Specific PCR-primers as a reliable tool for the detection of white truffles in mycorrhizal roots

During their symbiotic phase, white truffles are barely distinguishable morphologically, and molecular probes are needed for their identification. Here we report the design of species-specific primers for two white truffles ( Tuber magnatum and T. borchii ) on the basis of their ITS sequence. Their efficiency has been successfully tested on fruit bodies of the same species, many related and unrelated fungal species, as well as mycorrhizal roots in direct, nested and multiplex PCR experiments. They have allowed us to identify T. magnatum mycorrhizas for the first time.     

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.     

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.     

Calcareous amendments to soils to eradicate Tuber brumale from T. melanosporum cultivations: a multivariate statistical approach

Calcareous amendments are being used in Tuber melanosporum truffle plantations in attempts to eradicate Tuber brumale. However, there are no studies available which provide soil analysis and statistical data on this topic. We studied 77 soil samples to compare the values for carbonates, pH and total organic carbon in T. brumale truffières with the values for T. melanosporum truffières on contaminated farms and in natural areas. Statistical analyses indicate that the concentrations of active carbonate and total carbonate in the soil are significantly higher in T. brumale truffières than in T. melanosporum truffières, but that there are no significant differences in pH and total organic carbon. We conclude that liming would not suppress T. brumale ectomycorrhizas in contaminated T. melanosporum farms, and calcareous amendments do not therefore seem be a means of eradicating T. brumale in these farms.     

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.     

红皮云杉外生菌根菌对苗木生长的影响

2000年8～10月自大兴安岭新林林业局和兴隆林业局采集各种林型下的伞菌担子果,进行分离培养、纯化,获得36个菌株。经对红皮云杉室内接种试验获得6个可与红皮云杉共生形成外生菌根的菌株。室内接种试验结果表明,在红皮云杉播种苗上接种菌根菌的时期应在出苗后30d左右。菌根形成的适宜温度为20℃左右。筛选得到的6个菌株对红皮云杉幼苗的生长有明显的促进作用。接种林地菇(Agaricus silvaticus)、031和L15菌株的苗木叶绿素a含量明显高于其他菌株和对照;接种009、004、林地菇和L15菌株的苗木叶绿素b含量也明显高于其他菌株和对照;接种林地菇、L15、031、009和025菌株的苗木与对照苗木在重量上差异显著;其中接种林地菇和L15菌株的苗木比对照苗木分别增加了19.23%和23.08%;接种6个菌株的苗木与对照苗木高度比较差异非常显著,接种林地菇和L15菌株的苗木与对照相比分别增加17.83%和16.37%。室外接种结果表明,接种林地菇的苗木高生长最好,接种70d高于对照9.24%;接种菌株L15的苗木地径生长最好,接种70d高于对照9.92%;接种菌株009的苗木侧根数最多,接种70d高于对照51.91%;接种菌株009的苗木主根最长,接种70d高于对照3.36%;而接种菌株031的苗木全株感染菌根率最高,接种70d高于对照13.90%;接种菌株031的苗木光合作用最强,高于对照20.02%。从几项主要的指标综合来看,菌株L15、菌株009和菌株031是有潜在研究和应用价值的优良菌株。     

A rapid mini-prep method for isolation of ectomycorrhizal DNA from Tuber species

A rapid procedure has been developed to isolate DNA from the ectomycorrhizae of Tuber spp. for use in PCR experiments. The method described is fast and sensitive and can overcome the amplification problems that can arise in the presence of inhibitors. For this reason it can be used to type ectomycorrhizae even starting from a single root tip and make mycorrhizae identification much more rapid.     

Molecular identification of Tuber magnatum ectomycorrhizae in the field

Tuber ectomycorrhizae in a Tuber magnatum "truffière", located in Central Italy, were studied using molecular methods. Specifically, RFLP-ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the "truffière" under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively).     

中国块菌属一新种

本文描述了块菌属(Tuber)的一个新种,巨孢块菌(Tuber gigantosporum)。这一新种1989年采于四川省会东县。它的子囊孢子特殊大,一般105—115×75μm,最大可达120×80μm,小者亦可达80×55μm。而块菌属已知种的子囊孢子的大小一般在21—52×15—38μm范围内,最大也不超过90×60μm。仅此一点已使其明显区别于该属任何已知种。此外,该种孢子壁异常厚,可达14μm,有三层,也是别于其它块菌已知种的重要特征。该块菌生于地下,与云南松有共生关系。模式标本保存在中国科学院沈阳应用生态所植物标本室(IFS)。     

Tuber aestivum and Tuber uncinatum: two morphotypes or two species?

Tuber spp. are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, beta-tubulin and EF 1-alpha genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprint, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.     

Genetic structure of <Emphasis Type="Italic">Tuber mesentericum</Emphasis> Vitt. based on polymorphisms at the ribosomal DNA ITS

Tuber mesentericum fruit bodies are in increasing demand on the food market and are an important economic resource for southern Italy, their major production area. Because molecular studies on this truffle species are very scarce, we analyzed ITS1 and ITS2 nucleotide variability of 126 ascocarps of T. mesentericum collected in different European areas, mainly southern Italy. The results of haplotype distribution, analysis of molecular variance, and spatial analysis of molecular variance analyses show strong genetic structuring of the samples collected in the different geographic areas, confirmed by parsimony and distance analyses. In particular, the Italian samples seem to be a well-distinguished group.     

A global meta-analysis of Tuber ITS rDNA sequences: species diversity, host associations and long-distance dispersal

Truffles (Tuber) are ectomycorrhizal fungi characterized by hypogeous fruitbodies. Their biodiversity, host associations and geographical distributions are not well documented. ITS rDNA sequences of Tuber are commonly recovered from molecular surveys of fungal communities, but most remain insufficiently identified making it difficult to determine whether these sequences represent conspecific or novel taxa. In this meta-analysis, over 2000 insufficiently identified Tuber sequences from 76 independent studies were analysed within a phylogenetic framework. Species ranges, host associates, geographical distributions and intra- and interspecific ITS variability were assessed. Over 99% of the insufficiently identified Tuber sequences grouped within clades composed of species with little culinary value (Maculatum, Puberulum and Rufum). Sixty-four novel phylotypes were distinguished including 36 known only from ectomycorrhizae or soil. Most species of Tuber showed 1-3% intraspecific ITS variability and >4% interspecific ITS sequence variation. We found 123 distinct phylotypes based on 96% ITS sequence similarity and estimated that Tuber contains a minimum of 180 species. Based on this meta-analysis, species in Excavatum, Maculatum and Rufum clades exhibit preference for angiosperm hosts, whereas those in the Gibbosum clade are preferential towards gymnosperms. Sixteen Tuber species (>13% of the known diversity) have putatively been introduced to continents or islands outside their native range.