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Chapter 1: Introduction
1.1
Fungi Kingdom
Fungi are a group of eukaryotic organisms and heterotrophic organisms that have a number of phenotypic and genotypic characteristics such as to allow their classification in a separate kingdom. Elevated to the rank of kingdom by Whittaker in 1968, they differ from plant cells due to the lack of plastids, chlorophyll, differentiated and transport tissues (xylem and phloem), in addition to the presence of chitin in the plant wall. Other differences are related to the type of reserve substances (glycogen) and the biological cycle. From the genetic point of view, a further element of distinction is the difference in structure of their cytochromes and the size of the genome, on average, higher than in prokaryotes but average lower compared with Algae and Protozoa. Based on these characteristics, the fungi are treated as separate kingdom, and given the small size of the genome, are placed as the simplest taxon of Eukaryotes immediately after the Prokaryotes.
Fungi are an ancient group—not as old as bacteria, which fossil evidence suggests may be 3. 5 billion years old—but the earliest fungal fossils (non septate hyphae and spores) deposited in a presumed shallow marine are from the Ordovician, 460 to 455 million years old (Redecker et al., 2000). Based on fossil evidence, the earliest vascular land plants didn't appear until approximately 425 million years ago (Fig 1), although other Authors have somehow different hypothesis. Some scientists believe that fungi may have played an essential role in the colonization of land by these early plants (Redeker et al., 2000). Fungi exquisitely preserved in amber from the
2 Late Cretaceous (94 million years ago) tell us that there were mushroom-forming fungi remarkably similar to those that exist today when dinosaurs were roaming the planet (Hibbett et al., 2003).
Figure 1: A timescale of eukaryote evolution (from Hedges B. S. et al., 2004)
3 minimum time estimate for when different groups of fungi evolved. Molecular data suggest that fungi are much older than indicated by the fossil record, and may have arisen more than one billion years ago (Parfrey
et al., 2011). At first it seemed that the fungi could be assigned without question to the plant kingdom, since they are non-motile and draw their nourishment from the substratum. During the nineteenth century it was realized, however, that the most fundamental features of green plants are that they are phototrophs, utilizing energy from light, and autotrophs, synthesizing their organic components from atmospheric carbon dioxide. Animals on the other hand are chemotrophs, obtaining energy from organic materials, and heterotrophs, utilizing the same materials as the source of carbon for the synthesis of their own organic components. On these fundamental metabolic criteria it is clear that fungi, although non-motile, resemble animals rather than plants (Carris et al., 2012)
Fungi are now recognized as one of five eukaryotic kingdoms (Fig. 2), the others being Animalia (animals), Plantae (plants), Chromista (corresponding roughly to the algae and also known as Stramenopila) and Protozoa, which contains a wide variety of mainly phagotrophic unicellular organisms.
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Figure 2: A phylogenetic tree showing the relationships between the two Prokaryote and five Eukaryote kingdoms (from “The Fungi”, “2nd edition, Academic Press 2001)
Fungi Kingdom is a large group of eukaryotic organisms depending for their nutrition on the absorption of organic materials and producing a variety of spores. Most fungi reproduce by spores and have a body (thallus) composed of microscopic tubular cells called hyphae. Fungi are heterotrophs and, like animals, obtain their carbon and energy from other organisms. Some fungi obtain their nutrients from a living host (plant or animal) and are called biotrophs; others obtain their nutrients from dead plants or animals and are called saprotrophs (saprophytes, saprobes). Some fungi infect a living host, but kill host cells in order to obtain their nutrients; these are called necrotrophs (Carris et al., 2012).
It is now clear that the mainly studied fungi belong to three Kingdoms, the Chromista, the Protozoa and the Fungi, but only the kingdom Fungi consists exclusively of fungi. The features that distinguish these three Kingdoms are set out in Table 1.
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Table 1: Important groups of Fungi (from “The Fungi”, “2nd edition, Academic Press 2001)
Organisms traditionally studied as "fungi" belong to three very different unrelated groups: the true fungi in Kingdom Fungi (Eumycota), the Oomycetes, and the slime molds (Table 1).The group of fungi informally called Oomycetes or water moulds have structural, genetic and biochemical features that have now established them as a phylum, the Oomycota, within the Chromista. Eumycota division is made of one subkingdom (Dykaria) seven main groups (phyla) Blastocladiomycota, Chytridiomycota,
6 Glomeromycota, Microsporidia, Neocallimastigomycota, Ascomycota, Basidiomycota, (both in the subkingdom Dykaria) (Hibbett et al., 2007) (Fig 3).
Figure 3: Phylogeny and classification of Fungi. Basal Fungi and Dikarya. Branch lengths are not proportional to genetic distances. See Table 1 for support values for clades (from Hibbett et al., 2007)
Recent studies (Hibbett et al., 2007) get the support that the phylum Zygomycota is not accepted in this classification, pending resolution of
7 relationships among the clades that have traditionally been placed in the Zygomycota. The traditional Zygomycota are here distributed among the phylum Glomeromycota and four subphyla incertae sedis, including
Mucoromycotina, Kickxellomycotina, Zoopagomycotina and
Entomophthoromycotina.Glomeromycota represented an additional phyla, a group of fungi once placed in Zygomycota that form an association with the roots of most plants (Carris et al., 2012) (Fig 3). The Kingdom Fungi consists solely of species that are hyphal, or clearly related to hyphal species.
Slime molds are organisms that have a trophic (feeding) stage in their life cycle that lacks a cell wall, either uninucleate (amoeba) or multinucleate (plasmodium). The lack of a cell wall facilitates engulfment of food, in contrast to true fungi that must absorb their nutrients through a cell wall. The slime molds are now included in the Amoebozoa (Adl et al., 2005). As previously described, eumycetes are divided into seven phyla as shown in Fig. 3. Blastocladiomycota are saprotrophs as well as parasites of fungi, algae, plants and invertebrates, and may be facultatively anerobic (James et
al., 2006a). These zoosporic fungi are found in soil and fresh water habitats and are mostly detritivores, subsisting on decaying organic matter (Roberts 1999). Microsporidia are highly specialized eukaryotic unicells, living only as obligate intracellular parasites of other eukaryotes (Hirt et al., 1999). A characteristic feature of microsporidia is the polar tube or polar filament found in the spore used to infiltrate host cells. They are widely distributed in nature with over 1200 species characterized. Microsporidia are important parasites in fisheries, veterinary medicines and pest management. Neocallimastigomycota is a phylum of anaerobic fungi, found in the
8 digestive tracts of herbivores. It encompasses only one family (Hibbett et
al., 2007) and they have been recently studied because of their biotechnological potential, for more efficient animal nutrition and also biomass conversion/biofuel production (Griffith et al., 2010)
The Chytridiomycetes are the only group with motile cells (known as zoospores). The form taken by the sexual phase of the life cycle is an important criterion in the classification of fungi that lack zoospores. The sexual process leads to the production of characteristic spores in the different groups. The fungi that form ascospores are classified as Ascomycetes, and those forming basidiospores as Basidiomycetes. The hyphae of Ascomycetes and Basidiomycetes have numerous cross-walls. Another feature widespread in Ascomycetes and Basidiomycetes is that when hyphae within a fungal colony come into contact they may fuse with each other. Hyphal anastomosis, as indicated later, may be a major factor in permitting the mycelium of some Ascomycetes and Basidiomycetes to produce large fruit bodies. Cross-walls and hyphal anastomoses are largely lacking in the Chytridiomycetes and Glomeromycota. These organisms are sometimes termed the 'lower fungi', in contrast to the 'higher fungi', the Ascomycetes, Basidiomycetes and related forms. There is some justification for a loose distinction of this kind, in that the potentialities of hyphal and mycelial organization have been more fully exploited in the latter groups. Many Ascomycetes and Basidiomycetes, in addition to producing spores by a sexual process, form other types of spores asexually. There are also many species, recognizable as higher fungi through the presence of cross-walls in their hyphae that produce asexual spores but lack a sexual phase. These are known as anamorphic fungi, as all their spores are produced following
9 mitosis but none by meiosis. They were formerly termed the Deuteromycetes or Fungi Imperfecti, and a Deuteromycete was reclassified as an Ascomycete or Basidiomycete if a sexual phase was discovered. However, analysis of DNA sequences now allows these asexual fungi to be classified with their closest sexual relatives, and it appears that they have arisen from many different groups of fungi by the loss of sexuality.
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1.2 Ascomycota
Ascomycota, with approximately 64 000 known species (Kirk et al., 2008), is the largest phylum of Fungi and one of the most diverse and ubiquitous phylum of eukaryotes. Its species occur in numerous ecological niches and virtually all terrestrial and aquatic ecosystems. They function in the decay of organic substrates (e.g., wood, leaf litter, and dung) and act as mutualists, parasites, and pathogens of animals, plants, and other fungi (Schoch et al., 2009). More than 40% of all named Ascomycota are lichenized, covering approximately 8% of the Earth’s landmasses (Brodo et
al., 2001). In addition to their presence in most natural, industrial, and agricultural settings, they have been isolated from some of the most extreme environments on earth—from inside rocks on the frozen plains of Antarctica (Selbmann et al., 2005) to deep-sea wood (Kohlmeyer 1977) and sediments (Raghukumar et al., 2004). Thousands of basidiomycetes, and a quite a few ascomycetes, establish intimate mutualistic symbioses (mycorrhizas) with the roots of trees, especially conifers. Nearly 18,000 ascomycetes, and a few basidiomycetes, have domesticated algae, thus becoming lichens, which can live in some of the world's harshest climates, and colonize the barest and most inhospitable substrates. The phylum Ascomycota consists of three subphyla, Taphrinomycotina, Saccharomycotina and Pezizomycotina (Hibbet et al., 2007). Subphylum Pezizomycotina is the largest subphylum and contains all ascomycetes that produce ascocarps (fruiting bodies), except for one genus, Neolecta, in the Taphrinomycotina. Pezizomycotina is ecologically diverse with species functioning in numerous ecological processes and symbioses (e.g. wood and litter decay, animal and plant pathogens, mycorrhizae and lichens) and
11 occurring in aquatic and terrestrial habitats (Spatafora et al., 2006).The Saccharomycotina subphylum contains approximately 1500 species of yeasts, most of which live as saprotrophs in association with plants and animals, but also including a small number of plant and animal pathogens (Suh et al., 2006). Yeasts traditionally have been important in the production of beer, wine, single cell protein and baker's yeast, but their role in industry has expanded to the production of citric acid, fuel alcohol, and riboflavin (Kurtzman and Sugiyama 2001). The ascomycete subphylum Taphrinomycotina sensu Eriksson and Winka (1997) was based on the provisional class ‘‘Archiascomycetes’’ proposed by Nishida and Sugiyama (1993, 1994b). Subphylum Taphrinomycotina includes fungi that, with one known exception, do not form fruiting bodies—as examples, the fission yeast Schizosaccharomyces (Fig. 4a), plant parasites in the genera Protomyces and Taphrina (peach leaf curl; see Figs. 4b & 4c), and Pneumocystis, a yeast-like fungus previously mentioned (in "Fungi associated with animals") that causes pneumonia in animals including humans.
12 Recent molecular studies suggest that the group is monophyletic and basal to the rest of the Ascomycota. (Lutzoni et al., 2004, James et al., 2006b).
Among the Ascomycetes are such familiar and economically important fungi as yeasts, common molds, morels and truffles. Also included in this phylum are many serious plant pathogens, including the chestnut blight,
Cryphonectria parasitica, and Dutch elm disease, Ophiostoma ulmi.
The ascomycetes are named for their characteristic reproductive structure, the microscopic, saclike ascus (from Greek: σκός (askos), meaning "sac" or "wineskin"; plural, asci). The zygotic nucleus, which is the only diploid nucleus of the ascomycete life cycle, is formed within the ascus. The asci are differentiated within a structure made up of densely interwoven hyphae, corresponding to the visible portions of a morel or cup fungus, called the ascocarp.
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Figure 5: Asexual sporulation in Ascomycetes. A, Formation of arthrospores in Ascobolus
furfuraceus, by the separation of the constituent cells of an aerial hypha. B, Macroconidia of
Neurospora crassa. Alternate bulging and constriction occurs throughout the aerial hypha, septa develop at the constrictions and the resulting macroconidia detach at the slightest disturbance. C, Microconidia of Neurospora crassa. The tiny microconidia are budded from the conidiophore and form sticky clusters. D, Conidiophore of Aspergillus (Emericella)
nidulans bearing primary and secondary sterigmata (phialides). From the latter conidia are being produced, the basal ones not yet swollen to full size or separated from the phialides. E, Conidiophore of Penicillum expansum bearing metullae on which phialides are producing conidia. D and E are diagrammatic, phialides and conidia being more numerous than shown. (from “The Fungi”, “2nd edition, Academic Press 2001)
It takes place by means of conidia (singular, conidium), spores cut off by septa at the ends of modified hyphae called conidiophores. Conidia allow for the rapid colonization of a new food source. Manyconidia are
14 multinucleate. The hyphae of ascomycetes are divided by septa, but the septa are perforated and the cytoplasm flows along the length of each hypha. The septa that cut off the asci and conidia are initially perforated, but later become blocked.
The cells of ascomycete hyphae may contain from several to many nuclei. The hyphae may be either homokaryotic or heterokaryotic. Female gametangia, called ascogonia, each have a beaklike outgrowth called a trichogyne. When the antheridium, or male gametangium, forms, it fuses with the trichogyne of an adjacent ascogonium. Initially, both kinds of gametangia contain a number of nuclei. Nuclei from the antheridium then migrate through the trichogyne into the ascogonium and pair with nuclei of the opposite mating type. Dikaryotic hyphae then arise from the area of the fusion. Throughout such hyphae, nuclei that represent the two different original mating types occur. These hyphae are thus both dikaryotic and heterokaryotic. Asci are formed at the tips of the dikaryotic hyphae and are separated by the formation of septa. There are two haploid nuclei within each ascus, one of each mating type represented in the dikaryotic hypha. Fusion of these two nuclei occurs within each ascus, forming a zygote. Each zygote divides immediately by meiosis, forming four haploid daughter nuclei. These usually divide again by mitosis, producing eight haploid nuclei that become walled ascospores. In many ascomycetes, the ascus becomes highly turgid at maturity and ultimately bursts, often at a preformed area. When this occurs, the ascospores may be thrown as far as 30 cm, a remarkable distance considering that most ascospores are only about 10 micrometers long.
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1.2.1 Ascomycetes fruit bodies
The multicellular structures (ascomata) that produce the asci, are the platforms from which the spores are launched. They come in four different forms (aphotecial, perithecial, pseudothecial and cleistothecial ascomata) characterized by different design and functional properties.
1.2.1.1 Aphotecial ascomata
The Apothecia ascomata (Fig 6) are cup- or dish-shaped structures bearing a layer of asci. At maturity the asci become broadly exposed to the environment and are able to discharge their ascospores directly into the air. Some apothecia can be a centimeter or more in diameter and have the ability to shoot their spores a considerable distance. In some species a small burst of air, such as blowing gently on the apothecium, will cause a visible "smoke" of ascospores to be released. Apothecia can be obviously disc-shaped or may remain closed for a long time and appear elongated. Some may be large and compound and hardly resemble a cup at all.
1.2.1.2 Peritechial ascomata
Perithecia ascomata (Fig 6) differ from apothecia in that they completely enclose the asci, leaving only a small pore, the ostiole, for the escape of the spores. It contains unitunicate-inoperculate asci. Perithecia differ in how the asci inside are arranged. In Sordaria humana, the species in the photo, the asci are produced at the base of the perithecium and are oriented upwards toward the ostiole. In others the perithecium is larger and the asci line the bottom and the sides of the inner wall.
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Figure 6: Diagrams of Ascomycete fruit bodies. A, Apothecium of Aleuria vesiculosa. The upper surface is lined with asci and spacer hyphae, paraphyses. B, Asci and paraphyses of
of A. vesiculosa C, Perithecium of Sordaria fimicola. An ascus is shown protruding from the opening (ostiole) of the perithecium prior to discharging its ascospores and collapsing. It will be replaced by a sequence of other asci, shown in various stages of development. D; Cleistothecium of Eurotium repens. The ascus walls lyse and finally the perithecium ruptures to release ascospores. (From “The Fungi”, “2nd edition, Academic Press 2001
1.2.1.3 Pseudotechial ascomata
Pseudothecial ascomata (Fig 6) is also called ascostroma and is characterized by the fact that the asci are formed in cavities (loculi) within stromatical tissue. Lacks the hymenium. The asci in this fruiting body are bitunicati, with a double wall. Species in which one can find the
17 pseudotecio are, for example, the scab (Venturia inaequalis) or Guignardia
aesculi.
1.2.1.4 Cleistotechial ascomata
Cleistothecial ascomata (Fig 6) completely enclose their asci and have no ostiole or opening of any kind. At maturity the walls break or split and the spores can escape. With an ascoma such as this it is not surprising that most cleistothecia contain asci that are incapable of shooting their ascospores. These non-ballistic asci often tightly enclose their ascospores and are club-shaped or spherical. At maturity their wall dissolves, releasing the spores into the cavity of the cleistothecium.
1.2.2 Ascomycetes asci
Fungi belonging to Ascomycota division produce 4 different types of asci. The Unitunicate asci (Fig 7) have a single wall. Some have a built-in lid or operculum (stained blue, Fig 7 left), at maturity this pops open around a built-in line of weakness (Fig 7, right) so that the spores can be ejected. Unitunicate-operculate asci are found only in apothecial ascomata. Some of the operculate asci in the colour picture (above, left) have discharged their spores; one has not.
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Figure 7: Unitunicate asci (from http://www.mycolog.com/CHAP4a.htm)
The Unitunicate-inoperculate asci (Fig 8) have no operculum, but have a special elastic ring mechanism built into their tip. This is a pre-set pressure release valve, or sphincter, and the ring eventually stretches momentarily, or turns inside out, to let the spores shoot through. Such inoperculate asci are found in perithecial and some apothecial ascomata.
19 The third kind of asci, the Prototunicate asci (Fig 9a, 9b), have no active spore-shooting mechanism. These asci are usually more or less spherical, and are found in cleistothecial (occasionally perithecial), and hypogeous ascomata.
Figures 9a, 9b: Prototunicate asci (on the left) and Tem microscope image (on the right) (from http://www.mycolog.com/CHAP4a.htm)
Sometimes the wall of this kind of ascus dissolves at maturity and releases the ascospores, which can then ooze, rather than be shot, out of the ascoma; or they may wait inside until it decays or is ruptured. These asci are often called prototunicate. Yet perhaps because they are found in several otherwise rather different orders, it seems likely that they represent a secondary condition, and have evolved several times from unitunicate asci (as they clearly did in the truffles-Tuberaceae).
The last one, the Bitunicate asci (Fig 10) have a double wall; the exotunica and the endotunica. A thin, inextensible outer wall covers a thick, elastic inner wall.
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Figure 10: Bitunicate asci (from http://www.mycolog.com/CHAP4a.htm)
At maturity the thin outer wall splits, and the thick inner wall absorbs water and expands upward, carrying the ascospores with it. This design allows the ascus to stretch up into the neck of the ascoma to expel its spores. The bitunicate ascus is so different from the unitunicate ascus that we assume they diverged a long time ago. Note that the ascospores in the colour photomicrograph (Fig 10, right) are darkly pigmented dictyospores, with septa running both across and along the spore.
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1.3 The
Tuber genus
Tuber spp. are ascomycetes belonging to Pezizales, a large group of ectomycorrhizal fungi growing in symbiosis with the roots of several vascular plant species (angiosperms and gymnosperms) (Hilszczańska et al., 2008). Known as ectomycorrize (Trappe 1979), the ascoma of these fungi is a hypogeous complex apothecium, commonly known as a truffle. The Tuber genus represents one of the 5 genera belonging to Tuberaceae family (subphylum Pezizomycotina) and it’s widely estimated to comprise at least 180 species worldwide (Bonito et al., 2010) mainly covering the temperate zones of the northern hemisphere, with at least three areas of genetic differentiation: Europe, South East Asia and North America. Truffle species present common ecological features such as a relatively wide range of host species and the need for a calcareous soil (pH between 7 and 8), except T.
borchii tolerating slightly acidic soils (Mello et al., 2006).
The truffle life cycle, like that of other symbiotic filamentous fungi, begins with a limited extraradical phase of vegetative growth in which the hyphae proliferate before coming into contact with the roots of the host plant (phase 1-2, Fig 11). After this contact (phase 3) the symbiotic phase begins, leading to the development of the ectomycorrhiza (phase 4), a new organ which is functionally and morphologically distinct from the two individual partners. In the final stage, the mycelium is organised into the fruit body (phase 5-6), the role of which is to produce sexual fructifications later dispersed in the environment. Vegetative mycelia then develop from these fructifications, originating a new extra radical phase and closing the truffle life cycle (Kües and Martin 2011). Those hyphae that originate from a single
22 spore are mononucelate, while those that form the mycelium and fruiting body are provided with one or more cores (Ceruti 1990).
Figure 11: The T. magnatum Pico life cycle (Paolocci et al., 2006)
Within the genus Tuber there are numerous edible fungi characterized by a high economic value (Table 2). The following paragraph describes some of the main species.
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Table 2: Geographic distribution of the main truffle species (Jeandroz et al., 2008)
1.3.1 Tuber melanosporum
The Perigord black truffle (T. melanosporum) is endemic to calcareous soils in southern Europe and is found in mutualistic symbiosis with roots of deciduous trees, mostly oak (Quercus spp.) and hazelnut (Corylus avellana) trees (Martin et al., 2010). The main natural populations are found in Mediterranean areas (France, Italy, and Spain). The harvest and cultivation of truffles has expanded to other locations in Europe and also to New Zealand, Australia, China, the United States, Israel, and to other countries in recent.
Wild production of truffles has decreased sharply in the last century, and production from truffle orchards is increasing with keen interest in understanding production factors (Suz et al., 2008). The high prize of the Perigord truffle (more than € 1000 kg) (Garcia-Montero et al., 2007) has
Species Distribution
T. melanosporum Vittad. Southern Europe (Spain, France, Italy, Greece, Bulgaria, Romania, Turkey, between 40 and 48° North)
T. borchii Vittad All Europe (between 37 and 55° North)
T. aestivum Vittad. All Europe (between 37 and 57° North) and North Africa
T. uncinatum All Europe
T. mesentericum Vittad Southern and Central Europe (Spain, France, Italy, Austria, Germany, Poland, between 40 and 52° North)
T. brumale Vittad. Europe (between 40 and 61°North)
T. macrosporum Vittad Western Europe (between 41 and 51° North)
T. indicum Asia (India, central China and Tibet, Korea, Japan)
T. magnatum Pico Southern and central Europe (Italy, Switzerland, Croatia,
24 prompted the development of its culture through man-made inoculation of seedlings (Murat et al., 2004). The ascocarp is roundish, of size varying from 3 to 7 cm, covered with brownish-black with reddish warts basis. The gleba present a white colour during its maturation phase but soon black-brown tending to violet or the reddish (Fig 12), with thick and thin whitish venations becoming brown with the age.
Figure 12: T. melanosporum ascocarp (on the left) and spores (on the right) (from
http://tartufi-online.com/categorie/tuber-uncinatum)
An early indicator of Black truffle activity in soil is the appearance of a zone with diminishing vegetation surrounding the stem of the tree, known as the burn, caused by the phytotoxic effect of T. melanosporum mycelium (Suz et
al., 2008).
T. melanosporum among the black truffles and T. magnatum (Pico) among the white truffles are by far the two species with the highest economic value. The market for them is particularly active and represents a valuable source of agricultural income in some areas of southern Europe (Séjalon-Delmas et
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1.3.2 Tuber borchii
T. borchii Vitt., known as bianchetto or marzuolo is an edible species morphologically similar to T. magnatum Pico, the finest truffle species, but lacks the latter's typical sulphureous fragrance (Tirillini et al., 2000).
Figure 13: T. borchii ascocarp (on the left) and spores (on the right) (from http://tartufi-online.com/categorie/tuber-borchii)
Its wide areal distribution of the fruiting body (isolate or in aggregate form) is between 37 ° and 61 ° parallel of north latitude. The T. borchii areal extends from Europe, where it is present as ubiquitous, to a few non-European countries such as China and the USA. The Italian distribution is uniform, being present in almost all the Italian regions, from Trentino to Sicily. T. borchii, like other edible truffles is commonly found in calcareous soils with pH between 7 and 8 or in acidic soils. Its low host specificity joined to its good adaptation to a wide range of soils and climates allow to use this species for the truffles cultivation (Iotti et al., 2010). The outward appearance is similar to the T. magnatum; they share an irregular, smooth and white colour even if they become darker when ripe. Also the gleba at first is white (Fig 13), than later on becomes dark. What really distinguishes this kind of truffle from the white ones is the fragrance; at the beginning it
26 is fine and pleasant, but then, in a second moment, it becomes strong and garlicky.
1.3.3 Tuber aestivum
T. aestivum Vitt. is an hypogeous ascomycete that grows in ectomycorrhizal symbiosis with e.g. Quercus robur and Corylus avellana (Wedèn et al., 2004). Known as “summer truffle” or “Truffe de la Saint-Jean" (Fig 14), is less aromatic than black and white truffles (T. melanosporum and T. magnatum respectively), but is moderately priced and has a good aroma quality and is widely appreciated and distributed in Mediterranean countries (Cullerè et
al., 2010). T. aestivum grows in more compact and clayey soils, usually in sunny places and inhibits plants growing in the burn areas where mycelial colonies produce ascocarps, more conspicuous than T. melanosporum.
Figure 14: T. aestivum ascocarp (on the left) and spores (on the right) (from
http://www.mycolog.com/CHAP4b.htm http://www.trufamania.com/truffles.htm )
Soil and climatic requirements of the summer truffle can be met in many natural localities in Europe and this fungus is thus probably the easiest of all truffles to cultivate commercially (Gryndler et al., 2011). Black summer
27 truffles grow quite superficial and we can detect their presence through the cracks in the soil. They are sometimes found under leaf litter. Usually they are harvested from May to July, but we can find them until December if moisture conditions are suitable. In some countries (e.g. France, burgundy truffle), T. aestivum is harvested, cultivated and marketed, whereas in others, for example, the Czech Republic or Slovakia, this species is considered critically endangered and protected by law (Gryndler et al., 2011). There, collecting of T. aestivum fruit bodies in the wild is forbidden, which is detrimental for any economic evaluation of this species. However, the status of T. aestivum as an endangered species may not correspond to its real geographic distribution and abundance due to a lack of information (Streiblovà et al., 2010).
1.3.4 Tuber uncinatum
Also known as the ‘Grey Truffle’ or Burgundy truffle, with an appearance and skin similar to T. aestivum but characterized by a highest economic value (Mello et al., 2002).
Figure 15: T. uncinatum ascocarp (on the left) and spores (on the right) (from http://tartufi-online.com/categorie/tuber-uncinatum)
28 More Recent publications (Wedèn et al., 2005, Paolocci et al., 2004) assessed that because no stable genetic characters differentiate T. aestivum and T.
uncinatum, there is no basis for suspecting that the two names represent two varieties and the genetic dissimilarity existing within the ITS of T.
aestivum/ T. uncinatum samples could be confidently confined within intraspecific variability. The name of this truffle is derived from its hook shaped spores and it mainly grows in France (region of Burgundy as well as in Champagne, Lorraine and Franche-Comté) and Italy (central and northern zone). It presents a black skin, with a dark brown gleba and white veins at maturity (Fig 15).
1.3.5 Tuber mesentericum
T. mesentericum Vitt. is an ectomycorrhizal fungus that generates edible
fruit bodies that are a product in increasing demand on the food market. Its distribution is spread throughout Europe, with the center of production localized in Italy, in particular in the area surrounding Bagnoli Irpino (Province of Avellino, inland of Naples), where T. mesentericum is very abundant (Sica et al., 2007).
Figure 16: T. mesentericum ascocarp (on the left) and spores (on the right) (from
29 It grows and reaches maturity at the same time as T. uncinatum. Truffles are small, 2 to 5 cm with distinctly coarser (larger) pyramidal verrucae, the gleba is light hazel-brown at full maturity (Fig 16) and the smell is generally very strong in just collected fruit bodies, reminiscent of phenol and iodine (Bozac et al., 2012).
1.3.6 Tuber brumale
The T. brumale Vittad. fungus forms mycorrhizas with a variety of hosts, including the Tilia and Quercus genera (Giomaro et al., 2002). Known as violet truffle, it occurs naturally in France and Italy and in other parts of Europe. Despite its lowest economic value if compared with the more precious species (T. magnatum and T. melanosporum), it is one of the major competitors of T. melanosporum cultivated in truffle grounds and, to a lesser extent, T. magnatum (Mamoun and Oliver 1993). T. brumale size, colour, skin, spores and production period are identical to T. melanosporum. The gleba is white, greyish, then blackish-violet without the reddish and purple shade of the T. melanosporum (Fig 17) and becomes brownish and blackish but less dark than the black truffle.
Figure 17: T. brumale ascocarp (on the left) and spores (on the right) ( from http://www.trufamania.com/truffles.htm )
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1.3.7 Tuber macrosporum
T. macrosporum Vittad. or smooth black truffles, is not a common truffle
species, but with remarkable organoleptic qualities and much economic interest (Benucci et al., 2012a) with a similar aroma and taste to T.
magnatum. Probably the most common variety in Italy, it’s widely distributed in Europe and grown in forests, from 50 to 950 m above sea level, and preferentially forms associations with angiosperm symbionts, especially with Corylus avellana L., Quercus robur L., Quercus cerris L., Ostrya
carpinifolia Scop., Carpinus betulus L., Tilia cordata Miller, Tilia platyphyllos Scop., Populus spp. and Salix spp. (Vezzola 2005).
Ascomata present a subglobose or irregular in form and lobed, medium size of 1-5 cm with black and purple rough bark and small warts (Fig 18). The gleba is solid with numerous wide, white meandering veins.
Figure 18: T. macrosporum ascocarp (on the left) and spores (on the right) ( from http://www.trufamania.com/truffles.htm )
31
1.3.8 Tuber indicum
The Asian black truffle T. indicum is morphologically and phylogenetically similar to the European black truffle T. melanosporum.
Figure 19: T. indicum ascocarp (on the left) and spores (on the right) ( from http://www.trufamania.com/truffles.htm )
It grows in association with host species in the Betulaceae (Corylus spp.,
Alnus spp.), Fagaceae (Quercus spp., Castanea spp., Castanopsis spp.,
Lithocarpus spp.) and Pinaceae (Pinus spp., Keteleeria spp.) (Bonito et al., 2011). It’s also known under other names, Tuber himalayense, Tuber
pseudohimalayense and Tuber sinense and it’s available in large quantity during the winter season. As the price and taste of T. indicum are very inferior to those of T. melanosporum, and since the mycorrhizas and fruiting bodies looks alike, although the price is very inferior, they can be easily mistaken with the most precious black truffle (Mabru et al., 2001).
There is morphological similarity with T. melanosporum (Fig 19), but no flavour and the veins in the gleba are rose-red.
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1.3.9 Tuber magnatum Pico
The ectomycorrhizal fungus T. magnatum Pico is an hypogeous ascomycete greatly appreciated for its intense flavor and aroma (Splivallo et al., 2011), that forms specialized symbioses with fine roots of higher plants (Fig 20). The interest on this specific Tuber is undoubtedly related to its ecological role in boreal and temperate forests, though the use of its fruiting bodies (commonly known as truffles) as a cooking ingredient is certainly equally relevant. Due to its limited availability, T. Magnatum fruit bodies represent one of the most expensive delicacies on the market. While T. Melanosporum Vittad. is generally sold at € 30–40/100 g in France, T. Magnatum reached € 300–400/100 g in fall 2003 (Murat et al., 2005).
Figure 20: T. magnatum ascocarp (on the left) and spores (on the region) ( from http://www.trufamania.com/truffles.htm )
White truffle aroma is very unique and characteristic; its complex composition has been the object of several studies over the last 20 years, employing different technologies available for the analysis of volatile compounds (Pennazza et al., 2012).
33 Piedmont, Marche, Umbria, etc.), Istria and in several Balkan regions, growing in clayey-sandy soils. The quality of its fruiting bodies is variable and depends on the environment and the growth conditions.
Several attempts have been made to artificially grow this fungus, which to date have been unsuccessful, despite the success achieved with other species of edible truffles, such as T. melanosporum, T. aestivum and T.borchii
(Bertini et al., 2006). This is partly because it is difficult to obtain plants infected with T. magnatum that are uncontaminated by other species of white truffle, such as T. maculatum and T. borchii or Sphaerosporella brunnea (Danielson 1984) and Pulvinula constellatio (Amicucci et al., 2001). T.
maculatum, T. borchii and other Tuber species also seem to actively compete with T. magnatum when artificially infected plants are planted in ‘‘truffieres’’ (Bertini et al., 2006). Altough the advances in truffle biology (Paolocci et al., 2006) many aspect remain unclear and the factors which may control the T.
magnatum life cycle, such as spore germination, mycorriza and fruit body formation, are still unknown (Mello et al., 2006). It has been shown that bacteria can promote the establishment of ectomycorrizal symbiosis (Barbieri et al., 2007) and it’s also known that some bacteria groups influence the fruit body formation of saprotrophyc or pathogenic fungi such as Agaricus bisporus and Fomitopsis pinicola, but until now there is no a complete description of the microbial network that could promote the growth and development of T. magnatum ascoma.
As a result, the fruiting bodies must be obtained from their natural place of growth, in the woods. Knowing where to look for truffles requires years of training and experience, and often requires the help of specially trained animals, for example dogs. In addition, the fruiting bodies grow only in a
34 restricted number of locations and environments. The life cycle of a truffle can be divided into stages (Martin et al., 2010) and many attempts have been made to characterize and to distinguish them in species that are very similar from a morphological point of view (e.g., white truffles group). To reach this goal conventional classification methods have been used, as well as more sophisticated analytical tools. Polymerase chain reaction (PCR) has been used to try to identify molecular markers suitable for distinguishing truffle species during their life cycle (Stocchi et al., 1995). The use of Simple Sequence Repeat (SSR) also showed the possibility to use a molecular marker to trace the T. magnatum life cycle (Paolocci et al., 2006). The results are encouraging, but different environmental origins of the fruiting bodies cannot be determined using these methods.
A proteomic approach to resolving this problem therefore appeared suitable. Initial attempts were made by French groups (Mouchès et al., 1978) using fruiting body protein analysis as a taxonomic criterion in superior fungi. This method made it possible to differentiate several Tuber species. Later these investigations were extended to other French and Italian samples, again with the aim of distinguishing between species (Duprè et al., 1984). The results were also encouraging although most efforts were then concentrated on isoenzyme analysis (Gandeboeuf et al., 1994) and the problem of differentiating areas of origin within the same species remains unresolved. High-resolution two-dimensional gel electrophoresis (2-DE) has been used in several studies to generate fungal protein maps (de Oliveira et al., 2011). Protein variability may originate from alternative splicing, post-translational modifications, and amino and carboxy-terminal modifications. Also, the formation of disulfide bonds, glycosylation, the
35 addition of lipid groups, and partial proteolysis can vary. (Navarro et al., 2006). The interaction between a particular environment and the genome can generate different proteins, different protein structures (qualitative differences) or proteins in varying amounts (quantitative differences). Recently the haploid genome of Tuber melanosporum has been sequenced (Martin et al., 2010), marking a step forward in understanding of the biology and evolution of ectomycorrhizal symbiosis.
36
1.4 Proteomics
Proteomics can be viewed as an experimental approach to explain the information contained in genomic sequences in terms of the structure, function, and control of biological processes and pathways (Aebersold and Goodlett 2001). The term "proteomics" was first coined in 1997(James 1997) to make an analogy with genomics, the study of the genes. For semi-quantitative analyses and comparisons, proteomics scientists have now access to a variety of sophisticated techniques, including mass spectrometric approaches combined with stable isotopic labelling and 2-D gel based approaches combined with differential staining or traditional visualisation and comparison techniques (Wilkins et al., 2006). The variety of methods for the characterisation of protein co- and post-translational modifications is ever-expanding, as are the means of elucidating protein-protein interactions (Wilkins et al., 2006). 2-DE couples IEF in the first dimension with SDS-PAGE in the second dimension, and enables the separaration of complex mixtures of proteins according to pI and Mr. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (2000 proteins routinely) and can detect less than 1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or PTM (Görg et al., 2004).The former limitations of carrier ampholyte (CA) based 2-DE (Whittaker et al., 1999, Jazdzewski et al., 2000) with respect to reproducibility, resolution, separation of veryacidic and/or very basic proteins, and sample loading capacity have been largely overcome by the introduction of IPGs for the first dimension of 2-DE. Narrow-overlapping pH gradients provide increased resolution (DpI = 0.001) and detection of
37 low abundance proteins, whereas alkaline proteins up to pH 12 have been separated under steady-state conditions. The major steps of the 2-DE-MS workflow include: (I) sample preparation and protein solubilization; (II) protein reduction and alkylation; (III) separation by 2-DE; (IV) protein staining; (V) spot detection and quantitation trough computer assisted analysis of 2-DE patterns; (VI) protein identification and characterization by mass spectrometry analysis (Fig 21).
Figure 21: Flow diagram of Proteomics experiment
During the decade of the 1990s, changes in MS instrumentation and techniques revolutionized protein chemistry and fundamentally changed
38 the analysis of proteins. These changes were catalyzed by two technical breakthroughs in the late 1980s: the development of the two ionization methods Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI) (Aebersold and Goodlett 2001). Because of the lack or minimal extent of analyte fragmentation during the ESI and MALDI processes, they are also referred to as “soft” ionization methods (Aebersold and Goodlett 2001). In the last few years one of the most active proteomic approaches MS-based consisted of profiling isolated cell organelles. They stand for an important organizational level of the plant cell proteome. After an organelle has been biochemically purified, it can be analyzed by mass spectrometry, which has now almost completely replaced two-dimensional (2D) gel electrophoresis as a method for proteome analysis. Many mass-spectrometry based methods have been used in organellar proteomics, the most robust and powerful of which involve one-dimensional (1D) gel electrophoresis (Andersen and Mann 2006). Advantages of this approach are that 1D gel electrophoresis is an almost universal protein-separation method, and the use of sodium dodecyl sulphate (SDS) during this step allows the efficient denaturation of proteins and the removal of buffer constituents that are detrimental to mass spectrometry.
The increasing power of mass-spectrometry based proteomics now makes it possible to characterize organelles with more than 1,000 proteins and with a dynamic range in protein abundance of several orders of magnitude. In fact, obtaining mass-spectrometry data has already ceased to be the limiting step in organellar proteomics. Instead, the main challenges are purifying the organelle and removing the background proteins. (Andersen and Mann
39 2006). Proteomic techniques are then frequently used for the discovery of differentially expressed proteins, including biomarkers (Wilkins et al., 2006). These techniques can be used in a hypothesis-independent manner, making them attractive for this purpose. Clinically useful cancer biomarkers would provide earlier and better diagnosis of patients permitting treatments to be initiated at early stages of the disease where the chance of them being effective is greatest (Blonder et al., 2011). Over the past decade, the search for biomarkers has intensified with the development of technologies that enable large numbers of biomolecules to be identified with greater throughput. For the discovery of genetic markers, these developments included massively parallel sequencing methods such as 454 Sequencing (Blonder et al., 2011).
The availability of a protein Biomarkers is also searched for the traceability of food products. A biomarker is a measurable change related to a phenotype. Molecular biomarkers are, for example, a variation in mRNA, protein, or metabolite concentration, and these should be responsive, specific, and applicable. A valid nutritional biomarker can also function as a key measure linking a specific exposure of a dietary compound to a health outcome and thus offers great potential to understand the relationship between diet and health. Biomarkers help understand nutrient absorption, transport, and metabolism within an organism to produce an effective dose at target tissue. Biomarkers of susceptibility consider host, environmental and lifestyle factors, and in particular genetic predisposition. (Kussmann et al., 2010).
In this frame, proteomics has already contributed to accumulating a relevant body of knowledge, which might result not only in improving
40 food safety through enhancing selection of food quality and traceability markers but also in providing the end-user consumer with a unique tool to make a fully aware alimentary choice (D’Alessandro and Zolla 2012) (Fig 22).
As instance, adulteration of meat has been also performed through the addition of proteins of nonanimal origins, such as soybean proteins. In this field as well, mass spectrometry-based proteomics has provided some clues (Leitner et al., 2006). Proteomics has been also applied in wines to detect either the presence of fining agents or wine-specific proteins. The majority of wine proteins are in the range of 20-30 kDa (Pastorello et al., 2003).
Figure 22: Proteomics application to food quality and safety (from D’Alessandro and Zolla 2012)
Most of the contribution of proteomic experts in food research have been so far addressing the quality issue, through the depiction of the protein
41 portraits of specific aliments, the valorization of local/regional food, or the attempts to determine the mechanisms underpinning resistance to abiotic stresses as in plant research (D’Alessandro and Zolla 2012).
