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.     

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.     

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.     

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.     

Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina)

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.     

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.     

中国块菌要览及其保护策略

截至目前报道的中国块菌有28种,其中黑块菌4种,白块菌24种.具有重要经济价值和生态价值的黑块菌类有印度块菌复合种和中国夏块菌,其中印度块菌复合种与欧洲法国黑孢块菌属于姊妹类群,中国夏块菌与欧洲夏块菌也是姊妹类群.虽然欧洲意大利白块菌在中国尚未找到、波氏块菌在中国的存在尚待最终确认,但是,最近的更多白块菌新种的发现,说...     

中国块菌属研究概况

文献调查研究结果表明,自我国报道的块菌属(Tuber)有35个名称,其中21个具有标本引证的支持、有3个缺乏明确的标本引证、3个与国际上已有的描述存在明显差异1、个鉴定时使用的是暂定名而有待进一步查证、还有2个名称已被证明为异名、5个名称为不合格发表而不应再继续使用。作者对我国的块菌属研究概况进行了概述,并对有关种类的研究现状及其分布进行了介绍,同时也整理和修订了这些种的汉语学名。     

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.     

Genetic Diversity and Mating Type Distribution of Tuber melanosporum and Their Significance to Truffle Cultivation in Artificially Planted Truffieres in Australia

Tuber melanosporum is a truffle native to Europe and is cultivated in countries such as Australia for the gastronomic market, where production yields are often lower than expected. We assessed the genetic diversity of T. melanosporum with six microsatellite loci to assess the effect of genetic drift on truffle yield in Australia. Genetic diversity as assessed on 210 ascocarps revealed a higher allelic diversity compared to previous studies from Europe, suggesting a possible genetic expansion and/or multiple and diverse source populations for inoculum. The results also suggest that the single sequence repeat diversity of locus ME2 is adaptive and that, for example, the probability of replication errors is increased for this locus. Loss of genetic diversity in Australian populations is therefore not a likely factor in limiting ascocarp production. A survey of nursery seedlings and trees inoculated with T. melanosporum revealed that <70% of seedlings and host trees were colonized with T. melanosporum and that some trees had been contaminated by Tuber brumale, presumably during the inoculation process. Mating type (MAT1-1-1 and MAT1-2-1) analyses on seedling and four- to ten-year-old host trees found that 100% of seedlings but only approximately half of host trees had both mating types present. Furthermore, MAT1-1-1 was detected significantly more commonly than MAT1-2-1 in established trees, suggesting a competitive advantage for MAT1-1-1 strains. This study clearly shows that there are more factors involved in ascocarp production than just the presence of both mating types on host trees.     

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.     

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.     

Identification of internal transcribed spacer sequence motifs in truffles: a first step toward their DNA bar coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (< or = 50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.     

Tuber melanosporum: mating type distribution in a natural plantation and dynamics of strains of different mating types on the roots of nursery-inoculated host plants

? In light of the recent finding that Tuber melanosporum, the ectomycorrhizal ascomycete that produces the most highly prized black truffles, is a heterothallic species, we monitored the spatial distribution of strains with opposite mating types (MAT) in a natural truffle ground and followed strain dynamics in artificially inoculated host plants grown under controlled conditions. ? In a natural truffle ground, ectomycorrhizas (ECMs), soil samples and fruit bodies were sampled and genotyped to determine mating types. Simple sequence repeat (SSR) markers were also used to fingerprint ECMs and fruit bodies. The ECMs from nursery-inoculated host plants were analysed for mating type at 6 months and 19 months post-inoculation. ? In open-field conditions, all ECMs from the same sampling site showed an identical mating type and an identical haploid genotype, based on SSR analysis. Interestingly, the gleba of fruit bodies always demonstrated the same genotype as the surrounding ECMs. Although root tips from nursery-grown plants initially developed ECMs of both mating types, a dominance of ECMs of the same MAT were found after several months. ? The present study deepens our understanding of the vegetative and sexual propagation modes of T. melanosporum. These results are highly relevant for truffle cultivation.     

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.     

Discrimination of truffle fruiting body versus mycelial aromas by stir bar sorptive extraction

Stir bar sorptive extraction (SBSE) was applied in head space mode (HS), coupled with GC/MS, to compare the aroma profile of three truffle species. A total of 119 volatile organic compounds (VOCs) were identified from the fruiting bodies, of which 70 were not yet described in truffles and 60 in fungi. VOCs profile showed a high intra- and inter-specific variability, with alcohols and sulfur compounds dominating the HS of Tuber borchii and, alcohols, aldehydes and aromatic compounds the HS of T. melanosporum and T. indicum. Despite these variations, eight VOCs markers could be identified allowing the discrimination of the three species. Additionally, T. borchii and T. melanosporum both distinguished themselves from T. indicum due to higher aroma content and larger variety of sulfur containing compounds. Mycelial VOCs production was also investigated under two cultural conditions and led to the identification of eight VOCs. On one side, seven of them were also detected in the fruiting body, confirming their mycelial origin. On the other side, the total absence of some class of compounds (i.e. sulfur) in the mycelium raises questions about their origins in the fruiting bodies and confirms deep metabolic changes between the reproductive (fruiting body) and vegetative (mycelium) stages.     

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.     

成熟度对印度块菌香气成分的影响

研究成熟度对印度块菌Tuber indicum香气成分的影响,并测定成熟印度块菌的关键香气成分。将3种不同成熟度的印度块菌,以固相微萃取(SPME)技术为香气富集方法,利用气相色谱-质谱联用(GC-MS)分析其香气成分。结果表明未成熟印度块菌中仅检测出4种香气成分,中度成熟阶段检测出8种,成熟阶段检测出13种,而且成熟阶段检测出的香气成分大多都是前人报道过对块菌香气有贡献的成分;通过计算成熟块菌各香气组分的香气活度值(OAV),可知二甲基硫醚、2,3-丁二酮、3-甲基正丁醛、2-甲基正丁醛、己醛、1-辛烯-3-醇这6种物质是印度块菌的关键香气成分(OAV>1)。     

The combined effects of Pseudomonas fluorescens and Tuber melanosporum on the quality of Pinus halepensis seedlings

The ecological, economic and social values of the ectomycorrhizal fungi of the black truffle found in the rural Mediterranean are well known. The inoculation of Pinus halepensis seedlings with mycorrhizal fungi and rhizobacteria can improve the morphology and physiology of the seedlings and benefit the regeneration of arid regions and the reintroduction of inocula of mycorrhizal fungi into these areas. Some rhizobacteria can improve the establishment and functioning of ectomycorrhizal symbiosis. In this study, seedlings of P. halepensis were inoculated with the mycorrhizal fungus Tuber melanosporum and the rhizobacteria Pseudomonas fluorescens CECT 844 under non-limiting greenhouse conditions. Five months after inoculation, we analysed the growth, water parameters (osmotic potential at saturation, osmotic potential at turgor loss and modulus of elasticity), concentrations of mycorrhizal colonies, nutrient concentration and nutrient contents (N, P, K, Ca, Mg and Fe) in roots and aerial parts of the seedlings. Subsequently, tests were performed to estimate the root growth potentials. None of the treatments changed the water parameters or growth potentials of the roots. The inoculations improved the growth and nutrient uptake of the seedlings, although the combination of P. fluorescens CECT 844 and T. melanosporum did not generally lead to a significant improvement over the positive effects of a simple inoculation of T. melanosporum; however, the addition of P. fluorescens CECT 844 did double the rate of the mycorrhization of T. melanosporum. These results may be promising for enhancing the cultivation of truffles.