Truffle biogeography—A case study revealing ecological niche separation of different Tuber species

Ecology of hypogeic mycorrhizal fungi, such as truffles, remains largely unknown, both in terms of their geographical distribution and their environmental niches. Occurrence of true truffles (Tuber spp.) was therefore screened using specific polymerase chain reaction (PCR) assays and subsequent PCR amplicon sequencing in tree roots collected at 322 field sites across the Czech Republic. These sites spanned a wide range of climatic and soil conditions. The sampling was a priori restricted to areas thought to be suitable for Tuber spp. inasmuch as they were characterized by weakly acidic to alkaline soils, warmer climate, and with tree species previously known to host true truffles. Eight operational taxonomic units (OTUs) corresponding to Tuber aestivum, T. borchii, T. foetidum, T. rufum, T. indicum, T. huidongense, T. dryophilum, and T. oligospermum were detected. Among these, T. borchii was the OTU encountered most frequently. It was detected at nearly 19% of the sites. Soil pH was the most important predictor of Tuber spp. distribution. Tuber borchii preferred weakly acidic soils, T. foetidum and T. rufum were most abundant in neutral soils, and T. huidongense was restricted to alkaline soils. Distribution of T. aestivum was mainly dictated by climate, with its range restricted to the warmest sites. Host preferences of the individual Tuber spp. were weak compared to soil and climatic predictors, with the notable exception that T. foetidum appeared to avoid oak trees. Our results open the way to better understanding truffle ecology and, through this new knowledge, also to better‐informed trufficulture.     

Characterization of the reproductive mode and life cycle of the whitish truffle <Emphasis Type="Italic">T. borchii</Emphasis>

Truffles are the fruiting structures of ascomycetes in the genus Tuber. Because of their economic importance, truffles have been cultivated for many years using artificially inoculated host plants. Nevertheless, the life cycle and reproductive mode of Tuber spp. are still poorly understood. In filamentous ascomycetes, sexual reproduction is genetically controlled by the mating-type (MAT) locus. Among Tuber spp., the MAT locus has been recently characterized in the black truffles Tuber melanosporum and Tuber indicum. Here, by using sequence information derived from these species and from a Tuber borchii expressed sequence tag (EST) showing similarity to the mat1 gene of Alternaria brassicicola, we embarked on a chromosome-walking procedure to sequence the complete MAT region of T. borchii. This fungus produces highly commercialized whitish truffles and represents a model species for addressing basic questions concerning the life cycle of Tuber spp. We show that T. borchii is heterothallic, as its MAT locus is organized into two idiomorphs, each harbored by different mycelial strains. The alignment of the MAT locus from black truffles and T. borchii reveals that extensive sequence rearrangements and inversions occurred between these species. Moreover, by coupling mating-type analyses to karyological observation, we show that mycelia isolated from ascocarps and mycorrhizae are formed by homokaryotic hyphae.     

<Emphasis Type="Italic">Tuber</Emphasis> spp. biodiversity in one of the southernmost European distribution areas

The aims of this paper were to study the genetic diversity within and among 6 Tuber taxa, the phylogenetic relationships and the spatial distribution of truffles from the Basilicata region (southern Italy). Molecular analyses, using the internal transcribed spacer region, microsatellites, minisatellites and random amplified polymorphic loci revealed the highest expected intra-specific heterozygosity (He=0.30) in T. aestivum, followed by that of T. mesentericum (He=0.22). T. borchii (He=0.17), T. magnatum (He=0.16), T. aestivum fo. uncinatum (He=0.12) and T. brumale fo. moschatum (He=0.09) showed a lower average heterozygosity. The whole set of markers were demonstrated to be useful in clearly differentiating Tuber taxa, separating them in two distinct groups and in five sub-clusters, each corresponding to one single taxon. The first cluster (T. borchii, T. magnatum and T.␣brumale fo. moschatum) was more differentiated than the second one (T. aestivum, T.␣aestivum fo. uncinatum and T. mesentericum). T. brumale fo. moschatum was the most differentiated Tuber taxon. It was more related to the white truffles (the closely related T.␣borchii and T. magnatum) than to the remaining related black truffles T. aestivum and T.␣mesentericum. The amount of Tuber species richness was higher over the western side of Basilicata, in cold Lauretum and in Castanetum phytoclimatic Pavari’s zones. A spatial genetic structure was detected for T. aestivum, as shown by the significant correlation between geographic and genetic distances (r _s =0.32; P<0.0001). Integration of molecular and geographic diversity patterns can allow the selection of sites for Tuber and Tuber-related biodiversity conservation.     

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

Truffle diversity (<Emphasis Type="Italic">Tuber</Emphasis>, Tuberaceae) in British Columbia

To improve baseline data for the developing truffle industry in British Columbia, we compiled existing Tuber species sequences from published and unpublished studies and generated new ITS sequences for truffles belonging to Tuber collected in the province. In doing so, we obtained evidence that 13 species of Tuber occur in the province, including six introduced and seven native species, two of which are putative undescribed species. Of the native species, the Tuber anniae species complex is widely distributed in the province while Tuber beyerlei appears to be much more restricted in distribution. Four of the introduced species have commercial value (Tuber melanosporum, Tuber aestivum, Tuber brumale, and Tuber borchii) as do two of the native species (Tuber gibbosum and Tuber oregonense). Focused sampling on likely tree hosts, both hardwood and Pinaceae species, as well as in currently unexplored parts of the province seems likely to expand our knowledge of the diversity and distribution of Tuber species in British Columbia.     

Mycorrhizal detection of native and non-native truffles in a historic arboretum and the discovery of a new North American species, <Emphasis Type="Italic">Tuber arnoldianum</Emphasis> sp. nov.

During a study comparing the ectomycorrhizal root communities in a native forest with those at the Arnold Arboretum in Massachusetts (USA), the European species Tuber borchii was detected on the roots of a native red oak in the arboretum over two successive years. Since T. borchii is an economically important edible truffle native to Europe, we conducted a search of other roots in the arboretum to determine the extent of colonization. We also wanted to determine whether other non-native Tuber species had been inadvertently introduced into this 140-year-old Arboretum because many trees were imported into the site with intact soil and roots prior to the 1921 USDA ban on these horticultural practices in the USA. While T. borchii was not found on other trees, seven other native and exotic Tuber species were detected. Among the North American Tuber species detected from ectomycorrhizae, we also collected ascomata of a previously unknown species described here as Tuber arnoldianum. This new species was found colonizing both native and non-native tree roots. Other ectomycorrhizal taxa that were detected included basidiomycetes in the genera Amanita, Russula, Tomentella, and ascomycetes belonging to Pachyphlodes, Helvella, Genea, and Trichophaea. We clarify the phylogenetic relationships of each of the Tuber species detected in this study, and we discuss their distribution on both native and non-native host trees.     

Molecular characterisation of the small GTPase CDC42 in the ectomycorrhizal fungus Tuber borchii Vittad.

Summary. The small GTPase CDC42 is ubiquitously expressed in eukaryotes, where it participates in the regulation of the cytoskeleton and a wide range of cellular processes, including cytokinesis, gene expression, cell cycle progression, apoptosis, and tumorigenesis. As very little is known on the molecular level about mycorrhizal morphogenesis and development and these events depend on a tightly regulated reorganisation of the cytoskeleton network in filamentous fungi, we focused on the molecular characterisation of the cdc42 gene in Tuber borchii Vittad., an ascomycetous hypogeous fungus forming ectomycorrhizae. The entire gene was isolated from a T. borchii cDNA library and Southern blot analyses showed that only one copy of cdc42 is present in the T. borchii genome. The predicted amino acid sequence is very similar to those of other known small GTPases and the similar domain structures suggest a similar function. Real-time PCR analyses revealed an increased expression of Tbcdc42 during the phase preparative to the instauration of symbiosis, in particular after stimulation with root exudate extracts. Immunolocalisation experiments revealed an accumulation of CDC42 in the apical tips of the growing hyphae. When a constitutively active Tbcdc42 mutant was expressed in Saccharomyces cerevisiae, morphological changes typical of pseudohyphal growth were observed. Our results suggest a fundamental role of CDC42 in cell polarity development in T. borchii. Correspondence and reprints: Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino, Via Saffi 2, 61029 Urbino, Italy.     

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.     

Diversity and ecological distribution of endophytic fungi associated with medicinal plants

A total of 973 isolates of endophytic fungi were recovered from 1144 tissue fragments of the six medicinal plant species belonging to 4 families collected in the Beijing Botanical Garden. Of these isolates 778 sporulated and were identified into 21 taxa by morphological characteristics. Among the taxa 11 belonged to Coelomycetes, 6 to Ascomycetes, and 4 to Hyphomycetes. Various numbers of endophytic fungi (5–8 taxa) were obtained from each plant. Alternaria alternata was the dominant species in the 6 plants, and Microsphaeropsis conielloides was also dominant in Eucommia ulmoides. There were high colonization rates (47.9%–63.1%) and isolation rates (0.7–0.93) of endophytic fungi, and they were conspicuously higher in twigs than those in leaves in the 6 plants examined. The colonization and isolation rates of endophytic fungi increased with the twig age. The results based on the analyses of cluster and Sorenson’s similarity coefficients indicated that some endophytic fungi showed a certain degree of host and tissue preference.     

New Tuber species found in Poland

New information from a survey of truffles carried out in southern part of Poland in September 2007 is presented. The fruit bodies of Tuber aestivum, T. excavatum, T. rufum and one unidentified Tuber sp. were found. The soil chemistry of the five T. aestivum sites was analysed. Our inventory showed that T. aestivum prefers the mixed forest with host-trees such as: Quercus robur, Corylus avellana, Carpinus betulus, Fagus silvaticus and Tilia cordata. Selection of local inocula of T. aestivum for the propagation of truffles production could be an alternative to the Mediterranean truffles orchards in times when demand for the fungi will be increasing. Fruit bodies of truffles are conserved in the collection of biological material in the Forest Research Institute in Sękocin Stary.     

Diversity and ecological distribution of endophytic fungi associated with medicinal plants

A total of 973 isolates of endophytic fungi were recovered from 1144 tissue fragments of the six me-dicinal plant species belonging to 4 families collected in the Beijing Botanical Garden. Of these isolates 778 sporulated and were identified into 21 taxa by morphological characteristics. Among the taxa 11 belonged to Coelomycetes, 6 to Ascomycetes, and 4 to Hyphomycetes. Various numbers of endophytic fungi (5―8 taxa) were obtained from each plant. Alternaria alternata was the dominant species in the 6 plants, and Microsphaeropsis conielloides was also dominant in Eucommia ulmoides. There were high colonization rates (47.9%―63.1%) and isolation rates (0.7―0.93) of endophytic fungi, and they were conspicuously higher in twigs than those in leaves in the 6 plants examined. The colonization and isolation rates of endophytic fungi increased with the twig age. The results based on the analyses of cluster and Sorenson's similarity coefficients indicated that some endophytic fungi showed a certain degree of host and tissue preference.     

Keratinophilic fungi inhabiting floor dusts of student houses at the South Valley University in Egypt

Keratinophilic fungi include true fungi that vigorously degrade keratin as well as a number of important human pathogenic dermatophytes. We identified 41 species and one variety belonging to 19 genera in 50 floor dust samples following culture on Sabouraud’s dextrose agar medium at 28°C. Dermatophytes and closely related fungi were represented by six species—Aphanoascus fulvescens, Aphanoascus sp., Arthroderma cuniculi, Chrysosporium lucknowense, Gymnoascus uncinatus and Trichophyton rubrum. There were 35 species and one variety of other fungal species, with members of Aspergillus and Penicllium being the most prevalent. Twenty-seven species and 1 variety belonging to 14 genera were identified from 24 dust samples gathered at 2-week intervals from male student housing at El-Kenose during January–December 2005 that had been cultured on Sabauraud’s dextrose agar at 28°C. The monthly counts of keratinophilic fungi showed irregular fluctuations, with a peak in April and the lowest point in November. Dermatophytes and closely related fungi were represented by Aphanoascus fulvescens, Aphanoascus sp., Chrysosporium lucknowense, Gymnoascus uncinatus, and Trichophyton rubrum. In conclusion, the study reports the incidences of some dermatophytes found in the floor dust of student houses of South Valley University, Egypt.     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.     

Isolation and characterization of polysaccharides with the antitumor activity from Tuber fruiting bodies and fermentation system

Fifty-two polysaccharides were isolated from the fermentation systems of Tuber melanosporum, Tuber indicum, Tuber sinense, Tuber aestivum and the fruiting bodies of Tuber indicum, Tuber himalayense, Tuber sinense by elution with an activated carbon column. Polysaccharides from Tuber fermentation system exhibited relatively higher in vitro antitumor activity against HepG2, A549, HCT-116, SK-BR-3, and HL-60 cells than those from Tuber fruiting bodies. All polysaccharides were mainly composed of d-mannose, d-glucose, and d-galactose, which suggested that the polysaccharides from Tuber fruiting bodies and fermentation system have identical chemical compositions. The results of antitumor activity and structural identification indicated that the polysaccharide fractions could promote antitumor activity. Tuber polysaccharides from Tuber fermentation system exhibited relatively higher than that from Tuber fruiting bodies. These results confirm the potential of Tuber fermentation mycelia for use as an alternative resource for its fruiting bodies.     

Microsatellite primed-PCR to select molecular markers for Tuber species

The direct microsatellite-primed PCR and the RAMPO techniques were applied to detect inter-specific polymorphisms in Tuber species and to select species specific fragments. A T. borchii marker was identified and specific primers were selected.     

Somatic embryogenesis from stigmas and styles of grapevine

Summary An in vitro protocol has been developed for callus indiction, somatic embryogenesis, and plant regeneration from stigma-style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.: cvs. ‘Bombino Nero’, ‘Greco di Tufo’, ‘Merlot’, and ‘Sangiovese’) were tested. Exlants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA: 4.5 and 9.0 μM) and β-naphthoxyacetic acid (NOA; 5.0 and 9.9 μM). Sucrose (88 mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. after culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot’, which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma-style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 μM BA and 9.9 μM NOA). 14% in ‘Greco di Tufo’ (4.5 μM BA and 9.9 μM NOA), and 8% in ‘Sangiovese’ (9.0 μM BA and 9.9 μM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88 mM sucrose.     

Evaluation of the enzymes involved in primary nitrogen metabolism in Tilia platyphyllos-Tuber borchii ectomycorrhizae

No information is available about Tuber borchii Vittad. ammonium metabolism during its life cycle, which involves the succession of three distinct phases. In this direction, the levels of glutamine synthetase (GS; EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.1.13-14) and glutamate dehydrogenase (GDH; EC 1.4.1.2-4) were evaluated in Tilia platyphyllos Scop.-Tuber borchii Vittad. ectomycorrhizae, free living mycelium and non-inoculated roots. In the plant roots, GS shows high specific activity and only NADH-GDH (EC 1.4.1.2) is detectable; on the other hand, in free living mycelium GS and NADPH-GDH (EC 1.4.1.4) can be detected. Ectomycorrhizal metabolism was found to be deeply influenced by the two symbiotic partners. In fact, GS and both forms of GDH are present and their specific activities are higher than those found in the plant root and in the mycelial cells.     

First evidence for truffle production from plants inoculated with mycelial pure cultures

Truffle (Tuber spp.) cultivation is based on raising mycorrhizal trees in greenhouses that have been inoculated with suspensions of ascospores. The problem with this is that pests, pathogens, and other mycorrhizal fungi can contaminate the trees. Furthermore, because ascospores are produced sexually, each plant potentially has a different genetic mycorrhizal makeup from each other so tailoring the mycorrhizal component of plants to suit a particular set of soil and climatic conditions is out of the question. Here, we report on the production of Tuber borchii-mycorrhized plants using pure cultures, establishing a truffière with these and subsequent production of its fruiting bodies. This study opens up the possibility of producing commercial numbers of Tuber-mycorrhized trees for truffle cultivation using mycelial inoculation techniques. It also poses questions about the mechanism of fertilization between the different strains which were located in different parts of the experimental truffière.     

Genetic diversity amongst landraces of a dioecious vegetatively propagated plant,betelvine (<Emphasis Type="Italic">Piper betle</Emphasis> L.)

Betelvine (Piper betle L., family Piperaceae) is an important, traditional and widely cultivated crop of India. The cultivators and consumers recognize more than 100 cultivars (landraces) based on regional and organoleptic considerations, while in terms of phytochemical constituents only five groups have been identified for all the landraces. Since betelvine is an obligate vegetatively propagated species, genomic changes, if any, may have become ‘fixed’ in the landraces. We carried out random amplified polymorphic DNA (RAPD) analysis in several landraces considered in four groups, namely, ‘Kapoori’, ‘Bangla’, ‘Sanchi’ and ‘Others’ in order to ascertain their genetic diversity. On the basis of the data from eleven RAPD primers, we distinguished genetic variation within and among the four groups of landraces. The results indicate the’Kapoori’ group is the most diverse. The neighbour joining (NJ) tree after a bootstrap (500 replicate) test of robustness clearly shows the four groups to be well separated. Interestingly, all known male or female betelvine landraces have separated in the NJ tree indicating an apparent gender-based distinction among the betelvines.     

Effects of leguminous ground cover competition on red birch and soil nutrient status in the nursery

Legumes as ground cover are regularly planted to increase nitrogen economy of crops and to improve soil. In the present study various clover species were evaluated as vegetative ground cover in nursery field production of micropropagated red birch (Betula pubescensEhrh. f. rubraUlvinen f. nova) in two 2-year experiments. The clover species and cultivars, Trifolium pratenseL. ‘Bjursele’, T. repens L. ‘Jogeva’, T. repens L. ‘Sonja’, T. hybridum L. ‘Frida’, T. incarnatum L. ‘Opolska’, T. resupinatum L. and T. subterraneum L. were compared to grass sod Festuca rubra L. ‘Ensylva’ and to a coverless ground (control). The last one was kept weed free by hand hoeing. Birch (leaves, stems, branches and roots) and soil nutrient concentrations (N, P, K, Ca, Mg and Fe) were analysed and nutrient ratios in birch determined. The annual clovers, T. incarnatumL., T. resupinatumL. and T. subterraneumL., provided about the same nutrient status in birch as did the control. Perennial clovers and grass were strong competitors with trees. High levels of P and Mg in birch leaves relative to N concentration were typical for poorly growing seedlings. Neither annual nor perennial clovers did generally improve soil nutrient status. This revised version was published online in June 2006 with corrections to the Cover Date.