## 3 Discussion
However, many investigations demonstrated nootkatone activities against insects (for example, described in the previous studies [33; 34; 35; 36]). On the other hand, caryophyllene was reported to be an attractant to nematodes in nature [37; 38], which could explain the elevated parasitism of _H. schachtii_ on _Arabidopsis_ plants when exposed to this compound in our research. Since we discovered nootkatone to remarkably inhibit _H. schachtii_ parasitism, we further aimed to investigate the underlying mechanism. The mode of action of any antimematode compound can be categorized as (1) direct toxicity or repellent of nematodes or (2) indirect effects by activating plant defense mechanisms against nematodes. Our results revealed no lethal effect of nootkatone against nematodes even after two days of exposure. Therefore, we concluded that the effect was not direct. In contrast, previous studies demonstrated the direct effect of nootkatone against some arthropods--specifically, fleas, ticks and mosquitos--but still with an unclear mode of action [39]. To determine the mechanism against mosquitos, [PERSON] and [PERSON] (2010) challenged mutant strains of _Anopheles gambiae_ Giles harboring mutations at three different target sites known to mediate an insecticide resistance with nootkatone and compared the lethal dosages. The altered target sites evaluated were the sodium channel para-locus mutation (L1014 F KDR) that confers permethrin resistance, the ACE-1 gene that confers organophosphate and carbamate resistance and a \(\gamma\) aminobutyric acid receptor mutation of the Rdl locus conferring dieldrin resistance. In the study, they found that the _A. gambiae_ wild type and all three mutants were equally sensitive to nootkatone, as there were no significant differences in the required lethal doses determined. Therefore, they concluded that nootkatone acts through a different mode of action than existing chemicals currently used in mosquito control. As nootkatone had no nematocidal effect, we decided to analyze if nootkatone affects the nematode's behavior by causing positive or negative chemotaxis. Our results displayed that the presence of nootkatone in the medium did not affect the J2s' preference for the plant. We further demonstrated that the nematodes were equally attracted to both sides of the treatment in presence and absence of nootkatone. This means that nootkatone does not affect the nematode's orientation towards plants. However, the J2 penetration percentage of the plant roots was significantly decreased in the presence of nootkatone compared with the untreated control. This indicates that the plant defense was activated and inhibited J2 penetration and nematode development. To analyze the molecular changes nootkatone induces in the plant, we first determined whether the compound triggers a ROS burst. The production of ROS is one of the earliest defense responses observed in various plant-pathogen interactions [40; 41]. Additionally, the elevation of plant ROS plays a major role in plant resistance against nematodes [42]. Therefore, we measured the presence of ROS upon the exposure to several concentrations of nootkatone. Plant ROS levels were not elevated in nootkatone-treated leaf discs, demonstrating that nootkatone is functioning through another mechanism. Another defense option is the emission of volatile organic compounds (VOCs) that protect plants against herbivores and pathogen attacks. Sesquiterpenes, as a group of the emitted VOCs, have been shown to play a major role in the plant defense, including direct activity against pathogens (e.g., inducing oxidative stress) or triggering defense signaling pathways [16; 43]. Therefore, we also measured the plant sesquiterpene profile upon exposure to nootkatone to determine if the presence of nootkatone induces the emission of sesquiterpenes, which, in return, could play a role against nematodes. We performed a GC analysis of the volatile profile of _A.