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.     

Assessment of Abundance and Species Composition of Filamentous Fungi in the Underground Rzeczka Complex in Sowie Mountains (Lower Silesia,Poland)

Our study is the first mycological evaluation of the air and the rocks in the underground Rzeczka complex in Sowie Mountains (Lower Silesia, Poland). The density of filamentous fungi isolated from the air inside and outside the adit ranged between 245.5 to 1332.6 Colony Forming Units in 1 m³ of air. Sixteen species of filamentous fungi were isolated from the internal air sampled and 11 from the outside of the adit. Cladosporium spp. were the fungi most frequently isolated from the internal atmosphere of the adit and from the external air. From the rock walls and from the rock debris on the floor of the adit only seven species of fungi were isolated. The fungi most frequently isolated from the rock walls were the Aspergillus niger group and from the rock debris we also found A. niger group along with species of Mucor. The concentration of airborne fungi in the adit did not exceed official limits and norms present for a health risk to the tourists, but fungi species isolated from the rocks can cause their degradation.     

Biosynthesis of galactomannans found in filamentous fungi belonging to Pezizomycotina

The galactomannans (GMs) that are produced by filamentous fungi belonging to Pezizomycotina, many of which are pathogenic for animals and plants, are polysaccharides consisting of α-(1→2)-/α-(1→6)-mannosyl and β-(1→5)-/β-(1→6)-galactofuranosyl residues. GMs are located at the outermost layer of the cell wall. When a pathogenic fungus infects a host, its cell surface must be in contact with the host. The GMs on the cell surface may be involved in the infection mechanism of a pathogenic fungus or the defense mechanism of a host. There are two types of GMs in filamentous fungi, fungal-type galactomannans and O-mannose type galactomannans. Recent biochemical and genetic advances have facilitated a better understanding of the biosynthesis of both types. This review summarizes our current information on their biosynthesis.     

Root-tip cutting and uniconazole treatment improve the colonization rate of Tuber indicum on Pinus armandii seedlings in the greenhouse

The Chinese black truffle Tuber indicum is commercially valuable. The main factors influencing the success or failure of a truffle crop include the mycorrhizal colonization rate and host plant quality. The effects of a plant growth regulator (uniconazole) and plant growth management technique (root-tip cutting) on T. indicum colonization rate and Pinus armandii seedling growth were assessed under greenhouse conditions. The results indicated that 10 mg l⁻¹ uniconazole or the combination of 5 mg l⁻¹ uniconazole and root-tip cutting constitutes an effective method for ectomycorrhizal synthesis based on an overall evaluation of colonization rate, plant biomass, plant height, root weight, stem circumference and antioxidant enzyme activities (SOD and POD) of P. armandii. The abundance of Proteobacteria in the rhizosphere of colonized seedlings might serve as an indicator of stable mycorrhizal colonization. This research inspires the potential application of uniconazole and root-tip cutting treatments for mycorrhizal synthesis and truffle cultivation.     

Bacterial and fungal communities associated with Tuber magnatum‐productive niches

Abstract

Truffles are hypogeous ectomycorrhizal fungi of ecological interest for forestry in soils of the northern hemisphere, and of economical relevance for food markets worldwide. The molecular mechanisms that control truffle body formation are largely unknown, as well as the environmental factors that are likely involved. Among the latter, it has been hypothesized that soil‐borne communities may have an impact on truffle production. To address this question, we investigated bacterial and fungal communities resident in productive versus adjacent non‐productive grounds of the white truffle Tuber magnatum by using PCR‐DGGE. Although bacterial communities were generally highly similar across all samples within the grounds, profiles did cluster according to the productivity of circumscribed niches, and a Moraxella osloensis population appeared to be associated with productive sites. Fungal communities revealed several populations, yet showed no obvious patterns in relation to productivity, although Mortierella and Fusarium oxysporum appeared to be more abundant in the productive area. Our results offer a first glimpse into microbial communities thriving in truffle productive niches, and open the question as to whether microbe‐mediated mechanisms may facilitate/inhibit truffle fruiting‐body production or, vice versa, i.e. whether truffle sporocarps have an impact on the microbes living in the rhizosphere.     

Essential Elements as a Distinguishing Factor between Mycorrhizal Potentials of Two Cohabiting Truffle Species in Riparian Forest Habitat in Serbia

True truffles (Tuber sp.) that establish ectomycorrhizal symbiosis (ECM) with trees in the Mediterranean and temporal regions have species specific abilities to assimilate soil born elements. Suitable habitats are usually inhabited by few truffle species, while distinguishing their symbiotic potentials appeared very difficult. Two species that commonly inhabit riparian forests in Serbia are the most prized one, Tuber magnatum Pico (Piedmont white truffle) and not so highly valued Tuber brumale Vitt . In order to assess potential differences between their assimilation and accumulation abilities, the differences between contents of elements that may be the subjects of the symbiotic trade between the host plant and fungi were evaluated in accumulation target (ascocarps) and their source (the soil). Essential (K, Na, Ca, Mg, Fe, P, S, and Zn) and essential trace elements (Co, Cr, Cu, Mn, and Se) in truffles and soil samples were determined by means of inductively coupled plasma with optical emission spectrometry (ICP‐OES). Their concentrations (mg/kg) in ascocarps were in the range from 1.364±0.591 (Cr) to 10760.862±16.058 (K), while in soil ranged from 23.035±0.010 (Cr) to 20809.300±122.934 (Fe). Element accumulation potential (bioaccumulation factor) was calculated in the system truffle/soil. The statistical approaches were used for establishing the differences, while the possible differentiation between symbiotic potentials of two mycelia in the defined soil conditions was discussed.     

Secrets of the fungus-specific potassium channel TOK family

Several families of potassium (K⁺) channels are found in membranes of all eukaryotes, underlining the importance of K⁺ uptake and redistribution within and between cells and organs. Among them, TOK (tandem-pore outward-rectifying K⁺) channels consist of eight transmembrane domains and two pore domains per subunit organized in dimers. These channels were originally studied in yeast, but recent identifications and characterizations in filamentous fungi shed new light on this fungus-specific K⁺ channel family. Although their actual function in vivo is often puzzling, recent works indicate a role in cellular K⁺ homeostasis and even suggest a role in plant–fungus symbioses. This review aims at synthesizing the current knowledge on fungal TOK channels and discussing their potential role in yeasts and filamentous fungi.     

Identification of host plants and description of sclerotia of the truffle <Emphasis Type="Italic">Mattirolomyces terfezioides</Emphasis>

The truffle, Mattirolomyces terfezioides, is a hypogeous ascomycete with uncertain host relationships. The fungus has been regularly collected on sandy soils in the Carpathian Basin. During the study of the natural host plants of the fungus, strange, amorphous, belowground hyphal aggregates incorporating soil and sand particles have been found attached to the surface of the roots. The fruitbodies of M. terfezioides develop from these hyphal aggregates. This structure, similar to that formed by morels, could be interpreted as a sclerotium. Sclerotia were found both on roots of woody and herbaceous plants. To detect the roots colonized by M. terfezioides, a species-specific polymerase chain reaction was developed. Seven natural hosts of the fungus were identified by this method. No specificity regarding taxa or life form of the plants was found. The colonization of the roots by the septate hyphae of M. terfezioides was weak, particularly compared to the colonization by arbuscular–mycorrhizal fungi. This suggests that this fungus is not the dominant fungal partner of these plants. Therefore, using M. terfezioides as the only inoculum may be inappropriate in truffle cultivation experiments. Nevertheless, further in vitro experiments are needed to develop reliable knowledge on the still ambiguous symbiotic strategy of this fungus.     

Assessing ectomycorrhizal fungal spore banks of truffle producing soils with pecan seedling trap-plants

Background and Aims

Recently, the truffle species Tuber lyonii Butters was found to be dominant in ectomycorrhizal (EcM) fungal communities of cultivated pecan (Carya illinoinensis (Wangenh.) K. Koch). Many truffle fungi exhibit the trait of effectively colonizing plant roots via spores. We hypothesized that T. lyonii would be well represented in the spore bank of pecan orchard soils where it is found.

Methods

We used axenically-grown pecan seedlings as trap-plants to bait for EcM associates in soils collected beneath truffle-producing pecan trees. EcM fungi on seedlings were characterized through rDNA sequencing and were compared to EcM communities of adult trees in these orchards.

Results

Tuber lyonii mycorrhizas were well formed on seedlings inoculated with truffle spores, but were limited to just a few of the trap-plants grown in field soils. We compared EcM communities of adult pecan orchard trees to those on trap-plants and found distinct communities on each, with a high degree of similarity at the ordinal but not species level.

Conclusions

Although species of Pezizales are abundant in pecan EcM communities and as propagules in their soil spore banks, only a low level of T. lyonii was detected in soil spore banks beneath orchard trees naturally colonized by T. lyonii. Other factors including land-use history or orchard management may better explain this truffle species presence and abundance in pecan EcM communities.

     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

The eukaryotic protein kinase superfamily of the necrotrophic fungal plant pathogen,Sclerotinia sclerotiorum

Protein kinases have been implicated in the regulation of many processes that guide pathogen development throughout the course of infection. A survey of the Sclerotinia sclerotiorum genome for genes encoding proteins containing the highly conserved eukaryotic protein kinase (ePK) domain, the largest protein kinase superfamily, revealed 92 S. sclerotiorum ePKs. This review examines the composition of the S. sclerotiorum ePKs based on conserved motifs within the ePK domain family, and relates this to orthologues found in other filamentous fungi and yeasts. The ePKs are also discussed in terms of their proposed role(s) in aspects of host pathogenesis, including the coordination of mycelial growth/development and deployment of pathogenicity determinants in response to environmental stimuli, nutrients and stress.     

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.     

Otidea subterranea sp. nov.: Otidea goes below ground

Evidence suggests that truffle-like sporocarp forms have evolved many times in the Pezizales, but primarily from epigeous ancestors within ectomycorrhizal clades. There are several ectomycorrhizal clades, however, that contain no known hypogeous species. We collected specimens of an unusual unidentified truffle from mixed oak woodlands in Iowa. Although clearly a member of the Pezizales (Ascomycota), this hypogeous species did not belong to any of the described truffle genera. Based on a combination of ecological, phylogenetic, and morphological evidence we determined that this new truffle is a hypogeous member of the genus Otidea (Pyronemataceae), a lineage with no described truffle species. We describe it here as a new species, Otidea subterranea.     

Endocytosis in the plant-pathogenic fungus Ustilago maydis

Summary. Filamentous fungi are an important group of tip-growing organisms, which include numerous plant pathogens such as Magnaporthe grisea and Ustilago maydis. Despite their ecological and economical relevance, we are just beginning to unravel the importance of endocytosis in filamentous fungi. Most evidence for endocytosis in filamentous fungi is based on the use of endocytic tracer dyes that are taken up into the cell and delivered to the vacuole. Moreover, genomewide screening for candidate genes in Neurospora crassa and U. maydis confirmed the presence of most components of the endocytic machinery, indicating that endocytosis participates in filamentous growth. Indeed, it was shown that in U. maydis early endosomes cluster at sites of growth, where they support morphogenesis and polar growth, most likely via endosome-based membrane recycling. In humans, such recycling processes to the plasma membrane involve small GTPases such as Rab4. A homologue of this protein is encoded in the genome of U. maydis but is absent from the yeast Saccharomyces cerevisiae, suggesting that Rab4-mediated recycling is important for filamentous growth. Furthermore, human Rab4 regulates traffic of early endosomes along microtubules, and a similar microtubule-based transport is described for U. maydis. These observations suggest that Rab4-like GTPases might regulate endosome- and microtubule-based recycling during tip growth of filamentous fungi. Correspondence and reprints: MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Federal Republic of Germany.     

Kinetic model to describe the morphological evolution of filamentous fungi during their early stages of growth in the standard submerged and microparticle‐enhanced cultivations

Biosynthesis of metabolites and enzymes by filamentous fungi depends on their morphological form in submerged cultures. However, their early stages of growth lasting approximately 24 h, from the introduction of spores to the medium until the formation of stable morphological forms, such as clumps or pellets, have rarely been the objects of experimental and modeling studies. Microparticle‐enhanced cultivation (MPEC) has been applied only to a few fungal species, mainly Aspergilli. Therefore, the objective of this work was to formulate the kinetic model to describe the early stages of the fungal evolution in the standard cultivation and MPEC for Aspergillus terreus, Chaetomium globosum, Penicillium rubens, and Mucor racemosus. These fungi exhibit various mechanisms of agglomerates formation in submerged cultures. The experiments were performed in batch shake flasks (parameters identification) and a stirred tank bioreactor (model verification). In the balance equation for fungal cells, the mean projected area of hyphal objects measured by the digital analysis of microscopic images was used as the dependent variable. The analysis of the experimental data and model solution revealed that the effect of the microparticles (aluminum oxide at 6 g L^?1) in MPEC toward the studied filamentous fungi was to the high extent species dependent. This effect was most evident in the case of spore coagulative A. terreus and noncoagulative M. racemosus.     

嗜热毁丝霉高通量培养技术及其应用

【背景】丝状真菌是一类重要的工业发酵生产宿主菌,如何进行高通量纯菌培养和高效检测筛选性能优异的菌株是工业丝状真菌研究的重要方向。【目的】研究建立丝状真菌的高通量培养技术并测试应用效果。【方法】通过对丝状真菌培养过程中的制种、接种、培养和检测研究,建立基于孔板的高通量培养技术,并以嗜热毁丝霉为例对该技术进行验证。【结果】与传统的平板制种和摇瓶接种培养方式相比,高通量孔板的培养方式将制种通量提高24倍,单位面积产孢子能力提高350%,液体培养转接效率提高10-40倍,并建立96孔板测定乙醇含量的高通量检测技术。【结论】将丝状真菌的培养和检测通量提高1-2个数量级,为快速检测丝状真菌改造过程产生的大量性状不同菌株并获得目标菌株奠定基础,为丝状真菌高通量筛选研究提供应用指导价值。     

Presence of weed fungus in a non‐social beetle–fungus cultivation mutualism

1. Farming by non‐human organisms has arisen independently in several animal lineages, allowing them to survive on food sources that are otherwise difficult to access. However, agricultural gardens are prone to invasion by parasites that overgrow cultivars in the absence of host animals. The presence of garden parasites and associated host adaptations are well studied in advanced fungal agriculture practised by social insects (ants, termites, and ambrosia beetles), but the impact of garden parasites in more primitive forms of agriculture is poorly known. The aim of the present study was to report the presence of weed fungi in fungal gardens of the non‐social lizard beetle Doubledaya bucculenta Lewis (Coleoptera: Erotylidae: Languriinae), which cultivates the yeast Wickerhamomyces anomalus (E.C. Hansen) Kurtzman, Robnett & Bas.‐Powers in the internodes of recently dead bamboo culms. 2. The filamentous fungi Arthrinium spp., Fusarium spp., and an unidentified species were isolated from the inner wall and insect‐made wall debris of bamboo internodes inhabited by the beetle. 3. When cultured together with the yeast in the absence of host larvae, Arthrinium sp.1 outcompeted the yeast, suggesting that this filamentous fungus can invade yeast gardens but is normally suppressed under natural conditions. 4. Rearing experiments showed that beetle larvae grew faster when grown on yeast cultures than when fed only Arthrinium sp.1. 5. These results suggest that Arthrinium sp.1 acts as a weed in the fungal gardens of D. bucculenta, inhibiting both growth of the beetle larvae and proliferation of the beneficial yeast.     

Isolation and characterization of some mycelia inhabiting Tuber ascomata

Tuber spp. are ectomycorrhizal ascomycetes that produce subterranean ascomata known as truffles. Truffles can be regarded as complex microhabitats hosting bacteria and yeasts. In this paper we show that guest filamentous fungi are also associated to truffle ascomata, regardless of the Tuber spp., and report the morpho-molecular characterization of seven truffle-hosted mycelia isolated from healthy and intact Tuber ascomata. Some of these isolates were shown to be related to the fungal endophytes of plants. Interestingly, the truffle-hosted mycelia grew stuck to the hyphal wall of their partner when co-cultivated with the Tuber borchii mycelium, but not when co-cultivated with the test species Agaricus macrosporus. The present data suggest that guest filamentous fungi can be added to the list of truffle-interacting microorganisms.     

Actinobacteria may influence white truffle (Tuber magnatum Pico) nutrition,ascocarp degradation and interactions with other soil fungi

To test the hypothesis that truffle-associated bacteria may improve truffle nutrition, we isolated bacteria from white truffle ascocarps and tested Actinobacteria for their ability to solubilise phosphate and iron, nutrients that have limited availability in white truffle grounds. Two isolates with sequence similarities to Curtobacterium flaccumfaciens and Rhodococcus sp. were characterized in detail. Both solubilised Ca₃(PO₄)₂ in a way that was dependent on the nitrogen and carbon sources present. Neither strain broke down phytate, but both produced chelating compounds, performed ammonification, and broke down β-glucan. Additionally, C. flaccumfaciens decomposed chitin, pectin, lipids and proteins, while Rhodococcus sp. exhibited urease activity. Three potentially fungicolous fungi were isolated from diseased white truffle ascocarps and bioassayed against the isolated Actinobacteria. The Rhodococcus isolate inhibited Verticillium leptobactrum, neither bacterium affected Clonostachys rosea, while both isolates promoted growth of Trichoderma sp. The results suggest that Actinobacteria might be involved in improving truffle nutrition, ascocarp degradation and establishing relationships with other soil fungi.