Quantificazione dell'azotofissazione della sulla in Sardegna: azoto fissato nella fitomassa epigea ed effetto dell'inoculazione

An important and economic source of nitrogen for Mediterranean crop and forage systems arises from symbiotic nitrogen fixation, which contributes also to a high environmental protection and sustainability. This papers refers on the quantification of nitrogen fixation in sweetvetch, by the isotopic dilution and nitrogen balance methods. In addition, the effect of sweetvetch inoculation with a Sardinian strain of Rhizobium sullae was evaluated against an uninoculated treatment. The potential of nitrogen fixation in sweetvetch, estimated by the isotopic dilution method, resulted high, reaching 180 kg ha-1 of nitrogen fixed in the biennium. The nitrogen balance method underestimated the nitrogen fixation compared to the isotopic dilution. Very low differences between inoculated and uninoculated sweetvetch were due to a presence of resident rhizobia in the soil. This result outlines the need of evaluating where inoculation is necessary or useless

Nodule-associated microbiome diversity in wild populations of Sulla coronaria reveals clues on the relative importance of culturable rhizobial symbionts and co-infecting endophytes

Abstract The culturable bacteria from root nodules of Sulla coronaria growing in spontaneous conditions in Sardinia were characterized. This plant's peculiarity is to represent a legume still found in both wild and cropped statuses. We tested whether culturable bacteria would differ from those commonly isolated from its field-cropped varieties, to date exclusively represented by Rhizobium sullae. 63 isolates from 60 surface-sterilized nodules were analyzed by ARDRA and 16S rDNA sequencing. The official nitrogen-fixing symbiont Rhizobium sullae was found only in 25 nodules out of 60. The remaining nodules did not yield culturable rhizobia but a number of different endophytic genera including Pseudomonas sp. (17 nodules), Microbacterium sp. (15 nodules), Pantoea agglomerans (5 nodules). The situation appears therefore a hybrid between what is commonly observed in other Mediterranean legumes occurring only in wild status (featuring non-culturable rhizobia and arrays of culturable endophytes within nodules), as opposed to cropped legumes (endowed with fully culturable rhizobia and minimal endophytic occurrence). These findings, within a species bridging the ecology between native and cropped conditions, suggest insights on the relative importance of endophytic co-occupancy vs. true N-fixing symbiont culturability within nodules. The latter trait thus appears to accompany the domestication path of plants with a main trade-off of renouncing to interactions with a diversity of endophytic co-invaders; the relationships with those being critical in the non-cropped status. In fact, endophytes are known to promote plant growth in harsh conditions, which can be particularly stressful in the Mediterranean due to drought, highly calcareous soils, and pathogens outbreaks

Pangenomics of the Symbiotic Rhizobiales. Core and Accessory Functions Across a Group Endowed with High Levels of Genomic Plasticity

Pangenome analyses reveal major clues on evolutionary instances and critical genome core conservation. The order Rhizobiales encompasses several families with rather disparate ecological attitudes. Among them, Rhizobiaceae, Bradyrhizobiaceae, Phyllobacteriacreae and Xanthobacteriaceae, include members proficient in mutualistic symbioses with plants based on the bacterial conversion of N2 into ammonia (nitrogen-fixation). The pangenome of 12 nitrogen-fixing plant symbionts of the Rhizobiales was analyzed yielding total 37,364 loci, with a core genome constituting 700 genes. The percentage of core genes averaged 10.2% over single genomes, and between 5% to 7% were found to be plasmid-associated. The comparison between a representative reference genome and the core genome subset, showed the core genome highly enriched in genes for macromolecule metabolism, ribosomal constituents and overall translation machinery, while membrane/periplasm-associated genes, and transport domains resulted under-represented. The analysis of protein functions revealed that between 1.7% and 4.9% of core proteins could putatively have different functions.This work was supported in part by grant “Progetto di Ateneo PRAT CPDA154841/15” from the University of Padova

Draft genome sequence of the nitrogen-fixing Rhizobium sullae type strain IS123^T focusing on the key genes for symbiosis with its host Hedysarum coronarium L.

© 2017 Sablok, Rosselli, Seeman, van Velzen, Polone, Giacomini, La Porta, Geurts, Muresu and Squartini. The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments

PCR primers based on different portions of insertion elements can assist genetic relatedness studies, strain fingerprinting and species identification in rhizobia

Using the sequence of an insertion element originally found in Rhizobium sullae, the nitrogen-fixing bacterial symbiont of the legume Hedysarum coronarium, we devised three primer pairs (inbound, outbound and internal primers) for the following applications: (a) tracing genetic relatedness within rhizobia using a method independent of ribosomal inheritance, based on the presence and conservation of IS elements; (b) achieve sensitive and reproducible bacterial fingerprinting; (c) enable a fast and unambiguous detection of rhizobia at the species level. In terms of taxonomy, while in line with part of the 16S rRNA gene- and glutamine synthetase I-based clustering, the tools appeared nonetheless more coherent with the actual geographical ranges of origin of rhizobial species, strengthening the European–Mediterranean connections and discerning them from the asian and american taxa. The fingerprinting performance of the outward-pointing primers, designed upon the inverted repeats, was shown to be at least as sensitive as BOX PCR, and to be functional on a universal basis with all 13 bacterial species tested. The primers designed on the internal part of the transposase gene instead proved highly species-specific for R. sullae, enabling selective distinction from its most related species, and testing positive on every R. sullae strain examined, fulfilling the need of PCR-mediated species identification. A general use of other IS elements for a combined approach to rhizobial taxonomy and ecology is proposed

Nitrogen fixation of sulla under Mediterranean conditions

Sulla coronaria (L.) B.H. Choi & H. Ohashi (=Hedysarum coronarium L.) is a Mediterranean forage legume grown extensively in rainfed Italian environments. Here we report the field quantification of its seasonal and annual N fixation, using the 15N isotopic dilution (ID) method, the influence of various nonfixing reference species on these estimates, and the allocation of fixed N between the aerial and the belowground biomass. Parallel experiments were conducted over two biennial crop cycles at two Italian locations (Sassari and Ancona), characterized by sandy-loam to heavy clay-loam calcareous soils. The proportion of nitrogen derived from the atmosphere (%Ndfa) present in the aerial biomass reached 78.2% at Sassari and 82.7% at Ancona, with no significant effect of the identity of the nonfixing reference species. At Sassari, the amount of fixed N present in the aerial dry matter (DM) of sulla was 187 kg ha–1 in the first crop cycle, and 265 kg ha–1 in the second, while the equivalent N yields at Ancona were 350 and 170 kg ha–1. The relationship between the quantity of fixed N in the aerial biomass and the aerial DM yield of sulla was significant and not site-specific, realizing an equivalence of about 1.8 kg ha–1 of fixed N per 100 kg ha–1 of aerial DM. The relative contribution of the root biomass to the amount of fixed N was on average 4%. The N balance of sulla at the end of the second crop cycle was negative, but the N sparing effect compared with the nonfixing species ranged from 65 to 175 kg ha–1

Legumes of the Sardinia Island: Knowledge on Symbiotic and Endophytic Bacteria and Interactive Software Tool for Plant Species Determination

A meta-analysis was carried out on published literature covering the topic of interactive plant microbiology for botanical species of legumes occurring within the boundary of the Italian island Sardinia, lying between the Tyrrhenian and the western Mediterranean seas. Reports were screened for the description of three types of bacterial occurrences; namely, (a) the nitrogen-fixing symbionts dwelling in root nodules; (b) other bacteria co-hosted in nodules but having the ancillary nature of endophytes; (c) other endophytes isolated from different non-nodular portions of the legume plants. For 105 plant species or subspecies, over a total of 290 valid taxonomical descriptions of bacteria belonging to either one or more of these three categories were found, yielding 85 taxa of symbionts, 142 taxa of endophytes in nodules, and 33 in other plant parts. The most frequent cases were within the Medicago, Trifolium, Lotus, Phaseolus, and Vicia genera, the majority of symbionts belonged to the Rhizobium, Mesorhizobium, Bradyrhizobium, and Sinorhizobium taxa. Both nodular and extra-nodular endophytes were highly represented by Gammaproteobacteria (Pseudomonas, Enterobacter, Pantoea) and Firmicutes (Bacillus, Paenibacillus), along with a surprisingly high diversity of the Actinobacteria genus Micromonospora. The most plant-promiscuous bacteria were Sinorhizobium meliloti as symbiont and Bacillus megaterium as endophyte. In addition to the microbial analyses we introduce a practical user-friendly software tool for plant taxonomy determination working in a Microsoft Excel spreadsheet that we have purposely elaborated for the classification of legume species of Sardinia. Its principle is based on subtractive keys that progressively filter off the plants that do not comply with the observed features, eventually leaving only the name of the specimen under examination

Use of antioxidant treatments to enhance culturability of rhizobial and non-rhizobial endophytes from nodules and other plant tissues

When rescuing either symbionts or other endophytic bacteria from internal plant tissues, including root nodules, and attempting their isolation and culturing on laboratory media, failure to obtain colonies is often experienced. This phenomenon applies particularly to the rhizobia from wild legume species which frequently result in non-culturable status (1), possibly as a consequence of a biochemical stress inherent to the isolation procedure. Other taxa can co-occupy nodules along with rhizobia, mostly belonging to the Bacillaceae, Pseudomonadaceae and Enterobacteriaceae families, which appear much less sensitive to the putative stress as their isolation from nodules, is apparently not impaired. We hypothesized that the limited culturability of rhizobia, compared to that of other types of nodule invaders, could be related to an oxidative stress caused by the histological disruption of plant tissues when surface-sterilized nodules are squashed for bacterial isolation. In order to test this hypothesis we isolated bacteria from nodules of two mediterranean wild legumes (Hedysarum spinosissimum and Tetragonolobus purpureus) by squashing nodules either in control phosphate buffer saline (PBS) or in PBS supplemented with scavenging systems apt to prevent damage from reactive oxygen species. The treatments included: an antioxidant cocktail (Glutathione, Sodium Ascorbate and EDTA) or an enzyme cocktail (Catalase, Peroxidase, Superoxide Dismutase). The mixtures were tested either as modified squashing buffers or as additions to the plates on which bacteria were spread after squashing in control PBS. The latter option allowed to distinguish whether the oxidative stress were due to immediate damage from plant-derived reactions or as a consequence of the transfer in carbon-rich agar media from the plate culturing stage. Some of the combinations yielded orders of magnitude-different increases of culturability in terms of colony counts and also allowed the cultivation of otherwise undetectable taxa. The coexistence of up to three different rhizobiaceae within the same nodules was observed, two of which of supposedly non-symbiotic nature (unable to re-induce nodule formation in axenic nodulation tests). Isolates were subsequently tested for tolerance to H2O2 in liquid cultures, which showed differences in comparison with the levels displayed by non-alphaproteobacterial endophytes. Ecological and physiological implications are discussed

Molecular probe for identification of <i>Trichomonas vaginalis</i> DNA

Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic observation of vaginal discharge, cell culture, and immunological techniques. A 2.3-kb Trichomonas vaginalis DNA fragment present in strains from diverse geographic areas was cloned and used as a probe to detect T. vaginalis DNA in vaginal discharge by a dot blot hybridization technique. This probe was specific for T. vaginalis DNA. It recognized strains from two regions in Italy (Sardinia, Piemonte) and from Mozambique (Africa). In addition, our probe did not cross-react with bacterial (Escherichia coli, Enterococcus spp., group B streptococci, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Lactobacillus spp.), viral (herpes simplex virus type 2), fungal (Candida albicans), protozoan (Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Leishmania major, and Leishmania infantum), or human nucleic acids. The probe reacted with Pentatrichomonas hominis and Trichomonas foetus. The limit signal recognized by our probe corresponded to the DNA of 200 T. vaginalis isolates. The 2.3-kb probe was used in a clinical analysis of 98 samples. Of these, 20 samples were found to be positive both with the probe and by cell culture, and only 14 of these were positive by a standard wet mount method