Journal
ARCHIVES OF MICROBIOLOGY
Volume 195, Issue 6, Pages 385-391Publisher
SPRINGER
DOI: 10.1007/s00203-013-0886-6
Keywords
Endophytes; Reactive oxygen species; Culturability; Wild legumes
Categories
Funding
- Fondazione Banco di Sardegna [2010 p1293/2010.1163]
- University of Padova [CPDR093189]
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In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants.
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