Relationships between rhizobial culturability from legumes nodules and nitrite reductase activity

The culturability of rhizobia from nodules of wild legumes of the Mediterranean regions has been found to be very limited and their occupancy of nodules is often shared with that of a number of endophytic bacteria (Benhizia et al., 2005; Muresu et al., 2008). Among the factors to explain their scarce capability to form colonies on microbiological media, stress due to production of reactive oxygen species from the plant has been shown to be one of the issues (Tondello et al., 2010). Other aspects however could be contributing to this multifaceted phenomenon. In the present investigation we assessed their relationships with the presence of 3-nitropropionic acid (3NPA) a naturally occurring compound present in certain legumes as a deterrent against herbivores. It induces an irreversible inhibition of succinate dehydrogenase causing toxicity to eukaryotes. Plants that make 3NPA also contain the enzyme, 3NPA-oxidase to protect themselves in the actively growing tissues from the effects of the compound (Hipkin 1999). The legumes so far known to accumulate 3NPA belong to seven genera. Within Astragalus, Coronilla, Indigofera, and Lotus only some of the species tested produce the toxin, while all the tested species of Hippocrepis, Securigera and Scorpiurus are positive for 3NPA. None of the agriculturally grown legumes produce this toxin (Salem et al., 1995, Hipkin et al., 2004). 3NPA-oxydase converts 3NPA into two equivalents of nitrate and one of nitrite, constituting a rare example of in-planta nitrate production mechanism (Hipkin et al., 2004). In the present report we sought to investigate whether the failure to isolate bacteria from nodules of wild legumes could be in part related to consequences of the 3NPA metabolism. The instances that led us to consider such possibility stem from the following considerations: (1) Rhizobia could be sensitive targets to 3NPA when as they employ succinate dehydrogenase as main part of the dicarboxylate metabolism fueling their energy requirments in the nodules (Gardiol et al., 1982); (2) as the oxydation of 3NPA carried out by the plant generates nitrate and nitrite, some rhizobia that are proficient in denitrification could reduce nitrite up to stages known to cause them the entrance into the Viable But not Culturable State (Toffanin et al., 2000). We took into consideration eight spontaneous legumes, two of which, Scorpiurus muricatus and Hippocrepis unisiliquosa are known to be respectively a medium (9.4 μmol g -1 f.w.) and a heavy (61.5 μmol g -1 f.w.) producer of 3NPA (Hipkin 2004)