A first assessment of the biocidal efficacy of plant essential oils against lichens on stone cultural heritage, and the importance of evaluating suitable application protocols

Plant essential oils (EOs) are increasingly considered as potential tools to control biodeterioration of stone cultural heritage. The application of EOs was indeed shown to devitalize photo- and hetero-trophic constituents of lithobiontic biofilms, but their effects on lichens, which are other remarkable biodeteriogens of stone surfaces, is still unexplored. In this work, we analyzed the biocidal efficacy of a commercial mixture of essential oils (Syzygium aromaticum, Thymbra capitata, Cinnamomum zeylanicum) against crustose lichen species on stone walls in Persepolis (Iran) and Saluzzo (Italy), in semi-arid and temperate bioclimatic areas, respectively. In the former site (ST1), the efficacy of EOs, quaternary ammonium and isothiazolinone biocides, applied with cellulose poultice, was compared against Acarospora cervina, Calogaya biatorina and Protoparmeliopsis usbekica. In the latter (ST2), the efficacy of different protocols to apply EOs was compared against Candelariella vitellina and Protoparmeliopsis muralis. The devitalization effect was evaluated by measuring chlorophyll a fluorescence parameters of lichen thalli (basal fluorescence, F0, and maximum quantum efficiency, Fv/Fm) and with epifluorescence microscopy. In ST1, EOs poultice application remarkably affected lichen vitality, determining in all the targets a decrease of Fv/Fm values similar or even higher than that observed for the other biocidal products. F0 values and epifluorescence microscopy, however, indicated some persistence of viable cells in A. cervina and P. usbekica. Incomplete hydration and metabolic activation of thalli during EOs application, also related to anatomical traits, may account for such partial resistance of these species of semi-arid areas. In ST2, the zeroing of Fv/Fm and a strong decrease of F0 indicated the devitalization of both the target species when EOs were applied with cellulose poultice, and also when the EOs application by brush (requiring a minor quantity of product) was combined with the immediate covering of the surface with a paper tissue, a second EOs supply and the final covering with a plastic film. Both these application protocols assured a prolonged wet condition of the surface -at least in the humid autumn climate of the temperate ST2-, and thus the metabolic activity of thalli and their susceptibility to the chemicals. The simple EOs application by brush, even if combined with the plastic film covering, was instead ineffective, likely due to the lower product supply and thallus hydration. In conclusion, the biocidal potency of EOs was also confirmed for lichens, although some species-specific resistance may be encountered, as in the case of A. cervina and P. usbekica in ST1. The set up of the application protocol, particularly with respect to the assurance of thallus hydration, was shown as crucial to make the EOs treatments effective and should be taken into particular care when targeting lichens of semi-arid areas and, in general, to plan interventions in favourable, more humid seasons.