Dissertationen zum Thema „Truffle“

Title from PDF of title page (University of Missouri--Columbia, viewed on February 24, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Johann Bruhn. Vita. Includes bibliographical references.

The thesis focuses on ‘green’ sample preparation techniques that utilise minimal or no organic solvents thereby producing negligible volumes of organic waste, to ensure safety both to user and the environment. Volatile compounds were extracted and pre-concentrated from Kgalagadi desert truffles (kalaharituber) by headspace solid phase microextraction (HSSPME) and supercritical fluid extraction (SFE). PHWE was employed for the extraction of amino acids and fatty acids. Subsequent analysis of volatile compounds was carried out by gas chromatography coupled with mass spectrometry. Four types of HS-SPME fibers (PDMS 100 μm, PDMS 7 μm, Polyacrylate 85 μm, CAR/DVB/PDMS 50/30 μm) were evaluated. A total of 24 volatile compounds with a molecular weight range from 110 to 354, the most prominent peak being 2-t-Butyl-2,3-dimethyl-3-buten-1-ol (C₁₀H₂₀O, MW 156) were detected after sampling with a PDMS 100 μm fiber. Less volatile compounds were detected after SFE with CO2. A total of 16 amino acids were identified while 17 fatty acids (MW from 132.12 to 367.49) were also identified. The characteristic profile of the Kgalagadi desert truffle was found to contain mainly fatty acid methyl esters and unsaturated aliphatic hydrocarbons. The most prominent compound peaks identified were; 2-t-butyl-2,3-dimethyl- 3-buten-1-ol, disulfide, ethyl benzoic acid 2-4-dhydroxy-3,6-dimethyl-methyl ester, 8,11- octadecanoic acid methyl ester, benzoic acid, 2,4-dihydroxy-3,6-dimethyl-methyl ester, isoquinoline, 1 butyl-3,4-dihydro and 3-heptanone, 6 methyl. Optimization results indicated that fresh slices from the heart of truffles were the best to use for HS-SPME-GCMS volatile compound analysis as they showed a higher sensitivity

The black truffle (Tuber melanosporum Vitt.) industry in Australia is relatively new and has enormous potential but some truffières (truffle farms) fail to meet anticipated harvest projections. Inappropriate soil conditions and climate, and the management of such factors are suggested as the primary reasons for inadequate yield. In addition, requirements for ascocarp initiation and development and the role of the host plant in such processes are unknown. This study examines interactions between European hazel (Corylus avellana L.) and the ectomycorrhizal (ECM) black truffle symbiont in a commercial truffière (Hazel Hill) in south-western Australia. Specific studies were initiated to examine the interactions of host physiology, mycorrhizal infection and the interaction with abiotic factors. The study related specific physiological processes of the host plant to the known life cycle of the black truffle to determine the role of the host plant in ascocarp production, if any. The work also examined the effect of silvicultural treatments intended to increase truffle production. A review of existing literature was undertaken to determine the key soil and climatic factors required for successful truffle production. Climatic conditions appeared more important than soil chemistry and structure in Western Australia, with significant seasonal variation in air and soil temperatures required plus irrigation to supplement summer rainfall. This information was used to define areas with potential for truffle production in the south-west of Western Australia: the cooler, high rainfall regions (>1000 mm annual rainfall) where there is sufficient seasonal variation in soil temperature and availability of adequate quantities of quality water for irrigation. Subsurface soil acidification and salinity, as well as groundwater salinity, are constraining factors. Lime amendment is necessary to create sufficiently high pH and CaCO3 levels required by the truffle fungus. A field trial was established to monitor the seasonal C dynamics of European hazel in the context of the life cycle of the black truffle. Maximum translocation of sucrose in the phloem sap coincided with the period of anticipated rapid growth of the truffle ascocarp implicating the use of current photosynthate in C nutrition of the ascocarp. Sampling of non-structural carbohydrates (NC) of above and belowground plant material indicated maximum storage of C in the host coincides with maturation of the ascocarp. These observations provide evidence of a synchronous growth habit of the plant host and the ascocarp. The C allocation patterns of European hazel in response to liming a loamy soil, taken from near the Hazel Hill truffière, and inoculation with ECM fungi (T. melanosporum, Hebeloma sp. and Scleroderma sp.) were examined in a glasshouse pot trial. Liming increased biomass allocation to the shoot and induced deficiencies of phosphorus and manganese. Colonisation by ECM fungi significantly increased net photosynthesis, indicating the sink strength of these fungi, but there was no relationship between the level of mycorrhizal infection and fine root NC. The maximum rate (40 g lime kg-1 soil) reduced infection by Hebeloma and Scleroderma and had no impact on T. melanosporum. Further, infection rates of T. melanosporum did not increase in response to lime suggesting lime is not necessary for ECM development in this soil type. Fertiliser is widely used in commercial truffières in Australia but the consequences for truffle production are unknown. In a field trial, the growth and physiological response of European hazel to forms of phosphorus (34 and 68 kg ha-1 apatite-P and 68 kg ha-1 triple super phosphate -P) and nitrogen (50 kg ha-1 of NO3- and NH4-N) were examined as well as the mycorrhizal response to fertiliser. Apatite-P increased phloem sap sucrose concentrations which was attributed to increased root biomass and associated sink capacity. Fertiliser application did not change fine root NC concentrations suggesting no increase in allocation of C to ECM structures. The highest rate of apatite-P decreased mycorrhizal infection rates of T. melanosporum and, most likely, was the result of increased infection rates of Hebeloma. In contrast to the literature relating to indigenous Australian ECM fungi, the highest rate of soluble-P did not decrease ECM infection rates in T. melanosporum. Nitrogen treatments increased foliar N content and improved gas exchange efficiency of plants, and had no adverse impact on the level of ECM infection. Fertilisation with N significantly increased soil respiration rates suggesting N limits mineralisation at this site. Some truffières manage the canopies of the host tree to ensure maximum exposure of the soil surface in order to increase soil temperatures. As there are no published data on the effect of pruning on black truffle production, a field trial was established to document the impact of canopy pruning on host physiology and soil temperature. The removal of 65% of canopy leaf area reduced phloem sap sucrose concentrations, soil respiration rate and the soluble: insoluble NC ratio of fine roots in the short term (1-3 weeks). There was no compensatory response of leaf gas exchange parameters as a result of pruning. Generally, there was no long term impact on plant physiological parameters as a result of pruning. Long term effects on soil temperature were observed as a result of pruning. Mean annual temperature and amplitude increased significantly beneath pruned trees and spring, summer and autumn soil temperatures increased as did diurnal variation as a result of pruning. Pruning did not increase winter soil temperatures and therefore would probably not impact on ascocarp maturation during this period. This research has provided insight into the C physiology of hazel associated with the black truffle and the consequences for truffle production. The results provide anecdotal evidence of direct C transfer between the host and the developing truffle, contrary to the existing paradigm that the ascocarp is saprotrophic for the majority of its growth and development. There is a need to validate this finding as there are consequences for management of commercial truffières. Liming of loam duplex soils can reduce the abundance of the most common competitor ECM fungi and should be encouraged in commercial truffières. Applying phosphorus and nitrogen to commercial truffières will improve growth rates of planted trees without adversely impacting on ECM infection by black truffle fungi, although the impact on truffle production remains unknown. It is anticipated truffle production will improve in the longer term as a result of pruning and prudent canopy management. Management options should include tree removal to reduce planting density and increase soil exposure in truffières. There is a need for longer term trials to be established to determine the C nutrition of the truffle ascocarp and to clearly define the key stages of the black truffle life cycle in Western Australia.

The black truffle (Tuber melanosporum Vitt.) industry in Australia is relatively new and has enormous potential but some truffieres (truffle farms) fail to meet anticipated harvest projections. Inappropriate soil conditions and climate, and the management of such factors are suggested as the primary reasons for inadequate yield. In addition, requirements for ascocarp initiation and development and the role of the host plant in such processes are unknown. This study examines interactions between European hazel (Corylus avellana L.) and the ectomycorrhizal (ECM) black truffle symbiont in a commercial truffiere (Hazel Hill) in south-western Australia. Specific studies were initiated to examine the interactions of host physiology, mycorrhizal infection and the interaction with abiotic factors. The study related specific physiological processes of the host plant to the known life cycle of the black truffle to determine the role of the host plant in ascocarp production, if any. The work also examined the effect of silvicultural treatments intended to increase truffle production. A review of existing literature was undertaken to determine the key soil and climatic factors required for successful truffle production. Climatic conditions appeared more important than soil chemistry and structure in Western Australia, with significant seasonal variation in air and soil temperatures required plus irrigation to supplement summer rainfall. This information was used to define areas with potential for truffle production in the south-west of Western Australia: the cooler, high rainfall regions (>1000 mm annual rainfall) where there is sufficient seasonal variation in soil temperature and availability of adequate quantities of quality water for irrigation. Subsurface soil acidification and salinity, as well as groundwater salinity, are constraining factors. Lime amendment is necessary to create sufficiently high pH and CaCO3 levels required by the truffle fungus. A field trial was established to monitor the seasonal C dynamics of European hazel in the context of the life cycle of the black truffle. Maximum translocation of sucrose in the phloem sap coincided with the period of anticipated rapid growth of the truffle ascocarp implicating the use of current photosynthate in C nutrition of the ascocarp. Sampling of non-structural carbohydrates (NC) of above and belowground plant material indicated maximum storage of C in the host coincides with maturation of the ascocarp. These observations provide evidence of a synchronous growth habit of the plant host and the ascocarp. The C allocation patterns of European hazel in response to liming a loamy soil, taken from near the Hazel Hill truffiere, and inoculation with ECM fungi (T. melanosporum, Hebeloma sp. and Scleroderma sp.) were examined in a glasshouse pot trial. Liming increased biomass allocation to the shoot and induced deficiencies of phosphorus and manganese. Colonisation by ECM fungi significantly increased net photosynthesis, indicating the sink strength of these fungi, but there was no relationship between the level of mycorrhizal infection and fine root NC. The maximum rate (40 g lime kg-1 soil) reduced infection by Hebeloma and Scleroderma and had no impact on T. melanosporum. Further, infection rates of T. melanosporum did not increase in response to lime suggesting lime is not necessary for ECM development in this soil type. Fertiliser is widely used in commercial truffieres in Australia but the consequences for truffle production are unknown. In a field trial, the growth and physiological response of European hazel to forms of phosphorus (34 and 68 kg ha-1 apatite-P and 68 kg ha-1 triple super phosphate -P) and nitrogen (50 kg ha-1 of NO3- and NH4-N) were examined as well as the mycorrhizal response to fertiliser. Apatite-P increased phloem sap sucrose concentrations which was attributed to increased root biomass and associated sink capacity. Fertiliser application did not change fine root NC concentrations suggesting no increase in allocation of C to ECM structures. The highest rate of apatite-P decreased mycorrhizal infection rates of T. melanosporum and, most likely, was the result of increased infection rates of Hebeloma. In contrast to the literature relating to indigenous Australian ECM fungi, the highest rate of soluble-P did not decrease ECM infection rates in T. melanosporum. Nitrogen treatments increased foliar N content and improved gas exchange efficiency of plants, and had no adverse impact on the level of ECM infection. Fertilisation with N significantly increased soil respiration rates suggesting N limits mineralisation at this site. Some truffieres manage the canopies of the host tree to ensure maximum exposure of the soil surface in order to increase soil temperatures. As there are no published data on the effect of pruning on black truffle production, a field trial was established to document the impact of canopy pruning on host physiology and soil temperature. The removal of 65% of canopy leaf area reduced phloem sap sucrose concentrations, soil respiration rate and the soluble: insoluble NC ratio of fine roots in the short term (1-3 weeks). There was no compensatory response of leaf gas exchange parameters as a result of pruning. Generally, there was no long term impact on plant physiological parameters as a result of pruning. Long term effects on soil temperature were observed as a result of pruning. Mean annual temperature and amplitude increased significantly beneath pruned trees and spring, summer and autumn soil temperatures increased as did diurnal variation as a result of pruning. Pruning did not increase winter soil temperatures and therefore would probably not impact on ascocarp maturation during this period. This research has provided insight into the C physiology of hazel associated with the black truffle and the consequences for truffle production. The results provide anecdotal evidence of direct C transfer between the host and the developing truffle, contrary to the existing paradigm that the ascocarp is saprotrophic for the majority of its growth and development. There is a need to validate this finding as there are consequences for management of commercial truffieres. Liming of loam duplex soils can reduce the abundance of the most common competitor ECM fungi and should be encouraged in commercial truffieres. Applying phosphorus and nitrogen to commercial truffieres will improve growth rates of planted trees without adversely impacting on ECM infection by black truffle fungi, although the impact on truffle production remains unknown. It is anticipated truffle production will improve in the longer term as a result of pruning and prudent canopy management. Management options should include tree removal to reduce planting density and increase soil exposure in truffieres. There is a need for longer term trials to be established to determine the C nutrition of the truffle ascocarp and to clearly define the key stages of the black truffle life cycle in Western Australia.

The black truffle (Tuber melanosporum Vitt.) industry in Australia is relatively new and has enormous potential but some truffieres (truffle farms) fail to meet anticipated harvest projections. Inappropriate soil conditions and climate, and the management of such factors are suggested as the primary reasons for inadequate yield. In addition, requirements for ascocarp initiation and development and the role of the host plant in such processes are unknown. This study examines interactions between European hazel (Corylus avellana L.) and the ectomycorrhizal (ECM) black truffle symbiont in a commercial truffiere (Hazel Hill) in south-western Australia. Specific studies were initiated to examine the interactions of host physiology, mycorrhizal infection and the interaction with abiotic factors. The study related specific physiological processes of the host plant to the known life cycle of the black truffle to determine the role of the host plant in ascocarp production, if any. The work also examined the effect of silvicultural treatments intended to increase truffle production. A review of existing literature was undertaken to determine the key soil and climatic factors required for successful truffle production. Climatic conditions appeared more important than soil chemistry and structure in Western Australia, with significant seasonal variation in air and soil temperatures required plus irrigation to supplement summer rainfall. This information was used to define areas with potential for truffle production in the south-west of Western Australia: the cooler, high rainfall regions (>1000 mm annual rainfall) where there is sufficient seasonal variation in soil temperature and availability of adequate quantities of quality water for irrigation. Subsurface soil acidification and salinity, as well as groundwater salinity, are constraining factors. Lime amendment is necessary to create sufficiently high pH and CaCO3 levels required by the truffle fungus. A field trial was established to monitor the seasonal C dynamics of European hazel in the context of the life cycle of the black truffle. Maximum translocation of sucrose in the phloem sap coincided with the period of anticipated rapid growth of the truffle ascocarp implicating the use of current photosynthate in C nutrition of the ascocarp. Sampling of non-structural carbohydrates (NC) of above and belowground plant material indicated maximum storage of C in the host coincides with maturation of the ascocarp. These observations provide evidence of a synchronous growth habit of the plant host and the ascocarp. The C allocation patterns of European hazel in response to liming a loamy soil, taken from near the Hazel Hill truffiere, and inoculation with ECM fungi (T. melanosporum, Hebeloma sp. and Scleroderma sp.) were examined in a glasshouse pot trial. Liming increased biomass allocation to the shoot and induced deficiencies of phosphorus and manganese. Colonisation by ECM fungi significantly increased net photosynthesis, indicating the sink strength of these fungi, but there was no relationship between the level of mycorrhizal infection and fine root NC. The maximum rate (40 g lime kg-1 soil) reduced infection by Hebeloma and Scleroderma and had no impact on T. melanosporum. Further, infection rates of T. melanosporum did not increase in response to lime suggesting lime is not necessary for ECM development in this soil type. Fertiliser is widely used in commercial truffieres in Australia but the consequences for truffle production are unknown. In a field trial, the growth and physiological response of European hazel to forms of phosphorus (34 and 68 kg ha-1 apatite-P and 68 kg ha-1 triple super phosphate -P) and nitrogen (50 kg ha-1 of NO3- and NH4-N) were examined as well as the mycorrhizal response to fertiliser. Apatite-P increased phloem sap sucrose concentrations which was attributed to increased root biomass and associated sink capacity. Fertiliser application did not change fine root NC concentrations suggesting no increase in allocation of C to ECM structures. The highest rate of apatite-P decreased mycorrhizal infection rates of T. melanosporum and, most likely, was the result of increased infection rates of Hebeloma. In contrast to the literature relating to indigenous Australian ECM fungi, the highest rate of soluble-P did not decrease ECM infection rates in T. melanosporum. Nitrogen treatments increased foliar N content and improved gas exchange efficiency of plants, and had no adverse impact on the level of ECM infection. Fertilisation with N significantly increased soil respiration rates suggesting N limits mineralisation at this site. Some truffieres manage the canopies of the host tree to ensure maximum exposure of the soil surface in order to increase soil temperatures. As there are no published data on the effect of pruning on black truffle production, a field trial was established to document the impact of canopy pruning on host physiology and soil temperature. The removal of 65% of canopy leaf area reduced phloem sap sucrose concentrations, soil respiration rate and the soluble: insoluble NC ratio of fine roots in the short term (1-3 weeks). There was no compensatory response of leaf gas exchange parameters as a result of pruning. Generally, there was no long term impact on plant physiological parameters as a result of pruning. Long term effects on soil temperature were observed as a result of pruning. Mean annual temperature and amplitude increased significantly beneath pruned trees and spring, summer and autumn soil temperatures increased as did diurnal variation as a result of pruning. Pruning did not increase winter soil temperatures and therefore would probably not impact on ascocarp maturation during this period. This research has provided insight into the C physiology of hazel associated with the black truffle and the consequences for truffle production. The results provide anecdotal evidence of direct C transfer between the host and the developing truffle, contrary to the existing paradigm that the ascocarp is saprotrophic for the majority of its growth and development. There is a need to validate this finding as there are consequences for management of commercial truffieres. Liming of loam duplex soils can reduce the abundance of the most common competitor ECM fungi and should be encouraged in commercial truffieres. Applying phosphorus and nitrogen to commercial truffieres will improve growth rates of planted trees without adversely impacting on ECM infection by black truffle fungi, although the impact on truffle production remains unknown. It is anticipated truffle production will improve in the longer term as a result of pruning and prudent canopy management. Management options should include tree removal to reduce planting density and increase soil exposure in truffieres. There is a need for longer term trials to be established to determine the C nutrition of the truffle ascocarp and to clearly define the key stages of the black truffle life cycle in Western Australia.

Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle.
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Les truffes sont des champignons ectomycorhiziens du genre Tuber. De part leurs qualités organoleptiques, certaines espèces de truffes ont une forte valeur économique, c'est le cas de Tuber melanosporum et T. Magnatum. Malgré la multiplication des études, il persiste de nombreuses inconnues sur la biologie (par exemple: phylogéographie et l'écologie) de ces espèces. L'analyse de la diversité génétique de T. Melanosporum sur l'ensemble de son aire de répartition en France met en évidence un niveau de différenciation génétique non négligeable entre les populations de l'Est et de l'Ouest de la France. Une analyse phylogéographique a permis de suggérer deux voies de recolonisation post-glaciaire : la voie de la vallée du Rhône et la voie de l'Atlantique. L'analyse des ascocarpes de T. Magnatum récoltés dans une truffière naturelle a montre l'existence d'au moins deux génotypes dans cette population. D'autre part, la récolte d'apex mycorhiziens indiquent que les ectomycorhizes de T. Magnatum sont très rares (deux morphotypes sur 39). Les espèces ectomycorhiziennes majoritaires appartiennent aux Telephoraceae, Pezizales et Sebacina
Truffles are ectomycorrhizal fungi belonging to Tuber. Fructifications of some species, such as T. Melanosporum and T. Magnatum, are appreciated by consumers and have important economic value. Despite of numerous studies, some aspects of truffle biology (i. E. Phylogeography and ecology) are unknown. Wide range analysis of genetic diversity in T. Melanosporum showed important genetic differentiation between East and West French populations. Phylogeographic analysis allowed us to hypothesis that T. Melanosporum recolonized France by two main routes : Rhône Valley route and Atlantic route. T. Magnatum ascocarps analysis in a natural truffle ground identified at least two genotypes in this population. In order to characterize T. Magnatum mycorrhizosphere, root tips have been harvested. We found that T. Magnatum ectomycorrhizas are very rare (two out of 39 morphotypes). Moreover, more abundant species belong to Telephoraceae, Pezizales and Sebacina

Black Périgord truffle is highly appreciated for its unique aroma, however, its seasonal perishability restricts its year-round availability. To extend the availability of the natural truffle aroma throughout the year, this thesis aimed to explore the potential of extraction and encapsulation techniques to process the black truffle into a natural flavouring ingredient for food application.

La formation de l'ascocarpe des truffes conduit à des modifications de leur taille, de l'organisation cellulaire et de la production des arômes. La réalisation de deux banques d'ADNc à partir de fructifications à deux stades de maturation différent a permis la production de 2043 clones d'ADNc et de 641 ESTs. La comparaison de l'expression génique sur filtre d'ADNc dans le mycélium et dans différents stades de maturation de l'ascocarpe montre que : 1) la majorité des gènes régulés lors de la formation de l'ascocarpe ont une fonction inconnue ; 2) un certain nombre de transcrits codent pour des enzymes impliquées dans les voies du métabolisme primaire et secondaire ; 3) des gènes impliqués dans la résistance aux stress sont les plus stimulés. Enfin, la caractérisation d'un EST de T. Borchii présentant une similarité avec le gène bli-3 de Neurospora crassa suggère que la truffe perçoit la lumière
The ripening of a truffle fruitbody leads to drastic changes in its size, morphology, cellular organization and aroma production. To explore the gene expression in Tuber borchii during fruitbody development, cDNA libraries from fruitbodies at two stages of maturation were constructed, 3000 cDNA clones were isolated, 2043 were used for constructing cDNA arrays and 641 ESTs of high quality were produced. The results demonstrate that 1) the molecular machinery which allows the truffle ripening mostly consists of orphan genes; 2) truffles have an intense metabolism where pathways belonging to the primary and secondary metabolism are active; 3) some stress-related genes can be identified as markers for fruitbody formation. Furthermore the expression pattern of a cDNA sequence, isolated from ripe fruitbody, showing a good similarity to a Neurospora crassa blue-light inducible gene, was studied using Real-Time RT-PCR assay. The data obtained suggest a light-dependent response in T. Borchii

I tartufi sono un gruppo polifiletico di funghi ipogei. I funghi del genere Tuber, i cosiddetti “veri tartufi”, sono ascomiceti ectomicorrizici dell’ordine Pezizales con un ciclo vitale complesso, durante il quale il micelio fungino stabilisce interazioni simbiontiche con le radici di diversi alberi ed arbusti. Delle oltre 180 specie di Tuber attualmente conosciute, alcune (T. aestivum Vittad., T. borchii Vittad., T. magnatum Picco e T. melanosporum Vittad.) sono molto ricercate sul mercato alimentare per via delle loro straordinarie caratteristiche organolettiche, con un enorme valore commerciale. È noto che i tartufi ospitano complesse comunità microbiche di batteri, lieviti, funghi filamentosi e virus, con le quali interagiscono sia a livello della micorrizosfera che dell’ascocarpo. Tuttavia, molti aspetti legati alla diversità e al potenziale ruolo dei microrganismi, nonché agli effetti delle interazioni tra comunità microbiche sulla biologia del tartufo sono ancora poco conosciuti. Pertanto, l’obiettivo principale di questo lavoro è stato quello di analizzare e caratterizzare le comunità batteriche associate a due delle specie di Tuber di maggiore interesse commerciale: T. aestivum, tartufo nero estivo, e T. magnatum, tartufo bianco pregiato. Le analisi sono state condotte su ascocarpi raccolti in Molise (Italia centro-meridionale), una delle regioni italiane a più alta vocazione tartufigena (circa il 40% della produzione nazionale di tartufo). Tuttavia, ad oggi, il tartufo molisano ha ricevuto pochissima attenzione dal punto di vista scientifico e, di conseguenza, non è opportunamente valorizzato e tutelato. Le attività di ricerca illustrate nella presente tesi gettano quindi le basi per colmare la carenza di informazioni scientifiche sul tartufo molisano, rappresentando un importante punto di partenza per un’ulteriore e più approfondita caratterizzazione di questa risorsa fondamentale per l’economia locale. Nello specifico, in un primo studio, sono state analizzate le comunità microbiche associate a sei corpi fruttiferi di T. aestivum e a sei campioni di suolo raccolti nei pressi del Comune di Vastogirardi (Isernia), mediante sequenziamento degli ampliconi del gene codificante per l’rRNA 16S. In linea con i risultati ottenuti in altre ricerche, i principali phyla rinvenuti negli ascocarpi sono stati Proteobacteria ed Actinobacteria, con il genere Bradyrhizobium particolarmente rappresentato. Ciononostante, sono state osservate considerevoli differenze tra il microbiota del suolo e del tartufo ed un’inaspettata eterogeneità in termini di composizione, abbondanza relativa dei principali taxa ed α-diversità all’interno delle comunità microbiche del tartufo. Nelle altre due ricerche presentate sono state considerate due popolazioni di T. magnatum provenienti da diverse aree della regione Molise. Nel complesso, 21 tartufi bianchi sono stati caratterizzati da un punto di vista morfologico, genetico e microbiologico. Le indagini morfologiche hanno riguardato lo spessore del peridio (parametro per il quale non erano disponibili in letteratura scientifica informazioni dettagliate), mentre le analisi genetiche e microbiologiche si sono focalizzate rispettivamente su un marcatore a singolo locus (SCAR A21-inf) e sulle comunità batteriche della gleba. È emersa una notevole variabilità tra e all’interno delle popolazioni di T. magnatum esaminate, a conferma di un’interessante eterogeneità dei tartufi molisani che li rende ideali per studi più approfonditi.
Truffles are a polyphyletic group of fungi whose fruiting bodies sequester their spores and develop underground. Fungi of the genus Tuber, the so-called “true truffles”, are ectomycorrhizal ascomycetes of the Pezizales order that undertake a complex life cycle, during which the fungal mycelium establishes symbiotic associations with the roots of several trees and shrubs. Some of the more than 180 Tuber species currently known (including T. aestivum Vittad., T. borchii Vittad., T. magnatum Picco, and T. melanosporum Vittad.) are highly sought after on the food market due to their unique organoleptic properties, with a huge commercial value. It is known that truffles harbour complex microbial communities of bacteria, yeasts, guest filamentous fungi, and viruses, with whom they interact both in the mycorrhizosphere and in the ascocarp. However, many aspects related to the diversity and the potential role of truffle-associated microorganisms, as well as the effects of the interactions among microbial communities on the biology of truffles are still poorly understood. Accordingly, the main purpose of this work was to analyse and characterise the bacterial communities associated with two of the most commercially relevant truffle species: the summer black truffle T. aestivum and the prized white truffle T. magnatum. Analyses were carried out on Tuber ascomata from Molise region (Central-Southern Italy), one of the most important Italian areas suited to truffle collection (about 40% of the national production). Nevertheless, to date, Molise truffle has received very little attention from a scientific perspective and, consequently, it is not adequately valorised and preserved. Thus, the research activities illustrated in the present thesis lay the foundation to fill the lack of scientific data on the Molise truffles, representing an essential starting point for a further and more in-depth characterisation of this resource of utmost importance for the local economy. In detail, in a first study, the microbial communities associated with six T. aestivum ascomata and six soil samples collected in the municipality of Vastogirardi (Isernia province) were examined using the 16S rRNA gene amplicon high-throughput sequencing. Consistently with previous researches, the main phyla retrieved in the investigated ascocarps were Proteobacteria and Actinobacteria, with the genus Bradyrhizobium particularly represented. Nonetheless, considerable differences between soil and truffle microbiota and an unexpected heterogeneity within the truffle bacterial communities in terms of composition, relative abundance of the main taxa, and α-diversity values were observed. The other two reported researches focused on T. magnatum populations from different areas of Molise region. Overall, twenty-one white truffles were characterised from a morphological, genetic, and microbiological point of view. In particular, morphological investigations concerned the thickness of the peridium, a parameter for which no comprehensive information was available, whereas genetic and microbiological analyses focused on the Sequence-Characterised Amplified Region SCAR A21-inf (a single locus marker) and the gleba bacterial communities, respectively. A considerable variability between and within the examined T. magnatum groups emerged, confirming an interesting heterogeneity of Molise truffle populations that makes them ideal for further in-depth studies.

Ectomycorrhizal fungi form an integral tripartite relationship with trees and rodents whereby the fungi provide nutritional benefits for the trees, the trees provide carbohydrate for the fungi, and the rodents feed on the fruit bodies produced by the fungi and then disperse the fungal spores in their feces. When forests are harvested, new ectomycorrhizae must form. It has been assumed that dispersal beyond the root zone of surviving trees happens by way of animals dispersing the spores in their feces, but the importance of particular animal taxa to fungal spore dispersal into disturbed areas in the Interior Cedar Hemlock Biogeoclimatic zone of British Columbia has not previously been investigated. This study observed the occurrence and prevalence of hypogeous fruit bodies (truffles) of ectomycorrhizal fungi, and fungal spores in the feces of a range of rodent species. Truffles were excavated and sciurids (squirrels, chipmunks) and murids (mice, voles) were trapped on sites in a 7 to 102-year chronosequence, as well as unharvested sites adjacent to 7- and 25-year-old sites. The average truffle species richness in soil did not change significantly over the chronosequence. Rhizopogon species were present at all sites and treatments. Deer mice (Peromyscus maniculatus) and yellow-pine chipmunks (Tamias amoenus) were the most commonly trapped rodents across all site ages and were also the most likely to move between harvested and unharvested areas. Red-backed voles (Clethrionomys gapperi), red squirrels (Tamiasciurus hudsonicus), and flying squirrels (Glaucomys sabrinus) were also studied, but were trapped in much lower numbers and rarely, if ever, were detected moving between harvested and adjacent mature sites. However, all animal taxa studied carried fungal spores in their feces. Spores of Rhizopogon spp. and Hysterangium separabile were the most frequently consumed by all the animals studied. Because deer mice and chipmunks were the most likely to move between mature and harvested sites and they frequently carried fungal spores in their feces, they are likely the most important mammals for dispersal of ectomycorrhizal fungal spores in this area. This study highlights the importance of small mammal conservation when forest management is considered.

Tuber aestivum is an ectomycorrhizal ascomycete with underground fruit bodies. It is an economically important species, but has been regarded as endangered in Sweden. My inventory has increased the number of reported localities from 3 to 31. It has long been debated whether T. aestivum and T. uncinatum are conspecific or not, so a clarification would help conservation biology and cultivation. My study included 117 fruit bodies of both taxa from 8 countries. The phylogenetic (ITS) and microscopic analyses showed that the two taxa were synonyms and that the spore reticulum height, used to separate the taxa, is not diagnostic. T. aestivum was clearly different from T. mesentericum, which I reported new to Sweden. The Gotland T. aestivum population was genetically distinct (RAPD) from other European specimens. The genetic variation suggested sexual reproduction. The habitat of 18 T. aestivum sites on Gotland were analysed and compared with data from France. No striking functional differences in soil chemistry were found, so a possible T. aestivum ecotype on Gotland would rather be an adaptation to the colder and drier climate. Selecting local T. aestivum inoculum for truffle orchards in Northern Europe could be important for successful truffle production. In 1999, 10 experimental truffle orchards with a total of 240 oak and hazel seedlings were established on Gotland, and as a result of this project 3000 commercial oak seedlings were planted in 2000-2001. In 2004, T. aestivum mycorrhiza was still present in all of the 22 orchards studied on Gotland, some in soils different from natural habitats. In addition, the project has also generated a truffle cultivation association, a truffle company, truffle dog breeding and export of T. aestivum to France.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Fungi represent an extremely important group of organisms in terrestrial ecosystems. Among their several important ecological roles, is the mutualistic association with plant roots, forming different types of mycorrhiza. Most studies carried out so far focused on epigeous ectomycorrhizal fungi, which occur above ground. On the other hand, below ground, hypogeous ectomycorrhizal fungi, are still poorly studied. This research aimed to study the diversity of hypogeous ectomycorrhizal fungi (Basidiomycetes) collected in Eucalyptus spp. plantations in the Central Region of Rio Grande do Sul, Brazil, based on morphological and molecular characters. Samples were taken between May 2009 and July 2010. A total of five species belonging to three families were identified. The material was analyzed to determine the morphological and molecular characters. Among the species identified are: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway and Setchelliogaster tenuipes (Setch.) Pouzar. Additionally a second species of Chondrogaster sp. was collected. However, no identity was determined for this species. Furthermore, it is believed it represents a new undescribed species to science. Among the species studied, Descomyces albus is reported for the first time in the State of Rio Grande do Sul. Hysterangium affine and H. inflatum are newly recorded species from Brazil while Chondrogaster pachysporus is recorded for the first in South America.
Os fungos representam um grupo de organismos extremamente importante nos ecossistemas terrestres. Entre as muitas funções por eles desempenhadas está a associação mutualística com as raízes dos vegetais, formando diferentes tipos de micorrizas. A maioria dos estudos realizados até o momento está principalmente focada nos fungos ectomicorrízicos epígeos, os quais ocorrem acima do solo. Por outro lado, os fungos ectomicorrízicos hipógeos, que vivem abaixo da superfície do solo, são ainda pouco estudados. A presente pesquisa tem como objetivo estudar a diversidade de fungos ectomicorrízicos hipógeos (Basidiomycetes) coletados em plantações de Eucalyptus spp. na Região Central do Rio Grande do Sul, Brasil, com base em características morfológicas e moleculares. O período de coleta dos fungos foi de maio de 2009 a julho de 2010. Um total de cinco espécies pertencentes a três famílias foram identificadas. Este material foi analisado para verificar suas características morfológicas e moleculares. Dentre as espécies identificadas estão: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway e Setchelliogaster tenuipes (Setch.) Pouzar. Adicionalmente identificou-se uma segunda espécie de Chondrogaster sp. porém não se chegou a uma espécie conhecida, acreditando tratar-se de uma espécie ainda desconhecida para a ciência. Dentre as espécies estudadas, destacam-se a Descomyces albus que é citada pela primeira vez para o Estado do Rio Grande do Sul, Hysterangium affine e H. inflatum que têm sua ocorrência registrada pela primeira vez no Brasil, além de Chondrogaster packysporus citado pela primeira vez para a América do Sul.

Els fongs micorízics formen associacions simbiòtiques amb les plantes i juguen un paper important en els processos de l'ecosistema. Els fongs micorízics són rellevants per la seva funció d'obtenció de compostos de carboni al mateix temps que aporten nutrició mineral a la seva planta hoste simbiòtica, però també els fongs micorízics tenen un interès econòmic, al proporcionar esporocarps comestibles. Entre ells, Tuber melanosporum és un dels fongs més apreciats en el mercat i el seu cultiu i producció silvestre és important per a l'economia i el paisatge en les zones rurals del sud d'Europa. L'objectiu principal d'aquesta tesi va ser analitzar: i) la situació actual de el sector de la tòfona a l'àrea mediterrània; ii) la dependència de la productivitat de T. melanosporum de la variabilitat de les precipitacions; iii) les interaccions de T. melanosporum amb altres fongs del sòl en plantacions; i iv) la diversitat de tipus de compatibilitat sexual o "mating types" a les plantacions de T. melanosporum. Amb l'objectiu de dilucidar la situació actual del sector de la tòfona a l'àrea mediterrània, un total de 17 panelistes experts van participar en una enquesta Delphi on van respondre un qüestionari en dues rondes. Es van analitzar tres registres continus de 49 anys de la producció anual de T. melanosporum de les principals regions productores de tòfona del sud d'Europa al nord-est d'Espanya, el sud de França i el nord i centre d'Itàlia per comprendre la dependència de la producció de T. melanosporum de les condicions climàtiques. Finalment, es van dur a terme dos experiments en una plantació experimental d'1 ha consistent en 249 plantes de Quercus ilex de cinc anys inoculats amb T. melanosporum plantats en una pastura recentment abandonada envoltada de bosc situada al Prepirineu oriental; i en 29 plantacions de T. melanosporum de Terol (Espanya) de 3, 5, 7, 10, 14 i 20 anys. Utilitzant diverses tècniques moleculars com la PCR en temps real (qPCR), la seqüenciació massiva d'ADN (PacBio RS II) i un assaig de qPCR patentat (WO2012 / 032.098) per quantificar els dos tipus de compatibilitat sexual, descrivim els canvis de la composició de la comunitat fúngica i la distribució del tipus de compatibilitat sexual. Els resultats obtinguts a través de les dues rondes Delphi ens van permetre tenir una descripció completa de la cadena de valor actual de la tòfona i confirmem una evolució del sector a causa de l'èxit del cultiu de la tòfona. A causa de l'augment de la producció de T. melanosporum en els últims anys, s'ha observat una tendència a la baixa dels preus de la tòfona que han alertat el sector. Per reforçar el sector de la tòfona, es van identificar accions consensuades per desenvolupar en el futur. Pel que fa a la part ecològica d'aquesta tesi, revela com la producció de T. melanosporum, depèn significativament dels totals de precipitació anteriors de juny a agost, mentre que massa precipitació a la tardor afecta negativament la collita d'hivern. Una de les principals preocupacions d'aquesta tesi ha estat si altres fongs ectomicorízics podrien desplaçar T. melanosporum a les plantacions i afectar la producció de tòfones. Trobem una major abundància relativa de fongs ECM diferents de T. melanosporum associats amb arbres més propers a bosc, però els arbres amb major diàmetre del coll de l'arrel van tenir major biomassa de miceli de T. melanorporum i van ser aquells on la comunitat fúngica es va veure menys afectada per la distància a bosc. No trobem associació entre el miceli de T. melanosporum a terra i la distància a bosc o l'abundància de fongs ECM diferents de T. melanosporum. En l'estudi de cronoseqüència, trobem que T. melanosporum es va desenvolupar de manera constant al llarg dels anys, ja que el miceli extraradical de T. melanosporum no es va correlacionar amb canvis en altres fongs ECM. La freqüència del tipus de compatibilitat sexual no va canviar al llarg dels anys. En resum i amb base a aquests resultats, es pot concloure que (i) hi ha la necessitat de promoure el consum de tòfones, donat l'augment de la producció a causa del cultiu de tòfones; (ii) la producció de T. melanosporum depèn significativament de les pluges de l'estiu anterior, mentre que massa precipitació de tardor té efectes negatius; (iii) encara que el veïnatge del bosc influirà en la comunitat de fongs del sòl, això pot tenir un efecte limitat sobre el creixement del miceli de T. melanosporum; (iv) T. melanosporum pot mantenir la colonització dels arbres a llarg termini; (v) l'inici de la reproducció sexual no està limitat per una presència desproporcionada de qualsevol dels tipus de compatibilitat sexual, ni pel desplaçament per fongs ECM diferents de T. melanosporum.
Los hongos micorrícicos forman asociaciones simbióticas con las plantas y juegan un papel importante en los procesos del ecosistema. Los hongos micorrícicos son relevantes por su función de obtención de compuestos de carbono a la vez que aportan nutrición mineral a su planta huésped simbiótica, pero también los hongos micorrícicos tienen un interés económico, al proporcionarnos esporocarpos comestibles. Entre ellos, Tuber melanosporum es uno de los hongos más apreciados en el mercado y su cultivo y producción silvestre es importante para la economía y el paisaje en las zonas rurales del sur de Europa. El objetivo principal de esta tesis fue analizar: i) la situación actual del sector de la trufa en el área mediterránea; ii) la dependencia de la productividad de T. melanosporum de la variabilidad de las precipitaciones; iii) las interacciones de T. melanosporum con otros hongos del suelo en plantaciones; y iv) la diversidad de tipos de compatibilidad sexual o “mating types” en las plantaciones de T. melanosporum. Con el objetivo de dilucidar la situación actual del sector de la trufa en el área mediterránea, un total de 17 panelistas expertos participaron en una encuesta Delphi donde respondieron un cuestionario en dos rondas. Se analizaron tres registros continuos de 49 años de la producción anual de T. melanosporum de las principales regiones productoras de trufa del sur de Europa en el noreste de España, el sur de Francia y el norte y centro de Italia para comprender la dependencia de la producción de T. melanosporum de las condiciones climáticas. Finalmente, se llevaron a cabo dos experimentos en una plantación experimental de 1 ha consistente en 249 plantas de Quercus ilex de cinco años inoculados con T. melanosporum plantados en un pastizal recientemente abandonado rodeado de bosque ubicado en el Prepirineo oriental; y en 29 plantaciones de T. melanosporum de Teruel (España) de 3, 5, 7, 10, 14 y 20 años. Utilizando varias técnicas moleculares como la PCR en tiempo real (qPCR), la secuenciación masiva de ADN (PacBio RS II) y un ensayo de qPCR patentado (WO2012 / 032098) para cuantificar los dos tipos de compatibilidad sexual, describimos los cambios de la composición de la comunidad fúngica y la distribución del tipo de compatibilidad sexual. Los resultados obtenidos a través de las dos rondas Delphi nos permitieron tener una descripción completa de la cadena de valor actual de la trufa y confirmamos una evolución del sector debido al éxito del cultivo de la trufa. Debido al aumento de la producción de T. melanosporum en los últimos años, se ha observado una tendencia a la baja de los precios de la trufa que han alertado al sector. Para reforzar el sector de la trufa, se identificaron acciones consensuadas para desarrollar en el futuro. En cuanto a la parte ecológica de esta tesis, revela cómo la producción de T. melanosporum, depende significativamente de los totales de precipitación anteriores de junio a agosto, mientras que demasiada precipitación otoñal afecta negativamente la cosecha de invierno. Una de las principales preocupaciones de esta tesis ha sido si otros hongos ectomicorrícicos podrían desplazar a T. melanosporum en las plantaciones y afectar la producción de trufas. Encontramos una mayor abundancia relativa de hongos ECM distintos de T. melanosporum asociados con árboles más cercanos al bosque, pero los árboles con mayor diámetro de cuello de la raíz tuvieron mayor biomasa de micelio de T. melanorporum y fueron aquellos cuya comunidad fúngica se vio menos afectada por la distancia al bosque. No encontramos asociación entre el micelio de T. melanosporum en el suelo y la distancia al bosque o la abundancia de hongos ECM distintos de T. melanosporum. En el estudio de cronosecuencia, encontramos que T. melanosporum se desarrolló de manera constante a lo largo de los años, ya que el micelio extraradical de T. melanosporum no se correlacionó con cambios en otros hongos ECM. La frecuencia del tipo de compatibilidad sexual no cambió a lo largo de los años. En resumen y con base en estos resultados, se puede concluir que (i) existe la necesidad de promover el consumo de trufas, dado el aumento de la producción debido al cultivo de trufas; (ii) la producción de T. melanosporum depende significativamente de las lluvias del verano anterior, mientras que demasiada precipitación otoñal tiene efectos negativos; (iii) aunque la vecindad del bosque influirá en la comunidad de hongos del suelo, esto puede tener un efecto limitado sobre el crecimiento del micelio de T. melanosporum; (iv) T. melanosporum puede mantener la colonización de los árboles a largo plazo; (v) el inicio de la reproducción sexual no está limitado por una presencia desproporcionada de cualquiera los tipos de compatibilidad sexual, ni por el desplazamiento por hongos ECM distintos de T. melanosporum.
The mycorrhizal fungi form symbiotic associations with plants and play an important role in ecosystem processes. Mychorrizal fungi are relevant for their function for obtaining carbon compounds while providing mineral nutrition to their symbiotic host plant, but also, mycorrhizal fungi have an economic interest, by providing us with edible sporocarps. Among them, T. melanosporum is one of the most appreciated fungi in the market and its wild production and cultivation is important for rural livelihoods and landscapes in southern Europe. The main objective of this thesis was to analyse: i) the current situation of the truffle sector in the Mediterranean area; ii) the dependency of T. melanosporum productivity on rainfall variability; iii) the interactions of T. melanosporum with other soil fungi in plantations; and iv) the mating types diversity in T. melanosporum plantations. Aiming to elucidate the current situation of the truffle sector in the Mediterranean area, a total of 17 expert panellist participated in a Delphi survey where they answered a questionnaire in two of rounds. Three continuous, 49 year-long records of the annual T. melanosporum production from southern Europe’s main truffle producing regions in northeastern Spain, southern France and northcentral Italy was analysed to understand the dependency of T. melanosporum production on climate conditions. Finally, two experiments were carried out in a 1-ha experimental plantation consisting of 249 five-year-old Q. ilex inoculated with T. melanosporum planted in a recently abandoned pasture surrounded by forest located in the eastern Pre-Pyrenees; and in 29 T. melanosporum plantations from Teruel (Spain) with 3, 5, 7, 10, 14 and 20 years-old. Using several molecular techniques such as real-time PCR (qPCR), high-throughput DNA sequencing (PacBio RS II), and a patented qPCR essay (WO2012/032098) to quantify the two mating types, we describe the compositional changes of the fungal community and mating type distribution. The results obtained through the two Delphi rounds allowed us to have a complete description of the current truffle value chain and we confirmed an evolution of the sector due to the cultivation success of truffles. Due to the increase in T. melanosporum production in recent years, a trend of truffle prices decrease has been observed in the last years that have alerted the sector. To reinforce the truffle sector, highly agreed actions to develop in the future were identified. Regarding the ecological part of this thesis, it reveals how T. melanosporum production, significantly relies on previous June-August precipitation totals, while too much autumnal precipitation negatively affects winter harvest. One main concern of this thesis is if other ectomycorrhizal fungi could displace T. melanosporum in plantations and impair truffle production. We found a higher relative abundance of non-T. melanosporum ECM fungi associated with trees closer to the forest, but trees with larger root collar diameter had greater biomass of T. melanorporum mycelium and were those whose fungal community was less affected by the distance to the forest. We did not find association between T. melanosporum mycelium in the soil and distance to the forest or the abundance of non-T. melanosporum ECM fungi was observed. In the chronosequence study, we found that T. melanosporum developed steadily over the years as extraradical mycelium and T. melanosporum was not correlated with changes in other ECM fungi. Mating type frequency did not change across the years. In summary and based on these results, it can be concluded that (i) there is a need to promote the consumption of truffles, given the increase in production due to the cultivation of truffles; (ii) the T. melanosporum production significantly depends on previous summer rainfall, whereas too much autumnal precipitation has negative effects; (iii) even though the forest vicinity will influence the soil fungal community, this may have a limited effect on the growth of truffle mycelium; (iv) T. melanosporum is able to maintain colonization of the trees over the long term; (v) the initiation of the sexual reproduction is not limited by a disproportionate presence of either mating type, nor by displacement by non-T. melanosporum ECM fungi.

Řada programovacích jazyků byla schopna zvýšit svoji rychlost výměnou běhových systémů stavěných na míru za obecné platformy, které pro optimalizaci používají just-in-time překlad, jako jsou GraalVM nebo RPython. V této práci vyhodnocuji, zda je použití takovýchto platforem vhodné i pro jazyky se závislymi typy nebo důkazovými systémy. Tato práce představuje koncepty -kalkulu a teorie typů potřebné pro úvod do závislých typů s relevantními algoritmy, specifikuje malý závisle typovaný jazyk založený na $\lambda\Pi$ kalkulu, a prezentuje dva interpretery tohoto jazyka. Tyto interpretery jsou psané v jazyce Kotlin, první je jednoduchý, psaný ve funkcionálním stylu a druhý používá platformu GraalVM a Truffle. GraalVM je platforma založená na virtuálním stroji Javy (JVM), která přidává just-in-time překladač založený na částečném vyhodnocení (partial evaluation) a Truffle je knihovna pro tvorbu programovacích jazyků využívající tento překladač. Závěr práce vyhodnocuje běhové charakteristiky těchto interpreterů na různých zátěžových testech.Závěry práce jsou ale silně negativní. Vliv JIT překladu není znatelný ani přes snahu optimalizovat běžné algoritmy z teorie typů, které jsou zjevně nevhodné pro platformu JVM. Práce končí návrhy několika navazujících projektů, které by lépe využily možnosti Truffle a které by byly vhodnější pro implementaci závisle typovaných jazyků.

Magíster en Gestión para la Globalización
El objetivo de este proyecto es la elaboración de la primera etapa de un plan de negocios para la empresa Aphrodite Truffles, que iniciará actividades de truficultura en Chile, con la finalidad de comercializarlas en el exterior; específicamente en el mercado asiático, considerando a Hong Kong como puerta de entrada. Hoy en día, se ha dado un creciente interés por el cultivo de trufas, el cual puede realizarse en pocos lugares en el mundo, que cumplen con condiciones especiales. Europa, tradicional productor, por distintos factores ha disminuido en el tiempo su labor. Hoy en el hemisferio Sur, Australia, Nueva Zelandia y últimamente Argentina han incursionado con éxito desarrollando técnicas propias de producción. En Chile se ha logrado poner en marcha la producción de trufas negras gracias a la instauración de tecnologías agronómicas modernas junto a la elección acertada del lugar. La metodología utilizada para el desarrollo del presente estudio, considera la realización de un análisis de la industria y mercado de las trufas, investigando fuentes con experiencia acreditada en el rubro, asesoramiento técnico de instituciones locales, y revisión de regulaciones y certificaciones pertinentes, tanto en las plataformas gubernamentales de Chile como las de Hong Kong. El potencial del negocio se sustenta en la creciente demanda en países europeos, EEUU, China y Japón, en donde la oferta no es suficiente. Esto garantiza un acceso al mercado con altos precios, que van de 1500 2500 USD/kg. (Ville de Carpentras, 2012) a los 4000 USD/kg. (Oficina Económica y Comercial del Consulado de España en Hong Kong, 2012-2013). Por otra parte, Hong Kong ostenta una economía abierta y un poder adquisitivo y disposición a pagar por bienes de lujo que lidera el ranking mundial. La estrategia competitiva por diferenciación de producción en contra temporada respecto al Hemisferio Norte, es una ventaja. Por lo cual, son pocas las barreras de entrada y la competencia para la empresa. El tamaño del mercado, posee un nivel de importación actual cercano a los 28.500 Kgs. en volumen y de unos 74 millones de USD abasteciéndose sólo en temporada de invierno (Oficina Económica y Comercial del Consulado de España en Hong Kong, 2012-2013). Por lo tanto, teniendo que las proyecciones de oferta productiva de Aphrodite Truffles son cercanas a los 900 Kgs. en el peak de producción en la contra temporada insatisfecha, se calcula un match acorde a las capacidades del mercado. Por esto, se recomienda: continuar con la 2ª etapa del plan de negocios estableciendo y resaltando estándares de calidad con el fin de agregar valor al bien producido bajo técnicas rigurosas y amigables con el ambiente y; potenciar un enfoque hacia el cliente, sobre todo al tratarse de un mercado internacional con características particulares.

Magíster en Gestión para la Globalización
El propósito de este estudio, es evaluar y a la vez planificar la incursión exitosa de Aphrodite Truffles en el negocio de producción y posterior comercialización internacional de la trufa negra de perigord. En particular, corresponde a la segunda parte del plan de negocios, con foco en el plan de operaciones y plan financiero. El atractivo del negocio de la trufa negra de perigord, radica en la brecha entre la demanda y oferta mundial, los altos precios y la oportunidad de proveerla en contra-estación. Al respecto, las estimaciones más conservadoras, indican que la oferta actual satisface el 65% de la demanda, en tanto las más optimistas en torno al 20%. De ahí, que los precios en restaurant puedan llegar actualmente a los USD 2.500 por kilo de trufa. La metodología utilizada, está basada en el trabajo realizado en la primera parte del plan de negocios, el que identificó el atractivo del mercado de Hong Kong como destino para las exportaciones de Aphrodite Truffles. A partir de ahí, se planifican las operaciones, y la implementación de la empresa, para la posterior evaluación financiera y análisis de variables relevantes en el resultado del negocio. El plan de operaciones, proporciona los requerimientos técnicos para una producción eficiente y de calidad. Con ese fin, se contempla la adquisición de un predio en la zona de Rengo en el cual se cultivarán 15 Ha con árboles previamente micorrizados. Se requirió considerar el flujo de las operaciones productivas y comerciales que tendrá la empresa para establecer las necesidades de recursos humanos, activos e insumos para su operación. En cuanto a la implementación del proyecto, ésta se dividió en la adquisición de los activos, selección de personal y contratación, preparación del terreno y plantación. Posteriormente, el análisis económico, entrega una cuantificación de la rentabilidad del negocio, mediante una tasa de descuento de 14,5% se obtiene un VAN de USD 1.453.093.- en un horizonte de 25 años y una TIR de 25,6%. Luego, en la sensibilización por precio, monto de la inversión y rendimientos, se estimó los valores límite que pueden desviarse dichas variables, para entregar una rentabilidad al inversionista. Finalmente, como conclusión, se exponen los factores críticos de éxito, como la cadena logística y la asesoría experta para conseguir los rendimientos esperados y que si bien es un proyecto de largo plazo, tiene la ventaja de ser rentable en una escala de sólo 15 Ha, debido al alto margen de contribución del producto.

Les truffes sont des champignons ectomycorhiziens du genre Tuber, au sein des Pézizomycètes, vivant en symbiose avec de nombreux arbres et arbustes. Parmi les Pézizomycètes se retrouvent des espèces saprotrophes, pathogènes et symbiotiques ainsi que des champignons très connus comme les truffes et les morilles. Malgré leur intérêt, la structure et l'évolution des génomes des champignons de la classe des Pézizomycètes est encore mal connue. Les objectifs de ma thèse étaient de caractériser la structure et l'évolution des génomes de Truffes et d'autres Pézizomycètes. Une analyse de génomique comparative entre huit génomes de Pézizomycètes, dont trois de truffes, a montré que l'expansion de la taille des génomes des truffes, due principalement aux rétrotransposons gypsy, a probablement eu lieu chez l'ancêtre commun des Tuberaceae il y a environ 150 millions d'années. Ensuite, une réduction de leur contenu en gènes, et principalement en enzymes dégradant la paroi végétale, a eu lieu chez l'ancêtre des Tuber il y a environ 100 millions d'années. Des convergences et des divergences évolutives ont été mise en évidence entre les champignons ectomycorhiziens basidiomycètes et les truffes. Une analyse de génomique comparative au sein de l'espèce T. melanosporum a quant à elle permis de caractériser une ressource de plus de 400000 polymorphismes et de mettre en évidence des traces de sélection. Enfin une analyse plus détaillée des rétrotransposons gypsy présents dans le génome de T. melanosporum a montré que leur colonisation du génome est ancienne. Certains éléments semblent toutefois avoir transposé récemment suggérant que les rétrotransposons gypsy peuvent continuer de jouer un rôle dans l'évolution du génome de T. melanosporum
Truffles are ectomycorrhizal (ECM) fungi, belonging to Tuber genus in the Pezizomycete Class, that form symbiotic associations with numerous trees and shrubs. The Pezizomycetes constitute an early diverging lineage of Ascomycota composed of saprophytic, mycorrhizal and pathogenic species. Some Pezizomycetes, such as truffles and morels, are widely recognized by scientist and lay-person alike. Despite their importance, the genomic structure and evolution within the Pezizomycetes is largely unknown. The aims of my thesis were to characterize the structure and the evolution of the truffle and other Pezizomycete genomes. A comparative analysis performed using eight Pezizomycete fungi, among them three truffles, showed that the genome of truffles has evolved by a size expansion, mainly due to gypsy retrotransposons, in the common ancestor of Tuberaceae about 150 Mya. Then, a loss of the number of gene models, such as plant cell wall degrading enzymes occurred in the common ancestor of Tuber spp about 100 Mya. This study provides new insights into the evolution of the truffles, and ECM symbiosis in general, and highlights cases of divergence and convergence between Basidiomycota and truffle symbiotic species. A comparative analysis in T. melanosporum allowed characterizing a resource of more than 400,000 single nucleotide polymorphisms. This population genomic analysis allowed identifying candidate genomic regions with trace of selection. Finally, a more detailed characterization of T. melanosporum gypsy retrotransposons showed that the major invasive waves are old, but that more recently some elements have moved suggesting that retrotransposons continue to play an important role in the evolution of the truffle genome

Tesis para optar al grado de Magíster en Administración (MBA)
Guillermo Ponce [Parte I], Diego Pumarino [Parte II]
Maputruffle es una empresa que produce y exporta trufa negra del Perigord hacia mercados del hemisferio norte de demanda intensiva de este producto, tales como toda Europa y Norteamérica. Somos una empresa que tiene como visión posicionarse como el principal exportador de trufas del país, y ayudar a posicionar a Chile como referente en la venta de trufas frente a países competidores, como Nueva Zelanda o Australia. El mercado de las trufas se presenta como muy atractivo, debido a que la demanda supera la oferta hace al menos una década1. Esto se debe a que la demanda se eleva constantemente, como es la tendencia en el mercado del lujo, mientras que la producción se ha estancado, ya que la trufa silvestre –fuente principal histórica de trufas– se encuentra con producciones decrecientes, y los cultivos “artificiales” no han logrado cubrir la demanda. Chile se presenta como un país con condiciones de suelo y meteorológicas ideales para la producción de trufas. El análisis de fuerzas competitivas de Porter entrega el siguiente resultado: Potenciales Entrantes Rivalidad Sustitutos Poder Compradores Poder Proveedores Medio Baja Medio Bajo Alto Los potenciales entrantes son países que no cuentan con las condiciones ideales de suelo como Chile; la rivalidad es baja, por la escasez de oferta; los sustitutos son medios, si bien no existe un sustituto claro; el poder de los compradores es bajo por la escasez de oferta; el poder de proveedores es alto en Chile, ya que sólo hay uno que produce y vende árboles micorrizados e insumos necesarios. Nuestra propuesta de valor se define por la entrega de un producto de calidad comparable a los más tradicionales productores del hemisferio norte, en una temporada en que éste hemisferio no posee el producto fresco, con precios competitivos accesible oportunamente. La estrategia a implementar será de diferenciación, razón por la cual hemos elegido nuestra imagen corporativa: Maputruffles, que es una palabra compuesta entre el mapudungun y el inglés, resume el valor de procedencia que le otorga el país, reconocido históricamente por sus productos agrícolas de alta calidad, y el mercado objetivo de las trufas negras. Esto nos permitirá ingresar con un precio similar a la competencia (productores de Australia y Nueva Zelanda), lo que optimiza los resultados financieros. La operación requiere la adquisición de un terreno de 20 hectáreas, la plantación de árboles micorrizados, sistema de regadío y una bodega inicialmente. Debido a que los primeros cuatro años desde la plantación no se obtienen trufas, se considera un capital de trabajo para soportar la operación. El equipo principal cuenta de un gerente general y finanzas, un gerente de marketing y ventas, y un jefe de planta. De acuerdo a la producción anual se contratan operadores para la cosecha y cuidado. Los sueldos están de acuerdo al mercado, con una comisión del 1% de las ventas. Los resultados financieros son positivos y se resumen en la siguiente tabla: Inversión Inicial (USD) TIR VAN (USD) Payback Horizonte Proyecto Tasa de Descuento Margen Operacional / Ventas (Año 20) 1.055.751 23,8% 2.389.729 10 años 20 años 14,8% 92,8%

Les Truffes sont des champignons ascomycètes ectomycorhiziens appartenant à la famille des Tuberaceae et plus précisément au genre Tuber. On dénombre à ce jour plus d’une trentaine d’espèces de Tuber en Europe. Lors de ce travail de thèse, nous nous sommes plus précisément focalisés sur le modèle Tuber aestivum-uncinatum. Cette truffe communément appelée « Truffe de Bourgogne » présente un intérêt à la fois gastronomique et culturel.La première partie de ce travail de thèse a porté sur la clarification du statut taxonomique de la truffe de Bourgogne (Tuber uncinatum). Pour cela, nous avons utilisé une approche multi-marqueurs combinant des marqueurs génétiques couramment utilisés à l’échelle interspécifique. Nos analyses ont montré que les deux taxons Tuber aestivum (la truffe d’été) et Tuber uncinatum sont conspécifiques. Durant la deuxième partie, nous nous sommes intéressés à la diversité génétique de Tuber aestivum. Pour cela, nous avons tout d’abord développé des marqueurs microsatellites spécifiques par une approche de « direct shotgun pyrosequencing ». Cette méthode a permis le développement de 15 marqueurs microsatellites polymorphes. Nous les avons ensuite utilisés pour génotyper des individus provenant de différentes localisations en Europe. Nous avons pu identifier quatre sous populations différenciées qui ne correspondent pas, pour la majorité, à une répartition géographique. Cependant, un des clusters se différencie des autres à la fois par sa situation géographique (sud de la France) et ses caractéristiques génétiques (présence d’allèles rares). Ces résultats préliminaires pourraient indiquer l’existence d’un écotype particulier attaché à une écologie méridionale, Tuber aestivum sensu stricto.Nous nous sommes ensuite intéressés, dans la troisième partie de ce travail de thèse à la diversité aromatique de Tuber aestivum à l’échelle locale. Les résultats obtenus permettent de mettre en évidence l’existence d’une différenciation modérée entre les individus issus d’une truffière naturelle et les individus issus d’une truffière plantée. D’une saison de récolte à l’autre, une stabilité génotypique a été observée. Au niveau aromatique, seuls les composés C8 semblent être liés aux génotypes.Dans la dernière partie, nous nous sommes intéressés à l’analyse de données de récolte sur plus de trente ans au sein d’une truffière plantée de noisetiers inoculés initialement par Tuber melanosporum. Grâce à des analyses statistiques simples, nous avons pu noter les fluctuations tant en quantité qu’en poids des truffes récoltées suivant les saisons et les arbres truffiers. Il apparait que le remplacement de Tuber melanosporum par Tuber aestivum s’est fait de manière très rapide (trois ans). La disparition de Tuber melanosporum peut probablement être expliquée par la fermeture de la canopée des noisetiers, Tuber melanosporum n’appréciant pas un ombrage excessif
Truffles are ectomycorrhizal Ascomycota fungi belonging to the Tuberaceae family and more specifically to the Tuber genus. More than thirty Tuber species are currently described in Europe. In this thesis, we specifically focused on the Tuber aestivum-uncinatum model. This truffle is commonly called "Burgundy Truffle" and has a gastronomic and cultural interest.The first part of this thesis focused on the taxonomic status of the Burgundy truffle (Tuber uncinatum). For this, we used a multi-marker approach combining several genetic markers commonly used at the interspecific scale. Our analyses showed that the two taxa, Tuber aestivum (summer truffle) and Tuber uncinatum are conspecific.In the second part, we addressed the genetic diversity of Tuber aestivum. To do this, we firstly developed specific microsatellite markers by "direct shotgun pyrosequencing". This method has allowed the development of 15 polymorphic microsatellite markers. Then, we used those markers to genotype individuals from different European locations. We have identified four differentiated subpopulations that not correspond, for the majority, to a geographical distribution. However, one cluster differs from the others by its location (south of France) and its genetic characteristics (presence of rare alleles). These preliminary results may indicate the existence of a particular ecotype attached to a southern ecology: Tuber aestivum sensu stricto.We were then interested, in the third part of this thesis, to the aromatic diversity of Tuber aestivum at a local scale. Our results highlight the existence of a moderate differentiation between individuals from a natural truffle orchard and individuals from planted orchard. From one season to another, genotypic stability was observed. Only C8 volatile organic compounds seem to be related to the genotypes.In the last part, we analyzed harvesting data, over more than thirty years, from an hazelnut truffle orchard initially inoculated by Tuber melanosporum. Through simple statistical analyzes, we noted changes in both quantity and weight of truffles harvested according to the seasons and hazelnut trees. It appears that Tuber aestivum rapidly replaced Tuber melanosporum (in three years). The disappearance of Tuber melanosporum can probably be explained by the canopy closure; Tuber melanosporum not appreciating excessive shading

La composition chimique de l'arôme de la truffe noire a fait l'objet d'études scientifiques à partir des années 1980. Avec l'amélioration progressive des techniques d'analyse des effluves, nombre de composés volatils ont été identifiés. Afin d'élucider quels constituants de l'espace de tête de la truffe contribuent réellement à son parfum si célèbre, cette thèse présente une étude comportant 78 analyses sur la truffe fraîche en chromatographie en phase gazeuse (CPG) couplée à l'olfactométrie ainsi que 103 analyses en CPG couplée à la spectrométrie de masse. En plus des composés majoritaires déjà bien connus, douze nouveaux composés volatils à fort potentiel olfactif ont été décelés et identifiés. Les matrices de données instrumentales et olfactives ont ensuite été soumises à un traitement statistique dans l'objectif de mettre en évidence des facteurs explicatifs de la variance dans la composition des effluves de la truffe. L'influence du facteur origine géographique prévaut sur celle des autres paramètres en étude. L'origine « Sud-ouest de la France » affiche la plus grande différence dans le schéma de réponse. Le deuxième volet de cette thèse est consacré à la valorisation d'un sous-produit de l'industrie trufficole. Un jus de cuisson visqueux et aromatique a été mélangé à un matériau de support puis séché par atomisation et lyophilisation, respectivement. Un suivi aromatique s'appuyant sur quatre approches complémentaires d'analyse des effluves, a montré que les deux méthodes de séchage sont efficaces à l'égard de l'encapsulation des composés aromatiques
The chemical composition of black truffle aroma has been subject of scientific study since the early 1980ies. The development of new and efficient headspace trapping techniques has lead to the identification of a great number of volatiles. In order to shed light on the question which of these dozens of compounds really contribute to the famous truffle flavour, this thesis features a study comprising 78 runs of gas chromatography-olfactometry (GC-O) analysis on fresh truffles as well as another 103 GC runs with mass selective detection. In addition to the well known major compounds, twelve minor volatiles where identified as potent odourants. Instrumental and olfactory data matrices were then subjected to statistical analysis, in order to investigate factors explaining variance in the headspace data. Geographical origin was found to be the prevailing factor. Its significance is mainly due to the different response pattern of the sample class “south-western France”. The second part of this thesis is dealing with the utilisation of by-product of the truffle tinning industry. This highly aromatic and viscous cooking juice was mixed with maltodextrim and dired by spray-drying and freeze-drying, respectively. A follow-up study of aroma fidelity was based of four complementary analytical approaches and showed that both drying processes are efficient with regard to flavour encapsulation

Two species of desert truffles, Tirmania and Terfezia were collected from the Northern desert region in Saudi Arabia for antimicrobial and antioxidant activity testing. Both species were extracted with four types of extraction solutions, methanol, ethanol, ethyl acetate and water to test for antimicrobial activity and with three extraction solutions, methanol, ethanol and water for antioxidant activity. Using disc diffusion method, the extracts were subjected to twenty three different microorganisms to observe the antimicrobial activity by measuring clear zones. Methanol extract from Tirmania was the most effective, followed by those extracted with ethanol, water and ethyl acetate respectively. In Terfezia, ethanol extract was better than methanol extract in effectiveness. Ethyl acetate extracts were the least effective. The results indicate that truffles possess antimicrobial activity with broad spectrum effects against Gram positive, Gram negative, aerobic and anaerobic bacteria as well as Saccharomyces, while no effect was recorded with fungi. The results on antioxidant activity showed that truffles have very strong antioxidant property with 99.9% with ethanol extracts of Tirmania species and 95.5% with ethanol extract of Terfezia species using beta-carotene bleaching method and antioxidant property with 96.1% with ethanol extracts of Tirmania species and 95.3% with methanol extract of Terfezia species using DPPH free radical method.

Desert truffles are edible hypogenous ascocarps produced by Ascomycota (Pezizaceae) fungi characteristic of arid and semi-arid zones. Many of these desert truffle species are nowadays considered as valued Non-Wood Forest Products (NWFPs) endemic to the Mediterranean basin where their ascocarps, rich in proteins and poor in carbohydrates and lipids, constitute a potentially important food source for rural populations. They also represent a key component of the mycological flora on arid and semi-arid habitats, acting directly as symbiotic partners of diverse host plants, mainly Cistaceae, and indirectly as desertification and soil erosion prevention agents. Terfezia (Tul. & Tul.) Tul. & Tul. is undoubtedly the best known and most diversified of all desert truffle genera, but its taxonomy is far from resolved. In addition, cultivation of desert truffles is not trivial and despite all research efforts in the last two decades, only two Terfezia species were to date successful cultivated both with perennial and annual Helianthemum species in basic soils. Research on how to cultivate Terfezia spp. with different plant hosts and over a broader array of soil types and pH values is still necessary to find most suitable “fungal symbiont – plant host” combinations for sustainable and efficient cultivation over a wider range of habitats. With this in mind, this work aimed to: 1) expand the current knowledge on the diversity of genus Terfezia in southern Portugal; 2) study their putative mycorrhizal associations with Cistus spp.; and 3) develop methodologies to allow Cistus spp. to be used as host in desert truffle cultivation in acid soils. Overall, the present research clearly demonstrates that Cistus spp. are indeed promising candidates for desert truffle cultivation and that different Terfezia-Cistus combinations can be used to enable desert truffle cultivation over a wide range of situations depending on the plantation purposes; DIVERSIDADE DO GÉNERO TERFEZIA NO SUL DE PORTUGAL E AS SUAS ASSOCIAÇÕES MICORRÍZICAS COM CISTUS L.: UM ESTUDO PARA VIABILIZAR A PRODUÇÃO DE TRUFAS DO DESERTO EM SOLOS ÁCIDOS RESUMO: O termo trufas do deserto descreve os ascocarpos hipógeos comestíveis, produzidos por fungos Ascomycota (Pezizaceae), característicos de zonas áridas e semiáridas. Estes são atualmente considerados como importantes produtos florestais não lenhosos, endémicos da bacia do Mediterrâneo, e constituem uma fonte de alimento, rica em proteínas e pobre em hidratos de carbono e lipídios, para as populações rurais. As trufas do deserto representam ainda um componente-chave da flora micológica em habitats áridos e semiáridos, atuando como parceiros simbióticos de diversas plantas hospedeiras, principalmente Cistaceae, e como agentes de prevenção contra a desertificação e erosão do solo. Terfezia é indubitavelmente o género mais conhecido e diversificado de trufas do deserto, mas a sua taxonomia está longe de estar esclarecida. Também o seu cultivo não é trivial e, apesar de todos os esforços de pesquisa nas últimas duas décadas, apenas duas Terfezia foram, até o momento, cultivadas com sucesso, em solos básicos, com espécies perenes e anuais do género Helianthemum (Cistaceae). Falta ainda conhecer vários binómios “simbionte – planta hospedeira” que permitam o cultivo de Terfezia numa ampla variedade de solos e habitats. Assim, os principais objetivos deste trabalho foram: 1) ampliar o conhecimento atual sobre a diversidade do género Terfezia no sul de Portugal, 2) estudar as suas potenciais associações micorrízicas com Cistus spp. e 3) desenvolver metodologias para permitir a sua aplicação no cultivo de trufas do deserto em solos ácidos. No presente trabalho, é claramente demonstrado que Cistus spp. são candidatos promissores para o cultivo de trufas do deserto e que diferentes combinações de Terfezia-Cistus podem ser usadas para o cultivo de trufas do deserto numa ampla gama de situações, dependendo dos propósitos da plantação.

Etude de la croissance et du comportement in vitro en particulier de Tuber uncinatum. Mise au point de quatre milieux de base gélosés. Analyse des effets de la nutrition carbonée et azotée; influence des vitamines, des variations de température du PH, du taux d'oxygène du milieu; action des sels minéraux et des substances de croissance. La production d'arôme truffier in vitro est abordée

Contemporary scholars seeking to increase Tuber Melanosporum truffle production rely almost exclusively on technological advancements to increase yields, while failing to place the cultivation of truffles, trufficulture, in its historical or local landscape contexts. In this dissertation, I describe how truffle scholars’ conceptualization of trufficulture and landscapes has changed over 150 years in France, while focusing on the French département of Lot. I examine changing relations between humans and nonhumans and how they impact truffle harvests. I analyzed the history of French trufficulture through a close reading of historic truffle manuals, archival research and the classification of remotely sensed images. Shifting from the past to the present, from July 2014-August 2016, I conducted semi-structured survey interviews with working truffle-growers (trufficulteurs) and participant observation at meetings of trufficulteurs, truffle hunts and truffle markets. I utilize actor-network theory (ANT) as both a theory and methodology. Actor-network theory allowed me to follow the impacts made by both humans and nonhumans on trufficulture. I found that truffle harvests in the 1880s dropped by 90%. Highly populated, intensively worked landscapes of viticulture, silvopastoralism and cereal cultivation created conditions suitable to truffles. By the 1870s the phylloxera aphid ravaged grapevines, which made trufficulture an important source of revenue. These advantageous conditions would not last. Post-WWI, yields fell for decades because of an ongoing rural population exodus and consequent agricultural abandonment, which promoted reforestation and closed canopy forests in Lot, France. By the 1960s, French trufficulteurs organized associations to share knowledge and promote local truffle markets to revive production. Trufficulteurs’ utilization of tractors, ‘inoculated’ plants and irrigation systems produced a new form of “modern” trufficulture. State subsidies helped trufficulteurs adopt “modern” practices, in hopes of increasing yields. “Modern” trufficulture has not dramatically increased yields. A few highly-capitalized trufficulteurs dominate production in Lot. Many others practice trufficulture as a hobby. Instead of relying on “modern” technological fixes, my findings suggest that trufficulteurs, farmers and states should reinvigorate extensive polyculture farming practices that maintain open canopy forests, which were beneficial to trufficulture in the past. Actor-network theory allowed me to rethink human and nonhuman relations, and to propose alternatives to “modern” trufficulture.

La truffe noire du perigord, tuber melanosporum vitt. A suscite, de part ses qualites gastronomiques et sa croissance mysterieuse un interet economique mais egalement scientifique. L'evolution structurale et biochimique de ses trois derniers stades de maturation ont ete d'abord determinees puis la melanine des truffes matures a ete extraite, purifiee, caracterisee et finalement identifiee comme etant de la pheomelanine. En effet sa degradation chimique donne essentiellement de l'aminohydroxy-phenylalanine qui apparait resulter de l'oxydation de la benzothiazine, un intermediaire connu de la classe des pheomelanines. Ces resultats nouveaux peuvent ainsi servir pour une identification de la truffe noire et pour un dosage precis des melanines

La Truffe noire (Tuber melanosporum Vittad.) est un champignon ectomycorhizien spontanément présent dans les groupements végétaux ouverts en cours de reforestation. Alors que ces milieux ont fortement régressé au XXème siècle dans la zone méditerranéenne, 80 % de la production actuelle provient de boisements artificiels où des arbres inoculés par la Truffe sont plantés. Malgré un grand corpus de connaissances empiriques, la production reste souvent aléatoire et les connaissances fondamentales d'écologie et de biologie de la Truffe restent fragmentaires. Dans ce travail, nous avons d'abord étudié la distribution de la diversité fongique ectomycorhizienne sur les différents hôtes présents dans les garrigues pré-forestières à Truffe. Nous avons ensuite testé les interactions entre la Truffe et les plantes endo- ou non-mycorhiziennes, qui se matérialisent par la création d'un brûlé, où la flore est localement affectée. Dans un troisième volet, nous avons cherché à mieux comprendre la diversité génétique des populations de Truffes, et plus spécifiquement l'appariement sexuel et la dispersion de cette espèce à vie végétative haploïde et à fructification hypogée. Par une approche comparative entre truffières plantées et spontanées, nous avons finalement évalué les modifications liées au processus de proto-domestication en cours. Ainsi, en combinant écologie des communautés, expériences en conditions contrôlées et génétique des populations, nous avons montré qu'en région méditerranéenne :1) La Truffe est présente de façon fugace dans des communautés ectomycorhiziennes riches, avec de nombreuses espèces multi-hôtes, mais où la Truffe montre une préférence d'hôte marquée pour le chêne vert (Q. ilex). 2) Certaines plantes endo- ou non-mycorhiziennes, dont l'effet positif sur la Truffe a empiriquement été observé par les trufficulteurs, favorisent le développement du mycélium de Truffe dans le sol, agissant indirectement sur les interactions plante-plante (chêne – plantes endomycorhiziennes). L'effet inhibiteur de la Truffe observé sur la germination des graines peut-être une des causes précoces du brûlé. Par ailleurs, la Truffe semble effectivement coloniser les racines de plantes herbacées non-ectomycorhiziennes.3) Les flux de gènes sont limités à l'échelle de la truffière, l'appariement sexuel réunit des individus proches génétiquement et physiquement, et bien que la Truffe soit probablement hermaphrodite, les parents paternels sont peu détectables, probablement de taille plus réduite que les parents maternels (formant la gléba). Les pratiques culturales pourraient entraîner un brassage génétique plus important en plantation, mais à l'échelle régionale, aucune différence de diversité génétique n'a été détectée entre populations spontanées et cultivées. Ce travail montre la richesse des interactions biotiques impliquant la Truffe et les diversités végétale et fongique des truffières artificielles et spontanées de la région méditerranéenne. Les résultats acquis contribuent à lever le voile sur sa biologie reproductive, et jalonnent le chemin pour des pratiques intégratrices de la diversité biologique des truffières, ainsi que pour le développement de futures expérimentations in situ. Mots clés : appariement sexuel, Arbutus unedo, Cistus albidus, domestication, écologie des communautés, forêts méditerranéennes, génétique des populations, isolement par la distance, ITS, microsatellites, mésocosmes, mycorhizes, pratiques empiriques, qPCR, Quercus coccifera, Quercus ilex, successions secondaires
The Black Truffle (Tuber melanosporum Vittad.) is an ectomycorrhizal fungus spontaneously growing in open woodlands before canopy closure. Such open landscapes drastically regressed during the last century in the Mediterranean regions, and nowadays 80% of the production comes from man-made plantations where the Truffle is inoculated. Despite a large corpus of local knowledge and empirical practices, the production remains largely sporadic and unpredictable, and our knowledge of the biology and ecology of the Truffle is still fragmentary. In this work, we first analyzed the distribution of the ectomycorrhizal fungal diversity among host plants co-existing in the shrub-dominated landscapes where Truffle naturally occurs. We then analyzed the interactions between the Truffle and endo- or non-mycorrhizal plants, as they typically occur in the so-called brûlés, zones with scarce vegetation. A third part aimed at better understanding the genetic diversity of Truffle populations, with special focus on fertilization and dispersal process of this fungus with haploid lifecycle and hypogeous fruiting. In a multi-scale approach combining community ecology, experimentation and population genetics, we found that in the Mediterranean region:1) The Truffle is transiently present in rich ectomycorrhizal communities, showing a significant host preference for Q. ilex, in assemblies made of numerous multi-host fungal species. 2) Some endo- or non-mycorrhizal plants species, that were supposed to provide beneficial effect on the Truffle, can be experimentally shown to stimulate the development of T. melanosporum mycelium in soil, and indirectly trigger plant-plant interactions (between oak and endomycorrhizal plants). The early brûlé symptoms could at least partially result from a Truffle's inhibitor effect on seed germination.3) At truffle ground scale, gene flow is limited, and mating occurs between genetically and physically close parents. Despite probable hermaphroditism of the Truffle, paternal parents are poorly detectable, certainly of smaller size than maternal ones (these forming nourishing tissue of the ascocarp). Cultural practices could favor genetic mixing/diversity at brûlé scale, but at the regional scale, no difference in genetic diversity was found between spontaneous and planted compartments.This work revealed the richness of biotic interactions involving the black Truffle and the plant as well as the fungal diversity in both artificial and spontaneous truffle-ground of the Mediterranean region. These results enlighten the reproductive biology of the species, and pave the way for practices integrating the biological diversity of truffle-grounds and the development of further in situ experimentations. Key words: Arbutus unedo, Cistus albidus, community ecology, domestication, empirical practices, local knowledge, experimental approach, inbreeding, isolation by distance, ITS, Mediterranean forests, mesocosms, microsatellites, mycorrhiza, population genetics, qPCR, Quercus ilex, Q. coccifera, secondary successions

Depuis la plus haute antiquité jusquà nos jours, la truffe a suscité l'intérêt de nombreux scientifiques. Il s'agit d'un champignon de la classe des Ascomycètes qui correspond à la fructification d'un mycélium souterrain vivant de façon symbiotique au contact des racines de certains arbres, chênes et noisetiers. Ses zones de production se limitent principalement à l'Europe du sud, la France, l'Espagne et l'Italie. La 1ère partie de ce mémoire recense, sur la base d'une étude bibliographique exhaustive, l'ensemble des travaux réalisés sur les truffes et plus particulièrement l'espèce Tuber Melanosporum Vitt. Sont présentés ensuite les résultats d'une étude préliminaire dans laquelle est étudiée pour la 1ère fois, la relation entre la composition chimique de la truffe et le type de formation géologique sur lequel se développe le champignon. Le 2ème chapitre, est consacrée à l'identification chimique des composés volatils produits par les 12 principales espèces européennes du genre Tuber. Nous avons ainsi pu déterminer le ou les composés clés responsables de l'arôme ainsi que les éventuels marqueurs chimiques spécifiques d'une espèce. A la suite de ces travaux, une étude de faisabilité réalisée sur des jus de truffes industriels a permis de développer une méthode permettant de discriminer l'espèce de truffe employée. Enfin, la dernière partie de ce manuscrit, à visée industrielle, détaille l'optimisation d'un procédé d'aromatisation naturelle d'une huile à la truffe noire par la technique d'ultrasonification et la valorisation des résidus sous la forme de truffes reconstituées. La garantie de la naturalité du produit aromatisé est ensuite assurée par la mise au point d'une technique analytique basée sur la chromatographie chirale monodimensionnelle.

La truffe noire de Périgord, Tuber melanosporum Vitt. A suscité, de par ses qualités gastronomiques et sa croissance mystérieuse un intérêt économique mais également scientifique. Il est cependant surprenant de constater qu'après de nombreux travaux sur la systématique du groupe auquel appartient cet ascomycète, peu de recherches d'ordre physiologique n'ont été entreprises : c'est dans ce domaine que nous avons inscrit ce travail. En outre, si la plus grande partie des expérimentations portent sur la phase végétative qui correspond à la vie symbiotique que le mycélium entretient avec les radicelles d'un végétal hôte, le reste du cycle de la truffe était en revanche totalement inexploré. C'est pourquoi, nous nous sommes donnes pour objectif de définir les différentes étapes du développement de la truffe, d'un point de vue cytologique et physiologique, et d'en déterminer les facteurs externes limitants. Notre démarche reposait sur l'idée que la connaissance approfondie du cycle de la truffe permettrait d'améliorer et de réguler la production de ce champignon sur le terrain. Après avoir décrit dans un premier chapitre les techniques auxquelles nous avons eu recours, nous exposons les résultats les plus significatifs en montrant leur cohérence. Si les techniques analytiques sont classiques, en revanche, pour déterminer la dépendance de la croissance vis à vis des facteurs externes, nous avons eu recours à des observations et a des relevés de données sur plusieurs années.

La démarche suivie au cours de ce travail consiste en la micropropagation de chênes, la réalisation et la diversification des techniques de synthèses et enfin le contrôle de la mycorhization en microscopie photonique et électronique ainsi que l'étude des différentes étapes de la mycorhization. L'acclimatation des vitroplants est réalisée selon trois dispositifs: miniserre, pots horticoles et boites caubere, ce dernier donne de meilleurs résultats. L'étude du mycélium, en microscopie électronique à balayage et à transmission, a révélé la présence de bactéries. L'identification de quelques souches de bactéries a montré qu'elles appartiennent à bacillus polymixa. Plusieurs synthèses ont été réalisées en conditions gnotoxeniques en utilisant divers inoculums (spores, racines excisées inséminatrices et morceaux de gélose porteurs de mycélium), selon diverses techniques. La suspension sporale demeure le meilleur inoculum. En conditions axéniques, les synthèses sont effectuées en boites de pétri et en tubes. L'étude ultrastructurale nous a permis de montrer que l'infection se déroule en deux phases: la phase d'accrochage, par l'intermédiaire de fibrilles, et la phase d'installation des hyphes sur la surface racinaire. Les cellules corticales sont séparées des hyphes par de larges couches à tanins et les bactéries sont présentes. La progression des hyphes, à travers les couches à tanins, se fait par invagination pour la formation du manteau et par pression mécanique pour pénétration intercellulaire. La formation du manteau précède celle du réseau de Hartig. La dégénérescence des cellules fongiques suit celle des cellules corticales qui intervient précocement des l'édification du réseau de Hartig. Les relations entre les deux partenaires sont réduites comme le confirment les activités atpasiques

Kalaharituber pfeilii (Henn.) Trappe & Kagan-Zur commonly known as the “Kalahari truffle” is a desert truffle species identified from the Kalahari region of southern Africa. Two other species, Eremiomyces echinulatus (Trappe & Marasas) Trappe & Kagan-Zur and Mattirolomyces austroafricanus (Trappe & Marasas) Trappe & Kovacs are also known to occur in other parts of southern Africa. Truffles are hypogeous fruiting bodies of Ascomycetes, important to humans for their nutritional value and medicinal characteristics. These truffles are known as desert truffles as they prefer to occur under arid or semi-arid conditions characteristic of deserts. Truffle development depends on the presence of a mycorrhizal host, associated microorganisms as well as soil and climatic characteristics. It has been suggested that K. pfeilii has a suspected broad plant host range which includes herbaceous to woody trees and shrubs. However, these relationships have not been verified. Indigenous people of the Kalahari believe that truffles are found under grasses. In the Kalahari, truffle fruiting bodies are often found entangled in Stipagrostis ciliata (Desf.) De Winter var. capensis (Trin. & Rupr.) De Winter roots. S. ciliata, also known as the tall bushman-grass, is the most common grass found in the Kalahari. The objective of this study was to provide conclusive evidence that S. ciliata var. capensis is a host of the Kalahari truffle. Truffle fruiting bodies and grass roots from where the truffles were found were collected from Upington, South Africa. The fruiting bodies were identified by observing their morphological characteristics using the ‘Keys of Truffle genera’. All observed physical properties were similar to those of K. pfeilii and further identification was done using molecular techniques. DNA was extracted from the fruiting bodies, mycelial cultures, rhizosheaths and from the S. ciliata var. capensis grass roots, which were then amplified using the specific K. pfeilii specific primers TPF3 and TPR1 and sequenced. The obtained sequence results confirmed that the collected fruiting bodies were those of the K. pfeilii and the molecular techniques also confirmed that K. pfeilii DNA was present in the S. ciliata var. capensis rhizosheath and root cells. Microscopy showed an ectendomycorrhizal association between K. pfeilii and S. ciliata var. capensis. Mycorrhizal resynthesis experiments were conducted to establish this mycorrhizal association in-vitro. They were unsuccessful because of the structure of the grass and the availability of contaminants. And more...

La symbiose mycorhizienne est une interaction mutualiste formée entre les racines des plantes terrestres et des champignons du sol. Les changements morphoanatomiques associés au développement de cette symbiose sont accompagnés de modifications dans la régulation de l'expression génique. L'étude des profils transcriptomiques est donc fondamentale afin de caractériser les mécanismes moléculaires gouvernant la symbiose mycorhizienne. Le développement récent des approches de transcriptomique à haut débit offre de nouvelles perspectives pour la compréhension de ces mécanismes. Le travail entrepris dans le cadre de ce projet de thèse visait à caractériser in silico le transcriptome symbiotique du champignon ectomycorhizien Tuber melanosporum et du champignon endomycorhizien Glomus intraradices. Il s'agissait de mettre en place les outils et les protocoles bioinformatiques permettant l'exploitation des données transcriptomiques issues des nouvelles technologies de séquençage, afin de caractériser les transcrits exprimés par les symbiotes et d'identifier les gènes régulés au cours de la symbiose. Ce travail original a permis de souligner l'existence de traits communs aux profils d'expression des champignons mycorhiziens. De plus, la caractérisation du transcriptome de G. intraradices a permis d'établir le premier répertoire de gènes à l'échelle du génome pour un champignon endomycorhizien. Cette étude de génomique contribue à l'amélioration des connaissances sur les processus moléculaires qui sous-tendent la symbiose mycorhizienne et constitue une ressource unique pour de futures recherches sur les réseaux de gènes contrôlant la symbiose
Mycorrhizal symbiosis is a mutualistic interaction involving roots of terrestrial plants and soil fungi. Morphological changes associated with the development of this symbiosis are accompanied by changes in gene expression. The study of transcriptomic profiles is thus essential to characterize the molecular mechanisms that govern the mycorrhizal symbiosis. The recent development of high-throughput transcriptomic approaches provides new insights for the understanding of these mechanisms. The work undertaken during this thesis aimed to characterize in silico the transcriptome of the ectomycorrhizal fungus Tuber melanosporum and the endomycorrhizal fungus Glomus intraradices. In order to characterize transcripts expressed by the symbionts and to identify genes regulated during symbiosis, bioinformatic tools and protocols were implemented to process transcriptomic data derived from new sequencing technologies. This work has allowed to highlight common features in the expression profiles of mycorrhizal fungi. In addition, characterization of the G. intraradices transcriptome has allowed to establish the first genome-wide repertoire of genes for an endomycorrhizal fungus. The study helps to improve knowledge about the molecular processes underlying the mycorrhizal symbiosis and provides a unique resource for future research on the gene networks controlling symbiosis

Hazel (Corylus avellana L.) seedlings inoculated with the Perigord black truffle fungus (Tuber melanosporum Vitt.) are being planted in Tasmania in an attempt to culture truffles. Competition from other ectomycorrhizal fungi has a significant impact on truffle production in Europe and can be expected to have some effect on the Tasmanian industry. This thesis examines ectomycorrhizal fungi occurring in Tasmania with respect to their ability to form mycorrhizas with hazel and compete with T melanosporum under various soil treatments. Stands of hazel previously established for nut production or as ornaments were surveyed for the sporocarps of ectomycorrhizal fungi. Several species were found ; including species that are new either to Australia or Tasmania. The endemic species Descomyces albus (Klotzsch) Bougher & Castellano and Podohydnangium sp., previously thought to. be Eucalyptus specific were. fruiting under hazel. T melanosporum occurs naturally on calcareous soils in Europe. Truffieres in Tasmania are heavily limed to create a calcareous soil environment. The response of selected introduced and endemic ectomycorrhizal fungi to applied lime was studied in a glasshouse experiment. Some of the endemic species, which would normally inhabit acidic soils, were unable to survive high rates of lime application and therefore should not pose a threat to the truffle industry. The introduced species were generally more tolerant to lime application. A subsequent glasshouse experiment sought to separate the effects of pH and calcium on colonisation by T melanosporum. Seedling hazels were inoculated with T melanosporum. After twelve months, they were transplanted using soil amended with fourteen rates of either CaCO3 , CaSO4, or MgCO3 . The seedlings were then exposed to spores of two endemic fungal species. Applied CaCO 3 and MgCO3 increased level of colonisation by T melanosporum, whereas CaSO4 had little or no effect. Soil pH appears to have a stronger influence on colonisation by T melanosporum than the level of applied or exchangeable calcium. Colonisation by endemic species was low and sporadic across all treatments. Another glasshouse experiment of similar design to that above was established to observe the effect of lime and phosphorus interaction. Applied lime significantly increased the level of colonisation of T melanosporum, but phosphorus had no effect, even at very high rates of application (150 mg P / Kg soil). A commercial truffiere was surveyed for the level of colonisation by T melanosporum and other ectomycorrhizal fungi. Descriptions were compiled of the mycorrhizas of fungal species found in the glasshouse and field experiments to assist in their future identification. The morphological identification of Tuber mycorrhizas was confirmed using PCR and RFLP of DNA extracted from single mycorrhizal tips.

碩士
國立宜蘭大學
食品科學系碩士班
108
Tuber magnatum, also called as Alba white truffle, is known for its unique and characteristic aroma around the world. It has high economic value. Truffle fruitting body needs to be cultivated for several years and the yield is scarce. The large quantities of mycelia can be cultivated by solid-state fermentation and adding precursors can be coverted to increase volatile compounds by enzyme biosynthetic technology; therefore, it may probably meet the demand for truffles in market. Shio koji is a popular seasoning, and it can not only replace salt in cooking, but also add other different flavors. The production time of shio koji varies from season to season, indicating that the temperature will affect the finished time of shio koji. Shio koji contains amylase and protease, which have be considered to improve the texture of bread and rice bread, respectively. The objectives of this study were (1) to cultivate T. magnatum solid-state fermented product for 4 weeks, and then adding different precursors into the fermented product to make the enzyme of truffle mycelia by biosynthesizing characteristic aroma of the fruit body and to obtain kinetics model. And further truffle sauce was sterilized by high pressure processing (HPP). (2) The shio koji was cultured at different temperatures to find the optimum preparation temperature and time to shorten the preparation time of the shio koji, and then shio koji was used the rice breads which were spreaded by the truffle sauce. The results showed that the addition of precursor including leucine, isoleucine and methionine to the truffle solid-state fermented product allowed the enzymes in the truffle to biosynthesize its characteristic aroma such as 3-methyl-1-butanal, 2-methyl-1-butanal, methional, dimethyldisulfide (DMDS), dimethyltrisulfide (DMTS). Adding methionine as a precursor, the main volatile organic sulfur compounds (VOSCs) was methional, and the biosynthesize kinetics model of methional and total VOSCs were the zero-order reaction. The activation energy of methional and total VOSCs were 103.23 and 101.02 kJ/mol, respectively. DMDS and DMTS were produced in large amounts at high temperatures, both of which were the zero-order reaction; therefore, the VOSCs production by adding precursors in the white truffle fermented product were not only derived from enzyme biosynthesis, but also affected by the Maillard reaction. The truffle fermented product obtained from solid-state medium containing 0.1 M methionine could produce a large amount of volatile sulfur compounds than aroma biosynthesis by adding 0.2 M methionine into the truffle fermented product. Therefore, truffle sauce was prepared from truffle 4 weeks fermented products whose medium containing 0.1 M methionine. In addition, the activity of enzymes in shio koji was not affected by the incubation time and mainly based on temperature. Shio koji was treated at different temperatures, and its amylase and protease decreased while temperature and time increased. Amylase and protease activities were relatively stable at 40°C, and the higher temperature could accelerate the enzyme reaction. The sugar concentration at 40°C the was significantly higher than that at 25°C and reached the highest concentration on the third day; the increase rate of tyrosine was fast at 40°C, but the concentration was no significant difference between 25°C and 40°C after the balance reached on the third day. Thus, mixing the dry koji rice and 10% salt water at a ratio of 1:5, the optimal production was at 40° C for 3 days reaction. By adding shio koji into the bread could make the bread texture better, increase the bread volume, and slove the problem of easy hardening due to starch retrogradation in the rice bread. The 9-point hedonic sensory evaluation of rice bread showed that the overall preference of rice bread was 6 points, which indicated that shio koji had potential for development. The truffle solid-state fermented product, olive oil, salt and garlic were mixed at a ratio of 1:1:0.05:0.1 to prepare truffle sauce which was sterilized by 500 MPa HPP 5 min. The overall preference of truffle sauce shio koji rice bread in the 9-point hedonic sensory evaluation was 6.6 points, which had great potential for development.

碩士
國立中興大學
化學工程學系所
105
The truffle is not only one of the most delicious food around the world, but also the most expensive one. The truffle grows associated with the roots of pine, oaks and quercus trees. The nutrient compositions of truffle are abound in various proteins, amino acids (including eight essential ones), unsaturated fatty acid, multi-vitamins, essential trace elements, such as zinc, manganese, iron, calcium, phosphorus, selenium, etc, along with multiple metabolites, such as sphingolipid, cerebroside, neurotide, triterpenoids, -androstane, adenosine, truffle acid, polysaccharides, peptides and so on. Accordingly, the truffle possesses a highly nutritional value for health care. This study reviews the optimal cultivation conditions for the truffle mycelium in the solid and submerged fermentation. The collective data would provide the references for researchers interested in truffle mycelial cultivation. In the literature, the truffle can grow and produce the mycelia and its metabolites in the solid and submerged fermentations. In terms of submerged fermentation, the sucrose is the most applied carbon source, the second is the dextrose; As to nitrogen source, the yeast extract is the most used one, then followed by the peptone. Meanwhile, the most used pH is 6~7.5, where the suggested best pH value is 7. As to temperature, the most applied temperature is 25℃. The most used agitation speed is around 100~120 rpm. The addition of organic acids, lipids and trace elements would increase the yields of biomass and metabolites Keywords: Truffle, Mycelium, Culture condition, Submerged fermentation, Medium

碩士
國立臺灣大學
森林學研究所
93
The purposes of this study were to (1) confirm the effect of truffle inoculation on the seedlings growth of Cyclobalanopsis glauca, (2) observe the competition of Scleroderma areolatum to the mycorrhizae formed either by Tuber formosanom or T. indicum and (3) compare the competition of S. areolatum to the truffle cultured on YMT agar medium. After the seedlings of C. glauca were inoculated with T. formosanum and T. indicum , the height growth, root collar diameter, top dry weight and root dry weight were much more significantly stimulated than those of non-inoculated seedlings. The T/R ratio of both Tuber-inoculated seedlings was significantly lower than those of non-inoculated seedlings. S. areolatum could form typical white ectomycorrhizae , when it was inoculated to one year’s old C. glauca seedlings mycorrhized either with T. formosanum or T. indicum two months later after inoculation. The other uninoculated rootlets could be infected by the rhizomorph extended from white ectomycorrhiza. The rhizomorph could also infect the Tuber-infected ectomycorrhizae and finally enveloped it. Therefore, the inoculation of S. areolatum could reduce or impede the mycorrhiza formation formed by T. formosanum or T. indicum . The Tuber- mycorrhized C. glauca seedlings inoculated with S. areolatum two months later could also stimulate seedlings growth, as the seedlings were inoculated with truffle only. YMT agar medium was used to test the colony competition between T. formosanum and S. areolatum, T. formosanum and S. areolatum each other. The results showed that the colony growth of either T. formosanum or T. indicum grew much quicker than that of S. areolatum, and could impact the growth of S. areolatum. There was compete dominance for the colony growth of T. indicum , especially. The colony of T. indicum could cover the colony of S. areolatum at the end of the test. The colony of T. formosanum and S. areolatum grew mixedly each other at the late stage. Neither the hyphal anastomoses of T. indicum and S. areolatum, nor the hyphal anastomoses of T. formosanum and S. areolatum were observed.

CEMS
This Work Project is a part of the 2016 Business Project course of the CEMS Master in International Management program, done at Aalto School of Business in Helsinki, Finland. It was developed in cooperation with Nokia Networks, having two major goals: (1) the examination of the current state-of-art of mobile advertising within operators in a global level, as well (2) providing recommendations for Nokia on its sales strategy during the expansion to different mobile advertising markets. Additionally, this Work Project also discusses academic insights regarding the importance of managing business ecosystems and the crucial value given by dynamics of co-creation.

碩士
國立中央大學
電機工程學系
107
Understand the principles and operations of blockchain and smart contracts, understand the steps of algorithms, learn to develop Solidty language, write smart contracts, build blockchain technology, and build a real game that can be played in HTML/Javascript

博士
國立中興大學
化學工程學系所
103
The submerged fermentation process for the cultivation of mushroom mycelia and its secondary metabolites has been widely used for the production of pharmaceutical mushrooms. The mushroom’s metabolites could provide antitumor, hepatoprotective and pharmacological effects in the medical applications. In the first part of this study, exopolysaccharides (EPS) produced from Hypocreales sp. NCHU01 was carried out in the submerged cultivation. To find the optimal conditions, factors such as carbon and nitrogen source concentrations, pH, temperature, magnesium ion, and potassium ion levels were considered. The results showed that the optimal conditions were as follows: sucrose, yeast extract, magnesium sulfate and potassium dihydrogen phosphate were 10.6, 10.92, 1.0 and 1.01 g/L, respectively. The optimization conditions were set at 25 C with an initial pH 6.5. The maximum EPS production was about 1329 ± 23 mg/L, which was 3.34-fold to that of the original condition. The study displayed an increasing molecular weight distribution in the harvested EPS over a longer cultivation time. The quantitative relationship between mycelial biomass and EPS production was also built. In the second part, biomass and triterpenoids of the truffle endophytic fungus Hypocreales sp. NCHU01 were produced in the submerged culture. In the carbon source test, 10 g/L of sucrose yielded the highest triterpenoid production, reaching 132.26±1.77 mg/L. In the nitrogen source test, 15 g/L of yeast extract was identified as the most favorable source for crude triterpenoid production, reaching 157.68 mg/L. In the stimulatory test, the addition of 2 g/L linoleic acid or 125 mg/L chitosan to the medium was found to elevate triterpenoid production to 327.5 mg/L and 343.2 mg/L, respectively. The major sterol compositions of the fruiting body of Tuber magnatum and mycelia of Hypocreales sp. NCHU01 were identified by gas chromatography with a mass spectrometer. It was found that sterol composition in the fermentation mycelia of Hypocreales sp. NCHU01 was quite similar to that of the fruiting bodies of Tuber melanosporum, Tuber magnatum and Tuber sinense. Two kinds of sterols (i.e. barrigenol R1; and dehydroepiandrosterone) were identified for the first time from Tuber species. Finally, triterpenoids produced from Tuber borchii was carried out in the submerged cultivation. In the carbon source test, 20 g/L of glucose yielded the highest triterpenoid production (71.17±3.05 mg/L). In the nitrogen source test, 20 g/L of malt extract was identified as the most favorable source for crude triterpenoid production (123.27±4.22 mg/L). In the stimulatory effect, the addition of 2 % peanut oil to the medium was found to elevate triterpenoid production to 191.09±5.34 mg/L, which was 1.55-fold to that of the original condition.

碩士
國立宜蘭大學
食品科學系碩士班
107
Truffle, belonging to certain types of Tuber spp., is an edible fruiting body formed ectomycorrhiza with host plants. It has distinctive flavor and potential health benefits so that is popular all over the world. In recent years, inappropriate collection and deforestation has led to its production decreasing so that artificial cultivation has been attempted. The goal of this study is to select suitable farmland for truffle planting in Siji-Nanshan platform. We preliminarily used ITS sequence and MAT gene as molecular makers to establish the identifying method to confirm the truffle species including T. melanosporum, T. aestivum, T. indicum and T. pseudoexcavatum. The truffle spores were used to inoculate the roots of host seedling (Castanea mollissima and Quercus glauca). Results demonstrated that spores of T. melanosporum, T. aestivum and T. indicum were about 3×107/g and T. pseudoexcavatum was about 1×107/g, whose spores type compared with others was different obviously. Spores survival rate showed no significant difference under storage for one year at 4℃. Result demonstrated that the coral like ectomycorrhiza produced in the third month after inocutlated. The successful inoculation rate was 90%, 70%, 100% and 75%, respectively. In addition, root of host trees in test area of Siji-Nanshan (SJ810、SJ469、SJ416、NS1016 and NS1056) produced ectomycorrhizal primordial, but contamination are found by using ITS gene sequences for identification. Identification by DNA sequencing showed successful inoculation rate as 83.8%, 42.9%, 89.7%, 66.7%, and 80%. The tuber in the outplanted host trees are facing competition with other more original competitive species (T. formosanum、T. huidongense and T. furfuraceum) in the soil. The mating genes, MAT1−1 and MAT1−2, were found possible to be used as the second molecular maker for species identification in this study. Besides, alternative selection of MAT1−1 and MAT1−2 gene was found in some tuber host during growth, which could be the evidence to prove Tuber spp. sex life cycle as heterothallism.

碩士
明新科技大學
化學工程與材料科技系碩士在職專班
103
Abstract The objective of this study is to investigate the effect of addition of truffle extract into the body lotion formulation by a Taguchi method. Lanolin, sweet almond oil, the lecithin and water are the lotion base and truffle extract, the niacin, vitamin E and tranexamic acid are additives. Based on the Taguchi method, the moist level, oil level and softness of face skin, measured by 3 in 1 skin checker, can be set as the performance indices. In our test, the contents of the additive species, including truffle extract, niacin, vitamin E and tranexamic acid are parameters in the orthogonal. The lotion base were formulated with seven sets of recipe, and the best one was obtained by lanolin 3.16%, sweet almond oil 8.42%, lecithin 8.42%, and deionized water 85.26%. Subsequently, the naturally active materials were optimally determined by truffle extract 3%, niacin 42.17%, vitamin E 12.66% and tranexamic acid 42.17%.