Phylogenetic relationships between Tuber pseudoexcavatum, a Chinese truffle, and other Tuber species based on parsimony and distance analysis of four different gene sequences

Phylogenetic relationships between Tuber pseudoexcavatum and other Tuber species were investigated by studying the sequences of four genes: 5.8S-ITS2, beta-tubulin, protein kinase C and elongation factor 1alpha. The four phylogenetic trees allowed to differentiate the black truffle clade, composed of two subclades, one comprising the Asian black truffles (T. indicum, T. sinense, T. himalayense) and the Perigord black truffle (T. melanosporum), the second comprising T. pseudoexcavatum and T. brumale. These two subclades diverged relatively early. We propose a common ancestor, located between Europe and China, to all the black truffles. The T. brumale/pseudoexcavatum subclade would have started to diverge and migrate first, T. brumale towards Europe through a northern route and T. pseudoexcavatum towards China. Later the T. melanosporum subclade would have started to migrate through the same route, T. melanosporum towards Europe and T. indicum towards China, leading to vicariant species.     

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.     

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.     

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.     

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.     

Improved resolution of major clades within Tuber and taxonomy of species within the Tuber gibbosum complex

Tuber gibbosum Harkn., described from northern California, originally was thought to be a single, variable species that fruited from autumn through winter to spring. It has become popular as a culinary truffle in northwestern USA, where it is commercially harvested. Morphological studies suggested it might be a complex that includes at least two species. We conducted morphological and phylogenetic studies of the complex to determine how many species it might contain and how they differed morphologically, geographically and seasonally. We also provide the first LSU phylogeny for the genus Tuber. Phylogenetic analyses resolve nine major clades in the genus with high bootstrap support and distinguish the Gibbosum clade from the Aestivum, Excavatum, Macrosporum, Magnatum, Melanosporum, Puberulum, Rufum and Spinoreticulatum clades. Further analyses of ITS and LSU regions revealed four distinct species in the Gibbosum complex. Although morphologically similar the four species differ in spore size and shape and in peridial anatomy. These species share the synapomorphy of having suprapellis hyphae with distinctive, irregular wall swellings at maturity; we have not seen this hyphal type in any other Tuber spp. worldwide. The three new species are named and described as T. bellisporum Bonito & Trappe, T. castellanoi Bonito & Trappe and T. oregonense Trappe, Bonito & Rawlinson.     

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.     

Challenges in the delimitation of morphologically similar species: a case study of <Emphasis Type="Italic">Tuber brumale</Emphasis> agg. (Ascomycota,Pezizales)

Tuber brumale (winter truffle) is one of the most controversial true truffles, not only in regard to its ecological and economical role but also its taxonomy. Multilocus phylogenetic analyses have revealed that specimens identified earlier as T. brumale belong to two species. These species were deemed cryptic right away, because preliminary morphological measurements did not show any phenotypical differences. In this study, we measured the morphology of 119 T. brumale agg. specimens, identified by DNA-based phylogenetic tools. We found several continuous morphological characters which show strong statistical differences between the two species, albeit not without overlap. Using a combination of these characters, we show that efficient separation of the two species is possible. We describe T. cryptobrumale sp. nov. and present the environmental demands and the potential area reconstruction of both species. We argue that non-representative sampling is a major culprit in most failures to detect both the existence of morphologically similar species and their morphological differences. Our findings illustrate the benefits of integrative taxonomy: the use of a combination of molecular, morphological and ecological tools.     

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.     

Multilocus phylogenetic and coalescent analyses identify two cryptic species in the Italian bianchetto truffle, <Emphasis Type="Italic">Tuber borchii</Emphasis> Vittad.

Tuber borchii (Ascomycota, Pezizales) is a highly valued truffle sold in local markets in Italy. Despite its economic importance, knowledge on its distribution and genetic structure is scarce. The objective of this work was to investigate the factors shaping the genetic structure of T. borchii using 61 representative specimens with a broad distribution throughout Italy. In spite of the lack of morphological differences, phylogenetic and coalescent-based analyses using four loci identified two genetically isolated groups sympatrically distributed. The low levels of divergence between the two clades may be the result of recent range expansions from geographically distinct refugia, potentially mediated by reforestation using coniferous species that are common ectomycorrhizal symbionts for both groups.     

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.     

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)。     

中国大型高等真菌生物多样性的关键类群

由于生物多样性的关键类群在科学研究、社会经济及生态系统中所处的重要地位,有必要对其进行筛选和确定。在多年的野外考察和有关文献资料分析的基础上,参照植物和动物关键类群的划分原理及方法,结合高等真菌的特点将大型高等真菌划分为3种类型：濒危类群、重大科学价值类群和重要经济类群。其中,濒危类群指在近年的考察和报告中发现的数量急剧减少或由于过度的采集资源明显受到威胁的种类,其中有虫草属(Cordyceps)的中华虫草(C．siaensis)、块菌属(Tuber)的中华块菌(T．sinense)、假下陷块菌(T．pseu—doexcawatum)、喜马拉雅块菌(T.himalayense)、革菌属(Thelephora)的干巴菌(T．ganbajun)、口蘑属的松茸(Tricholoma matsuatke)及其近缘种和蒙古口蘑(T．mongolicum);重大科学价值类群指在真菌系统演化或与动、植物和其它真菌协同进化中的一些重要类群,或在应用研究领域具重要价值的类群,如鸡Zong菌属(Termitomyces)、鹅膏属(Amanita)、腹菌与伞菌的过渡类型如地红菇属(Macowanites)、粉褶包属(Richoniella)、轴腹菌属(Hydnangium)、腹牛肝菌属(Gastroboletus)等、牛肝菌类中的一些特殊单种属和寡种属如圆花孢牛肝菌属(Heimiel-la)、圆孔牛肝菌属(Gyroporus)等;重要经济类群指广为利用的食用菌、药用菌和外生菌根菌,其中主要有虫草属的中华虫草、块菌属的中华块菌、鸡Zong菌属盾尖鸡Zong菌(T．dypeatus)、鸡Zong菌(T.ewrhizus)、球形鸡Zong菌(T．gtoulus)、口蘑属的松茸群及蒙古口蘑、牛肝菌属(Bolaus)的美味牛肝菌(B．edulis)、小美牛肝菌(R．speciosus)、茶褐牛肝菌(B．brunneissimus)等,乳菇的红汁乳菇(L,hatsudake)、松乳菇(L．deliciosus)、多汁乳菇(L．volemus）、红菇属的变绿红菇(R．vir-escens)、蓝黄红菇(R．cyanoxantha)、鹅膏属的红黄鹅膏(A．hemibapha)、隐花青鹅膏(A．mang-iniana)、灵芝属(Ganoderma)的灵芝(G,lucidum)及紫芝(G,sinense)、竹荪属(Dictyophora)、豆马勃属(Pisdithus)的一些种。这些种由于具有重要的经济价值而受到过度的采集,故往往与濒危的种类一致。关键类群的筛选与提出为大型高等真菌的研究、应用和保护提供参考。     

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.     

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...     

Determination of 5alpha-androst-16-en-3alpha-ol in truffle fermentation broth by solid-phase extraction coupled with gas chromatography-flame ionization detector/electron impact mass spectrometry

A novel method using solid-phase extraction coupled with gas chromatography and flame ionization detector (FID)/electron impact mass spectrometry (EIMS) was developed for the determination of 5alpha-androst-16-en-3alpha-ol (androstenol), a steroidal compound belonging to the group of musk odorous 16-androstenes, in truffle fermentation broth. Comparison studies between FID and EIMS indicated two detectors gave similar quantitative results. The highest androstenol concentration of 123.5 ng/mL was detected in Tuber indicum fermentation broth, while no androstenol was found in Tuber aestivum fermentation broth. For the first time, this work confirmed the existence of androstenol in the truffle fermentation broth, which suggested truffle fermentation is a promising alternative for androstenol production on a large scale.