Spatial distribution and ecological variation of re-discovered German truffle habitats

Several truffle species (Tuber spp.) are highly prized by chefs and gourmets with some commanding prices of up to €9.000 kg^?1 on international markets. Their ecological drivers and geographical patterns, however, often remain a puzzle. Truffle species in Germany are classified as Very Rare or even Extinct on the national Red Lists, while historical literature described their sporadic existence. Here we present evidence of seven Tuber species (T. aestivum, T. brumale, T. excavatum, T. fulgens, T. macrosporum, T. mesentericum, T. rufum), discovered at 121 sites in Southwest Germany. The valuable Burgundy truffle (T. aestivum) occurred at 116 sites. An unexpected abundance of Tuber spp. associated with 13 potential host plants along wide ecological gradients in a region far outside the traditional Mediterranean truffle foci in France, Italy and Spain, is likely indicative of possible responses to climate change, and also suggests ample truffle cultivation potential north of the Alpine arc.     

Cultivation of Mediterranean species of Tuber (Tuberaceae) in British Columbia,Canada

Based on an assessment of soil and climatic conditions in British Columbia (BC), the Truffle Association of British Columbia (TABC) determined that the cultivation of Mediterranean Tuber melanosporum and Tuber aestivum might be possible in the warmer parts of the province. With the cooperation of independent truffle growers, TABC assessed the colonization of host tree roots collected from eight truffle orchards planted 2–7 years earlier using morphological and molecular criteria. Both Tuber species persisted on the roots of inoculated trees in six of the eight truffle orchards studied. The identity of Tuber ectomycorrhizas that had been characterized morphologically as differing from those of T. melanosporum and T. aestivum were determined using DNA sequence analysis to belong to three species of truffles native to the Pacific Northwest. One of those species, Tuber anniae, had been previously reported from BC, but the other two, Tuber menseri nom. prov. and Tuber beyerlei, are reported here from BC for the first time. Recently, production of three Périgord black truffles in one truffle orchard and one Burgundy truffle in another orchard demonstrates that these truffles are able to fruit in BC.     

The Role of the Microbiome of Truffles in Aroma Formation: a Meta-Analysis Approach

Truffles (Tuber spp.) are ascomycete subterraneous fungi that form ectomycorrhizas in a symbiotic relationship with plant roots. Their fruiting bodies are appreciated for their distinctive aroma, which might be partially derived from microbes. Indeed, truffle fruiting bodies are colonized by a diverse microbial community made up of bacteria, yeasts, guest filamentous fungi, and viruses. The aim of this minireview is two-fold. First, the current knowledge on the microbial community composition of truffles has been synthesized to highlight similarities and differences among four truffle (Tuber) species (T. magnatum, T. melanosporum, T. aestivum, and T. borchii) at various stages of their life cycle. Second, the potential role of the microbiome in truffle aroma formation has been addressed for the same four species. Our results suggest that on one hand, odorants, which are common to many truffle species, might be of mixed truffle and microbial origin, while on the other hand, less common odorants might be derived from microbes only. They also highlight that bacteria, the dominant group in the microbiome of the truffle, might also be the most important contributors to truffle aroma not only in T. borchii, as already demonstrated, but also in T. magnatum, T. aestivum, and T. melanosporum.     

Whose truffle is this? Distribution patterns of ectomycorrhizal fungal diversity in Tuber melanosporum brûlés developed in multi‐host Mediterranean plant communities

In the Mediterranean region, patches of vegetation recovering from disturbance and transiently dominated by shrubs produce one of the world's most prized fungi, the black truffle (Tuber melanosporum). In these successional plant communities, we have fragmentary knowledge of the distribution of T. melanosporum in space among ectomycorrhizal (ECM) host species and in time. Molecular identification of hosts (Restriction Fragment Length Polymorphism) and fungi (Internal Transcribed Spacer sequencing) and quantification of T. melanosporum mycelium (quantitative Polymerase Chain Reaction) were employed to evaluate the presence of T. melanosporum on four dominant ECM host species (Quercus ilex, Quercus coccifera, Arbutus unedo, Cistus albidus) and the extent to which their respective ECM communities shared fungal diversity, over the course of development of truffle grounds, from recent unproductive brûlés to senescent ones where production has stopped. We found that truffle grounds host rich communities in which multi‐host fungal species dominate in frequency. When considering both ECM tips and soil mycelia, we documented a dynamic and spatially heterogeneous pattern of T. melanosporum distribution in soils and a presence of ECM tips restricted to Q. ilex roots. This study advances our knowledge of the ecology of T. melanosporum, and provides insight into the extent of ECM fungal sharing among plant species that dominate Mediterranean landscapes.     

Tuber aestivum Vittad. mycelium quantified: advantages and limitations of a qPCR approach

A quantitative real-time PCR (qPCR) marker Ta0 with hydrolysis probe (“TaqMan”), targeted to the internal transcribed spacer region of the ribosomal DNA, has been developed for quantification of summer truffle (Tuber aestivum) mycelium. Gene copy concentrations determined by the qPCR were calibrated against pure culture mycelium of T. aestivum, enabling quantification of the mycelium in soil and in host roots from the fields. Significant concentrations of the fungus were observed not only in the finest roots with ectomycorrhizae but also in other root types, indicating that the fungus is an important component of the microbial film at the root surface. The concentration of T. aestivum in soil is relatively high compared to other ectomycorrhizal fungi. To evaluate the reliability of the measurement of the soil mycelium density using qPCR, the steady basal extracellular concentration of the stabilized T. aestivum DNA should be known and taken into account. Therefore, we addressed the stability of the qPCR signal in soil subjected to different treatments. After the field soil was sieved, regardless of whether it was dried/rewetted or not, the T. aestivum DNA was quickly decomposed. It took just about 4 days to reach a steady concentration. This represents a conserved pool of T. aestivum DNA and determines detection limit of the qPCR quantification in our case. When the soil was autoclaved and recolonized by saprotrophic microorganisms, this conserved DNA pool was eliminated and the soil became free of T. aestivum DNA.     

Variable fitness and reproductive effects of Wolbachia infection in populations of the two-spotted spider mite Tetranychus urticae Koch in China

Maternally inherited Wolbachia bacteria are widely distributed among insects, and their presence usually causes modifications of the host. To understand the evolutionary history of diverse host-Wolbachia associations, we investigated the symbiosis between Wolbachia and the two-spotted spider mite Tetranychus urticae Koch in China. The cytoplasmic incompatibility (CI) level, fecundity, female ratio, host longevity and host development time were examined. Our results indicate that Wolbachia bacteria had variable effects on the reproduction and fitness of Chinese populations of T. urticae. Variability of CI expression within T. urticae ranged from no CI to a strong level of CI in spite of the low variability of the wsp gene. Relative to uninfected mites, infected females in one of the three populations showed enhanced fecundity associated with the infection of Wolbachia. This is the first report of a Wolbachia infection promoting the fecundity of infected females in T. urticae. Furthermore, we found both positive and negative effects of Wolbachia infection on longevity and the development time. The differences in ecological characters may be attributed to both Wolbachia and host genotype.     

Ecological indicators of Tuber aestivum habitats in temperate European beech forests

Although Tuber aestivum is widely distributed across Europe, little is known about its biology and ecology. We assess the vegetation composition of wild T. aestivum sites and use this information to characterise suitable habitats in Fagus sylvatica, Carpinus betulus and Ostrya carpinifolia (FCO) forests. Plant species composition at 16 naturally colonised T. aestivum sites in southwestern Germany and Switzerland was compared with that of 232 reference sites in Swiss FCO forest communities. The vegetation composition of truffle sites exhibits a high proportion of herb and shrub species that are indicators of a warm and dry climate, alkaline soil, and open tree canopy and thus typically occur in thermophilous FCO communities on calcareous bedrock. We conclude that T. aestivum is associated with a set of key species in addition to known host species. Based on their floristic composition, we identified FCO forest communities with high and low suitability for T. aestivum.     

Potential and limitations of Burgundy truffle cultivation

Burgundy truffles (Tuber aestivum syn. Tuber uncinatum) are the highly prized fruit bodies of subterranean fungi always occurring in ectomycorrhizal symbiosis with host plants. Successful cultivation can be achieved through artificial mycorrhization and outplanting of mostly oaks and hazel on suitable terrain. Here, we review ecological requirements, the influence of environmental factors, and the importance of molecular techniques for a successful cultivation of T. aestivum across Europe. The historical background and current knowledge of T. aestivum cultivation are discussed in light of its socioeconomic relevance.     

A Pine Is a Pine and a Spruce Is a Spruce – The Effect of Tree Species and Stand Age on Epiphytic Lichen Communities

With an increasing demand for forest-based products, there is a growing interest in introducing fast-growing non-native tree species in forest management. Such introductions often have unknown consequences for native forest biodiversity. In this study, we examine epiphytic lichen species richness and species composition on the trunks of non-native Pinus contorta and compare these to the native Pinus sylvestris and Picea abies in managed boreal forests in northern Sweden across a chronosequence of age classes. Overall, we recorded a total of 66,209 lichen occurrences belonging to 57 species in the 96 studied forest stands. We found no difference in species richness of lichens between stands of P. contorta and P. sylvestris, but stands of P. abies had higher total species richness. However, species richness of lichens in stands of P. abies decreased with increasing stand age, while no such age effect was detected for P. contorta and P. sylvestris. Lichen species composition progressively diverged with increasing stand age, and in 30-year-old stands all three tree species showed species-specific assemblages. Epiphytic lichen assemblages in stands of 30-year-old P. contorta were influenced by greater basal area, canopy closure, and average diameter at breast height, P. abies stands by higher branch density and canopy closure, and stands of P. sylvestris by greater bark crevice depth. Differences in lichen species richness and composition were mainly explained by canopy closure and habitat availability, and the greater canopy closure in mature P. abies stands promoted the colonization and growth of calicioid lichen species. Our results indicate that the non-native P. contorta have similar species richness as the native P. sylvestris. The main difference in lichen species richness and composition is between P. abies and Pinus spp. in managed forests of boreal Sweden.     

Microbial and pigment profile of the reddish patch occurring within Tuber magnatum ascomata

Tuber magnatum Pico, the delectable white truffle, is the most prized truffle species. In this study, we examined the reddish pigmentation that frequently occurs in T. magnatum ascomata for the presence of pigment-producing bacteria.The inner part of the reddish-pigmented region of three T. magnatum ascomata collected in North–Central Italy was analysed. This reddish part was used to establish a bacterial culture collection and to extract the total genomic DNA in order to obtain a library of 16S rRNA genes representative of the bacterial community. The molecular approach revealed limited microbial diversity within the reddish-pigmented regions compared to the wider range of bacterial species commonly found at the same maturation stage and season in T. magnatum ascomata. The pigmented regions showed a prevalence of specific bacterial species belonging to α-, β- and γ- Proteobacteria, Actinobacteria and Firmicutes. From the tandem mass spectrometry analysis of the extracted pigment, four compounds were identified: i) bixin, ii) β-carotene, iii) cis-1-glycosyl-apo-8′- lycopene and iv) the fucoxanthin. Carotenoid producing species such as Microbacterium and Chryseobacterium emerged as the most likely cause of the peculiar reddish pigment production. Indeed, our findings suggest that the peculiar reddish pigment might be produced by these bacterial species.     

Magnetosome-containing bacteria living as symbionts of bivalves

Bacteria containing magnetosomes (protein-bound nanoparticles of magnetite or greigite) are common to many sedimentary habitats, but have never been found before to live within another organism. Here, we show that octahedral inclusions in the extracellular symbionts of the marine bivalve Thyasira cf. gouldi contain iron, can exhibit magnetic contrast and are most likely magnetosomes. Based on 16S rRNA sequence analysis, T. cf. gouldi symbionts group with symbiotic and free-living sulfur-oxidizing, chemolithoautotrophic gammaproteobacteria, including the symbionts of other thyasirids. T. cf. gouldi symbionts occur both among the microvilli of gill epithelial cells and in sediments surrounding the bivalves, and are therefore facultative. We propose that free-living T. cf. gouldi symbionts use magnetotaxis as a means of locating the oxic–anoxic interface, an optimal microhabitat for chemolithoautotrophy. T. cf. gouldi could acquire their symbionts from near-burrow sediments (where oxic–anoxic interfaces likely develop due to the host''s bioirrigating behavior) using their superextensile feet, which could transfer symbionts to gill surfaces upon retraction into the mantle cavity. Once associated with their host, however, symbionts need not maintain structures for magnetotaxis as the host makes oxygen and reduced sulfur available via bioirrigation and sulfur-mining behaviors. Indeed, we show that within the host, symbionts lose the integrity of their magnetosome chain (and possibly their flagellum). Symbionts are eventually endocytosed and digested in host epithelial cells, and magnetosomes accumulate in host cytoplasm. Both host and symbiont behaviors appear important to symbiosis establishment in thyasirids.     

The phylogenetic position of Tuber glabrum sp. nov. and T. sinomonosporum nom. nov., two Paradoxa-like truffle species from China

Based on morphological and molecular phylogenetic analysis, the new species Tuber glabrum is described from China, and the new name T. sinomonosporum is proposed for Paradoxa sinensis. Tuber glabrum is similar to T. gigantosporum but differs by its glabrous ascomata. The ITS sequences of the two Paradoxa-like truffle species, which invariably have one-spored asci, fall into the lineage of Tuber and cluster in a distinct clade together with the European T. macrosporum and the North American T. canaliculatum.     

Bacteria associated with truffle‐fruiting bodies contribute to truffle aroma

Truffles, symbiotic fungi renown for the captivating aroma of their fruiting bodies, are colonized by a complex bacterial community of unknown function. We characterized the bacterial community of the white truffle Tuber borchii and tested the involvement of its microbiome in the production of sulphur‐containing volatiles. We found that sulphur‐containing volatiles such as thiophene derivatives, characteristic of T. borchii fruiting bodies, resulted from the biotransformation of non‐volatile precursor(s) into volatile compounds by bacteria. The bacterial community of T. borchii was dominated by α‐ and β‐Proteobacteria. Interestingly, all bacteria phyla/classes tested in this study were able to produce thiophene volatiles from T. borchii fruiting body extract, irrespective of their isolation source (truffle or other sources). This indicates that the ability to produce thiophene volatiles might be widespread among bacteria and possibly linked to primary metabolism. Treatment of fruiting bodies with antibacterial agents fully suppressed the production of thiophene volatiles while fungicides had no inhibitory effect. This suggests that during the sexual stage of truffles, thiophene volatiles are exclusively synthesized by bacteria and not by the truffle. At this stage, the origin of thiophenes precursor in T. borchii remains elusive and the involvement of yeasts or other bacteria cannot be excluded.     

Comparison of Chemical Composition in Tuber aestivum Vittad. of Different Geographical Origin

Truffles are prized and nutrition‐rich edible hypogeous fungi. The aim of this study was a comprehensive investigation of chemical composition of Burgundy truffle (Tuber aestivum Vittad .). We tried to answer the question: what is the impact of the environment on the truffle quality. To know the nutritional value of Burgundy truffle we compared lipids, proteins, saccharides, polyphenolics, flavonoids, total sterols, ergosterol, volatile flavour and aroma compounds content in fruit bodies of the fungus collected in three different geographical regions, i.e., Poland, Slovakia, and Italy. A comparison of the above mentioned compounds is especially interesting due to environmental and climatic differences among the studied geographical regions. Results revealed that fruit bodies of T. aestivum from Poland and Slovakia possessed nearly similar content of proteins, total sterols, and saccharides. The fruiting bodies from Italy contained significantly larger amounts of most of the investigated compounds. In turn, Polish specimens had higher content of lipids and polyphenolics than Slovak and Italian ones. We have found higher similarity of volatile compounds composition between Polish and Italian specimens than those of Polish and Slovak origin.     

Eco-archaeological excavation techniques reveal snapshots of subterranean truffle growth

Despite its status as a highly-prized and coveted fungi in gastronomy, many aspects of the subterranean life cycle of the Burgundy truffle (Tuber aestivum) are still unknown, because in situ observations of the formation and maturation of truffle fruitbodies remain difficult. Here, we adopted a suite of archaeological fine-scale excavating techniques to provide unique spatiotemporal snapshots of Burgundy truffle growth at three sites in southern Germany. We also recorded the relative position, fresh weight, maturity level and genotype composition of all excavated fruitbodies. Varying by a factor of thousand, the fresh weight of 73 truffle ranged from 0.1 to 103.2 g, with individual maturity levels likely representing different life cycle stages from completely unripe to fully ripe and even decaying. While only a slightly positive relationship between fruitbody weight and maturity level was found, our results suggest that genetically distinct specimens can exhibit different life cycle stages at the same period of time and under the same environmental conditions. We therefore argue that truffles are likely able to grow, mature and ripe simultaneously between early summer and late winter of the following year. Our case study should encourage further eco-archaeological truffle excavations under different biogeographic settings and at different seasons of the year to gain deeper insights into the fungi's subterranean ecology. The expected cross-disciplinary findings will help truffle hunters and farmers to improve their harvest practices and management strategies.     

Assessment of ectomycorrhizal biodiversity in Tuber macrosporum productive sites

Tuber macrosporum Vittad. is a truffle with superb organoleptic properties, whose cultivation is still in its infancy. For the first time we have aimed to provide information on ectomycorrhizal communities in natural and cultivated T. macrosporum sites. Ectomycorrhizal morphotypes were identified using ITS nrDNA sequencing and sorted into molecular operational taxonomic unit (MOTU). We detected 16 MOTUs in the T. macrosporum cultivated plantation. Ascomycota were the most abundant (86.4 %) with Helvellaceae, Pyronemataceae and Pezizaceae the most common. Twenty-two MOTUs were collected in the natural T. macrosporum site. Basidiomycota morphotypes were plentiful (70.6 %) and Thelephoraceae dominated. Each site had different taxa belowground with only T. macrosporum in common, being more abundant in the natural (18.2 %) than in the cultivated (14.4 %) site. Species richness, Simpson and Shannon diversity indices, taxonomic diversity, distinctness and variation of taxonomic distinctness were lower in the cultivated than in the natural site.     

Bats,Trypanosomes, and Triatomines in Ecuador: New Insights into the Diversity,Transmission, and Origins of Trypanosoma cruzi and Chagas Disease

The generalist parasite Trypanosoma cruzi has two phylogenetic lineages associated almost exclusively with bats—Trypanosoma cruzi Tcbat and the subspecies T. c. marinkellei. We present new information on the genetic variation, geographic distribution, host associations, and potential vectors of these lineages. We conducted field surveys of bats and triatomines in southern Ecuador, a country endemic for Chagas disease, and screened for trypanosomes by microscopy and PCR. We identified parasites at species and genotype levels through phylogenetic approaches based on 18S ribosomal RNA (18S rRNA) and cytochrome b (cytb) genes and conducted a comparison of nucleotide diversity of the cytb gene. We document for the first time T. cruzi Tcbat and T. c. marinkellei in Ecuador, expanding their distribution in South America to the western side of the Andes. In addition, we found the triatomines Cavernicola pilosa and Triatoma dispar sharing shelters with bats. The comparisons of nucleotide diversity revealed a higher diversity for T. c. marinkellei than any of the T. c. cruzi genotypes associated with Chagas disease. Findings from this study increased both the number of host species and known geographical ranges of both parasites and suggest potential vectors for these two trypanosomes associated with bats in rural areas of southern Ecuador. The higher nucleotide diversity of T. c. marinkellei supports a long evolutionary relationship between T. cruzi and bats, implying that bats are the original hosts of this important parasite.