The biological activity of bacterial rhamnolipids is linked to their molecular structure
Authors: Sandra Bredenbruch , Conrad Müller , Henry Atemnkeng , Lukas Schröder , Till Tiso , Blank Lars M. , Grundler Florian M.W. , Schleker A. Sylvia S. 
Published: January 2023

Biosurfactants are amphiphilic compounds of microbial origin with a wide range of industrial applications. Rhamnolipids (RLs) offer a broad range of potential applications as biosurfactants in industry and agriculture. Several studies report a high capacity for controlling different plant pests and pathogens and consider RLs as promising candidates for bio-based plant protection agents. RLs are a class of glycolipids consisting of one (mRLs) or two (dRLs) rhamnose moieties linked to a single or branched β-fatty acid. Due to the diversity of RLs, so far little is known about the relation between molecular structure and biological activity.

Engineering the synthesis pathway allowed us to differentiate between the activities of mixtures of pure mRL and of pure dRL congeners and elaborated HPLC techniques further enabled to analyse the activity of single congeners. In a model system with the plantArabidopsis thalianaand the plant-parasitic nematodeHeterodera schachtiiwe demonstrate that RLs can significantly reduce infection, whereas their impact on the host plant varies depending on their molecular structure. While mRLs reduced plant growth even at low concentration, dRLs showed no or a beneficial impact on plant development. In addition, the effect of RLs on plant H2O2production was measured as an indicator of plant defense activity. Treatment with mRLs or dRLs at a concentration of 50 ppm increased H2O2production. Lower concentrations of up to 10 ppm were used to stimulate plants prior to a treatment with water or flagellin (flg22), a bacterial inducer of plant defense responses. At 10 ppm both mRLs and dRLs fostered an increased response to flg22. However, mRLs also led to an increased response to water, the non-inducing negative control, indicating a generally elevated stress level. Neither mRLs nor dRLs induced expression of plant defense marker genes of salicylic acid, jasmonic acid and ethylene pathways within a 1 hour and 48 hours treatment.

Due to the negative effect of mRLs on plants further studies were concentrated on dRLs. Treatment of pre-parasitic infective juveniles ofH. schachtiirevealed that dRLs did not increase mortality even at a very high concentration of 755 ppm. In order to analyse the effect of single dRL congeners nematode infection assays were performed. While dRL congeners with a C10-C8 acyl chain increased nematode infection, dRLs with C10-C12 and C10-C12:1 acyl chains reduced nematode infection even at concentrations below 2 ppm. Plant growth was not reduced by C10-C8 dRLs, but by C10-C12 and C10-C12:1 dRLs at concentrations of 8.3 ppm. H2O2production was increased compared to the water control upon treatment with C10-C8 dRLs at a concentration of 200 ppm, while C10-C12 and C10-C12:1 dRLs triggered the same effect already at 50 ppm.

Our experiments show a clear structure-effect relation. In conclusion, functional assessment and analysis of mode of action of RLs in plants require careful consideration of their molecular structure and composition.