Investigation of the Proteomes of the Truffles Tuber albidum Pico, T. aestivum, T. indicum, T. magnatum, and T. melanosporum

Truffles of the Tuber species are known as expensive foods, mainly for their distinct aroma and taste. This high price makes them a profitable target of food fraud, e.g., the misdeclaration of cheaper truffle species as expensive ones. While many studies investigated truffles on the metabolomic level or the volatile organic compounds extruded by them, research at the proteome level as a phenotype determining basis is limited. In this study, a bottom-up proteomic approach based on LC-MS/MS measurements in data-independent acquisition mode was performed to analyze the truffle species Tuber aestivum, Tuber albidum Pico, Tuber indicum, Tuber magnatum, and Tuber melanosporum, and a protein atlas of the investigated species was obtained. The yielded proteomic fingerprints are unique for each of the of the five truffle species and can now be used in case of suspected food fraud. First, a comprehensive spectral library containing 9000 proteins and 50,000 peptides was generated by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Then, samples of the truffle species were analyzed in data-independent acquisition (DIA) proteomics mode yielding 2,715 quantified proteins present in all truffle samples. Individual species were clearly distinguishable by principal component analysis (PCA). Quantitative proteome fingerprints were generated from 2,066 ANOVA significant proteins, and side-by-side comparisons of truffles were done by T-tests. A further aim of this study was the annotation of functions for the identified proteins. For Tuber magnatum and Tuber melanosporum conclusive links to their superior aroma were found by enrichment of proteins responsible for sulfur-metabolic processes in comparison with other truffles. The obtained data in this study may serve as a reference library for food analysis laboratories in the future to tackle food fraud by misdeclaration of truffles. Further identified proteins with their corresponding abundance values in the different truffle species may serve as potential protein markers in the establishment of targeted analysis methods. Lastly, the obtained data may serve in the future as a basis for deciphering the biochemistry of truffles more deeply as well, when protein databases of the different truffle species will be more complete.

Virtual Truffle Hunting—A New Method of Burgundy Truffle (Tuber aestivum Vittad.) Site Typing

The aim of this study was to enable searches for truffles (Tuber spp.), particularly the Burgundy truffle (T. aestivum Vittad.), to be carried out in forests based on a method that has been constantly developed since 2007 by the Forest Research Institute. The method is termed “Virtual Truffle Hunting” and it takes 12 parameters into account: bedrock, soil pH, Ca+ and CaCO3 content in soil, C/N ratio, soil structure, altitude of terrain, type of forest site, forest structure, the Burgundy truffle host trees, and the presence of particular species including orchids and insects. A simple “Virtual Truffle Hunting” software has also been developed, which makes the use of the method easy, fast, and effective. This method is to ascertain the truffle potential for all areas in which digital maps are not available. In 2015, the method was tested in 20 sites, representing forests in 5 Polish macroregions. Hunting for hypogeous fungi was conducted from June to October with the help of trained dogs. Thanks to this method, 14 new truffle sites were found. The knowledge of environmental conditions conducive to the Burgundy truffle growth enabled us to form an effective tool in order to identify new sites of truffle presence.

Biochemical and Health Properties of Truffles

Truffles are the most expensive edible mushrooms refer to genus tuber which grows symbiotically in plant roots such as oaks and hazels. Truffles are underground mushrooms also known for their characteristic earthy flavor which is the major reason for their special place in the culinary. Their characteristic intense aroma helps them in reproduction by attracting small animals. Truffles can survive in a wide range of environments such as deep forests as well as deserts. The most expensive varieties of truffles include Tuber melanosporum (Black truffle), Tuber magnatum (White truffle), Tuber aestivum (Burgundy truffle), Tirmania nivea, and Terfezia chlaveryi (Dessert truffles). Truffles vary in their composition and flavor profile from species to species. The major volatile components which are responsible for truffle aroma are aldehydes, ketones, sulfur compounds, alcohols, and esters. Truffles are highly nutritious, rich in antioxidants, and have therapeutic properties such as antimicrobial activity, antiviral activity, antimutagenic activity, anti-inflammatory activity, hepatoprotective activity, etc. The major active components present in truffle are tuberoside, phenolics, anandamide, and ergosterol.

Comparison of bacterial communities in roots of selected trees with and without summer truffle (Tuber aestivum) ectomycorrhiza

In this study, we examined the effect of the presence of mycorrhiza and ascomata of summer truffle (Tuber aestivum) on the bacterial composition of roots from small trees growing in selected sites of the Nida Basin. Qualitative DNA sequencing methods such as Sanger and next-generation sequencing (NGS) were used. The Sanger method revealed different bacterial species compositions between the samples where summer truffle ascomata was recorded and control samples. Five genera of bacteria could be distinguished: Bacillus, Erwinia, Pseudomonas, Rahnella and Serratia, among which the most numerous were Pseudomonas (Gammmaproteobacteria class) at 32.9%. The results obtained by the NGS method also showed differences in species composition of the bacteria depending on the study sample. Seven genera of bacteria were distinguished: Rhizorhabdus, Methylotenera, Sphingomonas, Nitrosospira, Streptomyces, Methyloceanibacter and Niastella, which dominated in roots from the truffle sites. Telmatobacter, Roseiarcus, Granulicella, Paludibaculum, Acidipila, Acidisphaera and Aliidongia dominated in roots from the control sites. With the NGS method, it is possible to identify the microbiome of a whole root, while only a root fragment can be analysed by the Sanger method. These results extend the scope of knowledge on the preferences of certain groups of bacteria associated with truffles and their influence on the formation of ascomata in summer truffles. Our results may also be useful in selecting and monitoring sites that promote ascomata of Tuber aestivum.

Ectomycorrhiza resilience and recovery to extreme flood events in Tuber aestivum and Quercus robur

Very little is known about the impact of flooding and ground saturation on ectomycorrhizal fungi (EcM) and increasing flood events are expected with predicted climate change. To explore this, seedlings inoculated with the EcM species Tuber aestivum were exposed to a range of flood durations. Oak seedlings inoculated with T. aestivum were submerged for between 7 and 65 days. After a minimum of 114-day recovery, seedling growth measurements were recorded, and root systems were destructively sampled to measure the number of existing mycorrhizae in different zones. Number of mycorrhizae did not display correlation with seedling growth measurements. Seven days of submersion resulted in a significant reduction in mycorrhizae numbers and numbers reduced most drastically in the upper zones. Increases in duration of submersion further impacted mycorrhizae numbers in the lowest soil zone only. T. aestivum mycorrhizae can survive flood durations of at least 65 days. After flooding, mycorrhizae occur in higher numbers in the lowest soil zone, suggesting a mix of resilience and recovery. The results will aid in furthering our understanding of EcM but also may aid in conservation initiatives as well as providing insight for those whose livelihoods revolve around the collection of EcM fruiting bodies or cropping of the plant partners.

Effect of earthworms on mycorrhization, root morphology and biomass of silver fir seedlings inoculated with black summer truffle (Tuber aestivum Vittad.)

Species of the genus Tuber have gained a lot of attention in recent decades due to their aromatic hypogenous fruitbodies, which can bring high prices on the market. The tendency in truffle production is to infect oak, hazel, beech, etc. in greenhouse conditions. We aimed to show whether silver fir (Abies alba Mill.) can be an appropriate host partner for commercial mycorrhization with truffles, and how earthworms in the inoculation substrate would affect the mycorrhization dynamics. Silver fir seedlings inoculated with Tuber. aestivum were analyzed for root system parameters and mycorrhization, how earthworms affect the bare root system, and if mycorrhization parameters change when earthworms are added to the inoculation substrate. Seedlings were analyzed 6 and 12 months after spore inoculation. Mycorrhization with or without earthworms revealed contrasting effects on fine root biomass and morphology of silver fir seedlings. Only a few of the assessed fine root parameters showed statistically significant response, namely higher fine root biomass and fine root tip density in inoculated seedlings without earthworms 6 months after inoculation, lower fine root tip density when earthworms were added, the specific root tip density increased in inoculated seedlings without earthworms 12 months after inoculation, and general negative effect of earthworm on branching density. Silver fir was confirmed as a suitable host partner for commercial mycorrhization with truffles, with 6% and 35% mycorrhization 6 months after inoculation and between 36% and 55% mycorrhization 12 months after inoculation. The effect of earthworms on mycorrhization of silver fir with Tuber aestivum was positive only after 6 months of mycorrhization, while this effect disappeared and turned insignificantly negative after 12 months due to the secondary effect of grazing on ectomycorrhizal root tips.

Microbial Safety of Black Summer Truffle Collected from Sicily and Umbria Regions, Italy

Background: Tuber aestivum Vittad., known as black summer truffle, represents high-value food especially used as garnishment in nouvelle cuisine. The aim of this study was to investigate on the viable microbial populations associated with T. aestivum ascomata collected in different sites of Sicily and one locality of Umbria (Italy). Methods: The ripe ascomata of black summer truffles were collected from Central Italy. Cell densities of spoilage bacteria, fecal indicators, potential pathogens, yeasts, and molds were analyzed. Statistical analysis was conducted with XLSTAT software. Results: The microbiological counts of truffles ranged between 6.00 and 9.63 log Colony Forming Unit (CFU)/g for total mesophilic count and between 6.18 and 8.55 log CFU/g for total psychrotrophic count; pseudomonads were in the range 6.98-9.28 log CFU/g. Listeria spp. and coagulase-positive streptococci detected in no samples. Coagulase-negative streptococci were found in some samples with 2.11-4.76 log CFU/g levels. Yeasts and filamentous fungi were detected at consistent levels of 3.60-7.81 log CFU/g. Significant differences (p<0.01) were found between samples and also for all microbial groups. Conclusion: This study evidenced that the common brushing procedure applied for preparation of truffles is not sufficient to eliminate microbial risks for consumers. The application of an efficient decontamination treatment is strongly suggested before consumption of fresh truffles.