Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules
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ORIGINAL PAPER
Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules Rosella Muresu • Alessandra Tondello • Elisa Polone • Leonardo Sulas • Barbara Baldan Andrea Squartini
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Received: 1 July 2012 / Revised: 24 February 2013 / Accepted: 22 March 2013 / Published online: 10 April 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract 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
Communicated by Jo¨rg Overmann. Rosella Muresu and Alessandra Tondello: equal contributors. R. Muresu L. Sulas C.N.R., Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo (ISPAAM), Traversa La Crucca 3, Localita` Baldinca, Li Punti, Sassari, Italy A. Tondello E. Polone A. Squartini (&) Dipartimento di Agronomia, Animali, Alimenti, Risorse Naturali e Ambiente, DAFNAE, Universita` di Padova, Viale dell’Universita` 16, 35020 Legnaro, Padova, Italy e-mail: [email protected] A. Tondello B. Baldan Dipartimento di Biologia, Universita` di Padova, Via Ugo Bassi 58/b, 35131 Padua, Italy
major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants. Keywords Endophytes Reactive oxygen species Culturability Wild legumes
Introduction When rescuing either symbionts or other endophytic bacteria from plant tissues, including root nodules, and attempting their isolation and culturing on laboratory media, failure to obtain colonies is often experienced. This condition applies particularly to the rhizobia from wild legume species which frequently result in non-culturable status (Muresu et al. 2008), possibly as a consequence of a stress induced from the isolation procedure. Other taxa can co-occupy nodules along with rhizobia, mostly belonging to the Bacillaceae, Pseudomonadaceae, and Enterobacteriaceae, which appear much less sensitive to the putative stress as their isolation from nodules is apparently less impaired (Tokala et al. 2002; Bai et al. 2003; Benhizia et al. 2004; Zakhia et al. 2006). Concerning oxygen radicals during development
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