Chapter 1: Diversity of the Fungi Kingdom: Molecular Tools to Distinguish Mushrooms Considered Safe and Unsafe for Human Health

Mushroom is a term that represents a short reproductive stage in the life-cycle of a macrofungi, being generally used to describe the fruiting body of the phyla Ascomycota and Basidiomycota.¹  Basidiomycetes are one of the four branches of Eumycota (true fungi), representing 35% of the total fungal species currently described, many of them having both ecological and industrial importance.²  Mushrooms can be either epigeous or hypogeous, also varying according to the ecological impact resulting from their lifestyles which can be saprotrophs, symbionts, or even pathogenic to fungi, plants and humans.³  Over the years, several investigations have demonstrated that mushrooms hold unique properties that have played major roles in human history, religion, and culture. These highly adaptive organisms survive by feeding mainly on matter from decomposing/death of other organisms and, occasionally, on living matter. These organisms are the most visible and colorful of the fungi kingdom, being recognized for their value as food and delicacies.⁴  Since mushroom fruiting is dependent on climatic conditions, the annual yields in the wild may vary from one year to another, depending on variables such as the average temperature or the precipitation rate.⁵  It is therefore predictable and already proved that domestication of some mushroom species provides stable productivity independent of climate events.⁶ 

Wild mushroom picking is generally a leisure activity that is very common in southern Europe, providing the seasonal income of some professional pickers.⁷  In Spain, for example, there are regulated mycological areas that require the purchase of picking licenses issued by the managing authority to ensure sustainable management of this resource.⁸  A good approach for the conservation of fungal resources requires undertaking management and regulatory processes for edible mushrooms in picking areas, thus quantifying and controlling the pressure on the most appreciated species.⁹  Northeast India is populated by over 45 million people, covers an area of 0.26 million km², and comprised of eight states: Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim, and Tripura.¹⁰  This region is rich in biodiversity, and characteristic for its heavy precipitation and high seismicity.^11,12  In Table 1.1 are presented 112 edible, medicinal, or poisonous mushrooms collected from all eight states of northeast India. The edible mushrooms foraged from northeast India include Lentinula edodes,^13,14  Lentinus sajor-caju,^15–17  Pleurotus djamor,¹⁷  Cantharellus cibarius,^18,19  Agaricus augustus,^1,20  and Lactarius deliciosus.²⁰  The poisonous mushrooms found in the same areas are Gyromitra infula,¹  Amanita phalloides,²⁰  and Polyozellus multiplex;¹³  and the medicinal ones are Ganoderma lucidum,^13,15,16  Pleurotus pulmonarius,¹⁷  Ganoderma applanatum,²¹  and Microporus xanthopus.¹³ 

The public demand for edible mushrooms continues to increase every year due to their rich nutritional value and massive industrial production. Mushroom growers increasingly require good-quality varieties with a fast maturity period, improved resistance to both pests and diseases, and a high production yield. Thus, the molecular characterization of fungi up to their species level is an essential task in mushroom research for more applications in different fields such as for agronomic and medicinal purposes.²² 

There is a growing industry of edible mushroom production based on a solid fermentation process of pasteurized or sterilized substrates inoculated with the appropriate spawn that proceeds under controlled temperature and humidity conditions.⁶  The market has been pushing to improve the yield, quality and quantity of mushrooms currently produced, propelling research aimed at developing breeding programs for edible fungi and to formulate appropriate substrates and culture conditions for new species. These proposals are based on three main factors: (i) the economic value of some highly demanded fungal species; (ii) their use to produce enzymes or chemicals that are useful in industry or pharmacy; and (iii) their application in processes aimed at recycling industrial and/or agricultural wastes.

Wild mushroom picking is a very healthful activity that requires specific knowledge about the mycological habitats and tools to discriminate among edible, toxic, and poisonous species.

Among the general rules for sustainable mushroom picking are:²³  (a) mushrooms must be harvested in an appropriate developmental stage to avoid misperception with toxic species and to take the best advantage of their culinary properties; (b) excessively trampled areas should be avoided, using only tracks and paths for vehicles to access the area; (c) collect the complete carpophores to facilitate identification; (d) clean the mushrooms immediately after harvesting to lengthen the shelf-life of the carpophores; (e) do not alter the surface of the ground by removing the leaf litter; (f) it is highly advisable to use a wicker basket instead of plastic bags to ensure spore dispersal while walking through the woods; (g) avoid mixing edible mushrooms with toxic species in the same basket and do not damage species that we are not interested in harvesting; and (h) as mushrooms can bioaccumulate toxic compounds, collection in contaminated areas should be avoided.

Depending on different variables, such as the chemical composition of the soil, flora, pluviometry, elevation above sea level, and temperature, different species associated with a particular mycological habitat can be found. Among others, in Spain, different habitats, such as pine trees on alkaline soils, pine trees on siliceous soils, forests of birches, gall oaks, Pyrenean oaks, holm oaks, junipers, different pines, riverside woods, peatlands, meadows, and grasslands are inhabited by countless mycological species.²³  The characterization and understanding of the mycological habitat can facilitate the identification of the different species. Figure 1.1 shows different types of mushrooms growing in multiple habitats.

Fungi identification requires two independent methodologies: morphological and molecular characterization. Mushrooms are macroscopic structures that conform to the sexual organs of the fungi. To prevent poisoning, mushrooms must be correctly identified before consumption. Of the visual characteristics, color, stipe, pileus, and hymenium morphologies stand out.²⁴  Novel tools such as mining data from mushroom shapes with tailor-made algorithms can be useful to identify edible and toxic species.^25,26  Additional characteristics such as the spore footprint, texture, and flavor are also useful tools to identify different mushroom species. It is very important that, in case of doubt, the mushroom is rejected or identified by an expert mycologist.

A variety of molecular genetic tools have been employed for the identification of fungi.²⁷  Molecular markers allow the selection of desired traits based on genotype rather than phenotype and can hence complement and accelerate plant breeding programs.²⁸  Here, we describe different types of molecular markers used for mushroom identification.

Random amplified polymorphic DNA (RAPD) is used to distinguish the genetic diversity of mushrooms²⁹  and to study the genetic and phylogenetic relationship between them³⁰  (Table 1.2). This is a simple methodology as it requires fewer DNA templates, is not susceptible to any hazardous contamination, and embraces a cheap technology.³¹  Apart from the genetic characterization of different organisms, RAPD analysis is also employed for the identification and study of genetic diversity in synthetic or newly found species with agricultural and industrial importance.^32,33  Even though the traditional RAPD technique has its limitations, such as low production and poor reproducibility, the new and improved RAPD technique known as RAMP-PCR (where the ramp time of traditional RAPD-PCR is increased from annealing to extension) is found to be more accurate and more consistent as compared to the traditional RAPD analysis.³¹ 

Inter-simple sequence repeat (ISSR) is an economical and reliable methodology that has been widely employed for the identification of different strains, populations, and genetic diversity of numerous fungi³⁴  (Table 1.2). ISSR markers are semi-arbitrary markers amplified by PCR in the presence of another primer corresponding to a target microsatellite.³⁵  This methodology has been successfully employed in the study of the genetic diversity of several mushrooms such as Agaricus species,³⁶  Auricularia auricula cultivars,³⁷  Lentinula edodes strains,³⁸  Pleurotus citrinopileatus,³⁹  Pleurotus eryngii cultivars,⁴⁰  and Volvariella volvacea.⁴¹ 

Li and Qurios⁴²  first introduced a novel molecular marker named SRAP (sequence-related amplified polymorphism), which has been widely used in genetic linkage mapping, genetic diversity analysis, gene tagging, and fungal genetic diversity studies⁴³  (Table 1.2). This approach has been used consecutively for the evaluation of genetic diversity in Agaricus bisporus,⁴⁴  Ganoderma population,⁴⁵  Lentinula edodes strains,⁴⁶  Pleurotus pulmonarius strains,⁴⁷  and Tricholoma matsutake.⁴⁸ 

Simple sequence repeats, also known as microsatellites, consist of only one or a few tandemly repeated nucleotides.⁴⁹  The SSR marker is a PCR-based molecular methodology with several advantages such as genetic co-dominance, high reproducibility, multi-allelic variation, and easy amplification by PCR.⁵⁰  These microsatellites are universally found in eukaryotic and prokaryotic genomes.⁵¹  SSR found in species with little or no DNA sequence information usually involves the construction and screening of partial genomic libraries and the sequencing of SSR-positive clones.⁵²  SSRs have been used to evaluate the genetic diversity of several mushrooms (Table 1.2) such as Lentinula edodes,⁵³  Auricularia auricula-judae,⁵⁴  Armillaria luteovirens⁵⁵  and Cordyceps militaris.⁵⁶ 

Amplified fragment length polymorphism (AFLP) is a molecular technique for DNA of any origin and complexity fingerprinting, including animal, microbe, and vegetal DNA.⁵⁷  The main advantages of the AFLP technique include its high reproducibility, reliability, and specificity. This technique has been employed to differentiate several types of mushrooms (Table 1.2) including Pleurotus eryngii strains,⁵⁸  Hypsizygus marmoreus,⁵⁹  Armillaria gallica,⁶⁰  and Agaricus bisporus.⁶¹  Mukhopadhyay et al.⁶²  successfully studied 30 Lentinula sp. (including L. edodes, L. lateritia, and L. novae-zelandiae) using 32 AFLP primer combinations.

The sequence characterized amplified region (SCAR) methodology was initially developed in 1991 and has been widely used since 1996 in studies on various aspects of basidiomycetes including breeding, population genetics, and genetic analyses.⁴⁶  SCAR markers have been employed for the identification of several mushrooms (Table 1.2) including Flammulina velutipes, Lentinula edodes, and Laccaria bicolor.⁶³  It also has been employed for the authentication and strain identification of Ganoderma lucidum⁶⁴  and Lentinula edodes.⁶⁵ 

DNA restriction fragment length polymorphisms (RFLPs) have been used as genetic markers in a wide variety of organisms, including humans, plants, fungi, and protozoa.⁶⁶  The most widely used RFLP method is a DNA–DNA hybridization technique that involves cutting genomic DNA with restriction endonuclease(s), followed by Southern hybridization with labeled specific probes.^67,68  This method yields highly reproducible fingerprint patterns, which are useful markers for genetic diversity studies among species⁶⁹  (Table 1.2). RFLP has been used to differentiate strains of Agaricus sp.,⁷⁰  phylogenetic analysis of Pleurotus spp., and DNA fingerprinting of Pleurotus eryngii complex.

The internal transcribed spacer (ITS) is a region of the nuclear ribosomal RNA which has been extensively used in molecular systematics.⁷¹  Variation between individual ribosomal DNA (rDNA) repeats can be observed within the ITS due to its higher degree of variation than that of the small subunit (SSU) and large subunit (LSU) of rRNA genes.^72,73  Phylogenetic analysis based on DNA sequencing, ITS1-5.8S-ITS2 of rDNA sequence analysis, is normally used for fungal identification.⁷⁴  ITS has been used to study the diversity of edible mushroom Pleurotus eryngii,²⁷  phylogenetic and biogeographic relationships of Suillus sp.,⁷⁵  molecular phylogeny of Japanese Amanita species,⁷⁶  and phylogenetic diversity of wild edible Russula genus⁷¹  (Table 1.2).