夏块菌与青刚栎形成外生菌根形态变化的研究

夏块菌（Tuber aestivum）是一种具有较高经济价值的菌根食用菌。对夏块菌与青刚栎（Cycloba-lanopsis glauca）在形成菌根过程中不同阶段的菌根形态变化进行了研究,结果显示：用夏块菌孢子液接种青刚栎苗后,第14天起开始形成淡乳色的外生菌根,外延菌丝刚毛状;第一至第二个月可形成黄褐色、褐色外生菌根,外延菌丝刚毛或羊毛状。外生菌根为单根,长1～4mm,直径150～250μm。菌套厚12～20μm,平坦或自菌根延伸出刚毛状菌丝,外延菌丝束黄绿色;哈替氏网菌丝直径1～1.5μm。菌根老化后变暗褐或萎缩。外延菌丝束呈黄绿色是夏块菌菌根区别于其它块菌菌根最重要的形态特征。     

Species-specific primers for the identification of the ectomycorrhizal fungus Tuber macrosporum Vittad

Limitations in the use of morphological traits to identify ectomycorrhizae have led to the development of species‐specific molecular markers. Herein, we report a PCR‐based technique for the reliable molecular identification of the ectomycorrhizal fungus Tuber macrosporum Vittad. Species‐specific primers were designed from an alignment of internal transcribed spacer rDNA sequences from Tuber spp. and from the most common ectomycorrhizal contaminants found in the root systems of truffle‐infected plants. The primers were tested for selective amplification using both different truffles and different ectomycorrhizae and were found to identify T. macrosporum successfully. The application of the primers in certifying the quality of truffle‐inoculated seedlings is discussed.     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

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.     

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.

     

Detection of summer truffle (Tuber aestivum Vittad.) in ectomycorrhizae and in soil using specific primers

Tuber aestivum is becoming an important commodity of great economical value in some European countries. At the same time, it is a highly protected organism in other countries, where it needs careful treatment. A reliable method of detection in roots and soil is thus needed for assessment of geographic distribution, ecological studies and inoculation efficiency testing in man-made experiments. A PCR-based method of detection of T. aestivum using specific primers was therefore developed. A pair of PCR primers Tu1sekvF/Tu2sekvR selective for T. aestivum and some genotypes of Tuber mesentericum was designed on the basis of the known internal transcribed spacer T. aestivum sequences. TaiI restriction cleavage was then used to distinguish the two species. The selectivity of the designed primer pair was evaluated using DNA extracted from specimens of a further 13 Tuber spp. Subsequently, the selectivity and robustness to false-positive results with nontarget DNA of the designed primers was compared with two other primer pairs (UncI/UncII and BTAE-F/BTAEMB-R). The occurrence of T. aestivum in soil and ectomycorrhizae collected in its native habitat has been successfully detected using the designed primers and nested PCR. The method is reliable and thus suitable for detection of T. aestivum in the field.     

New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation

Chinese truffles serve as a good complement to the market for Tuber melanosporum (Périgord black truffle). However, Chinese truffles could be introduced accidentally or fraudulently into the plantations of Mediterranean truffles, and they could have a negative effect on truffle production and natural ecosystems. The study of Tuber species from China which are commercialized in Europe began 14 years ago. Tuber pseudoexcavatum was proposed as a new species, and this has been validated by some authors based on molecular and phylogenetic studies. We synthesize their ectomycorrhizae using samples from the type collection, and we compare T. pseudoexcavatum and Tuber indicum ectomycorrhizae. The ectomycorrhizae of these species have a morphology which is related to the ectomycorrhizae of T. melanosporum. We provide useful information for the rapid screening of the above-mentioned Chinese truffles ectomycorrhizae, for the quality control of commercial plants mycorrhized with Tuber. Moreover, we analyze the soil tolerance and the host plant affinity of T. pseudoexcavatum and T. indicum, in order to assess the capacity of both Chinese truffles to penetrate T. melanosporum plantations and habitats.     

国产夏块菌(块菌科、子囊菌门)的比较研究

对在四川省会东县发现的夏块菌(TuberaestivumVittad.)标本进行了鉴别研究,并与产自欧洲的标本进行了宏微观比较,同时还讨论了该种与相近种类的主要区别。     

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.     

Synthesis and establishment of <Emphasis Type="Italic">Tuber melanosporum</Emphasis> Vitt. ectomycorrhizae on two <Emphasis Type="Italic">Nothofagus</Emphasis> species in Chile

Axenically germinated seedlings of two species of Southern beech (Nothofagus obliqua, N. glauca) from Chile were inoculated with spores of the Périgord black truffle (Tuber melanosporum). Ectomycorrhizal development was monitored for 6 months in the greenhouse and compared to the performance of the natural host species Quercus ilex and Quercus robur. Seedling survival and mycorrhization showed major differences in both Nothofagus species: T. melanosporum readily formed ectomycorrhizae with seedlings of N. obliqua, although at a lower rate than with Q. ilex but at a proportion very similar to Q. robur; survival and colonization rates were high, and seedling growth was not visibly affected by the high soil pH required by T. melanosporum. In contrast, more than 50% of N. glauca seedlings died after inoculation, and mycorrhiza formation was very sparse. In both species, no colonization by adventive ectomycorrhizal fungi could be observed, whereas both species of Quercus showed minor colonization by another fungus, probably Inocybe or Hebeloma. Our results show that it is possible to infect N. obliqua with the Périgord black truffle under greenhouse conditions, which opens up the possibility of cultivating this truffle as a secondary crop during reforestation with N. obliqua in Chile.     

Expression and purification of a Tuber borchii fruitbody-specific protein, TBF-1, from Escherichia coli: generation of polyclonal antibodies

TBF-1 is a fruitbody-specific protein present in the white truffle species Tuber borchii Vittad. A similar protein has been found only in the closely related species Tuber dryophilum (TDF-1), but not in other truffles. The protein from T. borchii was overexpressed as fusion protein in E. coli and was purified to homogeneity by affinity chromatography. Recombinant protein was used for generating polyclonal antibodies. The antiserum strongly reacted with TBF-1, weakly recognized TDF-1, and did not detect correlate band in the other white truffle species. The high level of expression of this protein in the fruitbody and the specificity of the antibody anti-TBF-1 make it possible to set up a diagnostic tool for detecting these species in natural samples and foodstuffs.     

Ectomycorrhizal communities in a productive Tuber aestivum Vittad. orchard: composition, host influence and species replacement

Truffles (Tuber spp.) and other ectomycorrhizal species form species-rich assemblages in the wild as well as in cultivated ecosystems. We aimed to investigate the ectomycorrhizal communities of hazels and hornbeams that are growing in a 24-year-old Tuber aestivum orchard. We demonstrated that the ectomycorrhizal communities included numerous species and were phylogenetically diverse. Twenty-nine ectomycorrhizal taxa were identified. Tuber aestivum ectomycorrhizae were abundant (9.3%), only those of Tricholoma scalpturatum were more so (21.4%), and were detected in both plant symbionts with a variation in distribution and abundance between the two different hosts. The Thelephoraceae family was the most diverse, being represented by 12 taxa. The overall observed diversity represented 85% of the potential one as determined by a jackknife estimation of richness and was significantly higher in hazel than in hornbeam. The ectomycorrhizal communities of hornbeam trees were closely related phylogenetically, whereas no clear distribution pattern was observed for the communities in hazel. Uniform site characteristics indicated that ectomycorrhizal relationships were host mediated, but not host specific. Despite the fact that different plant species hosted diverse ectomycorrhizal communities and that the abundance of T. aestivum differed among sites, no difference was detected in the production of fruiting bodies.     

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.     

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.     

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.     

Selection of a set of specific primers for the identification of Tuber rufum: a truffle species with high genetic variability

Tuber rufum is a truffle widely distributed throughout Europe, which forms mycorrhizal associations with numerous species of broadleaf and coniferous trees. The possibility of T. rufum contamination in commercial truffle-infected plants makes its detection important. To facilitate the identification of T. rufum from mycorrhiza and fruitbodies, species-specific primers were designed and tested. To overcome the high intraspecific genetic variability within the internal transcribed spacer (ITS) regions of T. rufum, as demonstrated by phylogenetic analysis, two forward primers, Ru1f and Ru2f, located on the ITS1 region were designed to be used in concert with the reverse primer ITS4. Only T. rufum was amplified with this primer combination, while DNA of Tuber magnatum, Tuber brumale, Tuber maculatum, Tuber borchii, Tuber excavatum and Tuber melanosporum was not. These primers give a specific amplicon ranging between 566 and 572 bp and are able to discriminate between T. rufum, T. borchii and T. magnatum in multiplex PCR. In addition, T. rufum-specific amplicons were obtained from both spore suspensions and mycorrhiza by direct PCR. Tuber rufum mycorrhiza obtained in the greenhouse using mycelial inoculation techniques had morphological features similar to those of other species of Tuber, stressing the importance of molecular tools for their identification.     

The cultivation of oak seedlings inoculated with Tuber aestivum Vittad. in the boreal region of Finland

Despite recent findings, truffles are rarely found in Finland. In 2006, we began to explore the cultivation potential of Tuber aestivum/uncinatum in Finland. In 2006–2008, roughly 1,200 Quercus robur seedlings and 200 Q. pubescens seedlings were planted in 20 orchards. We aimed to challenge the Southern European (France) tree provenances of oak seedlings in a boreal climate. Additional winter coverings made up of fabric or plastic and twigs prevented the seedlings’ mortality even when the air temperature was below ?30 °C during the second winter. The results showed that the top soil temperature at 15 cm depth has to be above ?5 °C to guarantee the survival of seedlings. Q. pubescens was more sensitive to low soil temperatures than Q. robur. Morphological and PCR analysis of root samples collected over 2007–2010 confirmed the presence of T. aestivum in all orchards despite unfavorable temperatures during the winter time. The first T. aestivum sporocarps were found under Q. robur in October 2012 in the orchards established in 2006 on old agricultural land, showing truffle cultivation to be successful in the boreal climate.     

Competition between Tuber melanosporum and other ectomycorrhizal fungi under two irrigation regimes

A glasshouse experiment was conducted to simulate the competition between artificially introduced Tuber melanosporum (Vitt.) and other symbionts, occurring on outplanted truffle-producing trees. Hazel (Corylus avellana L.) seedlings, previously inoculated with Tuber melanosporum, were rapidly infected with the competing truffle Tuber brumale (Vitt.), added to the soil as spores. Coexistence of both species on the root system was observed. Tuber melanosporum survived and continued to spread. Tuber brumale, which is naturally present in many truffle-orchard soils, protects the roots from other ectomycorrhizal symbionts. Although high water content is unfavourable for the development of T. melanosporum in the absence of other Tube species, Tuber brumale stimulates the development of T. melanosporum under these conditions.     

Cloning and characterization of PKC-homologous genes in the truffle species Tuber borchii and Tuber magnatum

The protein kinases C (PKCs) define a growing family of ubiquitous signal transducting serine/threonine kinases that control ion conductance channels, release of hormones and cell growth and proliferation. Degenerated oligonucleotides were used as primers for polymerase chain reactions to amplify PKC-related sequences from the white truffle species Tuber magnatum and Tuber borchii. The deduced amino acid sequences of cloned sequences reveal domains homologous to the regulatory and kinase domains of PKC-related proteins, but lack typical Ca(2+)-binding domain and therefore should be classified as nPKCs. Both contain a large extended N-terminus which is found exclusively in fungi PKCs. Phylogenetic analysis of the kinase domain demonstrates high homology with known filamentous fungi isoenzymes.     

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.