The effect of different hydrodistillation pretreatments, namely, reflux extraction, reflux extraction with the addition of cell wall-degrading enzymes, and ultrasound, on the yield and chemical composition of essential oils of sage, bay laurel, and rosemary was examined. All pretreatments improved essential oil yield compared to no-pretreatment control (40–64% yield increase), while the oil quality remained mostly unchanged (as shown by statistical analysis of GC-MS results). However, enzyme-assisted reflux extraction pretreatment did not significantly outperform reflux extraction (no-enzyme control), suggesting that the observed yield increase was mostly a consequence of reflux extraction and enzymatic activity had only a minute effect. Thus, we show that ultrasound and reflux extraction pretreatments are beneficial in the production of essential oils of selected Mediterranean plants, but the application of enzymes has to be carefully re-evaluated.

Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.