Rapid detection of cultivated truffle fungi (Tuber) from truffles and ectomycorrhizal root tips using loop-mediated isothermal amplification.

UNLABELLED: Truffle fungi, such as Tuber melanosporum and T. aestivum, are prized luxury food products that have been historically cultivated in Mediterranean regions of Europe. Recent advances in truffle cultivation have led to the establishment of productive truffle orchards in North America, South America, China, and Australia. Despite these advances, a consistent issue has been the introduction and accidental cultivation of non-target truffle species. Non-target truffle species (e.g., T. indicum and T. brumale) often morphologically resemble target species (e.g., T. melanosporum) and may unintentionally be included as an inoculum and introduced to seedlings. Detecting inoculum and seedling contamination is difficult and often relies on inconsistent morphological analyses or labor-intensive and time-consuming PCR-based methods. Furthermore, quantitative PCR (qPCR) assays have only been developed for T. melanosporum and T. aestivum, leaving truffle growers without rapid tools to detect important contaminants such as T. brumale. Here, we develop a rapid loop-mediated isothermal amplification (LAMP) assay to detect three globally cultivated Tuber species (T. melanosporum, T. borchii, and T. aestivum) and the most common introduced contaminant species (T. brumale). The assays presented here are sensitive to at least 10-3 ng of DNA and are species-specific. These assays should be of special interest to both truffle orchard owners and truffle testing facilities given the low time commitment, high sensitivity, high specificity, and cost comparable to PCR and qPCR-based approaches. IMPORTANCE: Despite the global success of truffle cultivation, the accidental cultivation of undesirable truffle species has been a persistent issue. To address this issue, we developed four loop-mediated isothermal amplification (LAMP) assays for the rapid detection of Tuber aestivum, T. borchii, T. brumale, and T. melanosporum from ectomycorrhizal root tips and truffle fruiting bodies. These assays are alternatives to the widely used T. aestivum and T. melanosporum qPCR assays and are the first rapid detection assays for T. borchii and T. brumale.