## 3 Results and Discussion  
Our study aimed to evaluate the water deficit effects on target metabolites (trigonelline and abscisic acid--ABA) in fenugreek, colonized by both AM fungi and nodule-forming symbionts in combination with exogenous ethephon application. Leguminous plants can in fact host within their roots both symbionts concomitantly, forming the so-called tripartite symbioses largely studied to elucidate the complex dialogue among the partners [31]. Over the last decade, several studies have been performed on the impact of a single symbiont in plant physiology features considering several environmental factors [32; 33; 34]. Conversely, poor studies are available on tripartite symbiosis effects in response to abiotic stressful events. In detail, after about six weeks from inoculation, control not-inoculated (NI) and inoculated (I) plants were subjected to several combined treatments: well irrigated (NS) and not-watered (S), both treated with different ethephon concentrations (E0, E350, E550, E850 ppm). Results (Table 1) showed that biometric parameters were not affected by the considered factors (Stress, I, E), with the exception of inoculation (I) factor for the fruit number where not -inoculated (NI) plants showed higher number of fruits with respect to the inoculated (I) ones. The only statistically significant difference was found in the nodules that were significantly higher in inoculated stressed (IS) plants than in inoculated non-stressed (INS) ones (Supplementary Materials Figure S1). Results suggest a similar trend for AM fungal colonization (Supplementary Materials Figure S2) in response to ethephon, although it will need further observations to confirm it due to the absence of a sufficient number of replicates to perform a statistical analysis. Ethylene was reported as an inhibitor of nodule and AM development at early stages of symbiosis formation [31]. In addition, exogenous ethylene application induced much lower heights in maize-treated plants with respect to their untreated controls [35]. In this study, exogenous ethephon application and water deficit conditions were applied when symbioses were already established. This could unfold the absence of significant differences in the morphometric measurements recorded. It is worth noting that nodule development was higher under S conditions irrespective of the ethephon concentration used, which opens new questions on symbioses development mechanisms that deserve further investigations, at least in the fenugreek model system. In terms of the SMs production, our results provide evidence that the interaction between the application of exogenous ethylene and the use of an AM-based inoculum significantly increases trigonelline content in fenugreek plants independently of the water deficit treatment (Figure 1 and Supplementary Materials Table S1). The content of trigonelline was significantly affected by symbioses, both in NS and S conditions. Looking at ethephon application, while inoculated plants (I) showed a dose-dependent trend, independent from the water regimes, not-inoculated (NI) plants showed a significant difference only in NISE550. The fact that trigonelline levels were significantly higher in NISE550 with respect to NINSE550 suggests a combined effect of the E550 treatment and water deficit at least in the absence of root colonization (Figure 1 and Supplementary Materials Table S1). Differently from not-inoculated (NI) plants, inoculated (I) ones responded proportionally to the different concentrations of ethephon, and the highest amount of trigonelline was determined in inoculated non-stressed (INS) plants treated with the highest concentration of ethephon (850 ppm) (\(P\leq 0.05\); Figure 1). An average increase of approximately 3000 ug g\({}^{-1}\) was measured in inoculated compared to not-inoculated plants (Figure 1  
Valuable secondary metabolites often can be accumulated by application of synthetic elicitors acting as molecular signals such as ethephon.