Molecular tools for rapid and accurate detection of black truffle (Tuber melanosporum Vitt.) in inoculated nursery plants and commercial plantations in Chile

Cecilia Cordero1, Pablo Cáceres1, Gloria González1, Karla Quiroz1, Carmen Bravo1, Ricardo Ramírez2, Peter D.S. Caligari2, Basilio Carrasco3, and Rolando García-Gonzales1*

Truffle (Tuber melanosporum Vitt.) culture is an agroforestry sector in Chile of increasing interest due to the high prices that truffles fetch in the national market and the recent evidence that its commercial production is possible in Chilean climatic and soil conditions. In this study, the efficiency of three methods of DNA extraction from a mix of 5 g of soil and roots from both nursery and field plants of Quercus ilex L. mycorrhized with T. melanosporum were evaluated, and a simple and reproducible protocol was established. Detection of T. melanosporum was performed by the technique of cleaved amplified polymorphic sequence (CAPS) from amplicons generated with the primers ADL1 (5´-GTAACGATAAAGGCCATCTATAGG-3´) and ADL3 (5´-CGTTTTTCCTGAACTCTTCATCAC-3`), where a restriction fragment of 160 bp specific for T. melanosporum was generated, which allows the discrimination of this species from the rest of the species belonging to the Tuber sp. genus. Direct detection of T. melanosporum in one step was also obtained by polymerase chain reaction (PCR) from total DNA isolated from mycorrhized roots and with the primers ITSML (5´-TGGCCATGTGTCAGATTTAGTA-3´) and ITSLNG (5´-TGATATGCTTAAGTTCAGCGGG-3´), generating a single amplicon of 440 bp. The molecular detection of T. melanosporum by the methods presented here will allow the rapid and accurate detection of mycorrhization of trees, both under nursery and field conditions. This technology will also provide more security to farmers by controlling the quality of the mycorrhized trees they will plant and also by following the mycorrhization status of established orchards.

Keywords: Tuber melanosporum, ectomycorrhizas, molecular markers, PCR, CAPS.
1Universidad Católica del Maule, Facultad de Ciencias Agrarias y Forestales, Av. San Miguel 3650, Talca, Chile. *Corresponding author (rgarciag@ucm.cl).
2Empresa Agrobriotruf SA, Talca, Chile. 3Pontificia Universidad Católica de Chile, Facultad de Agronomía e Ingeniería Forestal, Vicuña Mackenna 4860, Casilla 306, Santiago, Chile.