A novel assay for the detection of bioactive volatiles evaluated by screening of lichen-associated bacteria

Abstract

Volatile organic compounds (VOCs) produced by microorganisms are known both for their effect on pathogens and their role as mediators in various interactions and communications. Previous studies have demonstrated the importance of VOCs for ecosystem functioning as well as their biotechnological potential, but screening for bioactive volatiles remained difficult. We have developed an efficient testing assay that is based on two multi-well plates, separated by a sealing silicone membrane, two tightening clamps, and variable growth media, or indicators. The experiment design as presented here is a novel and robust technique to identify positive as well as negative VOC effects on the growth of a target organism and to test for specific substances e.g., hydrogen cyanide which can be detected with a suitable indicator. While the first pre-screening assay is primarily based on indicator color change and visible growth diameter reduction, we also introduce an advanced and quantitatively precise experiment design. This adaptation involves qPCR-based quantification of viable target cells after concluding the treatment with VOCs. Therefore, we chose preselected active isolates and compared the partial 16S rRNA gene copy number of headspace-exposed E. coli with non-treated controls. Separately obtained headspace SPME and GC/MS-based profiles of selected bacterial isolates revealed the presence of specific and unique signatures which suggests divergent modes of action. The assay was evaluated by screening 100 isolates of lung lichen-associated bacteria. Approximately one quarter of the isolates showed VOC-based antibacterial and/or antifungal activity; mainly Pseudomonas and Stenotrophomonas species were identified as producers of bioactive volatiles.

Inhibitory Abilities of Bacillus Isolates and Their Culture Filtrates against the Gray Mold Caused by Botrytis cinerea on Postharvest Fruit

“…Naturally occurring VOCs of endophytic bacteria are regarded as sources of new antifungal agents that are safe for humans and the environment and do not have a harmful influence on the host plants (Gao et al, 2017). Microbial VOCs inhibit the growth of pathogenic fungi; improve plant growth (Park et al, 2015); mediate relationships, interactions, and communications between organisms (Cernava et al, 2015); identify bacterial species; and induce systemic resistance in plants (Zamioudis et al, 2015). GC-MS has revealed that 29 unique VOCs produced by Bacillus velezensis ZSY-1 isolated from Chinese catalpa exhibit significant antifungal activity against plant pathogenic fungi (Gao et al, 2017).…”

Differential sharing and distinct co‐occurrence networks among spatially close bacterial microbiota of bark, mosses and lichens‬‬

“…Burkholderia and Pseudomonas isolated from lichens exhibit highly effective antagonists of certain bacterial and fungal pathogens (Cernava et al . ,b). Thus, an essential role of this bacterial fraction in pathogen defence and microbial community stabilization is envisioned.…”

Micro‐organisms growing on rapeseed during storage affect the profile of volatile compounds of virgin rapeseed oil

“…The analysis of volatile compounds from bacteria was performed according to the methods published by Verginer et al . and Cernava et al . In brief, 8 mL of autoclaved rapeseed meal agar was poured into a 20‐mL headspace vial, which was positioned at a slope to increase the surface of the agar.…”