Microbial Communities of Ascocarps and Soils in a Natural Habitat of Tuber Indicum

Truffles are the fruiting bodies of hypogeous fungi in the genus Tuber. Some truffle species usually grow in an area devoid of vegetation, called brûlé, which knowledge about the microbial composition and structure is still limited. Here, we investigated the bacterial and fungal communities of Tuber indicum ascocarps and soils inside and outside a characteristic brûlé from a poplar plantation with no truffle production history in northeastern China using a high-throughput sequencing approach. A predominance of bacterial phylum Proteobacteria was observed in all samples, with Bacillus among the main genera in the ascocarps, while members of Lysobacter and unidentified Acidobacteria were more abundant in the soil. In addition, Gibberella, Fusarium and Absidia were the dominant fungi in the ascocarps, while Tuber were enriched in the ascocarps and soils inside the brûlé. Soil samples from inside the brûlé had a lower bacterial diversity and a greater fungal diversity than did those from outside the brûlé. Furthermore, some mycorrhization helper bacteria (Rhizobium) and ectomycorrhiza-associated bacteria (Lysobacter) were detected, indicating their potential roles in the complex development of underground fruiting bodies and brûlé formation. These findings may contribute to the protection and cultivation of truffles.

On the Origin of Feces: Fungal diversity, distribution, and conservation implications from feces of small mammals

Fungi are extremely diverse, but only a small fraction of the total estimated species have been characterized. Often, the extent of diversity and distribution of fungal communities is difficult or near impossible to assess. This is due to the fact that many Fungi are cryptic and persist predominantly hidden within substrates such as soil or plant material. This is particularly true for hypogeous sporocarps, including truffles, which are extremely difficult to survey in a systematic manner. However, fungi with hypogeous sporocarps have evolved traits that make them highly attractive to animals, such as small mammals, which ingest and disperse fungal spores through defecation. Here, samples of feces from 138 small mammals collected in the western United States were assessed for total fungal diversity using a dual-index metabarcoding, high-throughput Illumina sequencing approach. Our findings exhibit many identifications within Agaricomycetidae, with 65 of the 138 samples containing sequences belonging to several species of the hypogeous truffle genus Rhizopogon. Metadata, such as geospatial coordinates, for each captured small mammal can be used as a proxy for the presence or absence of Rhizopogon species identified in their feces. Utilizing these proxy data, along with publicly available data on observation and occurrence from data repositories such as GBIF and MycoPortal, it is possible to augment our current knowledge of the extent of occurrence and area of occupancy of cryptic hypogeous fungi without direct observation, further enhancing our ability to assess their conservation status.

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.

Phytonutrients and Antimicrobial Activities of Methanolic Extract from Hafr Al-Batin Truffles

The desert truffle is a wild mushroom, also referred to as Kamah or Fagaa. Kamah is a rich source of polysaccharides that have medicinal, antitumoral, antibacterial, and immune-stimulant effects. Studies of hypogeous fungi, especially desert truffles, have recently entered traditional studies of epigeous higher Basidiomycetes. Based on the tasty desert truffle Kamah obtained from Hafr Al-Batin Governorate, Saudi Arabia, as a source of potential antimicrobial agents with both the aim of obtaining novel agents toward bacteria and Fungi of clinical significance. We specifically tested the antibacterial and antifungal efficacy of methanol extracts of Kamah against the Gram-negative bacterial pathogens reference strains E. coli ATCC® 8739, P. Aeruginosa ATCC®9027, S. aureus ATCC®6538, Enterococci NCTC®775 and opportunistic fungus C. albicans ATCC®1231.The extract had MIC (minimum inhibitory concentrations) varying from 100 g/ml to 500 g/ml against the pathogens examined. The LC-QTOF-MS (liquid chromatography coupled to quadrupole time of flight mass spectrometry) phytoconstituents assay chromatogram indicated that the methanol extracts of Kamah comprises 264 with retention periods varying from 1.04 to 18.86, which were categorized as unsaturated and saturated natural ingredients sch as aromatic compounds, carboxylic acids, oxygenated hydrocarbons, fatty acids, amino acids, and vitamins).The main compounds were discovered to be 21 with peak areas larger than 2X10-5 and retention periods varying from 2.3 to 9.13.The main known substances with the maximum peaks were adenosine (11.724), phenylalanine (7.711), phenprobamate (7.711), and 5-hydroxytryptophan (5.711). Such preliminary findings, we assume, are encouraging in terms of obtaining a beneficial antibiotic substitute to battle antibiotic-resistant pathogens especially eye infections.

New Records of Rare Hypogeous Fungi from Poland (Central Europe)

Investigations carried out in recent years have provided new data on the localities of some rare hypogeous fungi in Central Europe. In this study, we present new localities of <em>Leucangium carthusianum</em>, <em>Melanogaster luteus</em>, and <em>Rhizopogon </em><em>melanogastroides</em>, two of which are new for Poland. Sporocarps of <em>L. carthusianum </em>were found in seven new localities in mixed and coniferous forests in the Western Carpathians as well as in Sudetes. In Poland, the species was also recorded in a mixed forest in the Kraków-Częstochowa Upland. Fir, <em>Abies alba</em>, accompanied the fungus in almost all known localities. Three localities of <em>M. luteus </em>were found in the Polish Western and Eastern Carpathians in <em>Alnus incana </em>communities mostly associated with streams. Knowledge of the distribution of this species in Europe is incomplete due to the complicated taxonomic history; nevertheless, it is regarded as rare, despite its wide distribution. One new locality of <em>R. melanogastroides </em>recorded in the Tatra Mts, Western Carpathians, is the fourth known to date. This species is mostly associated with <em>Pinus mugo </em>in high mountain localities (the Alps, the Tatra Mts). In this study, detailed descriptions and illustrations of the macro- and micromorphological features of the species are provided.

Food Authentication: Identification and Quantitation of Different Tuber Species via Capillary Gel Electrophoresis and Real-Time PCR

Truffles are hypogeous fungi mainly found in Europe and Asia. Due to their special aroma and taste, some truffle species are sold on the international market at an extremely high price. Among the economically relevant species, the white Alba truffle (Tuber magnatum) and the black Périgord truffle (T. melanosporum) are the most appreciated species. The fruiting bodies of the Asian black truffle are morphologically very similar to T. melanosporum, and those of the Bianchetto truffle (T. albidum Pico) are similar to T. magnatum, but are of little economic value. Highly valued species are adulterated with cheaper ones, especially. Because of this problem, the aim of this study was the development of methods for detecting possible admixtures to protect consumers from fraud. This study is based on seven different truffle species (117 fruiting bodies) from different growing regions. Additionally, selected truffle products were included. Using this material, a real-time PCR (polymerase chain reaction) assay allowing the detection and quantitation of Asian black truffles in T. melanosporum up to 0.5% was developed. In addition, a capillary gel electrophoresis assay was designed, which allows the identification and quantitation of different species. The methods can be used to ensure the integrity of truffle products.

A novel approach for tracing mycophagous small mammals and documenting their fungal diets

We developed a method combining passive baiting (animals that are not trapped) with DNA meta-barcoding of the feces acquired, to study fungi in the diet of small mammals. Mammal and fungal species were identified using genomic DNA of 596 fecal samples collected in five regions of the eastern Canadian boreal forest. For identification of the small mammal species, the cytochrome b region was used. A total of eight species of small mammals displayed hypogeous fungi consumption, with northern flying squirrels (Glaucomys sabrinus) and red-backed voles (Myodes gapperi) as the top consumers. For identification of their fungal diets, the ribosomal internal transcribed spacer (ITS) region was used. We recovered 722 taxa of Ascomycota, 429 Basidiomycota, 81 Zygomycota, 4 Chytridiomycota, 1 Glomeromycota, and 44 unidentified fungal taxa. Of these, 28 were hypogeous sequestrate fungi (underground fructification), which presumably are dug out by small mammals for consumption. Otherwise, for the remaining fungi [epigeous (above ground fructification) or microscopic fungal species], it is unclear which ones are selected by the animal as a dietary source or result from incidental contamination. Our paper presents a promising approach for tracing mycophagy in small mammals, and our results suggest that fungal diversity is important for the diet of some small mammals.

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.