Soil analysis reveals the presence of an extended mycelial network in a Tuber magnatum truffle-ground

Truffles are hypogeous ectomycorrhizal fungi. They belong to the genus Tuber and are currently considered a hot spot in fungal biology due to their ecological and economic relevance. Among all the species, Tuber magnatum is the most appreciated because of its special taste and aroma. The aim of this work was to set up a protocol to detect T. magnatum in soil and to assess its distribution in a natural truffle-ground. We used the β-tubulin gene as a marker to identify T. magnatum in the soil. This gene allowed us to trace the distribution of the fungus over the entire truffle-ground. Tuber magnatum was found, in one case, 100 m from the productive host plant. This study highlights that T. magnatum mycelium is more widespread than can be inferred from the distribution of truffles and ectomycorrhizas. Interestingly, a new haplotype – never described from fruiting body material – was identified. The specific detection of T. magnatum in the soil will allow to unravel the ecology of this fungus, following its mycelial network. Moreover, this new tool may have practical importance in projects aimed to increase large-scale truffle production, checking for T. magnatum persistence in plantations.     

Phylogenetic study of two truffles, Tuber formosanum and Tuber furfuraceum, identified from Taiwan

Truffles are one of the most valuable edible fungi and have drawn extensive research interests worldwide. In Taiwan, two species of truffle, Tuber formosanum and Tuber furfuraceum , have been identified and reported. Although the morphological features of these two truffles have been described, lack of molecular identification has led to difficulties with firmly establishing their relatedness to other truffles. In this study, we utilized the internal transcribed spacer (ITS) and β-tubulin gene sequences to generate the phylogenetic relationship of T. formosanum and T. furfuraceum with other taxonomic relatives. Our analysis revealed five/three major phylogenetic clades according to the 5.8S-ITS2/β-tubulin gene sequences and corroborated with their morphological characterization. Tuber formosanum highly resembles the Tuber indicum B complex, while T. furfuraceum is most similar to Tuber huidongense . Based on a molecular clock, we estimated that T. furfuraceum and T. formosanum would have diverged from their close relatives in mainland China between 10.2 and 4.1 Ma, respectively. Based on the results, we propose that these two Tuber species found in Taiwan might originate from the common ancestors with some truffle species in China. However, due to a long divergence time and geographical separation, they have evolved into indigenous species of Taiwan.     

块菌名实考证及其资源保护

块菌是指Tuber属的真菌种类,属于子囊菌门(Ascomycota),生在地下,其中包含着世界上最美味和价值最昂贵的食用菌.中国食与药用真菌文化悠久,历代本草都不乏各种菌类的记载,但在本草中记载的与块菌有关系的菌类唯有1245年陈仁玉编撰的“菌谱”中称之为“麦蕈”和俗名为“麦丹蕈”的一种地下真菌.日本本草对“麦蕈”也屡有记载,并把“麦蕈”叫“地肾”和“松露”.这是“松露”一名的最早记载,“麦蕈”或“松露”可能是被现代菌物学称之为须腹菌(Rhizopogon spp.)的真菌种类.因而,“松露”一名虽是汉字,但是所指并非真正的块菌,应是须腹菌(Rhizopogon spp.),也称“假块菌”(false truffle),所以松露和块菌寓意各异.产于我国西南地区的食用黑块菌主要是印度块菌(Tuber indi-cum)或中华块菌(T.sinernse)、夏块菌(T.aestivum=T.uncinatu)和拟喜马拉雅块菌(T.pseudohimalayense=T.pseudoexcavatum),近年来身价倍增,成为中国野生食用菌出口种类中名列前三甲的菌类,在世界块菌市场上也颇受欢迎.喜马拉雅块菌(T.himalayense)在我国尚未发现它的踪迹.印度块菌和中华块菌形态上有差异,若把中国产的类似黑孢块菌的黑块菌统称为印度块菌(T.indicum)尚值得商榷.中华块菌或印度块菌形态和遗传变异多样,尚需进一步研究.2010年在云南和四川相继发现的大型香味浓郁的白块菌,近似于波氏块菌(T.borchii)组的块菌,颇具研究和巨大的商业价值,表明我国块菌资源比原有记载的要丰富的多.由于利益之驱动和疏于管理,掠夺式的采收方法造成了块菌产区生态环境毁灭性的破坏,一些商业化采集区的块菌已濒临灭绝,中国西南地区的块菌资源的保护问题迫在眉睫.本文对产自欧洲、美洲的块菌、沙漠块菌,以及块菌的生态意义和种植作了简要介绍和讨论.     

Effect of slug mycophagy on Tuber aestivum spores

Truffles in the genus Tuber produce subterranean fruiting bodies that are not able to actively discharge their spores in the environment. For this reason, truffles depend on mycophagous animals for reproduction. Fungus consumption (mycophagy) is a behaviour typical of both vertebrates and invertebrates. Mammals, especially rodents, are the most studied group of mycophagists and have been found to consume a great variety of fungi. Among invertebrates, mycophagy is documented in arthropods, but rarely in molluscs. In our study we assessed the effect on the morphology and mycorrhizal colonization of Tuber aestivum spores after passage through the gut of slugs (Deroceras invadens) and, for comparison, of a house mouse (Mus musculus). Light, scanning electron and atomic force microscopy revealed that the digestion, especially by slugs, freed spores from the asci and modified their morphology. These are believed to be the reasons why we observed an improvement in oak mycorrhization with the slug and rodent ingested spores in comparison to a fresh spore inoculation. We also demonstrated by molecular barcoding that slugs’ guts sampled on a Tuber melanosporum truffle ground contain spores from this species and Tuber brumale, further suggesting that some invertebrates are efficient Tuber spore dispersers.     

Transcript profiling reveals novel marker genes involved in fruiting body formation in Tuber borchii

cDNA arrays were used to explore mechanisms controlling fruiting body development in the truffle Tuber borchii. Differences in gene expression were higher between reproductive and vegetative stage than between two stages of fruiting body maturation. We suggest hypotheses about the importance of various physiological processes during the development of fruiting bodies.     

Olfactory orientation of the truffle beetle, Leiodes cinnamomea

Although the truffle beetle, Leiodes cinnamomea, inflicts substantial damage to the ripe stage of fruiting bodies of the economically important black truffle (Tuber melanosporum), it is not attracted by ripe truffle odours. Rather, male beetles are attracted to infested truffles only in the presence of female beetles, suggesting that the former employ a pheromone to locate truffles over short distances. In contrast, female beetles show no attraction to infested or uninfested truffles, suggesting that they employ other cues, possibly linked to odours emitted by truffles prior to the ripe stage. We hypothesize that the chemical composition of truffle volatiles changes over the life of the truffle fruiting body, being attractive to insects early on and to mammals just prior to decomposition.     

Viscosinamide-producing Pseudomonas fluorescens DR54 exerts a biocontrol effect on Pythium ultimum in sugar beet rhizosphere

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.     

Mycorrhization of Pecan trees (Carya illinoinensis) with commercial truffle species: Tuber aestivum Vittad. and Tuber borchii Vittad

Pecan (Carya illinoinensis) is an economically important nut tree native to the Mississippi basin and cultivated worldwide. In North America, species of truffles are regularly found fruiting in productive pecan orchards and the truffle genus Tuber appears to be abundant in pecan ectomycorrhizal (EM) communities. As an initial step to determine the feasibility of co-cropping European truffle species with pecan, we evaluated whether mycorrhizae of highly esteemed European truffle species (Tuber aestivum Vittad. T. borchii and T. macrosporum) could be formed on pecan seedlings. Seedlings were inoculated with truffle spores and were grown in a greenhouse for 10?months. Levels of EM colonization were estimated visually and quantified by counting EM tips. Ectomycorrhizae were identified both morphologically and molecularly with species-specific amplification and by sequencing of the ITS region of the nuclear ribosomal DNA (nrDNA). Both T. borchii and T. aestivum spores produced well-formed ectomycorrhizae on pecan seedlings with average root colonization levels of about 62% and 42%, respectively, whereas no ectomycorrhizae of T. macrosporum were formed. The anatomy and morphology of these truffle ectomycorrhizae on pecan was characterized. The co-cropping of T. aestivum and T. borchii may hold promise as an additional stream of revenue to pecan growers, although, further studies are needed to assess whether this symbiosis is maintained after planting in the field and whether truffle production can be supported by this host species.     

Fine-scale genetic structure of natural <Emphasis Type="Italic">Tuber aestivum</Emphasis> sites in southern Germany

Although the Burgundy truffle (Tuber aestivum) is an ectomycorrhizal fungus of important economic value, its subterranean life cycle and population biology are still poorly understood. Here, we determine mating type and simple sequence repeat (SSR) maternal genotypes of mapped fruiting bodies to assess their genetic structure within two naturally colonized forest sites in southern Germany. Forty-one genotypes were identified from 112 fruiting bodies. According to their mating types, the maternal genotypes were aggregated only in one population. Genotypic diversity of individuals that mostly were small and occurred in 1 out of 2 years of sampling was high. Although these results suggested a ruderal colonization strategy, some genets spread several hundred meters. This result indicates that, besides sexual spore dispersal, vegetative growth or spreading by mycelial propagules contributes to dissemination. In one site, fewer individuals with a tendency to expand genets belonging to only one genetic group were observed. In the second site, numerous small individuals were found and were grouped into two clearly differentiated genetic groups that were spatially intermingled. Forest characteristics and disturbances are possible reasons for the observed genetic patterns. Our findings contribute to a better understanding of the biology of one of the most widespread and commercially important truffle species. This knowledge is critical for establishing and maintaining sustainable long-term truffle cultivations.     

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.     

Morphological and molecular typing of the below-ground fungal community in a natural Tuber magnatum truffle-ground

The aims of the work were to elucidate the distribution of the ectomycorrhizal fungus Tuber magnatum Pico during its symbiotic stage, and to identify the root-associated fungi in a natural truffle-ground located in North Italy. Ectomycorrhizal root tips were harvested in the truffle ground, sorted in morphotypes and analyzed by ITS. Morphological and molecular analyses revealed that (i) T. magnatum mycorrhizae were rare and independent on the fruitbody productions and (ii) the dominant fungal species belonged to Thelephoraceae, followed by Tuberaceae and Sebacinaceae.     

Genetic variability of Tuber uncinatum and its relatedness to other black truffles

Genetic variability is one of the major survival strategies developed by symbiotic fungi. We focused on the ectomycorrhizal fungus Tuber uncinatum Chatin that produces edible ascomata. In order to understand the degree of its variability and its relatedness to another morphologically-similar truffle, T. aestivum Vittad., ascomata of T. uncinatum were collected from a single natural truffle-ground located in the north of Italy and compared with samples from other Italian sites, as well as with T. aestivum ascomata from other European regions. We used multi-locus approaches, such as microsatellite-primed PCR (polymerase chain reaction), and single locus markers, such as mitochondrial and nuclear ribosomal DNA on 30 samples. The results demonstrate that the level of genetic polymorphism among isolates of T. uncinatum was higher than in other Tuber species, like T. melanosporum. Neighbour-joining analyses were carried out on a binary data matrix on 12 ascomata of T. uncinatum and T. aestivum, and on 15 internal transcribed spacer (ITS) sequences of these species and 5 from other Tuber species. Taken together, they clustered T. uncinatum and T. aestivum in two separate groups. The mitochondrial rDNA primers, NMS1 and NMS2, were not able to differentiate morphologically related and unrelated truffles. Moreover, a pair of primers, intentionally designed to differentiate isolates of T. aestivum and T. uncinatum from other Tuber species, successfully amplified DNA from all the samples of T. aestivum and T. uncinatum considered in our analysis. In conclusion, different molecular approaches separate T. aestivum and T. uncinatum according to their spore reticulum and their taste and smell.     

Comparison of ectomycorrhizal communities in natural and cultivated Tuber melanosporum truffle grounds

Truffles are hypogeous ectomycorrhizal (EM) fungi belonging to the genus Tuber. Although outplanting of truffle-inoculated host plants has enabled the realization of productive orchards, truffle cultivation is not yet standardized. Therefore, monitoring the distribution of fungal species in different truffle fields may help us to elucidate the factors that shape microbial communities and influence the propagation and fruiting of Tuber spp. In this study, we compared the fungal biodiversity in cultivated and natural Tuber melanosporum truffle fields located in Central Italy. To this end, ectomycorrhizas (ECM) and soil samples were molecularly analyzed, and an inventory of the fungi associated with Quercus pubescens plants colonized by T. melanosporum, Tuber aestivum or Tuber brumale was compiled. T. melanosporum and T. aestivum were dominant on the cultivated plants, and the number of EM species was markedly lower in the cultivated sites than in the natural sites. However, in the same site, EM biodiversity was higher in T. brumale-colonized plants than in T. melanosporum-colonized plants. These results suggest that different Tuber spp. may have different competitive effects on the other mycobionts. Additionally, in keeping with our previous findings, we found that the number of T. melanosporum genotypes recovered from the soil samples was higher than that of the underlying ECM.     

Species recognition and cryptic species in the Tuber indicum complex

Morphological delimitation of Asian black truffles, including Tuber himalayense, T. indicum, T. sinense, T. pseudohimalayense, T. formosanum and T. pseudoexcavatum, has remained problematic and even phylogenetic analyses have been controversial. In this study, we combined five years of field investigation in China with morphological study and DNA sequences analyses (ITS, LSU and β-tubulin) of 131 Tuber specimens to show that T. pseudohimalayense and T. pseudoexcavatum are the same species. T. formosanum is a separate species based on its host plants and geographic distribution, combined with minor morphological difference from T. indicum. T. sinense should be treated as a synonym of T. indicum. Our results demonstrate that the present T. indicum, a single described morphological species, should include at least two separate phylogenetic species. These findings are of high importance for truffle taxonomy and reveal and preserve the richness of truffle diversity.     

Molecular markers for the identification of the ectomycorrhizal fungus Tuber borchii

Five species of white truffle were classified using PCR-based techniques. RAPD (random amplified polymorphic DNA) fingerprints and specific pairs of primers were used. A RAPD fragment was constant in Tuber borchii Vittad. isolates and polymorphic among the other species. Two molecular markers specific for T. borchii were developed from the sequence of the non-polymorphic RAPD fragment and from regions flanking the 5'-3' ends of a truffle gene. These markers were applied in the identification of T. borchii fruit bodies, mycelia and mycorrhizas, allowing us to monitor the development of this fungus during its entire life cycle.     

Ectomycorrhizal fungi with edible fruiting bodies 3.Tuber magnatum, tuberaceae

The Italian white truffle(Tuber magnatum Pico) forms mycorrhizal relationships, with the roots of, for example, poplars, willows, oaks, aspen, alder and hazelnut in Northern Italy and in small areas of Southern France, Switzerland and Yugoslavia. Its fruiting bodies, which are harvested in autumn and early winter, have a strong aroma and taste and are much sought-after by chefs and gourmets. Because they do not preserve well, good quality Italian white truffle is unavailable for much of the year. The vegetation, climate and soils where T. magnatum grows in Italy are described and compared with those found in similar areas in New Zealand. Market information and methods for cultivatingT. magnatum are also presented.     

Spatio-Temporal Dynamic of Tuber magnatum Mycelium in Natural Truffle Grounds

Tuber magnatum produces the world''s most expensive truffle. This fungus produces very rare ectomycorrhizas which are difficult or even impossible to detect in the field. A “real-time” PCR assay was recently developed to quantify and to track T. magnatum mycelium in soil. Here, this technique was used to investigate the spatial distribution of T. magnatum extra-radical mycelium in soil productive patches and its dynamic across seasons. This study was carried out in four different natural T. magnatum truffle grounds located in different Italian regions. During the fruiting seasons, the amount of T. magnatum mycelium was significantly higher around the fruiting points and decreased going farther away from them. Moreover, T. magnatum mycelium inside the productive patches underwent seasonal fluctuations. In early spring, the amount of T. magnatum mycelium was significantly higher than in summer. In summer, probably due to the hot and dry season, T. magnatum mycelium significantly decreased, whereas in autumn it increased again and was concentrated at the putative fruiting points. These results give new insights on T. magnatum ecology and are useful to plan the most appropriate sampling strategy for evaluating the management of a truffle ground.     

Truffle volatiles: from chemical ecology to aroma biosynthesis

Truffles (Tuber spp.) are symbiotic fungi that develop underground in association with plant roots. Food connoisseurs describe their scent as sensual, seductive and unique. These mysterious fungi, however, do not produce their aroma for the mere pleasure of humans. Truffle volatiles act as odorant cues for mammals and insects which are thus able to locate the precious fungi underground and spread their spores. They also freely diffuse in the soil and mediate interactions with microorganisms and plant roots, potentially regulating a complex molecular dialogue among soil fauna and flora. The aim of this review is to synthesize 30 yr of research on truffle volatiles, spanning fields of study from chemical ecology to aroma biosynthesis. Specific aspects of truffle volatile ecology and biology will be discussed, including which species have been studied so far and for what purpose, what ecological role has been demonstrated or speculated to exist for specific truffle volatiles, which volatiles are common or unique to certain species and what their biosynthetic route might be. Future challenges in truffle aroma research will also be addressed, focusing on how high-throughput post-genomic technologies may advance our understanding of truffle aroma biosynthesis and chemical ecology.     

Detection of Tuber melanosporum DNA in soil

Our objectives were (i) to develop a molecular method to detect mycelia of Tuber melanosporum (black truffle) in soil and (ii) to test for mycelial distribution around two truffle-bearing Quercus ilex trees in a truffle orchard. Isolation of total DNA from soil was performed, followed by PCR amplification with T. melanosporum-specific primers and restriction analysis. To address the detection sensitivity level, soil samples were inoculated with known amounts of gleba of T. melanosporum. The detection limit was >/=11.4 mug of hyphae g(-1) of soil. Mycelium was detected primarily within the area defined by the truffle burn and within the top 35 cm of the soil in all directions from the trees.