Tequila and mezcal are Mexican spirits prepared from Agave plants. The regulations for the designation ‘tequila’ are very strict with respect to used Agave species, the geographical origin, and the manufacturing process. Therefore, admixtures with cheaper and less regulated mezcal spirits are frequent. These counterfeit tequila products represent a serious problem for consumer health and the liquor market economy. With this motivation, we developed two high-throughput workflows for the classification of tequila and mezcal spirits. Both strategies are based on chemical fingerprinting by mass spectrometry (MS). The first method uses direct-injection electrospray ionisation (DIESI) with automated sampling, the second method employs low-temperature plasma (LTP) ionisation. For the ambient sampling with LTP we used a 3D-printed probe, as well as a home-build robot based on RepRap (3D printer) technology. Both methods are fully automated. With DIESI-MS, a sample analysis takes 6 min, with LTP-MS only 20 seconds. The data of both methods permit the discrimination between tequila and mezcal products, and thus are suitable for the quick screening of spirits for authenticity. We also found that pre-processing the raw spectra data with Random Forest models greatly supports the separation of informative signals from noise. This data preparation strategy is therefore especially useful for analyzing noisy data from ambient ionisation analyses.

The response to injury represents an important strategy for animals and plants to survive to mechanical damage and predation. Plants respond to injury by activating a defense response that includes the production of an important variety of compounds that help them withstand predator attack and recover from mechanical injury. Similarly, the filamentous fungus Trichoderma atroviride responds to mechanical injury (MI) by strongly modifying its transcriptional profile and producing asexual reproduction structures (conidia). Here we analyzed if the response to MI in T. atroviride is related to a possible predator defense mechanism from a metabolic perspective. We found that the production of specific groups of secondary metabolites increases in response to MI but is reduced after fungivory by Drosophila melanogaster larvae. We further show that fungivory results in repression of the expression of genes putatively involved in regulation of secondary metabolite production in T. atroviride. Activation of the secondary metabolite production appears to depend on the MAPK Tmk3. Interestingly, D. melanogaster larvae preferred to feed on a tmk3 gene replacement mutant rather than on the wild type strain. Consumption of the mutant strain, however, resulted in increased larval mortality. IMPORTANCE Fungi like other organisms have natural predators including fungivorous nematodes and arthropods that use them as an important food source. Thus, they require mechanisms to detect and respond to injury. Trichoderma atroviride responds to mycelial injury by rapidly regenerating its hyphae and developing asexual reproduction structures. Whether this injury response is associated with attack by fungivorous insects, is unknown. Therefore, determining the possible conservation of a defense mechanism to predation in T. atroviride and plants, and elucidating the mechanisms involved in the establishment of this response is of major interest. Here we describe the chemical response of T. atroviride to mechanical injury and fungivory and the role of a MAPK pathway in the regulation of this response.