## 3 Results and Discussion
Etephon treatment increased the accumulation of proline and antioxidant enzymes activity in maize subjected to water stress by maintaining water status, membrane stability and modulate cuticular wax biosynthesis [35]. In addition, foliar-applied ethephon markedly improve anthocyanin and total phenolic content in black carrots [36]. Several lines of evidence indicated the stimulating effect of ethylene on diosgenin content and enzyme activities also in fenugreek [37; 38]. During tripartite interaction in fenugreek, water stress did not significantly influence trigoneline accumulation. However, both inoculated (I) and not-inoculated (NI) plants seemed to accumulate higher amount of trigoneline during water deficit compared to non-stressed plants. It has been recently reported that fenugreek tolerated water deficit by increasing the endogenous melatonin and trigoneline molecules [39]. Interestingly, moderate drought conditions can lead to positively modulate secondary metabolites useful for food, cosmetic and pharmaceutical industries as previously reported in the model medicinal and aromatic sage plant [40]. In our experimental context, with respect to not-inoculated (NI) plants, trigoneline was generally higher in inoculated (I) ones with a peak recorded when ethephon was delivered at 550 and 850 ppm in both NS and S conditions. Here, an AM-based inoculum also leading to nodulation was used, rendering it impossible to verify the effect of the single microorganism. Although further experiments might be also needed to verify the efficiency of the rhizobia-forming nodules, we preferred to take in account their presence in the root apparatus of the inoculated plants. Our findings could be explained by the biochemical, hormonal and physiological crosstalk taking place as a consequence of the tripartite association between AM fungi, rhizobia and legumes. It was already reported that these complex interactions lead to a functional diversity of the root-associated symbionts, which can complement each other in acquiring different limiting nutrients and in driving important ecosystem functions [41]. Figure 1: Boxplots representing trigoneline production in the different conditions (NI, I: not-inoculated and inoculated plants, respectively; S, NS: water stressed and non-stressed plants; E0, E350, E550, E850: different ethephon concentrations in ppm). Different conditions were color coded as follows: red and brown boxes represent not-inoculated plants (NI) non-stressed (NS) and water stressed (S), respectively; blue and dark-blue boxes represent inoculated plants (I) non-stressed (NS) and water stressed (S), respectively. Letters are plotted according to outcomes of [PERSON]’s test. Previously, it has been proposed that trigonelline may be a regulatory factor during early signal events in the establishment of the AM symbiosis in _Prosopis laevis_[42]. In this context, we can hypothesize that the over production of trigonelline in fenugreek plants used in our study can be explained by the combinations of both ethephon application and presence of the symbionts. The use of AM-based inoculants in fenugreek cultivation has started to be considered as an effective and useful agronomic practice, showing an efficacy in improving tolerance of fenugreek to salinity [43] and to oxidative stresses [44]. The plant hormone ABA is a chemical signal involved in the plant response to various environmental stresses. Here, the content of ABA in the inoculated (I) plants was in general significantly higher than in not-inoculated ones, although no differences were found in the non-stress condition (NS) and in the absence of ethephon treatment (E0). On the contrary, it was significantly affected by interaction between different ethephon concentration and irrigation regimes. The highest accumulation of ABA was measured when inoculated non-stressed plants (INS) were subjected to high concentrations of ethephon (550 and 850 ppm, Figure 2 and Supplementary Materials Table S1).