Moreover a clear separation between above-ground (stem and leaves) and below-ground environments (soil and nodules) was detected. An analysis of the clone libraries, prepared from above-ground and below-ground pooled samples, revealed an uneven distribution of bacterial classes, with a marked pattern highlighting the class of Alphaproteobacteria as the more abundant in plant tissues (this class represented
half of the clones in the stem + leaf library). The same uneven pattern PD 332991 was then observed, at lower taxonomic ranks, within the Alphaproteobacteria, with sequences of clones belonging to members of the Methylobacteriaceae and Sphingomonadaceae families being more abundant in stem than in soil and nodules. Methylobacteria and Sphingomonadaceae have been found as endophytes in a number of plants [8, 12, 31, 33, 42–45] and it is believed that this group of bacteria may take advantage from living as plant-associated, thanks to its ability to utilize the one-carbon alcohol methanol discharged by wall-associated pectin metabolism of growing plant cells. Concerning root nodule bacterial communities, obtained
data indicated that very diverse see more bacterial taxa are associated with nodules, the most represented being the specific rhizobial host of M. sativa, the alphaproteobacterium S. meliloti. However, additional taxa have been found, including members of Actinobacteria Flavobacteria Gammaproteobacteria and Betaproteobacteria, which may have some additional plant growth-promoting activities (see for
instance [46, 47]). In soil, Amrubicin Acidobacteria was one of the most important divisions (in terms of number of clones in the library) and was present exclusively in the soil clone library, in agreement with many previous observations [48, 49]. A relatively high presence of Archaea (Thermoprotei) was also found. Checking the 16 S rRNA gene sequences present in the Ribosomal Database for 799f/pHr primer annealing, we found that PCR amplification from Thermoprotei was theoretically possible with this primer pair (data not shown). The presence of Archaea in the soil is not unexpected  and could be linked also to the anoxic or nearly anoxic conditions present in the bottom of the pot. However, since the low coverage of soil clone library, the presence of many other additional taxa, as well of different proportions of those found here cannot be excluded. In addition, it should be mentioned that differences between soil and plant-tissues bacterial communities could also be ascribed to the different DNA extraction protocols we were obliged to use, since a unique protocol (bead-beading protocol for both soil DNA and plant DNA) failed in a successful extraction of DNA from both soil and plant tissues (data not shown). A similar technical need was encountered by other authors also , which renders the study of the relationships between plant-associated and soil bacterial communities still at its beginning.