## 4 Discussion  
This study highlights two major findings. Firstly, it demonstrates that _Cff_ grown in vitro emits a distinct VOC profile that can effectively differentiate this bacterium from  
Figure 4: Percentage of single VOC out of the total sum of the most abundant VOCs produced in vitro by _Cff_ strains (TS, 50R, P990, C7), _Psp_ and _Xpp_, in a _P. vulgaris_ Cannellino flour naturalized medium at different concentrations: (**a**) 9 g/L, (**b**) 18 g/L, (**c**) 36 g/L. Compounds: (blue) 2-methyl-1-butanol + 3-methyl-1-butanol; (red) benzaldehyde; (green) phenylmethanol; (violet) 6,10-dimethyl-5,9-undecadien-2-one; (orange) 2-methoxy-4-vinylphenol. Values are the mean of nine replicates \(\pm\) percentage uncertainty. Figure 5: VOCs produced by Cannellino bean seeds artificially infected with _Cff_, _Psp_, and _Xpp_. (**a**) GC–MS vials with Cannellino bean seeds (1) mock-inoculated, or inoculated with (2) _Cff_ TS, (3) _Cff_ 5 OR. (**b**) Specific PCR test [28] on Cannellino bean seeds infected with _Cff_ TS and _Cff_ P990 (lanes 2-4 and 5-7, respectively), C+ positive control _Cff_ pure DNA (20 ng/reaction), (C-) negative control, Marker GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA). (**c**) Percentage of single VOC out of the total sum of the most abundant VOCs produced by Cannellino bean seeds artificially infected with _Cff_ strains (TS, 50R, P990, C7), _Psp_ and _Xpp_. Compounds: (blue) 2-methyl-1-butanol + 3-methyl-1-butanol; (red) benzaldehyde; (green) phenylmethanol; (violet) 6,10-dimethyl-5,9-undecadien-2-one; (orange) 2-methoxy-4-vinylphenol. Values are the mean of nine replicates \(\pm\) percentage uncertainty. other legume bacterial pathogens. Notably, phenylmethanol could serve as a diagnostic marker, even when considered on its own. Additionally, 2-methoxy-4-vinylphenol is produced by all the representative _Cff_ strains tested, except for the _Cff_ 50R strain. This suggests that some VOCs produced by _Cff_ strains could also be useful for mapping the evolution of these bacteria. This finding highlights the variability in metabolic pathways among different strains and such differences in VOC production could reflect genetic divergence or adaptation processes. Therefore, identifying and analyzing these variations may provide insights into the evolutionary trajectories and ecological niches of _Cff_ strains. Furthermore, our results showed that specific VOCs emitted in vitro by pure culture (i.e., phenylmethanol, 2-methoxy-4-vinylphenol) were also released from _Cff_-infected symptomatic and asymptomatic Cannellino bean seeds. Since VOC production from microbial species may strongly depend on the culture conditions [45], our results confirm that _Cff_ emits a similar blend of VOCs when grown in legume flour-based media and in _Cff_-infected Cannellino beans. Conversely, these VOCs are absent when _Cff_ is grown on naturalized media made using flour from plants reported as non-hosts for this bacterium (i.e., durum wheat) or on synthetic media (i.e., LB, NBY, TBY). The analysis focused exclusively on those VOCs associated with the _Cff_-legume interaction, without considering VOCs that are constitutively and exclusively released by legumes and, thus, are not potential markers of _Cff_ infection [46; 47]. The remarkable similarity in the mass spectrum of the 2-methyl-1-butanol + 3-methyl-1-butanol compounds, as well as their similar retention indexes, did not allow an unbiased individual quantification of these two compounds, therefore the peak at 17.6 min was integrated and considered to be the sum of the two compounds. This sum is significant in comparison to the control for _Cff_, as well as for _Xpp_ and _Psp_. To address the practicality of VOC-based detection, it is important to recognize both its potential and limitations. The challenge in identifying VOCs as specific markers lies in the fact that, during plant-pathogen interactions, a complex combination of VOCs is generated and released by both the plant and the pathogen, as well as a consequence of their interaction.