Elisa Polone

LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia

Parasponia uses a mycorrhizal signaling receptor essential for arbuscle formation to control rhizobium nodule symbiosis. Rhizobium–root nodule symbiosis is generally considered to be unique for legumes. However, there is one exception, and that is Parasponia. In this nonlegume, the rhizobial nodule symbiosis evolved independently and is, as in legumes, induced by rhizobium Nod factors. We used Parasponia andersonii to identify genetic constraints underlying evolution of Nod factor signaling. Part of the signaling cascade, downstream of Nod factor perception, has been recruited from the more-ancient arbuscular endomycorrhizal symbiosis. However, legume Nod factor receptors that activate this common signaling pathway are not essential for arbuscular endomycorrhizae. Here, we show that in Parasponia a single Nod factor–like receptor is indispensable for both symbiotic interactions. Therefore, we conclude that the Nod factor perception mechanism also is recruited from the widespread endomycorrhizal symbiosis.

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication

Cell-to-cell bacterial communication via diffusible signals is addressed and the conceptual framework in which quorum sensing is usually described is evaluated. By applying equations ruling the physical diffusion of the autoinducer molecules, one can calculate the gradient profiles that would occur either around a single cell or at the center of volumes of increasing size and increasing cell densities. Water-based matrices at 25 degrees C and viscous biofilms at colder temperatures are compared. Some basic consequences relevant for the field of microbial signalling arise. As regards induction, gradient-mixing dynamics between as little as two cells lying at a short distance appears to be sufficient for the buildup of a concentration reaching the known thresholds for quorum sensing. A straight line in which the highest concentrations occur is also created as a consequence of the gradient overlap geometry, providing an additional signal information potentially useful for chemotactic responses. In terms of whole population signalling, it is shown how the concentration perceived by a cell in the center is critically dependent not only on the cell density but also on the size of the biofilm itself. Tables and formulas for the practical prediction of N-acyl homoserine lactones concentrations at desired distances in different cell density biofilms are provided.

Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness.

The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.

Chemical elemental distribution and soil DNA fingerprints provide the critical evidence in murder case investigation

Background The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. Methodology/Principal Findings The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA). Conclusions Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

Characterization of endophytic and symbiotic bacteria within plants of the endemic association Centaureetum horridae Mol

Abstract We investigated the internal association of bacteria with Astragalus terraccianoi and Centaurea horrida, two endemic plants of the Mediterranean islands, forming the phytosociological association Centaureetum horridae, typical of windswept cliffs on the rocky shores of Asinara (Sardinia, Italy) and other limited locations. Sampling occurred in the protected natural park of the Asinara island. Roots and stems of the two plants and the root nodules of A. terraccianoi were surface sterilized in order to remove external and rhizospheric microbiota and to subsequently isolate the culturable bacterial communities. Plate counts revealed densities of endophytes between 3.7 × 102 and 2.8 × 104 colony forming units per gram of fresh weight. 16S rDNA sequencing revealed the occurrence of bacteria displaying high similarity with Actinobacterium sp., Paenibacillus sp., Rhizobium sp., Methylobacterium sp., Pedobacter panaciterrae, Aerococcus viridans, Stenotrophomonas rhizophila, Bacillus sporothermodurans, Bacillus pumilus, Bacillus simplex, Bacillus flexus, Streptomyces ciscaucasicus and Dyella sp. The putative nitrogen-fixing rhizobium symbiont of A. terraccianoi was identified for the first time. It turned out to belong to the slow-growing Bradyrhizobium genus and to share a 97% similarity with Bradyrhizobium canariense. It was found to be nonculturable and to coexist in nodules with a number of different endophytes.

Shovel roots: A unique stress-avoiding developmental strategy of the legume plant Hedysarum coronarium L

Hedysarum coronarium (sulla) is a legume native to the Mediterranean basin, known for its broad tolerance to various environmental stresses, and its ability to thrive without signs of chlorosis when growing in arid and alkaline soils up to pH 9.6. A unique but poorly known morphological feature of its root system is the production of “shovels”, modified lateral roots that acquire a curved and flattened shape. A combined structural and functional analysis was undertaken to define the nature and role of the shovel roots using various microscopy techniques, histochemical stains, STEM - energy dispersive X-ray microanalysis, infrared spectroscopy, and plant cultivation in different conditions. We found that sulla displays remarkable unique rhizosphere-buffering properties at both ends of the pH scale, and that shovels act as efficient calcium-absorbing organs that accumulate this cation intracellularly as insoluble crystalline salts. Such bioaccumulation results in a localized depletion of CaCO3 from the soil. As a consequence of this removal of the pivotal carbonate buffering system, the iron-solubilizing acidification activities of the roots can become effective. Further tests revealed that the factor triggering shovel development is exposure of roots to iron oxide. This signal, reporting at once both iron presence and alkalinity, assures the availability of iron nutrient reserves upon acidification of the local microenvironment surrounding the roots. These findings, besides casting light on a novel and unique botanical phenomenon, offer the potential to exploit sulla’s model and genes for the improvement of other crops to sustain productivity in a scenario of climate warming and increasing desertification.

Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules

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.