## 3 Discussion  
Secondary metabolites and mixtures such as essential oils are complex natural active agents formed by plants to be involved in several biological functions. Terpenoids are the major components of plant essential oils, and they are responsible for most of their biological properties [28; 29; 30]. Essential oils extracted from plants represent an invaluable source of bioactive compounds that were and still are used in pharmaceuticals, as well as in agrochemicals. Many of these essential oils and their main components are active against microbes, insects and nematodes. Particularly, terpenids are very active and heavily studied components [12; 13; 14]. In this study, we tested several compounds belonging to the terpenids for their activities against the nematode _H. schachtii_. We first evaluated the effects of caryophyllene oxide, nootkatone, zerumbone, \(\alpha\) amyrin, \(\beta\) amyrin, taraxasterol acetate, saffron and crocetin dialdehyde (Figure 1) on _A. thaliana_ to find a suitable concentration for the infection assay. Subsequently, we performed infection assays using an optimized, gnotobiotic system to study the interaction of _A. thaliana_ and _H. schachtii_ on Knop agar [31]. The ability of _H. schachtii_ to infect the model host plant _A. thaliana_ opens up a wide perspective of detailed studies of compounds against nematode parasitism [20; 32]. Furthermore, we investigated the possible mode of action for the effective candidate terpenoid nootkatone. The effect of terpenids on plant growth is variable and has been reported in several studies to be related to the applied concentration. Therefore, testing the effect of the chosen compound concentration on the plant phenotype first was necessary. In our study, we found that _Arabidopsis_ growth was affected in the presence of 10 ppm of nootkatone, zerumbone or crocetin dialdehyde in the growth medium. The inhibitory effect was abolished by decreasing the used concentration to 5 ppm. Similar observations were reported earlier using other terpenoids. In a previous study, four sesquiterpenoids, eight diterpenes and nine triterpenes were extracted from Tectona grandis and tested for phytotoxicity on several crop plants. Out of the 21 terpenes, 2 oxokovalenic acid and 19 hydroxyferruginol were phytotoxic. It was also shown that the toxicity was concentration-dependent and totally vanished for some terpenoids at low concentrations [17]. The same results were shown when trans-caryophyllene was tested against several plants, including _Arabidopsis_. The study showed  
Figure 7: Contour plot of a headspace solid-phase microextraction (HS-SPME)–gas chromatography (GC)\(\times\)GC–time-of-flight mass spectrometer (TOF–MS) chromatogram (TIC) demonstrating the separation of sesquiterpenes isolated from the headspace of a 30-day-old _Arabidopsis thaliana_ plant grown on nootkatone-containing Knop agar. The \({}^{1}\)tR (X-axis) corresponds to the retention time on the first dimension (\({}^{1}\)D) column and \({}^{2}\)tR (Y-axis) to the retention time on the secondary (\({}^{2}\)D) column. The color gradient reflects the intensity of the TOF-MS signal on white background from low (violet) to high (red). Numbers at peaks refer to compound numbers, as defined in Table 1 and Figure S2. The 3D view of the chromatogram section shown can be found in Supplementary Figure S3. that the effect was dependent on the plant species, as well as the tested concentration [15]. The study demonstrated that the effect is a result of changing the plant water status accompanied by oxidative damages [15]. Based on the results of the phytotoxicity tests, we conducted an infection assay with the plant parasite \(H\). _schachtii_ using the Knop medium containing 5 ppm of either nootkatone, zerumbone or crocetin dialdehyde or 10 ppm of the other terpenoids. Nootkatone was the only compound that decreased parasitism. There are no previous reports showing an effect of nootkatone against nematodes.