Specific primers for rapid typing of Tuber borchii mycorrhizal roots

Truffles are Ascomycetous fungi that need to form a symbiotic association (mycorrhiza) with the roots of a host plant to be able to produce their fruitbodies. Many of the morphological characters used to identify a truffle fruitbody, however, are lost during this phase. DNA analyses have therefore been devised for the typing of truffle species. The paper describes an application of this technique to identify the mycorrhizas of Tuber borchii (a commercial species) by means of the polymerase chain reaction (PCR), this being the most effective way of typing from mycorrhizas or mycelium. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was employed as the target, since it displays a high degree of variability and is highly repeated. The ITS region of T. borchii was cloned and sequenced. A primer pair (TBA/TBB) designed by comparing three Tuber species sequences and employed for PCR amplification of DNA isolated from T borchii fruitbodies, mycelia and mycorrhizas proved to be specific for this species at all three levels of its life cycle. It also failed to amplify DNA from T. maculatum and thus ensured the unambiguous differentiation of a species whose morphology is very similar to that of T borchii.