Rosella Muresu

Gamma proteobacteria can nodulate legumes of the genus Hedysarum.

The bacteria hosted in the root nodules of the three Mediterranean wild legume species Hedysarum carnosum, Hedysarum spinosissimum subsp. capitatum, and Hedysarum pallidum, growing in native stands in different habitats in Algeria were isolated. Bacteria were recovered on yeast-mannitol-agar or on minimal media from a total of 52 nodules. Isolates were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) using the enzyme CfoI, and further sorted by RAPD fingerprinting. A total of ten different types were found and their amplified 16S rDNA was sequenced and compared to databases. The BLAST alignment indicates that all the species whose sequences share 98 to 100% identity to the bacteria found in these nodules belong to the class Gammaproteobacteria and include Pantoea agglomerans, Enterobacter kobei, Enterobacter cloacae, Leclercia adecarboxylata, Escherichia vulneris, and Pseudomonas sp. No evidence of any rhizobial-like sequence was found even upon amplifying from the bulk of microbial cells obtained from the squashed nodules, suggesting that the exclusive occupants of the nodules formed by the three plants tested are members of the orders Enterobacteriales or Pseudomonadales. This is the first report of Gammaproteobacteria associated with legume nodules. Despite the presence of the related crop plant Hedysarum coronarium, specifically nodulated by Rhizobium sullae, these three Hedysarum species demonstrate to have undergone a separate path in terms of endophytic interactions with bacteria. An hypothesis to account for differences between the symbiotic relationships engaged by man-managed legumes, and those found in plants whose ecology is independent from human action, is discussed.

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.

Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco

A total of 274 bacterial strains were isolated from the root nodules of Prosopis juliflora, growing in two arid soils of the eastern area of Morocco. A physiological plate screening allowed the selection of 15 strains that could tolerate NaCl concentrations between 175 and 500 mM. These were compared with 15 strains chosen from among the ones which did not tolerate high salinity. The diversity of strains was first assessed by rep-PCR amplification fingerprinting using BOXA1R and ERIC primers. An analysis of the PCR-amplified 16S rDNA gene digestion profiles using five endonucleases indicated the presence of different lineages among the taxa associated with P. juliflora nodules in the soils studied. Nucleotide sequencing of the small subunit rRNA gene and BLAST analysis showed that P. juliflora could host at least six bacterial species in this region and that the identity of those associated with high salt tolerance was clearly distinct from that of the salt-sensitive ones. Among the former, the first type displayed 99% similarity with different members of the genus Sinorhizobium, the second 97% similarity with species within the genus Rhizobium, while the third ribosomal type had 100% homology to Achromobacter xylosoxidans. Within the salt-sensitive isolates the prevailing type observed showed 98% similarity with Rhizobium multihospitium and R. tropici, a second type had 98% similarity to R. giardinii, and a further case displayed 97% colinearity with the Ensifer group including E. maghrebium and E. xericitae. All of the thirty strains encompassing these types re-nodulated P. juliflora in microbiologically controlled conditions and all of them were shown to possess a copy of the nodC gene. This is the first report detecting the betaproteobacterial genus Achromobacter as nodule-forming species for legumes. The observed variability in symbiont species and the abundance of nodulation-proficient strains is in line with the observation that the plant always appears to be nodulated and efficiently fixing nitrogen in spite of a wide range of soil and environmental conditions.

Bacteria colonizing root nodules of wild legumes exhibit virulence-associated properties of mammalian pathogens

Bacteria not proficient in nitrogen fixing symbiosis were proven able to invade root nodules of three wild legumes of the genus Hedysarum in Algeria and to be multiplying in these in place of the natural rhizobium symbionts. The involved species featured taxa known as human pathogens including: Enterobacter cloacae, Enterobacter kobei, Escherichia vulneris, Pantoea agglomerans and Leclercia adecarboxylata. A direct screening of the phenotypic determinants of virulence using human cultured cells tested positive for the traits of cytotoxicity, vital stain exclusion and adhesion to epithelia. Antibiogram analyses revealed also a complex pattern of multiple antibiotic resistances. The data suggest that legume root nodules can be a site of survival and of active multiplication for populations of mammalian pathogens, which could thus alternate between the target animal and a number of neutral plant hosts. The worldwide distribution of as yet uninvestigated legumes raises the concern that these represent a general niche that could enhance the hazards posed by microorganisms of clinical nature.

Microbial immigration across the Mediterranean via airborne dust.

Dust particles lifting and discharge from Africa to Europe is a recurring phenomenon linked to air circulation conditions. The possibility that microorganisms are conveyed across distances entails important consequences in terms of biosafety and pathogens spread. Using culture independent DNA-based analyses via next generation sequencing of the 16 S genes from the airborne metagenome, the atmospheric microbial community was characterized and the hypothesis was tested that shifts in species diversity could be recorded in relation to dust discharge. As sampling ground the island of Sardinia was chosen, being an ideal cornerstone within the Mediterranean and a crossroad of wind circulation amidst Europe and Africa. Samples were collected in two opposite coastal sites and in two different weather conditions comparing dust-conveying winds from Africa with a control situation with winds from Europe. A major conserved core microbiome was evidenced but increases in species richness and presence of specific taxa were nevertheless observed in relation to each wind regime. Taxa which can feature strains with clinical implications were also detected. The approach is reported as a recommended model monitoring procedure for early warning alerts in frameworks of biosafety against natural spread of clinical microbiota across countries as well as to prevent bacteriological warfare.

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.

Colutea arborescens is nodulated by diverse rhizobia in Eastern Morocco

Eighteen isolates of rhizobia isolated from root nodules of Colutea arborescens (Bladder senna) grown in different soils of the eastern area of Morocco were characterized by phenotypic and genomic analyses. All the isolates characterized were fast growers. This is may be due to the isolation procedures used. The phenotypic, symbiotic and cultural characteristics analyzed allowed the description of a wide physiological diversity among tested isolates. The results obtained suggest that the phenotype of these rhizobia might have convergent evolved to adapt the local conditions. The genetic characterization consisted in an analysis of the rep-PCR fingerprints and the PCR-based RFLP of the 16S rDNA patterns. The 16S rDNA of six isolates representing the main ribotypes obtained by the PCR-based RFLP was sequenced. A large diversity was observed among these rhizobia, and they were classified as different species of the genera Rhizobium, Sinorhizobium and Mesorhizobium. The nodC gene was also sequenced, and the results confirmed the three lineages corresponding to the three genera. The results of the sequencing of nodC and 16S rDNA genes suggest that the nodulation genes and chromosome might have co-evolved among these bacteria.

Associative effects of poor-quality forages combined with food industry byproducts determined in vitro with an automated gas-production system

Thisexperiment aimedtoinvestigatetheassociativeeffectsamongtwolow-qualityforages(crowndaisy,milk thistle) and three agro-industrial byproducts (apple pomace, citrus pulp, tomato peel), by means of an automated gas production(GP) system.All feedswere incubated alone or as 50:50 mixtures of eachforagewitheach byproduct.Samples (0.500 � 0.0010g)ofsinglefeedsormixtureswereincubatedfor96h,inthreereplicatesinindividualbottles(310mL),with 75 mLof buffered rumen fluid. Bottles were ventedby anopen-close valve whenthe internal pressure reached 3.4kPa.The metabolisable energy content of single feeds and mixtures was computed from GP at 24 h and feed chemical composition. Feed substrates were ranked for GP in the following way: byproducts, mixtures, and forages. The two forages did not differ for GP and metabolisable energy content, although differences were observed among byproducts and among mixtures. Both forages interacted positively with apple pomace from 6 h (P < 0.001) to 24 h (P= 0.029) of incubation and with citrus pulp at 12 h (P = 0.005) and 24 h (P = 0.012), whereas no associative effects were detected when forages were incubated with tomato peels. Results suggest that in vitro fermentability of low-quality forages could be efficiently improved by combining these two forages with apple pomace or citrus pulp. These findings are relevant, because the use of low-quality forages and byproducts in ruminant feeding is considered important for improving the environmental and economic sustainability of forage systems in arid and semi-arid areas.

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.

Draft Genome Sequence of the Nitrogen-Fixing Rhizobium sullae Type Strain IS123T Focusing on the Key Genes for Symbiosis with its Host Hedysarum coronarium L.

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.