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Open Access Methodology article

Development and validation of a real-time PCR assay for detection and quantification of Tuber magnatum in soil

Mirco Iotti1, Marco Leonardi2, Marilena Oddis2, Elena Salerni3, Elena Baraldi1 and Alessandra Zambonelli1*

Author Affiliations

1 Dipartimento di Protezione e Valorizzazione Agroalimentare, Alma Mater Studiorum Università di Bologna, via Fanin 46, 40127, Bologna, Italy

2 Dipartimento di Scienze Ambientali, Università dell’Aquila, via Vetoio, Coppito 1, 67100, L’Aquila, Italy

3 Dipartimento di Scienze Ambientali “G. Sarfatti”, Università degli Studi di Siena, via Mattioli 4, 53100, Siena, Italy

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BMC Microbiology 2012, 12:93  doi:10.1186/1471-2180-12-93

Published: 6 June 2012

Abstract

Background

Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions.

Results

The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA.

Conclusions

Taken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières.

Keywords:
Real-time PCR; Taq-man probe; Tuber magnatum DNA concentration; Soil DNA extraction; ITS primers; Truffle production