Truffle Research Articles

A way to mechanically activate a suspension of black truffle spores

True truffles, or fungi of the genus Tuber, are among the most valuable, rare, expensive, and mysterious fungi on the planet. The reproduction of truffle fungi in the wild is a complex process that depends on many factors, for example, the presence of suitable host trees, as well as the optimal parameters of temperature, moisture, soil pH, soil symbionts, and other organisms. Successful production and reproduction of truffles require a large number of conditions to be met, primarily the release of their spores from ascii, germination under favorable conditions, and distribution in the environment. The present study aims to develop a method for mechanically breaking ascii in order to release the spores of black truffle. It is shown that homogenization by means of an immersion blender together with a vibrating chopper is a highly effective way to break ascii. The microscopic examination of the specimens at all stages of the experiment revealed that this method leads to the breakdown of at least 85% of ascii and the release of truffle spores. The use of centrifugation approaches enables the separation of black truffle spores from hyphal debris, fungal symbionts, and various associated organisms. Such spores can form mycelium on growth media. The study results can be used to prepare Tuber sp. inoculum and obtain their axenic cultures. Thus, the introduction of methods for releasing spores from ascii improves the efficiency of black truffle cultivation.

Optimising in vitro culture conditions for the truffle Tuber brumale

The vegetative propagation of ectomycorrhizal truffle fungi is limited by their slow mycelial growth. Many factors including media, isolate genotypes and environmental conditions can alter fungal mycelial growth rates. This study aimed to improve the in vitro growth rate of Tuber brumale by determining the optimal carbohydrate and nitrogen sources, temperature and pH. After 8 weeks, the highest level of growth and densest hyphal branching were recorded in the medium containing glucose as the main carbohydrate. For nitrogen, glutamine (200 mg N l-1) provided the greatest hyphal growth and density compared to the other amino acid treatments. Regarding temperature, 16?C proved to be optimal for T. brumale growth and branching. Media of pH 6 and pH 7 were most favourable for the growth of T. brumale. The results from this research provide baseline data on the vegetative nutrition of T. brumale and have implications for the in vitro culture of winter truffle hyphae.

Natural infection of Chiromyzinae larvae (Diptera: Stratiomyidae) in southern Chile by Tolypocladium valdiviae sp. nov.

A new species from the fungal genus Tolypocladium (Hypocreales: Ophiocordycipitaceae) that infects Stratiomyidae larva from the genus Hylorops is described: Tolypocladium valdiviae Gallardo-Pillancari, Montalva & González. The description is based on both genomic data and morphological characteristics. The sexual stage of T. valdiviae presents fleshy and visible stromata; unlike Tolypocladium ophioglossoides, it is smaller and emerges directly from its host and resembles Tolypocladium longisegmentis and Tolypocladium capitatum, both of which are parasites of deer truffle fungi of the genus Elaphomyces (Ascomycota: Eurotiales). In the anamorphic state, T. valdiviae presents conidiogenous cells similar in shape and arrangement to those of Tolypocladium inflatum, however T. valdiviae produces larger conidiogenous cells and, occasionally, produces chlamydospores. Phylogenetic evidence suggested that T. valdiviae is in a clade close to T. longisegmentis, T. inflatum and T. ophioglossoides, species also recognized to be parasites of fungi of the genus Elaphomyces. The new species is known so far only from Valdivia, southern Chile.

Fungal ecology: Truffle-guzzling birds

A new study of truffle fungi in Patagonia reveals the importance of native bird species in spore dispersal. This has wide implications for the study of animal-fungal symbioses in the Southern Hemisphere.

Effects of Field Fumigation and Inoculation With the Pecan Truffle (Tuber lyonii) on the Fungal Community of Pecan (Carya illinoinensis) Seedlings Over 5 Years.

Truffle fungi are esteemed for their aromatic qualities and are among the most widely cultivated edible ectomycorrhizal fungi. Here we document a successful method for establishing Tuber lyonii, the pecan truffle, on pecan (Carya illinoinensis) seedlings in a field setting. We assessed the impacts of soil fumigation and varying concentrations of truffle spore inoculum on the ectomycorrhizal fungal and the complete fungal communities as well as the colonization of T. lyonii on pecan roots at three nurseries in Georgia, United States. To identify fungal communities on pecan seedlings, we performed high-throughput amplicon sequencing of the fungal ITS1 rDNA region. Our 5-year long field experiment demonstrates that fumigation and inoculation together resulted in the highest persistence of T. lyonii on pecan roots. While fungal OTU numbers fluctuated over the years of our experiments, there was no statistical support to demonstrate diversification of communities when Shannon diversity metrics were used. However, we did find that older seedlings were less likely to be dominated by T. lyonii compared to younger ones, suggesting successional changes in the fungal community over time. This suggests that transplanting inoculated seedlings after 2 or 3 years post-inoculation is optimal for future truffle propagation efforts. Our results demonstrate that T. lyonii can be established in situ with methods that are compatible with current pecan nursery industry practices and that fungal communities on pecan seedlings vary depending on the experimental treatments used during planting. While the pecan truffle is not yet widely cultivated, our results provide insights for future large-scale cultivation of this and perhaps other Tuber species.

Report on Tuber huidongense, a Truffle Species Previously Unrecorded in Korea

Report on Tuber huidongense, a Truffle Species Previously Unrecorded in Korea

Orchard Conditions and Fruiting Body Characteristics Drive the Microbiome of the Black Truffle Tuber aestivum.

Truffle fungi are well known for their enticing aromas partially emitted by microbes colonizing truffle fruiting bodies. The identity and diversity of these microbes remain poorly investigated, because few studies have determined truffle-associated bacterial communities while considering only a small number of fruiting bodies. Hence, the factors driving the assembly of truffle microbiomes are yet to be elucidated. Here we investigated the bacterial community structure of more than 50 fruiting bodies of the black truffle Tuber aestivum in one French and one Swiss orchard using 16S rRNA gene amplicon high-throughput sequencing. Bacterial communities from truffles collected in both orchards shared their main dominant taxa: while 60% of fruiting bodies were dominated by α-Proteobacteria, in some cases the β-Proteobacteria or the Sphingobacteriia classes were the most abundant, suggesting that specific factors (i.e., truffle maturation and soil properties) shape differently truffle-associated microbiomes. We further attempted to assess the influence in truffle microbiome variation of factors related to collection season, truffle mating type, degree of maturation, and location within the truffle orchards. These factors had differential effects between the two truffle orchards, with season being the strongest predictor of community variation in the French orchard, and spatial location in the Swiss one. Surprisingly, genotype and fruiting body maturation did not have a significant effect on microbial community composition. In summary, our results show, regardless of the geographical location considered, the existence of heterogeneous bacterial communities within T. aestivum fruiting bodies that are dominated by three bacterial classes. They also indicate that factors shaping microbial communities within truffle fruiting bodies differ across local conditions.

Are bacteria responsible for aroma deterioration upon storage of the black truffle Tuber aestivum: A microbiome and volatilome study

Truffle fungi, luxurious food items with captivating aromas, are highly valued in the culinary world. However, truffles are perishable and their aroma undergoes deep changes upon storage. Additionally, truffle aroma might be partially derived from microbes. Hence, we investigated here the influence of storage on two factors, namely the volatile profile and bacterial community composition in the black truffle Tuber aestivum. The possible linkage among those factors was further explored.Our results demonstrate important changes in the volatile profiles of truffles over nine days of storage at room temperature. In the same time frame, dominant bacterial classes characteristic of fresh truffles (α-Proteobacteria, β-Proteobacteria, and Sphingobacteria classes) were gradually replaced by food spoilage bacteria (γ-Proteobacteria and Bacilli classes). Freshness and spoilage volatile markers (i.e. dimethyl sulfide (DMS), butan-2-one, 2- and, 2- and 3-methylbutan-1-ol, and 2-phenylethan-1-ol) were identified. Lastly, network analysis showed correlations between those markers and specific bacterial classes typical of fresh and spoiled truffles.Overall, our results demonstrate the profound effect of storage on the aroma and bacterial community composition of truffles and highlight how the gradual replacement of the commensal microbiome by spoilage microbes mirrors shifts in aroma profile and the possible loss of fresh truffle flavor.

A Survey of the Knowledge of Truffles among Polish Foresters and Implications for Environmental Education

While the use of truffles in Poland has a long tradition, for historical reasons this knowledge was almost lost. Currently, truffles and truffle orchards are again receiving public attention. For example, the Polish State Forests supported the establishment of truffle orchards by the Forestry Research Institute. In recent years, knowledge concerning these unique hypogeous fungi has been disseminated systematically through scientific and popular publications, films, and electronic media. This study investigates the awareness of economically and culinary valued truffle fungi (Tuber spp.) among more than 1400 Polish foresters. The results show that 70% of interviewees were familiar with historical and contemporary information about growing and using truffles in Poland. Based on respondents’ age, education, type of work, and gender we attempted to identify whether these elements were associated with the state of knowledge about truffles. The results indicated that younger foresters were better informed about the presence of truffles in Poland and also about their use in the past in Polish cuisine. Environmental education was an important source of knowledge about truffle harvesting and the soils that are conducive to truffle development. Foresters who have provided forest ecology education and who are 36–65 years of age generally possessed better knowledge about truffles than other age cohorts. More than 30% of respondents expressed interest in educational courses to improve their knowledge of truffles. The results point to the need for forestry education concerning truffles and indicate the need for fostering sustainable agroforestry-centered initiatives disseminating this knowledge to the public.

Beyaz trüf mantarında (Tuber magnatum) mikrosatelit markörlerinin tüm genom düzeyinde tanımlanması ve anotasyonu

The white truffle Tuber magnatum Pico is an ectomycorrhizal fungus, which produces the most economically valuable edible ascomata, referred to as truffles. Highly appreciated sensory properties and exceptional market prices make truffle fungi an important focus of research. Accordingly, there is increasing interest to characterize truffle aroma volatiles, fully understand their reproduction and, characterize their genomes, analyze their population structure and molecular genetic diversity. While production of truffle species was confined before to natural woodlands, development of host seedling inoculation protocols in 1970s established truffle production as a managed agricultural activity. On the other hand, T. magnatum inoculated seedling production was achieved only recently, since morphological criteria were insufficient for the precise identification of T. magnatum mycorrhizas without DNA-based identification protocols. The present research describes the development and functional annotation of novel microsatellite markers in T. magnatum genome. A bioinformatics workflow was applied in order to mine for microsatellite sequences in T. magnatum genome assembly, convert the loci to PCR markers, map marker amplification on genomic sequences and calculate allele sizes. As a result, 11 189 microsatellite markers specific to T. magnatum were developed. Annotation analysis was performed for marker sequences, resulting in 3377 marker loci matching with T. magnatum proteins. Thus, these markers have high potential to tag genes of interest in functional genomic analyses. As a result of the present work, a large database of T. magnatum specific DNA markers was introduced as a useful genomic resource for the valuable ectomycorrhizal fungus species.

The endangered northern bettong,Bettongia tropica, performs a unique and potentially irreplaceable dispersal function for ectomycorrhizal truffle fungi

Organisms that are highly connected in food webs often perform unique and vital functions within ecosystems. Understanding the unique ecological roles played by highly connected organisms and the consequences of their loss requires a comprehensive understanding of the functional redundancy amongst organisms. One important, yet poorly understood, food web is that between truffle-forming ectomycorrhizal fungi and their mammalian consumers and dispersers. Mammalian fungal specialists rely on fungi as a food source, and they consume and disperse a higher diversity and abundance of fungi than do mycophagous mammals with generalist diets. Therefore, we hypothesize that mammalian fungal specialists are functionally distinct because they disperse a set of fungal taxa not fully nested within the set consumed by the combined generalist mammalian community (i.e., functional redundancy of fungal dispersal is limited). Using high-throughput sequencing, we compared the fungal composition of 93 scats from the endangered fungal specialist northern bettong (Bettongia tropica) and 120 scats from nine co-occurring generalist mammal species across three sites and three seasons. Compared with other generalist mammals, B.tropica consumed a more diverse fungal diet with more unique taxa. This aligns with our hypothesis that B.tropica performs a unique dispersal function for ectomycorrhizal truffle fungi. Additionally, modelling of mammalian extinctions predicted rapid loss of food web connections which could result in loss of gene flow for truffle taxa. Our results suggest that this system is sensitive to the extinction of highly connected specialist species like B.tropica and their loss could have consequences for ectomycorrhizal truffle fungal diversity. This suggests that the conservation of fungal specialists is imperative to maintaining ectomycorrhizal fungal diversity and healthy plant-mycorrhizal relationships.

Truffle mediated (Terfezia claveryi) synthesis of silver nanoparticles and its potential cytotoxicity in human breast cancer cells (MCF-7)

Terfezia claveryi is a species that belongs to the genera of Terfeziaceae or desert truffles, which is a family of truffles. In the present study, silver nanoparticles were synthesized from aqueous extract of T. claveryi which are in the range of 25 to 60 nm. The synthesized nanoparticles were characterized by ultraviolet-visible (UV-Vis) spectroscopy, fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). The effect of the silver nanoparticles on human breast cancer cell line has been tested. Peak absorption was recorded at 440 nm in UV-Vis spectra of silver nanoparticles. The XRD data reports that the silver nanoparticles are crystalline in nature and have face centered cubic geometry. FESEM showed the size range of synthesized silver nanoparticles as 25 to 50 nm. The TEM image represents that the majority of silver nanoparticles are in spherical shape with sizes ranging between 40 and 60 nm. The aim of the present study was to report for the first time fruit mediated synthesis of silver nanoparticles using the extract of T. claveryi and showed remarkable cytotoxicity activity against human breast MCF-7 cancer cell line. Key words: Silver nanoparticles, Terfezia claveryi, fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (FESEM), transmission electron microscopy (TEM), MCF-7 cancer cell line.

Characterizing root-associated fungal communities and soils of Douglas-fir (Pseudotsuga menziesii) stands that naturally produce Oregon white truffles (Tuber oregonense and Tuber gibbosum).

Many truffle species in the genus Tuber are endemic to North America. Some of these have commercial value such as Tuber oregonense and Tuber gibbosum, commonly known as Oregon white truffles. Most of what is known about the ecology of these truffles comes from observational data. These truffle species form ectomycorrhizas with Douglas-fir (Pseudotsuga menziesii) and sometimes fruit abundantly in early successional forest regrowth. The goal of this study was to characterize fungal communities and soils associated with truffle-producing Douglas-fir sites. We extracted DNA from roots of five trees at four different truffle-producing Douglas-fir sites (n = 20). We amplified the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) and sequenced amplicons with 454 pyrosequencing. After quality filtering, we assembled 15,713 sequences into 150 fungal operational taxonomic units (OTUs). Pezizomycetes (Tuber and Pyronemataceae) were the most abundant taxa detected followed by Helotiales. Agaricomycetes represented most by Thelephoraceae, Russulaceae, and Inocybaceae were also abundant. A total of five Tuber species were detected. T. oregonense was the most abundant OTU, followed by T. gibbosum and Wilcoxina mikolae. Fungal root endophytes were also detected and well represented by Chalara and Phialocephala spp. Fungal community structure and soil chemistry differed between sites. This study represents the first characterization of the fungal communities in Douglas-fir stands producing Oregon white truffles. We found that Tuber species can be dominant ectomycorrhizal symbionts of Douglas-fir. Truffle fungi are also important in forest health, food webs, and as a non-timber forest resource that can contribute to rural economies.

Turkish truffles I: 18 new records for Turkey

We report the first records of 18 truffle species in Turkey. Three belong to the Ascomycota: Elaphomyces leucocarpus, E. muricatus, and Genea sphaerica; and 15 to the Basidiomycota: Alpova corsicus, Gautieria otthii, G. retirugosa, G. trabutii, Hymenogaster citrinus, H. hessei, H. luteus, H. lycoperdineus, Hysterangium clathroides, H. epiroticum, H. fragile, H. nephriticum, Leucogaster tozzianus, Octaviania asterosperma, and Protoglossum aromaticum. We also report new localities within Turkey for Picoa juniperi, P. lefebvrei, Geopora cooperi, Terfezia arenaria, T. claveryi, Tuber aestivum, and T. nitidum in the Ascomycota; and Leucogaster nudus, Hymenogaster thwaitesii, H. vulgaris, and Melanogaster broomeanus in the Basidiomycota.

The therapeutic potential of truffle fungi: a patent survey

The purpose of this article is to research and retrieve patent information regarding the therapeutic use of truffles. Truffles have a unique value as a foodstuff and impact positively on human health and well-being. They are applied in such industries as the pharmaceutical industry and the cosmetic industry. Patent documentation available in the Espacenet network and the Patentscope service were analyzed by key word and patent specifications were examined to describe state of the art and to identify scientific research trends in therapeutic applications of truffles. Medicinal properties of truffles such as the anticancer or cardiovascular effect, a reduction in blood lipids, immunological resistance and increased energy were identified. Other therapeutic benefits include sedative action, prevention of hormonal imbalances in women, pre-menopause symptom relief, senile urethritis and prostate disorders, sleep disorders and increased absorption of calcium from milk. Truffles can also be used to alleviate symptoms of milk intolerance such as diarrhoea or bloating, to ease rheumatic pains and to treat and prevent further development or recurrence of senile cataract.

Forty years of inoculating seedlings with truffle fungi: past and future perspectives.

The first commercialization of seedlings inoculated with truffle fungi occurred in 1973. Over the last 40 years, considerable progress has been made relative to quality control for inoculated seedlings. A recently published paper by Andrés-Alpuente and colleagues (Mycorrhiza 24:29-37, 2014) reviewed and tested the different methods of mycorrhization assessment currently used in Europe. The aim of this paper is to augment their findings by adding information to the discussion about the most important steps in seedling quality control. Additionally, the history of seedlings inoculated with truffles, procedures currently used in France for seedling control quality, and a reflection on future research aimed at increasing truffle production will be presented.

New records of some Ascomycete truffle fungi from Turkey

We report the first records of 6 Ascomycete truffle taxa in Turkey: Genea verrucosa Vittad., Genea klotzschii Berk. & Broome, Stephensia bombycina (Vittad.) Tul. & C.Tul., Terfezia olbiensis Tul. & C.Tul., Tuber excavatum Vittad., and Tuber rufum Pico. We also report new localities within Turkey for Picoa juniperi Vittad., Terfezia leptoderma Tul. & C.Tul., Choiromyces meandriformis Vittad., Tuber brumale Vittad., Tuber nitidum Vittad., and Tuber mesentericum Vittad.

A quantitative and molecular examination of Tuber melanosporum mycorrhizae in Quercus ilex seedlings from different suppliers in Spain

Aim of study: The aim of the work was to determine the degree of mycorrhization of Quercus ilex L. subsp. ballota (Desf.) Samp. by the black truffle fungus T. melanosporum Vittad. by quantitative and molecular analyses.Area of study: seedlings inoculated by different Spanish suppliers.Material and methods: The internal transcribed spacers (ITS) of mycorrhizae from different plants were amplified by nested PCR involving fluorescently-labelled primers, and the amplicons either directly sized by ARISA or analysed by TRFLP following their digestion with restriction endonucleases. TRFLP analysis distinguished between mycorrhizae of T. melanosporum, T. indicum Cooke & Massee and T. borchii Vittad., as suggested possible by virtual (in silico) TRFLP analysis and real TRFLP analysis of the ascomata of these species.Main results: Significant differences between suppliers were detected in terms of the mean number of mycorrhizae established per plant and percentage mycorrhization. These results allowed the following quality standards for 2 year-old plants to be proposed: a) good quality: >3000 mycorrhizae/plant, >40% mycorrhization, b) medium (acceptable): >3000 mycorrhizae/plant, >30% mycorrhization, c) low quality: <3000 mycorrhizae/plant or <30% mycorrhization, always supposing the mycorrhizae counted represent the species of interest as confirmed by the presence of its DNA and the absence of DNA belonging to contaminating species. Finally, a new microsatellite allelic map obtained from the analysis of several T. melanosporum populations across Spain was used to provide a tool capable of determining the geographic origin of the fungi used to inoculate plants.Research highlights: The proposed quality standards can be useful for the evaluation and certification of commercialized Q. ilex plants mycorrhized with T. melanosporum.Keywords: mycorrhiza; certification; truffle; TRFLP; fungi.

Consumption of Truffles and other Fungi by the American Red Squirrel (<em>Tamiasciurus hudsonicus</em>) and the Eastern Chipmunk (<em>Tamias striatus</em>) (Sciuridae) in Northwestern Ontario

Faecal matter collected from the American Red Squirrel (Tamiasciurus hudsonicus) and the Eastern Chipmunk (Tamias striatus) on the campus of Lakehead University in Thunder Bay, Ontario, was examined for the presence of consumed fungal tissue. A total of 14 faecal samples were collected from live-trapped animals over six trapping periods (19 June to 25 September 2010). Eight samples contained intact remains of spores representative of hypogeous truffle fungi found in the genera Elaphomyces, Gautieria, Hymenogaster, Hysterangium, and Leucangium, as well as spores of epigeous mushrooms found in the orders Boletales and Pezizales and possibly in the family Tricholomataceae of the Agaricales. The results of this brief survey suggest not only the importance of mycophagy in the diet of American Red Squirrels and Eastern Chipmunks in the boreal mixed wood forests of northwestern Ontario but also the important role played by sciurids in vectoring spores of truffle fungi in this region.

New records of truffle fungi (Basidiomycetes) from Turkey

We report the first records of 5 truffle taxa in Turkey: Gymnomyces xanthosporus (Hawker) A.H.Sm., Hymenogaster griseus Vittad., Hymenogaster olivaceous Vittad., Hymenogaster thwaitesii Berk. & Broome, and Hymenogaster vulgaris Tul. & C.Tul. We also report a new locality within Turkey for Melanogaster broomeanus Berk.