Intercellular communication in bacteria nodulating plants of the family Leguminosae

In order to achieve a better understanding of the nodulation patterns of rhizobia in their symbiosis with legume hosts, and with the aim to examine their signalling behaviour in cell-to-cell communication, a series of experimental projects were carried out. In first instance the microbial inhabitants of 831 pea root nodules formed on nine plants, sown in different field soil parcels, were isolated and characterized by PCR-based electrophoretic fingerprinting using the BOXA1R primer. Band profiles were analyzed by GelComparII image analysis software converting differences into a numerical matrix yielding their similarity dendrogram in terms of genetic fingerprint distances. The level of strain-specific association with individual plant or soil plots has been assessed. As 85% of the profiles result singletons, having been found in only one nodule, the overall diversity of the site appears particularly high. Estimates of the total diversity at biovar level were obtained by nonparametric estimators pointing to a value over 1300 types. Such richness was compared with the much lower one recorded eight years earlier on the same plots and was put in relation with the repeated host cropping occurred in between. Moreover, the position of each nodule within the root apparatus, in terms of root rank order and distance from the crown, had been recorded in digitized images and the existence of topological and temporal patterns in each strain's nodulation process has been inspected. The fingerprinting quality of BOX-PCR in terms of reproducibility and sensitivity, was compared to that obtainable by other primers as ISRh1 outbound primers. The same fingerprint-characterized strains were screened for the production of Quorum Sensing signals consisting in short-medium- (C4-C8) and long- (C14) chained N-acyl homoserine lactones (AHLs) using, respectively, the two reporter systems: Chromobacterium violaceum CV026 (violacein pigment induction) and Rhizobium leguminosarum A34 (colony growth inhibition). The majority of the natural Rhizobium leguminosarum strains were found to be quorum-signalling positive. The occurrence of isolates negative to one or both phenotypes however shows that those traits are not absolute requirements for host nodulation. In a different study we examined the root nodule symbionts of eight species of wild legumes collected in Sardinia. Interestingly, unlike the case of cultivated legumes, the recovery on plates of the rhizobial occupant could not be obtained under any of the conditions used, while at the same time a number of different endophytic taxa were rescued and their taxonomic identity was determined by 16S nucleotide sequencing. By direct PCR analysis from the nodule tissue, we were also able to show the presence of the nonculturable rhizobia inside the same nodules. In parallel, other two studies were conducted. AHL-mediated quorum sensing communication was quantified at single cell resolution trough a red-fluorescing AHLproducing and a green-fluorescing AHL-sensor strain in a 3-dimensional system by using computer-assisted microscopy (CMEIAS). The average effective "calling distance" from the single cell producer capable of inducing the gfp-tagged reporter cells, resulted 46.8 ?m. Moreover, in relation to the possible involvement of AHL signals in different phenotypes, a series of plant-interacting strains, among which Rhizobium leguminosarum, were tested for their ability to maintain viability in stressful situations (nutrient and oxygen limitations). Some of the tested strains lost culturability in different of the imposed conditions. However viable cells could be detected by staining microscope-based techniques (BacLight®, acridine orange and CTC), demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; none of the AHL Quorum Sensing signals tested was effective in promoting the transition to the VBNC state nor in recovering cells to culturability, suggesting that the two phenotypical frameworks of QS and VBNC do not share signalling paths.