.

Despite these numerous peculiarities Bao & Wang (2016) have established an efficient molecular marker-assisted cross-breeding technique for generating improved Volvariella volvacea cultivars. Whilst He et al. (2018) used a protoplast fusion technique to make interfamily hybridisations between V. volvacea and Pleurotus eryngii in attempts to improve the low-temperature tolerance and biological efficiency of Volvariella. Their hybrid strains showed higher biological efficiency (31.5%), compared with the 9.4% of the parental strain, and longer storage life at 16° C. The authors suggest that protoplast fusion provides an effective way to hybridise distantly-related edible fungi.

Updated August, 2021