When rescuing either symbionts or other endophytic bacteria from internal plant tissues, including root nodules, and attempting their isolation and culturing on laboratory media, failure to obtain colonies is often experienced. This phenomenon applies particularly to the rhizobia from wild legume species which frequently result in non-culturable status (1), possibly as a consequence of a biochemical stress inherent to the isolation procedure. Other taxa can co-occupy nodules along with rhizobia, mostly belonging to the Bacillaceae, Pseudomonadaceae and Enterobacteriaceae families, which appear much less sensitive to the putative stress as their isolation from nodules, is apparently not impaired. We hypothesized that the limited culturability of rhizobia, compared to that of other types of nodule invaders, could be related to an oxidative stress caused by the histological disruption of plant tissues when surface-sterilized nodules are squashed for bacterial isolation. In order to test this hypothesis we isolated bacteria from nodules of two mediterranean wild legumes (Hedysarum spinosissimum and Tetragonolobus purpureus) by squashing nodules either in control phosphate buffer saline (PBS) or in PBS supplemented with scavenging systems apt to prevent damage from reactive oxygen species. The treatments included: an antioxidant cocktail (Glutathione, Sodium Ascorbate and EDTA) or an enzyme cocktail (Catalase, Peroxidase, Superoxide Dismutase). The mixtures were tested either as modified squashing buffers or as additions to the plates on which bacteria were spread after squashing in control PBS. The latter option allowed to distinguish whether the oxidative stress were due to immediate damage from plant-derived reactions or as a consequence of the transfer in carbon-rich agar media from the plate culturing stage. Some of the combinations yielded orders of magnitude-different increases of culturability in terms of colony counts and also allowed the cultivation of otherwise undetectable taxa. The coexistence of up to three different rhizobiaceae within the same nodules was observed, two of which of supposedly non-symbiotic nature (unable to re-induce nodule formation in axenic nodulation tests). Isolates were subsequently tested for tolerance to H2O2 in liquid cultures, which showed differences in comparison with the levels displayed by non-alphaproteobacterial endophytes. Ecological and physiological implications are discussed
