Tuber melanosporum and other truffle species - Microscopic identification; Molecular identification of Tuber spp. (PCR and sequencing)

Information 25-11-2017.

Among the fungi, at least 400 species of them live in symbiotic association with tree roots and are known as mycorrhizal or ectomycorrhiza fungi.

What we know as "truffles" are the sexual fruiting bodies (ascocarps) of edible ectomycorrhizal ascomycetes of the genus Tuber.

These mushrooms have a complex life cycle. Like all filamentous fungi, its mycelium, that is, the set of filaments composed of cells (called hyphae), establishes a symbiotic interaction with the roots of trees. These mycelia, in the case of these fungi are heterothalics, that is to say they exist some with masculine sexual sign and others with feminine sexual sign and when they conjugate produce a body of reproduction (body of fructification or ascocarps). In the interior of these bodies of fructification (ascocarps) the forms of sexual reproduction called ascas are generated. When the ascas mature, inside the spores (ascospores) appear, which are the final elements of dissemination from their sexual reproduction. Fruiting bodies (ascocarps) generate volatile substances that attract insects and mammals, thanks to which they are located by dogs, and seduce people by their aroma and flavor.

The black truffle (Tuber melanosporum) is one of the most commercialized species due to its flavor and aroma. Natural production is almost exclusive to France, Spain and Italy. Given that the production of truffles declined spectacularly in the 20th century and that the ecological requirements of this species are well known, cultivation practices have been developed in these and other countries.

Many efforts have been made to cultivate this ectomycorrhizal fungus. The disintegrated fruiting bodies allow to obtain a paste with the spores they contain and are used to inoculate seedbeds. From these, the inoculation of the fields where there are host trees of this ectomycorrhizal fungus is carried out. Truffle cultivation requires long-term investments, since production generally begins 6 to 10 years after inoculation in the fields. The fruiting body (ascocarp or truffle) is usually collected in an area without vegetation, near or around the host trees, where the truffle competes with other ectomycorrhizal fungi, so the potential for truffle production is very unpredictable.

Tuber melanosporum (black truffle, Perigord) has been cultivated since the 19th century, but the white truffle (Alba truffle, Tuber magnatum) has resisted domestication and has very high prices. In addition to these two species, there are other morphologically similar but less aromatic species that are sold as Tuber melanosporum or Tuber magnatum.

Because Tuber melanosporum has a distinctive flavor, its commercial value is much higher than that of other black truffles. There are truffles of Asian species such as Tuber indicum (syn. Tuber sinense), Tuber himalayense, Tuber pseudohimalayense (= Tuber pseudoexcavatum) and another European species (Tuber brumale) with ascocarps (truffle) with morphological characteristics very similar to those of Tuber melanosporum, although with a lower aroma. The European market for Asian (Chinese) black truffles is increasing. The presence of ascocarps of Asian species in the same stocks as those of the true black truffle has the risk that plants artificially mycorrhized by Asian truffles may have unpredictable ecological consequences.

The differentiation of species can be done through the characteristics of the fruiting bodies (ascocarps) observing its peridium (external part), its glebe (internal part) and fundamentally by the microscopic characteristics of its spores (ascospores). These characteristics are recognized by experienced specialists and sometimes the identification is not reliable.

The microscopic characteristics of the ascospores, especially the ornamentation on the surface of the mature ascospores, are important to differentiate the species. According to these characteristics the Tuber species can be divided into three groups: with reticulated spores, spiny spores and with reticulate-spiny spores.

The correct identification of the fruiting bodies of Tuber melanosporum to differentiate them from those of other closely related species of lower economic value, such as those of Tuber indicum (Asian species) and Tuber brumale (European species), can be difficult especially when the fruiting bodies have not fully matured.

Due to the need to avoid frauds in the markets of fresh truffles, in the foods that contain them (juices, creams, butters, preserves, ...), in the sampling of soils where they are produced and in seedbeds inoculated for plantations in areas of host trees, affordable species identification methods are required.

The problems of misidentification of truffles have occurred in several countries such as Italy and the US, with the introduction of Tuber indicum (Asian variety) and in New Zealand with Tuber brumale.

All this entails the need to have a fast and simple method that allows to discriminate Tuber melanosporum from other species that can be confused macroscopically observing its fructification body (ascorcarp).

Molecular methods are useful for the study, diagnosis and identification of fungi. Tuber species can be differentiated using ITS (Internal Transcribed Spacer) sequences. Given the difficulties and experience required for the macroscopic identification (appearance of the ascorcarp, the truffle), and microscopic (the spores – ascospores-) to limit the frauds, molecular methods of amplification have been developed with specific primers.

Recommended tests for identification:

  • Microscopic observation of ascospores.
  • Molecular test with primers of genus and species.

Tests carried out in IVAMI:

  • Microscopic observation of ascospores.
  • Molecular test with primers of the genus Tuber and species Tuber melanosporum.

Recommended sample:

  • Sample of truffle, of food preparation that contains it, of earth where it may exist, of seedbed, ...

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 2 days.
  • Frozen: more than 2 days.

Delivery of results:

  • Microscopic and molecular test (PCR): 2 to 4 days..

Cost of the test:

  • Microscopic examination: 60.- €.
  • Molecular test for detection of Tuber melanosporum (PCR): Consult to
  • Molecular test for detection and identification of Tuber spp. (PCR and sequencing): Consult to