## 3 Results
#### 3.3.2 Antimicrobial Activities against Plant Growth-Promoting Bacteria  
As the previous investigations showed antifungal properties against several phytopathogenic fungi, the use of the sesquiterpenoids as natural compound-based plant protection products could be considered. To investigate potential toxic off-target effects on bacteria that promote plant growth, we next examined whether the addition of \(\beta\)-caryophyllene/oxide affects the growth of bacteria at concentrations used in the hyphal growth assay. For this purpose, the growth of representatives of the plant growth-promoting bacteria (PGPB) group [98; 99; 100], including the two diazotrophic bacteria _Rluizobium rhizogenes_ and _Rlodobacter capsulatus_[101; 102], the two bacilli _Bacillus subtilis_[103; 104] and _Paenibacillus polymyca_[105], as well as the pseudomonads _Pseudomonas fluorescens_[104] and _Pseudomonas putida_[106] was analyzed in presence of \(\beta\)-caryophyllene and \(\beta\)-caryophyllene oxide. Both compounds were added to diluted bacterial cultures in increasing concentrations. After overnight incubation, the MICs were determined according to the respective optical density of the cell cultures (Figure 4).  
The bacteria _R. rhizogenes_, _R. capsulatus_, _B. subtilis_, and _P. polymyva_ did not show reduced cell growth in comparison to the solvent control upon the addition of the two terpenes \(\beta\)-caryophyllene and \(\beta\)-caryophyllene oxide (Figure 4A-D). These bacteria showed an increase in growth, which could be explained by the metabolization of the terpenes. For _P. putida_, no effect of \(\beta\)-caryophyllene oxide was detected compared to the solvent control (Figure 4F). \(\beta\)-caryophyllene showed an influence on _P. putida_, which was concentration independent since all tested concentrations led to comparable cell growth. The cell densi  
Figure 3: The effect of \(\beta\)-caryophyllene and \(\beta\)-caryophyllene oxide on the hyphal growth of plant pathogenic fungi. The effect of \(\beta\)-caryophyllene and \(\beta\)-caryophyllene oxide against _S. sclerotium_ (**A). _F. oxysporum_ (**B**) and _A. brassicola_ (**C**). Final concentrations of 62.5 \(\mu\)g mL\({}^{-1}\), 125 \(\mu\)g mL\({}^{-1}\), and 250 \(\mu\)g mL\({}^{-1}\) of \(\beta\)-caryophyllene (grey bars) and \(\beta\)-caryophyllene oxide (blue bars) in PDA growth medium were used. Medium mixed with the solvents DMSO and Tween 20 (final concentrations, 0.5% and 1% \(\
u/\
u\), respectively) was used as the control (black bars). An equally sized disk with fungal mycelium was placed in the center of each plate and incubated for seven days at 24 \({}^{\circ}\)C. Subsequently, the diameter of each fungal colony was measured, and the relative growth compared to the solvent control was calculated. Each bar represents the mean \(\pm\) standard deviation of three independent biological measurements with three technical replicates each (\(n=9\)). Different letters on the top of the bars indicate significant differences between the treatments based on ANOVA and Holm-Sidak post-hoc method (\(p<0.05\)), while the same letters represent no significant differences.  
ties were about 40% lower compared to the solvent control. This effect was also observed for _P. fluorescens_, where the two terpen reduced growth by up to 40% (Figure 4E).  
In summary, for \(\beta\)-caryophyllene and \(\beta\)-caryophyllene oxide, no MIC could be determined for any of the tested PGPB, but a reduction of cell growth could be observed for both Pseudomonas. As a diverse group of different representative soil bacteria was tested, the results nevertheless indicate that \(\beta\)-caryophyllene and \(\beta\)-caryophyllene oxide do not exhibit strong broad-spectrum antibacterial activities at concentrations which considerably inhibit the hyphal growth of _S. scleroorum_ and _F. oxysporum_ (63 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\)).