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Truffle
From Wikipedia, the free encyclopedia Jump to: navigation, search For other uses, see Truffle (disambiguation). This article needs additional citations for verification. Please help improve this article by adding citations to reliable sources. Unsourced material may be challenged and removed. (April 2010)

Black Prigord Truffle

Crispy veal sweetbreads filled with truffle A truffle is the fruiting body of a subterranean mushroom; spore dispersal is accomplished through fungivores, animals that eat fungi. Almost all truffles are ectomycorrhizal and are therefore usually found in close association with trees. There are hundreds of species of truffles. The fruiting body of some (mostly in the genus Tuber) are highly prized as a food: Brillat-Savarin called them "the diamond of the kitchen". Edible truffles are held in high esteem in French, Spanish, northern Italian and Greek cooking, as well as in international haute cuisine.

Contents
1 Etymol ogy 2 Biology 3 Types 3 . 1 W h i t e t r u f f l e 3 . 2 B l a c k t r u f f l e 3 . 3 S u m m

Etymology
The origin of the word truffle appears to be the Latin term tuber, meaning "swelling" or "lump", which became tufer- and gave rise to the various European terms: French truffe, Spanish trufa, Danish trffel, German Trffel, Swedish tryffel, Dutch truffel, Polish trufel, Serbian /tartuf and Croatian tartuf. In Portuguese, the words trufa and tbera are synonyms, the latter closer to the Latin term. The German word Kartoffel ("potato") is derived from the Italian tartufo (truffle) because of superficial similarities.[1]

Biology
The mycelia of truffles form symbiotic relationships with the roots of several tree species including beech, poplar, oak, birch, hornbeam, hazel, and pine.[2][3] They prefer argillaceous or calcareous soils which are well drained and neutral or alkaline.[4][5] Truffles fruit throughout the year, depending on the species and can be found buried between the leaf litter and the soil.

Types
White truffle

White truffle washed and cut The "white truffle" or "Alba madonna" (Tuber magnatum) comes from the Langhe and Montferrat areas[6] of the Piedmont region in northern Italy and, most famously, in the countryside around the cities of Alba and Asti;[7] in Italy it can also be found in Molise and in the hills around San Miniato, in Tuscany. It is also found in Croatia, on the Istria peninsula in the Motovun forest alongside Mirna river.[8] Growing symbiotically with oak, hazel, poplar and beech and fruiting in autumn, they can reach 12 cm diameter and 500 g, though are usually much smaller. The flesh is pale cream or brown with white marbling.[9] Italian white truffles are very highly esteemed (illustration, left) and are the most valuable on the market: The white truffle market in Alba is busiest in the months of October and November when the Fiera del Tartufo (truffle fair) takes place. In 2001, the Tuber magnatum truffles sold for between 10002200 USD per pound (20004500 USD per kg);[10] as of December 2009 they were being sold at 14,203.50 USD per kilogram. Giancarlo Zigante and his dog Diana found one of the largest truffles in the world near Buje, Croatia. The truffle weighed 1.31 kilograms (2 lb 14 oz) and has entered the Guinness Book of Records.[11] The record price paid for a single white truffle was set in December 2007, when Macau casino owner Stanley Ho paid 330,000 USD (165,000) for a specimen weighing 1.5 kilograms (3.3 lb), discovered by Luciano Savini and his dog Rocco. One of the largest truffles found in decades, it was unearthed near Pisa and sold at an auction held simultaneously in Macau, Hong Kong and Florence.[12] This record was then matched on November 27, 2010 when Ho again paid 330,000

USD for a pair of white truffles, including one weighing nearly a kilogram. The Tuber magnatum pico white truffle is found mostly in northern and central Italy, while the Tuber borchii, or whitish truffle, is found in Tuscany, Romagna, the Marche and Molise. Neither of these is as aromatic as those from Piedmont, although those from Citt di Castello come quite close. [9]

Black truffle

Black Prigord Truffle, cut The "black truffle" or "black Prigord truffle" (Tuber melanosporum) is named after the Prigord region in France and grows with oak and hazelnut trees. Specimens can be found in late autumn and winter, reaching 7 cm in diameter and weighing up to 100 g.[9] Production is almost exclusively European, with France accounting for 45%, Spain 35%, Italy 20%, and small amounts from Slovenia, Croatia and the Australian states of Tasmania and Western Australia (see below). Recently large amounts have been found in Serbia.[13] In 1937, France produced around 1,000 metric tonnes (1,100 short tons) of Tuber melanosporum. Production has considerably diminished in the past century, and is now around 20 metric tons (22 short tons) per year, with peaks at 46 metric tons (50 short tons) in the best years. About 80% of the French production comes from southeast France: upper Provence (dpartements of Vaucluse and Alpes-de-Haute-Provence), part of Dauphin (dpartement of Drme), and part of Languedoc (dpartement of Gard); 20% of the production comes from southwest France: Quercy (dpartement of Lot) and Prigord. The largest truffle market in France (and probably also in the world) is at Richerenches in Vaucluse. The largest truffle market in southwest France is at Lalbenque in Quercy. These markets are busiest in the month of January, when the black truffles have their highest perfume. As of December 2009, black truffles were sold for about 1,000 per kilo in a farmer's market[14] and 3,940 per kilo in a retail seller. The genome sequence of the Prigord black truffle was published in March 2010.[15]

Summer or burgundy truffle


Main article: Summer truffle

Black summer truffle (in Italian: Tartufi Neri Estivi) in a shop window in Rome, Italy

Desert truffle - Terfezia spp. from Avanos, Turkey The black summer or burgundy truffle (Tuber aestivum/uncinatum) is found across Europe and is prized for its culinary value. Two varieties are distinguished within this species: burgundy truffles, harvested in autumn until December, and summer truffles, harvested in summer, whose flesh is of paler color and whose aroma is less pronounced.

Other species
Two lesser-used truffles include the "black truffle" (Tuber macrosporum) and the "Scorzone truffle" (Tuber aestivum). In the U.S. Pacific Northwest, several species of truffle are harvested both recreationally and commercially, most notably, the "Oregon white truffles", Tuber oregonense and Tuber gibbosum. The "pecan truffle" (Tuber lyonii)[16] syn. texense[17] is found in the Southern United States, usually associated with pecan trees. Chefs who have experimented with them agree "they are very good and have potential as a food commodity".[18] Although pecan farmers used to find them along with pecans and discard them, considering them a nuisance, they sell for about $100 a pound and have been used in some gourmet restaurants.

Truffle-like species
The term "truffle" has been applied to several other genera of similar underground fungi. The genera Terfezia and Tirmania of the family Terfeziaceae are known as the "desert truffles" of Africa and the Middle East. "Hart's truffle" is a name for Elaphomycetaceae. Pisolithus tinctorius, which was historically eaten in parts of Germany, is sometimes called "Bohemian truffle".[19]

History
Antiquity
The first mention of truffles appears in the inscriptions of the neo-Sumerians regarding their Amorite enemy's eating habits (Third Dynasty of Ur, 20th century BCE)[20] and later in writings of Theophrastus in the fourth century BC. In classical times, their origins were a mystery that challenged many; Plutarch and others thought them to be the result of lightning, warmth and water in the soil, while Juvenal thought thunder and rain to be instrumental in their origin. Cicero deemed them children of the earth, while Dioscorides thought they were tuberous roots.[19] Italy in the Classical period produced three kinds of truffles: the Tuber melanosporum, the Tuber magnificanus and the Tuber magnatum. The Romans, however, only used the terfez (Terfezia bouderi), a fungus of similar appearance, which the Romans called truffles, and which is sometimes called "desert truffle". Terfez used in Rome came from Lesbos, Carthage, and especially Libya, where the coastal climate was less dry in ancient times.[19] Their substance is pale, tinged with

rose. Unlike truffles, terfez have no taste of their own. The Romans used the terfez as a carrier of flavour, because the terfez have the property to absorb surrounding flavours. Indeed, Ancient Roman cuisine used many spices and flavours, and terfez were perfect in that context.

Middle Ages
Truffles were rarely used during the Middle Ages. Truffle hunting is mentioned by Bartolomeo Platina, the papal historian, in 1481, when he recorded that the sows of Notza were without equal in hunting truffles, but they should be muzzled to prevent them from eating the prize.[21]

Renaissance and modern times


During the Renaissance, truffles regained popularity in Europe and were honoured at the court of King Francis I of France. However, it was not until the 17th century that Western (and in particular French) cuisine abandoned "heavy" oriental spices, and rediscovered the natural flavour of foodstuffs. Truffles were very popular in Parisian markets in the 1780s. They were imported seasonally from truffle grounds, where peasants had long enjoyed their secret. Brillat-Savarin (1825) noted characteristically that they were so expensive they appeared only at the dinner tables of great nobles and kept women. A great delicacy was a truffled turkey.

Cultivation
Truffles long eluded techniques of domestication, as Jean-Anthelme Brillat-Savarin (1825) noted:

Mushroom and truffle output in 2005

Statue of Joseph Talon in Saint-Saturnin-ls-Apt

Planted truffle groves near Beaumont-du-Ventoux

"The most learned men have sought to ascertain the secret, and fancied they discovered the seed. Their promises, however, were vain, and no planting was ever followed by a harvest. This perhaps is all right, for as one of the great values of truffles is their dearness, perhaps they would be less highly esteemed if they were cheaper." However, truffles can be cultivated. As early as 1808, there were successful attempts to cultivate truffles, known in French as trufficulture. People had long observed that truffles were growing among the roots of certain trees, and in 1808, Joseph Talon, from Apt (dpartement of Vaucluse) in southern France, had the idea to sow some acorns collected at the foot of oak trees known to host truffles in their root system. The experiment was successful: Years later, truffles were found in the soil around the newly grown oak trees. In 1847, Auguste Rousseau of Carpentras (in Vaucluse) planted 7 hectares (17 acres) of oak trees (again from acorns found on the soil around truffle-producing oak trees), and he subsequently obtained large harvests of truffles. He received a prize at the 1855 World's Fair in Paris. These successful attempts were met with enthusiasm in southern France, which possessed the sweet limestone soils and dry, hot weather that truffles need to grow. In the late 19th century, an epidemic of phylloxera destroyed many of the vineyards in southern France. Another epidemic destroyed most of the silkworms there, too, making the fields of mulberry trees useless. Thus, large tracts of land were set free for the cultivation of truffles. Thousands of truffle-producing trees were planted, and production reached peaks of hundreds of tonnes at the end of the 19th century. In 1890, there were 75,000 hectares (190,000 acres) of truffle-producing trees.

Truffle market in Carpentras In the 20th century, however, with the growing industrialization of France and the subsequent rural exodus, many of these truffle fields (champs truffiers or truffires) returned to wilderness. The First World War also dealt a serious blow to the French countryside, killing 20% or more of the male working force. As a consequence of these events, newly acquired techniques of trufficulture were lost. Also, between the two world wars, the truffle groves planted in the 19th century stopped being productive. (The average life cycle of a truffle-producing tree is 30 years.) Consequently, after 1945, the production of truffles plummeted, and the prices have risen dramatically. In 1900, truffles were used by most people, and on many occasions. Today, they are a rare delicacy reserved for the rich, or used on very special occasions. In the last 30 years, new attempts for mass production of truffles have been started. Eighty percent of the truffles now produced in France come from specially planted truffle groves. Nonetheless, production has yet to recover its 1900s peaks. Local farmers are opposed to a return of mass production, which would decrease the price of truffles. There are now truffle-growing areas in the United Kingdom, United States, Spain, Sweden, New Zealand, Australia and Chile.

In New Zealand and Australia


The first black truffles (Tuber melanosporum) to be produced in the southern hemisphere were harvested in Gisborne, New Zealand in 1993.[22] In 1999, the first Australian truffles were harvested in Tasmania,[citation needed] the result of eight years of work. Trees were inoculated with the truffle fungus in the hope of creating a local truffle industry. Their success and the value of the resulting truffles has encouraged a small industry to develop. A Western Australian venture, The Wine and Truffle Company, had its first harvest in 2004, and in 2005 they unearthed a 1 kg truffle. In 2008, an estimated 600 kilograms (1,300 lb) of truffles were removed from the rich ground of Manjimup. Each year The Wine and Truffle Company has expanded its production, moving into the colder regions of Victoria and New South Wales. In June 2010, Tasmanian growers Michael and Gwynneth Williams harvested Australia's largest truffle from their property at Myrtle Bank, near Launceston. It weighed in at 1.084 kilograms (2 lb 6.2 oz).[23] Mrs. Williams told ABC Radio in Australia[24] that it is valued at approximately A$1,500 per kg. New Zealand's first burgundy truffle was found in July 2012, at a Waipara truffle farm. It weighed 330 g, and was found by Rosie, the farm owner's beagle.[25]

Extraction

Trained pig in Gignac, Lot, France

Trained dog in Mons, Var Looking for truffles in open ground is almost always carried out with specially trained pigs (truffle hogs) or, more recently, dogs. The Lagotto Romagnolo is currently the only dog breed recognized for sniffing out truffles (although virtually any breed could be trained for this purpose).[26] Truffle Hog Truffle Dog Keen sense of smell Keen sense of smell Innate ability to sniff out truffles Must be trained Tendency to eat truffles once found Easier to control

The female pig's natural truffle seeking, as well as her usual intent to eat the truffle, is due to a compound within the truffle similar to androstenol, the sex pheromone of boar saliva, to which the sow is keenly attracted. In Italy the use of the pig to hunt truffles is prohibited since 1985 due to damage caused by animals to truffle's mycelia during the digging that drop the production rate of the area for some years.

Culinary use

Truffle oil (olive oil with Tuber melanosporum). Because of their high price and their pungent taste, truffles are used sparingly. Supplies can be found commercially as unadulterated fresh produce or preserved, typically in a light brine. White truffles are generally served raw, and shaved over steaming buttered pasta or salads or fried eggs (this last is recommended by many gourmets as the best way to enjoy the flavour). White or black paper-thin truffle slices may be inserted into meats, under the skins of roasted fowl, in foie gras preparations, in pts, or in stuffings. Some speciality cheeses contain truffles as well. The flavor of black truffles is far less pungent and more refined than that of white truffles. Its strong flavor is often described as syrupy sweet. While in the past chefs used to peel truffles, in modern times most restaurants brush the truffle carefully and shave it or dice it with the skin on so as to make the most of this valuable ingredient. A few restaurants, such as Philippe Rochat in Switzerland, still stamp out circular discs of truffle flesh and use the skins for sauces.

Truffle oil
Main article: Truffle oil Truffle oil is often used as a lower cost and convenient substitute for truffles, to provide flavoring or to enhance the flavor and aroma of truffles in cooking. Most "truffle oil," however, does not contain any truffles.[27] The vast majority is olive oil which has been artificially flavoured using a synthetic agent such as 2,4-dithiapentane.[27] Daniel Patterson reported in the New York Times that "even now, you will find chefs who are surprised to hear that truffle oil does not actually come from real truffles."

Truffle vodka
Main article: Truffle vodka The bulk of truffle oil on the market is made with a synthetic ingredient like 2,4-dithiapentane, as are many other truffle products. However, alcohol is now being used to carry the truffle flavour without the need for synthetic flavourings. The first truffle vodka, Black Moth Vodka, is a natural vodka infused with black Prigord truffles (Tuber melanosporum). Although primarily used as a spirit in its own right and mixed in a range of cocktails, truffle vodka is also used by various chefs to flavour dishes by evaporating the alcohol through cooking whilst retaining the truffle aroma.[28]

Notes
1. ^ Simpson J, Weiner E.(eds) (1989) Oxford English Dictionary, 2nd edition, Clarendon Press, ISBN 0-19-861186-2 2. ^ "finds registered at Royal Botannical Gardens, Kew". Truffle UK Ltd.. Retrieved 200805-17. 3. ^ "Non-cultivated Edible Fleshy Fungi". Retrieved 2008-05-17. "...it has been known for more than a century that truffles were mycorrhizal on various trees such as oak, beech, birch, hazels, and a few others" 4. ^ Karen Hansen (Spring 2006). "Basidiomycota truffles: Cup fungi go underground". In K. Griffith (PDF). Newsletter of the FRIENDS of the FARLOW. Harvard University. Retrieved 2008-05-17. "Generally, truffles seems to prefer. warm, fairly dry climates and calcareous soils" 5. ^ "Mushroom Production". Mycology - Uses of Fungi. University of Sydney. June, 2004. Archived from the original on 2008-05-01. Retrieved 2008-05-17. "The soil of the truffiere tends to be alkaline, calcareous, and well drained." 6. ^ "White truffles from Alba". Lifeinitaly.com. Retrieved 2012-06-16. 7. ^ "Wine and Truffles Adventure - Piemonte". Savoryadventures.com. Retrieved 2012-06-16. 8. ^ etina. "Gastro.croatia.hr". Gastro.croatia.hr. Retrieved 2012-06-16. 9. ^ a b c Carluccio A (2003). The Complete Mushroom Book. Quadrille. ISBN 1-84400-040-0. 10.^ "Education & Networking | National Restaurant Association | National Restaurant Association". Restaurant.org. Retrieved 2012-06-16. 11.^ "Largest truffle". Guinnessworldrecords.com. 1999-11-02. Retrieved 2012-06-16. 12.^ "Giant truffle sets record price". BBC News. 2007-12-02. Retrieved 2007-12-02. 13.^ "KURIR". Arhiva.kurir-info.rs. Retrieved 2012-06-16. 14.^ "Jean-Michel Fabre, "Lalbenque. 1 000 le kg: la truffe de Nol au prix du diamant", 23/12/2009". Ladepeche.fr. 2009-12-23. Retrieved 2012-06-16. 15.^ Martin, Francis; Kohler, Annegret; Murat, Claude; et al. (2010), "Prigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis", Nature 464 (7291), doi:10.1038/nature08867, PMID 20348908 16.^ Fred K. Butters. "A Minnesota Species of Tuber". Botanical Gazette 35 (6): 427431. JSTOR 2556357.

17.^ J.M. Trappe, A.M. Jumpponen and E. Czares (1996). "NATS truffle and truffle-like fungi 5: Tuber lyonii (=T. texense), with a key to the spiny-spored Tuber species groups". Mycotaxon 60: 365372. 18.^ Tim Brenneman. "Pecan Truffles". Retrieved 2010-06-03. 19.^ a b c Ramsbottom J (1953). Mushrooms & Toadstools. Collins. ISBN. 20.^ Chiera, E. (1934), "Nos. 58 and 112", Sumerian Epics and Myths, Chicago 21.^ Benjamin, D. R. (1995), "Historical uses of truffles", Mushrooms: Poisons and Panaceas A Handbook for Naturalists, Mycologists and Physicians, New York: WH Freeman and Company, pp. 4850, ISBN 0716726009 22.^ "Truffles in New Zealand". Southern_truffles.co.nz. Retrieved 2012-07-19. 23.^ "Northeast growers break record with 1084g truffle find". The Examiner. 27 June 2010. 24.^ Australia's ABC Radio, Local Radio network, "Australia All Over" program, 27 June 2010 25.^ "Beagle digs up a New Zealand first". stuff.co.nz. Retrieved 2012-07-26. 26.^ Krista Simmons (28 August 2009). "On the hunt for truffles in Western Australia". Los Angeles Times. Retrieved 2009-08-31. "Traditionally, truffle hunters the Aussies call them "punters" have used pigs to track their prey. More recently, punters have started using dogs, which, unlike pigs, will settle for a biscuit instead of chowing down on the truffle." 27.^ a b Daniel Patterson (16 May 2007). "Hocus-Pocus, and a Beaker of Truffles". New York Times. Retrieved 2008-05-17. "Most commercial truffle oils are concocted by mixing olive oil with one or more compounds like 2,4-dithiapentane" 28.^ "Truffle vodka article". Mycorrhizalsystems.com. 2010-04-21. Retrieved 2012-06-16.

References
Trappe, Matt; Evans, Frank; Trappe, James M. (2007). Field Guide to North American Truffles: Hunting, Identifying, and Enjoying the World's Most Prized Fungi. Natural History Series. Ten Speed Press. ISBN 9781580088626. Brillat-Savarin, Jean Anthelme (1838) [1825]. Physiologie du got. Paris: Charpentier. pp. 109118. English translation

External links
Wikimedia Commons has media related to: truffle Website of the North American Truffling Society "In the throes of truffle fever" by Tyrone Beason, Seattle Times, January 4, 2007 Sorting truffle names

Letter
Nature 464, 1033-1038 (15 April 2010) | doi:10.1038/nature08867; Received 20 August 2009; Accepted 28 January 2010; Published online 28 March 2010

Prigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis
Francis Martin1, Annegret Kohler1, Claude Murat1, Raffaella Balestrini2, Pedro M. Coutinho3, Olivier Jaillon4,5,6, Barbara Montanini7, Emmanuelle Morin1, Benjamin Noel4,5,6, Riccardo

Percudani7, Bettina Porcel4,5,6, Andrea Rubini8, Antonella Amicucci9, Joelle Amselem10, Vronique Anthouard4,5,6, Sergio Arcioni8, Franois Artiguenave4,5,6, Jean-Marc Aury4,5,6, Paola Ballario11, Angelo Bolchi7, Andrea Brenna11, Annick Brun1, Marc Bue1, Brandi Cantarel3, Grard Chevalier12, Arnaud Couloux4,5,6, Corinne Da Silva4,5,6, France Denoeud4,5,6, Sbastien Duplessis1, Stefano Ghignone2, Benot Hilselberger1,10, Mirco Iotti13, Benot Marais1, Antonietta Mello2, Michele Miranda14, Giovanni Pacioni15, Hadi Quesneville10, Claudia Riccioni8, Roberta Ruotolo7, Richard Splivallo16, Vilberto Stocchi9, Emilie Tisserant1, Arturo Roberto Viscomi7, Alessandra Zambonelli13, Elisa Zampieri2, Bernard Henrissat3, Marc-Henri Lebrun17, Francesco Paolocci8, Paola Bonfante2, Simone Ottonello7 & Patrick Wincker4,5,6 1. INRA, UMR 1136, INRA-Nancy Universit, Interactions Arbres/Microorganismes, 54280 Champenoux, France 2. Istituto per la Protezione delle Piante del CNR, sez. di Torino and Dipartimento di Biologia Vegetale, Universit degli Studi di Torino, Viale Mattioli, 25, 10125 Torino, Italy 3. Architecture et Fonction des Macromolcules Biologiques, UMR 6098 CNRS-Universits Aix-Marseille I & II, 13288 Marseille, France 4. CEA, IG, Genoscope, 2 rue Gaston Crmieux CP5702, F-91057 Evry, France 5. CNRS, UMR 8030, 2 rue Gaston Crmieux, CP5706, F-91057 Evry, France 6. Universit dEvry, F-91057 Evry, France 7. Dipartimento di Biochimica e Biologia Molecolare, Universit degli Studi di Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy 8. CNR-IGV Istituto di Genetica Vegetale, Unit Organizzativa di Supporto di Perugia, via Madonna Alta, 130, 06128 Perugia, Italy 9. Dipartimento di Scienze Biomolecolari, Universit degli Studi di Urbino, Via Saffi 2 - 61029 Urbino (PU), Italy 10.INRA, Unit de Recherche Gnomique Info, Route de Saint-Cyr, 78000 Versailles, France 11.Dipartimento di Genetica e Biologia Molecolare & IBPM (CNR), Universit La Sapienza, Roma, Piazzale, A. Moro 5, 00185 Roma, Italy 12.INRA, UMR Amlioration et Sant des Plantes, INRA-Universit Blaise Pascal, INRA Clermont-Theix, 63122 Saint-Genes-Champanelle, France 13.Dipartimento di Protezione e Valorizzazione Agroalimentare, Universit degli Studi di Bologna, 40 126 Bologna, Italy 14.Dipartimento di Biologia di Base ed Applicata, 15.Dipartimento di Scienze Ambientali, Universit degli Studi dellAquila, Via Vetoio Coppito 1 - 67100 LAquila, Italy 16.University of Goettingen, Molecular Phytopathology and Mycotoxin Research, Grisebachstrasse 6, D-37077 Goettingen, Germany 17.INRA, UMR BIOGER-CPP, INRA-Grignon, av Lucien Brtignires - 78850 Thiverval Grignon, France Correspondence to: Francis Martin1 Correspondence and requests for materials should be addressed to F.M. (Email: fmartin@nancy.inra.fr). This article is distributed under the terms of the Creative Commons Attribution-Non-CommercialShare Alike licence (http://creativecommons.org/licenses/by-nc-sa/3.0/), which permits distribution, and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation, and derivative works must be licensed under the same or similar licence. Top of page

Abstract
The Prigord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate todays truffle market1, 2. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont3 endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a symbiosis toolbox4. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species5. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at ~125megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ~58% of the genome. In contrast, this genome only contains ~7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosisthe symbiosis toolboxevolved along different ways in ascomycetes and basidiomycetes. The 125-megabase (Mb) genome of T. melanosporum is the largest sequenced fungal genome to date6, but no evidence for whole-genome duplication or large scale dispersed segmental duplications was observed (Supplementary Table 1 and Supplementary Information section 2). The approximately fourfold larger size of the truffle genome compared with other sequenced ascomycetes is accounted for by multi-copy transposable elements (TE) which constitute about 58% of the assembled genome (Fig. 1, Supplementary Figs 5, 6 and 8, Supplementary Information section 3). Estimated insertion times suggest a major wave of retrotransposition at <5 million years ago (Supplementary Fig. 7). TEs are not uniformly spread across the genome, but are clustered in gene-poor or gene-lacking regions (Fig. 1 and Supplementary Fig. 8). The expansion of regions between blocks of protein-coding genes results from an increased density of TEs. The proliferation of TEs within the truffle genome may result from its low effective population size7 during postglaciation migrations8 (Supplementary Information section 2.5).
Figure 1: Genomic landscape of T. melanosporum.

a, The area chart quantifies the distribution of transposable elements (TE) and protein-coding genes (Gene models) along supercontig 5. The y axis represents the percentage of base pairs corresponding to TE (red), genes (blue), and other regions and gaps (white) in 10,000-bp sliding windows. b, Heat maps display the distribution of selected elements, including simple sequence repeats (SSR), gene models, all TE, long terminal repeat retrotransposons (class I LTR), long interspersed elements (class I LINE), terminal inverted repeats (class II TIR), and unknown TE classes (TE no cat.). Abundance of TE, protein-coding genes and other sequences is represented by a colour scale from 0 (white) to 9 occurrences (black) per 10kbp window. High resolution image and legend (217K)Download Power Point slide (602K) Slides may be downloaded for educational use, according to the terms described in Nature

Publishing Group's licensing policy. The predicted proteome is in the lower range of sequenced filamentous fungi6, as only 7,496 protein-coding genes were identified (Supplementary Information section 4). They are mainly located in TE-poor regions and the gene density is heterogeneous when compared with that of other ascomycetes (Fig. 1, Supplementary Figs 8 and 9). Among the predicted proteins, only 3,970, 5,596 and 5,644 showed significant sequence similarity to proteins from Saccharomyces cerevisiae, Neurospora crassa and Aspergillus niger, respectively (Supplementary Fig. 10). This agrees with the predicted ancient separation (>450Myr ago) of the Pezizomycetes from the other ancestral fungal lineages (Supplementary Fig. 4)9. Of the ~5,650 T. melanosporum genes that have an orthologue, very few show conservation of neighbouring orthologues (synteny) in at least one of the other species (Supplementary Fig. 11, Supplementary Information section 5.2). The T. melanosporum genome shows a structural organization strikingly different from other sequenced ascomycetes; the largest syntenic region (with Coccidioides immitis) only contains 99 genes with 39 orthologues (Supplementary Fig. 12). TE proliferation probably facilitated genome rearrangements. Some regions of meso-synteny were however detected, suggesting that T. melanosporum could be used for assessing the genome organization of ancestral ascomycete clades. Expression of most predicted genes was detected in free-living mycelium, ectomycorrhizal (ECM) root tips and/or fruiting body by custom oligoarrays, expressed-sequence-tag pyrosequencing and Illumina RNA-Seq (Supplementary Information sections 2.4 and 8, Supplementary Table 2, Supplementary Fig. 26). Only a low proportion of transcripts (7.6%) is differentially expressed (fold-ratio 4.0, P<0.05) in either ectomycorrhiza or fruiting body by comparison to free-living mycelium (Table 1, Supplementary Table 4). Only 61 transcripts unique to ectomycorrhiza, fruiting body or free-living mycelium were detected (Supplementary Table 5). A few transcripts coding for a H-type lectin, an arabinogalactan protein, a LysM-domain containing protein, major facilitator superfamily (MFS) transporters, laccase/tyrosinase, a lipase and polysaccharide-degrading enzymes are strikingly enriched (>1,000-fold) in symbiotic tissues (Table 1). They may play a role in adhesion to host cells, detoxication of plant defence metabolites, nutrient exchange, and colonization of root apoplast through the deconstruction of cell walls.
Table 1: The most highly upregulated transcripts in T. melanosporum/Corylus avellana ECM root tips

Full table A process that is crucial to the success of ECM interactions is the mutualistic exchange of nutrients between the microsymbiont and its host plant. A comparison with other fungi revealed that the total number of predicted transporters is lower in T. melanosporum (381 members) compared with L. bicolor (491 members) as well as with saprotrophic and pathogenic ascomycetes (481781 members) (Supplementary Table 26). However, 64 predicted membrane transporters showed an upregulated expression in truffle ectomycorrhizas, suggesting increased fluxes of carbohydrates, oligopeptides, amino acids and polyamines at the symbiotic interface (Supplementary Table 27). PFAM classification of fungal genes induced in symbiotic tissues of either L. bicolor or T. melanosporum ECM root tips revealed strikingly divergent fungal symbiotic proteomes (Supplementary Fig. 15). However, the PFAM categories corresponding to the MFS transporters (PFAM00083), aquaporin-related major intrinsic proteins (PFAM00230) and amino acid permeases (PFAM000324) were among the most strongly overrepresented in genes that were transcriptionally upregulated in both L. bicolor and T. melanosporum ectomycorrhizas.

Orthologous genes (that is, reciprocal best hits, BLASTP e-value 10-5) significantly induced in the symbiosis represent only 1.5% and 4.1% of the ectomycorrhiza-upregulated genes in both T. melanosporum and L. bicolor, respectively. Most of these rare transcripts code for membrane transporters involved in sugar, amino acid or sulphate uptake (Table 2). This transcriptome trait appears to be a hallmark of the mycorrhizal symbiosis. The resulting increased nutrient flux probably explains the beneficial effect of the symbionts on the growth of their host seedlings (Supplementary Information section 1 and Supplementary Fig. 3). Other overrepresented PFAM categories displayed different patterns in the two symbionts. None of the effector-like small secreted MiSSP proteins specifically expressed in L. bicolor ectomycorrhizas4 were detected among ectomycorrhiza-regulated T. melanosporum transcripts.
Table 2: Orthologous symbiosis upregulated genes of L. bicolor and T. melanosporum

Full table One of the most striking characteristics of the T. melanosporum genome is the almost complete absence of highly similar gene pairs. Of the predicted 7,496 protein-coding genes, only seven pairs share >90% amino-acid identity in their coding sequence, whereas 30 pairs share >80% identity (Supplementary Information section 5.3, Fig. 2). This feature was also observed in the ascomycetous saprotroph N. crassa10. In striking contrast to the ECM L. bicolor4, multigene families in T. melanosporum are limited in number and comprise only 19% of the predicted proteome; most families have only two members (Supplementary Fig. 13). The rate of gene family gain is much lower than the rate of gene loss and among the 11,234 gene families found in ascomycetes, 5,695 appear to be missing in T. melanosporum (Supplementary Information section 5.4, Fig. 2). This compact gene coding space may reflect the genome organization of an ascomycete common ancestor, as the Pezizomycetes clade is the earliest diverging lineage within the Pezizomycotina (Supplementary Fig. 4). By comparison to other ascomycetes, gene families predicted to encode metabolite transporters (for example, amino acid and sugar permeases) and secondary metabolism enzymes (such as polyketide synthases and cytochrome P450s) are much smaller. Only 465 genes encoded by expanding gene families of L. bicolor are also found in the T. melanosporum genome (BLASTP, e-value 10-5) and 154 orthologues are shared between expanding gene families of both symbionts. None of them is differentially expressed in ectomycorrhizas. Differences in gene family expansion, in particular dynamic repertoires of genes encoding symbiosis-regulated effector-like proteins and sugar-cleaving enzymes (see below), are probably responsible for different symbiotic traits between T. melanosporum and L. bicolor, such as altered host specificity. The compact genome of T. melanosporum might be a product of selection for specialization; this is because genome expansion, as observed in L. bicolor, is probably driven by selection on the symbiont to exploit a diversity of encountered substrates provided by multiple potential hosts and by their diverse soils4, 5.
Figure 2: Genome redundancy in the truffle genome.

a, The percentage of amino-acid identity of the top-scoring self-matches for protein-coding genes in T. melanosporum, Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, Magnaporthe grisea, and Botrytis cinerea. For each fungus, the protein-coding regions for each gene were compared with those of every other gene in the same genome using BLASTX. b, The

figure represents the total number of protein families in each species or node. The numerals on branches show numbers of expanded (left, red), unchanged (middle, black) or contracted (right, blue) protein families along lineages by comparison to the putative pan-proteome. High resolution image and legend (127K)Download Power Point slide (514K) Slides may be downloaded for educational use, according to the terms described in Nature Publishing Group's licensing policy. The volatiles released by truffles are attractive to rodents and truffle flies11, which disperse their spores, but also to humans who consider this elusive mushroom a delicacy. T. melanosporum is the first sequenced fungus producing highly flavoured hypogeous fruiting bodies (Supplementary Information section 6.4, Fig. 3). Genomic signatures of the long-standing (>2,000-year-old) reputation of the black truffle as a gastronomic delicacy are its extremely low allergenic potential (Supplementary Fig. 18), coupled with the lack of key mycotoxin biosynthetic enzymes (Supplementary Information section 6.2, Supplementary Table 14), and the preferential overexpression of various flavour-related enzymes in the fruiting body (Supplementary Figs 19 21). Among the latter are specific subsets of sulphur assimilation and S-amino acid interconversion enzymes. These include cystathionine lyases known to promote the side-formation of methyl sulphide volatiles abundant in truffles12 as well as various enzymes involved in amino acid degradation through the Ehrlich pathway which are giving rise to known truffle volatiles and flavours, for example, 2-methyl-1-butanal (Fig. 3, Supplementary Information section 6.4, Supplementary Figs 20 and 21). Also notable, given the subterranean habitat of this fungus, is the presence of various putative light-sensing components (Supplementary Information section 6.6), which might be involved in light avoidance mechanisms and/or in the control of seasonal developmental variations, especially those related to fruiting body formation and sexual reproduction.
Figure 3: Outline of sulphur metabolism in T. melanosporum fruiting body.

Numbers identify enzymes and gene models as specified in Supplementary Table 15. Reactions (arrows) catalysed by enzymes whose mRNAs are upregulated in fruiting bodies are shown in red; mRNAs downregulated by at least twofold in fruiting bodies, or whose expression levels differ by less than twofold compared to free-living mycelia and ectomycorrhizas are represented by green and black arrows, respectively. Off-pathway cystathionine--lyase (no. 22)- and cystathionine lyase (no. 20)-supported reactions, and spontaneous (non-enzymatic) breakdown reactions are indicated by grey and dashed arrows, respectively. APS, adenosine phosphosulphate; PAPS, phosphoadenosine phosphosulphate; PAP, phosphoadenosine phosphate; Trx, thioredoxin; SAM, Sadenosylmethionine; SAH, S-adenosylhomocysteine; DMS, dimethylsulphide; DMDS, dimethyldisulphide; DMTS, dimethyl-trisulphide. High resolution image and legend (275K)Download Power Point slide (660K) Slides may be downloaded for educational use, according to the terms described in Nature Publishing Group's licensing policy. The analysis of genes implicated in the mating process, including pheromone response, meiosis and fruiting body development showed that most sex-related components identified in other ascomycetes are also present in T. melanosporum (Supplementary Table 11). Sexual reproduction in ascomycete filamentous fungi is partly controlled by two different mating-type (MAT) genes that

establish sexual compatibility13: one MAT gene codes for a protein with an -box domain, whereas the other encodes a high mobility group (HMG) DNA binding protein (Supplementary Information section 6.5). It was widely believed that T. melanosporum was a homothallic or even an exclusively selfing species14. The sequenced Mel28 strain contains the HMG locus, and the opposite linked MAT locus was identified in another natural isolate (Supplementary Fig. 22), confirming recent hints that T. melanosporum is heterothallic and thus an obligate outcrossing species15. This result has major implications for truffle cultivation, which will be improved by the use of host plants harbouring truffle strains of opposite mating types. In most ascomycetes, the genomic regions flanking the MAT locus show an extended conservation13, but there is no synteny of the MAT loci between T. melanosporum and other sequenced fungi (Supplementary Fig. 23). To determine whether T. melanosporum sugar-cleaving capabilities resemble those of other fungi, we have undertaken a comparison of the glycoside hydrolase (GH) and polysaccharide lyase (PL) repertoires16 of 18 completely sequenced fungi (Fig. 4). As expected for a symbiotic fungus living in the root apoplast, T. melanosporum has a relatively small number of GH-encoding genes (91 members; Supplementary Tables 23 and 24); much fewer than phytopathogens (for example, Magnaporthe grisea, Fusarium graminearum) and saprotrophs (for example, N. crassa, Podospora anserina). The T. melanosporum GH repertoire bears some similarity with that of L. bicolor4, especially a reduced spectrum of enzymes targeting the plant cell wall compared to saprobes, culminating in both fungi with the absence of cellulases from families GH6 and GH7. There are however significant differences in the spectrum of enzymes present in these two symbiotic fungi. For instance, T. melanosporum has hemicellulases from families GH10 and GH43, whereas L. bicolor has none. Similarly, T. melanosporum has a family GH45 cellulase that is absent from the L. bicolor genome. Other differences include different strategies to cleave pectin: whereas L. bicolor utilizes six hydrolytic GH28 pectinases, T. melanosporum has only two, but these are complemented by three pectin lyases and a pectin methylesterase that are missing in L. bicolor. Both fungi have a set of proteins, few in number, bearing cellulose-binding domains, but differences appear here too: the single cellulose-binding CBM1 motif of L. bicolor is appended to a GH5 endoglucanase, whereas T. melanosporum has two CBM1 motifs attached to a GH61 enzyme and to a protein of unknown function. GH61 enzymes have been reported to display weak cellulolytic activity17.
Figure 4: Double clustering of the carbohydrate-cleaving families from representative fungal genomes.

Top tree: the fungi named are Aspergillus nidulans (A_nidu), Aspergillus niger (A_nige), Aspergillus oryzae (A_oryz), Cryptococcus neoformans (C_neof), Gibberella zeae (G_zeae), Hypocrea jecorina (H_jeco), Laccaria bicolor (L_bico), Magnaporthe grisea (M_gris), Malassezia globosa (M_glob), Nectria haematococca (N_haem), Neurospora crassa (N_cras), Penicillium chrysogenum (Pe_chr), Phanerochaete chrysosporium (Ph_chr), Podospora anserina (P_anse), Postia placenta (P_plac), Saccharomyces cerevisiae (S_cere), Schizosaccharomyces pombe (S_pomb), and Tuber melanosporum (T_mela). Left tree: the enzyme families are represented by their class (GH, glycoside hydrolase; PL, polysaccharide lyase) and family number according to the carbohydrate-active enzyme database16. Right side: known substrate of CAZy families (most common forms in brackets): BPG, bacterial peptidoglycan; BEPS, bacterial exopolysaccharides; CW, cell wall; ESR, energy storage and recovery; FCW, fungal cell wall; PCW, plant cell wall; PG, protein glycosylation; U, undetermined; a-gluc, -glucans (including starch/glycogen); b-glyc, glycans; b-1,3-gluc, -1,3-glucan; cell, cellulose; chit, chitin/chitosan; dext, dextran; hemi, hemicelluloses; inul, inulin; N-glyc, N-glycans; N-/O-glyc, N- / O-glycans; pect, pectin; sucr, sucrose; and tre, trehalose. Abundance of the different enzymes within a family is represented by a

colour scale from 0 (black) to 20 occurrences (red) per species. High resolution image and legend (420K)Download Power Point slide (805K) Slides may be downloaded for educational use, according to the terms described in Nature Publishing Group's licensing policy. An extended comparison with other sequenced fungi (Fig. 4) shows that T. melanosporum clusters neither with L. bicolor nor with saprotrophic ascomycetes, most probably because of its limited overall number of GHs and PLs that make it closer to yeasts and fungi that do not interact with plants, but rather with animals (Cryptococcus neoformans, Malassezia globosa). Differences between the enzyme repertoires of T. melanosporum and L. bicolor suggest differences in the mode of interaction of the two symbionts with their respective host plants. A striking difference is the presence of an invertase gene in T. melanosporum, whereas L. bicolor has none and is therefore completely dependent on its host for its provision of glucose5. In contrast, T. melanosporum could access and hydrolyse the plant-derived sucrose. This would suggest that although both fungi develop symbiotic relationships with plants, T. melanosporum is probably less dependent than L. bicolor. The overall pattern of induction of genes coding for enzymes acting on polysaccharides is similar in both L. bicolor and T. melanosporum symbiotic transcriptomes, although a larger number of carbohydrate-cleaving enzyme transcripts are upregulated for some familiesfor example, GH16 (-1,6-glucanases), GH18 (chitinases) and GT20 (,-trehalose-phosphate synthase) in L. bicolor (Supplementary Table 25 and Supplementary Fig. 24). Intriguingly, a GH5 cellulase and a GH28 pectinase are among the rare transcripts that are highly upregulated in both L. bicolor and T. melanosporum ectomycorrhizas, suggesting that they play a key role in the symbiosis. On the other hand, the -glucan synthesis-associated protein present in both ectomycorrhizas is involved in fungal cell wall remodelling16 and may play a role in the alteration of cell wall surface during infection to conceal the hyphae from the host. The ability to establish ECM symbioses is a widespread characteristic of various ascomycetes and basidiomycetes3. The truffle genome reveals features of an ancestral fungal lineage that diverged from other lineages >450Myr ago9. Despite their similar symbiotic structures and similar beneficial effects on plant growth, the ascomycete T. melanosporum and the basidiomycete L. bicolor encode strikingly different proteomescompact with very few multigene families, versus large with many expanded multigene familiesand symbiosis-regulated genes. Effector-like proteins, such as the L. bicolor ECM-induced SSP MiSSP7 (ref. 4), are not expressed in T. melanosporum ectomycorrhizas. On the basis of our results, the ECM symbiosis appears as an ancient innovation that developed several times during the course of Mycota evolution using different molecular toolkits18. Sequencing of the T. melanosporum genome has provided unprecedented insights into the molecular bases of symbiosis, sex and fruiting in a most popular representative of the only lifestyle not yet addressed by Ascomycota genomics19. This sequencing will be a major step in moving truffle research into the realm of ecosystem science, and a deeper understanding of the genome of the Prigord black truffle is expected to have substantial social and cultural impact. Top of page

Methods Summary
A whole-genome shotgun strategy was adopted for sequencing and assembling the T. melanosporum genome (Supplementary Information section 2). All genomic DNA was obtained from the homokaryotic haploid strain Mel28. All data were generated by paired-end sequencing of cloned 3kb and 10kb inserts using Sanger technology. The pool of data available for the assembly consisted of 1,262,177 reads, with ~1,250Mb of sequence. The data were assembled using the

ARACHNE assembler. The 4,464 contigs (N50 = 62kb) were assembled in 398 supercontigs (N50 = 638kb) corresponding to 124.946Mb of sequence. The main genome scaffolds were at a depth of 10. Assemblies and annotations are available at INRA (http://mycor.nancy.inra.fr/IMGC/TuberGenome/) and Genoscope (http://www.genoscope.cns.fr/tuber). The GAZE pipeline selected a best representative gene model for each locus on the basis of expressed-sequence-tag support and similarity to known proteins from other organisms, and predicted 7,496 protein-coding gene models (Supplementary Information section 4). All predicted genes were annotated using Gene Ontology and KEGG pathways. Protein domains were predicted using InterProScan. Gene families were built from proteins using Tribe-MCL. Single dye labelling of cDNAs, hybridization procedures, data acquisition, background correction and normalization of custom-exon expression arrays were performed at the NimbleGen facilities following their standard protocol. A Student t-test with false discovery rate was applied to the data using the ARRAYSTAR software (DNASTAR). Transcripts with a significant P value (<0.05) and 4-fold change in transcript level were considered as differentially expressed in ECM root tips or fruiting body. Top of page

References
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Supplementary Information
Supplementary information accompanies this paper. Top of page

Acknowledgements
We thank the late L. Riousset and C. Dupr for providing the Mel28 isolate, and acknowledge F. Le Tacon and J. Weissenbach for continuous support. The genome sequencing of T. melanosporum was funded by the Gnoscope, Institut de Gnomique, CEA, and Agence Nationale de la Recherche (ANR). Genome annotation and transcriptome analysis were supported by INRA, the European FP6 Network of Excellence EVOLTREE, Rgion Lorraine, the ANR FungEffector project, Fondazione Cariparma, Compagnia di San Paolo-Torino, the Italian Ministry of Education, University and Research (MIUR), Regione Umbria and Instituto Pasteur Fondazione Cenci Bolognetti. We thank D. Hibbett and J. Heitman for comments on an early draft of the manuscript, and J. Plett for a critical reading of the paper. Author Contributions B.H., M.-H.L., F.P., P. Bonfante, S.O. and P.W. contributed equally to this work as senior authors; A.K., C.M., R.B., P.M.C., O.J., B.M., E.M., B.N., R.P., B.P. and A.R. contributed equally to this work as second authors. F.M. initiated the project and coordinated the genome annotation, data analysis and manuscript preparation; P.W. coordinated the sequencing and automated annotation at Genoscope. F.M. and S.O. wrote the manuscript with input from P. Bonfante. R.B., P.M.C., B.H., O.J., A.K., M.H.L., B.M., E.M., C.M., B.N., F.P., R.P., A.R. and P.W. also made substantial contributions (listed in alphabetical order). All other authors are members of the genome sequencing consortium and contributed annotation, analyses or data throughout the project, and are listed in alphabetical order. We also thank A. Bonfigli, M. Buffalini, S. Colafarina, T. Flutre, S. Kamal, P. Ceccaroli, C. Roux, R. Saltarelli, S. von Pall di Tolna and O. Zarivi for their assistance in annotation. Top of page

Author Information
Genome assemblies together with predicted gene models and annotations have been deposited at DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank under the project accession numbers CABJ01000001CABJ01004455 (whole genome shotgun sequencing data) and FN429986FN430383 (scaffolds and annotations). The complete expression dataset is available as series (accession number GSE17529) at the Gene Expression Omnibus at NCBI.

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Reevaluation of the Life Cycle of Tuber magnatum


Francesco Paolocci, Andrea Rubini, Claudia Riccioni, and Sergio Arcioni* Author information Article notes Copyright and License information This article has been cited by other articles in PMC. Go to:

Abstract
Tuber spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of Tuber biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single Tuber magnatum truffles. We then used these truffles

to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that T. magnatum outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the T. magnatum life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other Tuber spp. Tuber spp. are Ascomycetes fungi that establish an ectomycorrhizal symbiosis with trees and shrubs. As a result of this mutualistic symbiosis, ascocarps known as truffles are produced. Some Tuber spp. produce edible truffles that, given their distinctive taste and aroma, are highly valued by gourmets. Research on these fungi has focused on promoting the cultivation of these fungi to meet increasing worldwide demand and to provide replacements for the catastrophic decline in their natural production (10). Truffle cultivation is no longer an agronomic practice confined to Europe, where the most profitable species are endemic. Truffle plantations have been established in various countries worldwide, including New Zealand and Israel. Nevertheless, the understanding of many basic aspects of truffle biology is still in its infancy, and the ecological requirements for some of these species are still not known. One of the most elusive goals has been discerning the reproductive system of Tuber spp. The reproductive structures of these species in pure cultures have not been reported, and axenic spore germination remains an unresolved problem (26). Furthermore, the mating-type genes have never been characterized in truffles. Molecular markers are being developed to type each truffle species to overcome the difficulty of identifying these species solely on their morphological traits (1, 8, 11, 17, 18, 19, 22, 23). By combining molecular markers with an appropriate sampling strategy, we may be able to critically evaluate the truffle reproductive system and life cycle even without reproducing the entire life cycle in the lab. To date, Tuber melanosporum Vittad. and Tuber magnatum Pico, the finest black and white truffle species, respectively, have been regarded as selfing species. When codominant markers were evaluated, heterozygous ascocarps were not detected (2, 3, 7, 15, 16). These studies proceed from the assumption that the ascocarps are diploid (dikaryotic) structures. We recently used simple sequence repeat (SSR) markers and a large survey of natural populations to show that extensive genetic exchange occurs within T. magnatum populations, which suggests that this truffle outcrosses (24). We interpreted the lack of heterozygotes to mean that haploid, maternal tissue is the dominant component of truffle ascocarps, while paternal DNA is not easily recoverable. Such partitioning of genetic material typifies many ascomycetes (4, 12). Our objectives in this study were to use SSR markers (i) to provide direct genetic evidence of outcrossing in T. magnatum and (ii) to determine the ploidy of the T. magnatum mycorrhizae, gleba, and spores. We tested the hypothesis that truffles are primarily haploid and reproduce by outcrossing. Our results led us to significantly reinterpret the T. magnatum life cycle, which also has implications for other Tuber spp., and to urge further reconsideration of existing data and strategies concerning truffle population genetic studies and growth management. Go to:

MATERIALS AND METHODS


Sample source.
Ten fresh mature ascocarps of T. magnatum, each characterized by a gray-brown gleba, were used for DNA isolation, ascus purification, and plant inoculation (Table (Table1).1). Asci were isolated by grinding small pieces (10 to 20 mg) of a fresh ascocarp in a ceramic mortar in sterile distilled water. Two hundred microliters of the ground material was layered over a 10 to 50% (wt/vol) sucrose density gradient (total volume, 1.5 ml) in a 2-ml centrifuge tube and centrifuged (800 g,

10 min, room temperature). The band containing asci, which was free of hyphal fragments, was recovered from the gradient with a Pasteur pipette, and the asci were washed twice in sterile distilled water and used immediately for DNA isolation.

TABLE 1. SSR patterns of the gleba and asci from single ascocarps The ma453 and ma455 ascocarps were used to inoculate two sets of 8 to 10 Quercus pubescens Willd. plantlets grown under semisterile conditions, as previously described (22). The inoculated plants were grown spaced in the greenhouse under ambient environmental conditions. After ~6 months, individual T. magnatum ectomycorrhizal root tips were collected and processed for DNA isolation or frozen in liquid N2 and stored in microcentrifuge tubes at 80C. The position of each mycorrhiza on the root branch was recorded.

DNA isolation.
DNAs were isolated from the following different sources: small portions of internal gleba, pools of purified asci collected from single ascocarps, and a number of single mycorrhizae collected from plants inoculated with genetically typed ascocarps. DNA was isolated from small pieces of gleba basically as described by Paolocci et al. (18), i.e., by grinding the truffle either in a ceramic mortar with liquid N2 or in a microcentrifuge tube with a sterile glass pestle. DNAs were extracted from pools of purified asci (about 200) with a FastPrep apparatus (QBIOgene, Carlsbad, CA) according to the manufacturer's instructions. Spores were disrupted with ceramic spheres (diameter, 1.4 mm) in the presence of 300 l of NTE buffer (200 mM Tris-HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA). Nucleic acids were precipitated with cold isopropanol and resuspended in sterile distilled water. DNAs from individual mycorrhizae were isolated according to the method of Paolocci et al. (18).

PCR amplification.
The molecular characterization of ascocarps, asci, and mycorrhizae was performed with T. magnatum species-specific internal transcribed spacer (ITS) and universal ITS1/ITS4 primer pairs as previously described (22). The resulting DNA fragments were sequenced directly and analyzed as reported previously (22). Primer pairs specific to the T. magnatum SSR loci MA2, MA4, MA5, MA7, MA12, and MA19 (25) were used to analyze the gleba and the purified asci from the T. magnatum ascocarp (Table (Table1).1). The primers specific to the MA12, MA19, and MA4 loci were also used to amplify DNAs from individual ectomycorrhizal root tips. All of the resulting SSR DNA fragments were separated in an ABI 310 genetic analyzer and analyzed with Genescan and Genotyper software (Applied Biosystems, Foster City, CA). Go to:

RESULTS
DNA isolation from asci and the gleba.
Asci are surrounded by a network of hyphae, arranged as a pseudoparenchyma, in truffle ascocarps. In the white truffles we evaluated, the asci contained primarily two or three pale yellow ascospores or, more rarely, one or four ascospores. Asci and ascospores were largely undamaged during the

gleba DNA isolation process, regardless of the tissue grinding procedures followed (data not shown). Even if a few ascospores were broken, their contribution in terms of relative amount of DNA was expected to be very low with respect to the DNA derived from the hyphae that composed the ascocarp. Thus, screening aimed at genetically differentiating the spores from the surrounding hyphae could be difficult even if a PCR approach is used. To analyze DNA from only the spores/asci, the glebae from mature T. magnatum ascocarps were ground and then centrifuged through a sucrose density gradient to selectively recover asci. Purified asci, based on microscopic observation, were layered on top of the sucrose gradient. A pool of purified asci was recovered from each of the 10 ascocarps (Table (Table1).1). DNAs from ~200 asci were then isolated in the presence of ceramic beads with a FastPrep apparatus, which ensured that most of the spores were broken. The resulting DNAs sufficed for several PCRs. Amplification and direct sequencing analysis of the DNAs isolated from the gleba and purified asci, with both T. magnatum ITS species-specific and universal ITS1/ITS4 primer pairs, confirmed the species identity and the absence of any other fungi.

SSR amplification from ascocarps and purified asci.


For the 10 ascocarps analyzed, the DNAs from the gleba produced a single peak (allele) for each of the six SSR loci, while 7/10 DNA samples from the purified asci and ascospores had two peaks (alleles) for one or more of the SSR loci (Table (Table1).1). Note that one of the two alleles found in the ascospores was also always found in the gleba, regardless of the locus analyzed (Table (Table1).1). Locus MA7 was monomorphic (allele 211), regardless of the tissue or ascocarp analyzed. Repeated DNA isolation and amplification with material from different portions of the gleba never resulted in multiple alleles/loci, confirming the results previously reported for a larger sample of T. magnatum ascocarps (25).

Plant inoculation and molecular characterization of ectomycorrhizae.


Ascospores from two of the ascocarps, ma453 and ma455, were used to inoculate separate lots of Quercus pubescens plantlets. ma453 was selected because its asci had multiple alleles, while the gleba and ascospore DNA from ma455 was monomorphic. Successful plant inoculation was confirmed by microscopic observations of all the inoculated plantlets. Sixty-two mycorrhizal root tips from two plants inoculated with ma453 ascospores and 35 root tips from a plant inoculated with ma455 ascospores were collected and molecularly typed. The MA12 and MA19 primer pairs were selected because the loci were both polymorphic in the ma453 ascospores, while the MA4 primer pair amplification product was monomorphic. Based on amplification with the T. magnatum ITS species-specific primers and the sequences of the DNA fragments resulting from amplification with the universal ITS1/ITS4 primers, the mycorrhizae produced were free of mycelia from other fungal species. The mycorrhizae from plants inoculated with asci from ma455 all had the same SSR pattern and only one allele per locus. This haplotype was the same as that of the gleba of ma455, as expected. Mycorrhizae from plants inoculated with ascospores from ma453 were individually monomorphic, but collectively all of the alleles present in the ascospore pool were present among the progeny. All four possible genotypes were also represented among the progeny, with the genotype of the gleba (130/109, MA12/MA19) being the most common (46/62 ascospores) and that of the other imputed parent (134/121) being the least common (2/62 ascospores). The two recombinant types, 130/121 and 134/109, were also relatively rare (8/62 and 6/62 ascospores, respectively). Mycorrhizae with different haplotypes could be recovered from the same root branch. Go to:

DISCUSSION
The results from this study clearly support the hypothesis that T. magnatum outcrosses and that its mycorrhizae originate from primary monokaryotic mycelia. These genetically different mycelial strains can coexist on the same root branches, but probably not on the same mycorrhizal root tip. The current model for the truffle life cycle has a very closed mating system, with homothallism or exclusive selfing as the precursor to sexual reproduction. This model was based on evidence that neither SSR, single-nucleotide polymorphism, nor allozyme markers were heterozygous in either T. magnatum or T. melanosporum truffles (2, 3, 7, 15, 16). However, linkage disequilibrium analyses of microsatellite loci showed that extensive gene flow occurs in T. magnatum both within and among geographically discrete populations (24). Extensive genetic exchange without the formation of detectable heterozygotes could occur but would be difficult to model. The asci and, more importantly, the ascospores within them are largely undamaged by the protocols commonly adopted for isolating nucleic acids from Tuber spp. Thus, the DNAs extracted from the ascocarps are almost exclusively that of the maternal parent. If this maternal tissue is haploid, which is typical of many ascomycetes, then the lack of heterozygosity observed when whole truffles are ground reflects this haploid DNA content and not necessarily a homothallic or self-fertilizing lifestyle. In this model, gleba tissue should always be monomorphic when evaluated with codominant markers. Heterozygosity, however, is expected among the ascospores unless the fertilizing parent is the same as the maternal parent (homothallism or selfing) or the maternal and the fertilizing parents are closely related (inbred). The fact that only 7/10 of the truffles analyzed had progeny that were polymorphic for the SSR markers suggests that either inbreeding or selfing occurs to at least some degree. Our sampling of truffles for this study does not suffice to distinguish between these hypotheses since fungi that reproduce homothallically, e.g., Aspergillus nidulans and Gibberella zeae, can also outcross under both laboratory and field conditions (5). We followed meiosis from one heterozygotic truffle and characterized 62 mycorrhizal root tips. If each of these root tips represents a single ascospore, then we recovered the four expected progeny classes (two parental and two recombinant). The relative frequencies of these classes should be equal if the two loci are unlinked, Mendelian segregation occurs, and there is no selection for the genotype that colonizes the root tips. The heterokaryotic truffle we analyzed yielded an excess of the maternal genotype and similar numbers of the fertilizing and two recombinant genotypes among the progeny. Thus, we think that the two loci are unlinked and that Mendelian segregation occurs and, for the moment, attribute the excess of the maternal genotype to selection for the ability to form mycorrhizae on the root tips or to foster growth in the soil or along the root (if not all of the root tips were colonized by mycelia from distinct ascospores). As a further hypothesis, we can envisage that the fragments of gleba present in the inocula also contribute to the synthesis of mycorrhizae. Tuber ascospores are reported to be multinucleate (9, 15, 20). We think that these nuclei are all copies of a single meiotic product, as found in other ascomycetes, and are not representatives of different meiotic products. If the ascospores are initially heterokaryotic, then these nuclei must segregate from one another mitotically during vegetative growth since the mycorrhizal mycelia we recovered from the root tips were all monomorphic. Thus, it is unlikely that truffle mycorrhizae are formed only following the establishment of heterokaryotic mycelia (13, 21). The nonexistence of such heterokaryons suggests that this species may also contain a vegetative compatibility system, as found in many other ascomycetes (14), that serves to keep haploid individuals discrete. We can now use molecular markers to trace the truffle life cycle (Fig. (Fig.1).1). Existing data show that outcrossing occurs, but the proportion of ascocarps that do so (or are even fertilized) is unknown. The morphology of the fertilization process in the truffle life cycle still remains elusive. Reports of ascogonia within truffles are not common (6), but antheridia have never been described. Interestingly, an anamorphic phase was recently described for Tuber borchii Vittad. and Tuber

oligospermum (Tul. and C. Tul.) Trappe (26), and asexual conidia could act as fertilizing agents in Tuber, as also occurs in many other Ascomycetes, e.g., most of the Pyrenomycetes.

FIG. 1. Revised life cycle of Tuber magnatum. (1) Asci are released following ripening of the ascocarp. (2) In close proximity to host plant root tips, ascospores germinate to produce primary homokaryotic (haploid) mycelia. (3) The primary mycelia colonize the ... In summary, we think that our study substantially deepens our fundamental understanding of Tuber biology and could lead to a reinterpretation of existing data and to altered strategies for the development and management of both artificial Tuber plantations and native populations. Go to:

Acknowledgments
This research was supported in part by a grant from the Regione Umbria/CNR-IGV-Perugia. Go to:

Footnotes
Contribution

no. 67 from the Institute of Plant Genetics.

Go to:

REFERENCES
1. Amicucci, A., A. Zambonelli, G. Giomaro, L. Potenza, and V. Stocchi. 1998. Identification of ectomycorrhizal fungi of the genus Tuber by species-specific ITS primers. Mol. Ecol. 7:273-277. 2. Bertault, G., M. Raymond, A. Berthomieu, G. Callot, and D. Fernandez. 1998. Trifling variation in truffles. Nature 394:734. 3. Bertault, G., F. Rousset, D. Fernandez, A. Berthomieu, M. E. Hochberg, G. Callot, and M. Raymond. 2001. Population genetics and dynamics of the black truffle in a man-made truffle field. Heredity 86:451-458. [PubMed] 4. Bistis, G. N. 1981. Chemotropic interactions between trichogynes and conidia of opposite mating-type in Neurospora crassa. Mycologia 73:959-975. 5. Bowden, R. L., and J. F. Leslie. 1999. Sexual recombination in Gibberella zeae. Phytopathology 89:182-188. 6. Callot, G. 1999. La truffe, la terre, la vie. INRA Editions, Paris, France. 7. Frizzi, G., G. Lalli, M. Miranda, and G. Pacioni. 2001. Intraspecific isozyme variability in Italian populations of the white truffle Tuber magnatum. Mycol. Res. 105:365-369. 8. Gandeboeuf, D., C. Dupr, P. Roeckel-Drvet, P. Nicolas, and G. Chevalier. 1997. Typing Tuber ectomycorrhizae by polymerase chain amplification of the internal transcribed spacer of rDNA and the sequence characterized amplified region markers. Can. J. Microbiol. 43:723-728. [PubMed] 9. Gross, G. 1987. Zu den europischen Sippen der Gattung Tuber, p. 79-99. In H. Derbsch and J.

A. Schmitt (ed.), Atlas der Pilze des Saarlandes. Teil 2. Nachweise, kologie, Vorkommen und Beschreibungen. Derlattinia, Saarbruken, Germany. 10. Hall, I. R., W. Yun, and A. Amicucci. 2003. Cultivation of edible ectomycorrhizal mushrooms. Trends Biotechnol. 21:433-438. [PubMed] 11. Henrion, B., G. Chevalier, and F. Martin. 1994. Typing truffle species by PCR amplification of the ribosomal DNA spacers. Mycol. Res. 98:37-43. 12. Johnson, T. E. 1978. Isolation and characterization of perithecial development mutants in Neurospora. Genetics 88:27-47. 13. Lanfranco, L., M. Arlorio, A. Matteucci, and P. Bonfante. 1995. Truffles: their life cycle and molecular characterization, p. 139-149. In V. Stocchi, P. Bonfante, and M. Nuti (ed.), Biotechnology of ectomycorrhizae. Molecular approach. Plenum Press, New York, N.Y. 14. Leslie, J. F. 1993. Fungal vegetative compatibility. Annu. Rev. Phytopathol. 31:127-151. 15. Mello, A., C. Murat, A. Vizzini, V. Gavazza, and P. Bonfante. 2005. Tuber magnatum, a species of limited geographical distribution: its genetic diversity inside and outside a truffle ground. Environ. Microbiol. 7:55-65. [PubMed] 16. Murat, C., J. Dez, P. Luis, C. Delaruelle, C. Dupr, G. Chevalier, P. Bonfante, and F. Martin. 2004. Polymorphism at the ribosomal DNA ITS and its relation to postglacial recolonization routes of the Perigord truffle Tuber melanosporum. New Phytol. 164:401-411. 17. Paolocci, F., P. Angelini, E. Cristofari, B. Granetti, and S. Arcioni. 1995. Identification of Tuber spp. and corresponding ectomycorrhizae through molecular markers. J. Food Sci. Agric. 69:511-517. 18. Paolocci, F., A. Rubini, B. Granetti, and S. Arcioni. 1999. Rapid molecular approach for a reliable identification of Tuber spp. ectomycorrhizae. FEMS Microbiol. Ecol. 28:23-30. 19. Paolocci, F., A. Rubini, B. Granetti, and S. Arcioni. 1997. Typing Tuber melanosporum and Chinese black truffle species by molecular markers. FEMS Microbiol. Lett. 153:255-260. [PubMed] 20. Parguey-Leduc, A., M. C. Janex-Favre, and C. Montant. 1987. Formation et volution des ascospores de Tuber melanosporum Vitt. (truffe noire du Prigord, Discomyctes). Can. J. Bot. 65:1491-1503. 21. Rouquerol, T., and H. Payre. 1974. Observations sur le comportement de Tuber melanosporum dans un site naturel. Rev. Mycol. 39:107-117. 22. Rubini, A., F. Paolocci, B. Granetti, and S. Arcioni. 2001. Morphological characterization of molecular-typed Tuber magnatum ectomycorrhizae. Mycorrhiza 11:179-185. 23. Rubini, A., F. Paolocci, B. Granetti, and S. Arcioni. 1998. Single step molecular characterization of morphologically similar black truffle species. FEMS Microbiol. Lett. 164:7-12. 24. Rubini, A., F. Paolocci, C. Riccioni, G. G. Vendramin, and S. Arcioni. 2005. Genetic and phylogeographic structure in the symbiotic fungus Tuber magnatum. Appl. Environ. Microbiol. 71:6584-6589. [PMC free article] [PubMed] 25. Rubini, A., F. Topini, C. Riccioni, F. Paolocci, and S. Arcioni. 2004. Isolation and characterization of polymorphic microsatellite loci in white truffle (Tuber magnatum). Mol. Ecol. Notes 4:116-118. 26. Urban, A., I. Neuner-Plattner, I. Krisai-Greilhuber, and K. Haselwandter. 2004. Molecular studies on terricolous microfungi reveal novel anamorphs of two Tuber species. Mycol. Res. 108:749-758. [PubMed]

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TUBER MAGNATUM Pico


Melethemata inauguralia. De fungorum generatione et propagatione 79, without illustration (1788)

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Synonyms: Tuber griseum Persoon (1801), Synopsis Methodica Fungorum 127 Tuber griseum Persoon: Fries (1823), Systema Mycologicum 2, Pt. II: 292 Macroscopic characters: Ascomata: hypogeous, irregular in form, lobed, gibbous, sometimes flattened, 2-6 (-15) cm in size, smooth in appearance but minutely papillose under the lens, pale ochre, sometimes greenish. Gleba: firm, solid, soapy texture, whitish at first, becoming pale yellow, ochre brown, reddish brown, often with flesh-red spots, marbled with numerous, thin, whitish, meandering, anastomosing veins.

Odour: strong, a complex mixture of methane gas, fermented cheese and garlic. Taste: strong, pleasant, garlicky Habitat: Marly calcareous soils with high macroporosity, in generally well drained alluvial plain forests. Soils are usually grey and stones are rare. Always in shady woods with a limited temperatures fluctuation. Tuber magnatum are harvested in areas with lots of vegetation and without burn and with a microclimate characterized by a short dry season. July and August rainfalls are essential for their production. Tuber magnatum season is very short, from late September to late November, according to region. They are associated with poplars (Populus), willows (Salix), hazels (Corylus), oaks(Quercus) and lindens (Tilia). The Italian white truffle has not yet been cultivated. Notes: Tuber magnatum is the most expensive truffle in the world, reaching a price of more than 3,000 per kg. It is harvested mainly in Italy and so far it has only been found in Italy, Croatia (Istria), Slovenia and Hungary. They must be eaten raw. Once you cook them they lose all their aroma

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Microscopic characters: Asci: subglobose, ellipsoid, sessile or short-stalked, 60-90 x 50-60 m, 1-3 (-4)-spored (usually 2spored). Ascospores: 20-33 x 20-30 m excluding ornament, size variable depending on number of spores in the ascus, Q range = 1.05-1.33, subglobose to broadly ellipsoid, light yellow, yellow ochre at maturity, ornamented with a coarse irregular reticulum 4-5 (-8) m high, meshes variable, usually 2-3 across width of spore. Peridium: pseudoparenchymatous, composed of subglobose to ovoid cells

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