Microbial Dysbiosis and Macrofungal Outbreaks Induced by Long-term Plant Essential Oil Biocide Application in a Humid Archaeological Earthen Site

Abstract Aims The long-term application of plant essential oil (EO)-based biocides is a popular “green” strategy for microbial control in heritage science. However, its ecological consequences in enclosed, humid environments remain poorly understood. This study investigated the unintended ecological shifts following prolonged use of an oregano-cinnamon EO mixture at the Wangjingmen earthen site, aiming to elucidate the mechanisms behind a subsequent macrofungal outbreak. Methods and Results We employed a multi-faceted approach combining environmental monitoring, high-throughput amplicon sequencing of bacterial (16S rRNA) and fungal (ITS) communities, and geochemical analysis. Our findings revealed that the stable, high-humidity microenvironment (>95% RH, 20–32°C), coupled with the selective pressure from continuous EO application, significantly altered the microbial community structure between 2022 and 2024. This led to a decrease in microbial richness and a community shift favouring resistant taxa. Specifically, bacterial communities shifted towards Pseudomonadota and Actinomycetota dominance, while fungal communities saw an enrichment of Ascomycota and Basidiomycota, culminating in the proliferation of Leucocoprinus at temperatures above 28°C. Geochemical analysis indicated that evaporation on vertical surfaces created nutrient and salt enrichment zones (e.g. NO₃⁻, SO₄²⁻, gypsum), which further facilitated fungal colonization. Predictive functional profiling suggested an enrichment of metabolic pathways related to EO degradation (e.g. limonene and pinene degradation), indicating microbial adaptation to the biocide. Conclusions Prolonged application of EO-based biocides in stable, humid heritage environments can induce significant microbial dysbiosis, creating an ecological vacuum that facilitates opportunistic outbreaks of resistant macrofungi. Our findings highlight the potential risks of relying on broad-spectrum biocides without considering their long-term ecological impacts.