Rosella Muresu

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.

A next-generation sequencing (NGS)-based taxonomic analysis was carried out on airborne bacteria sampled at ground level in two periods (May and September) and two opposite locations on the north–south axis of the island of Sardinia. Located in a central position of the Mediterranean basin, Sardinia constitutes a suitable outpost for revealing possible immigration of bacterial taxa during transcontinental particle discharge between Africa and Europe. With the aim of verifying relative effects of dust outbreaks, sampling period, and sampling site on the airborne bacterial community composition, we compared air collected during dust-carrying meteorological events to that coming from wind regimes not associated with long-distance particle lifting. Results indicated that (a) higher microbial diversity and richness (118 vs. 65 orders) and increased community evenness were observed in the campaign carried out in September in comparison to the one in May, irrespective of the place of collection and of the presence or absence of dust outbreaks; (b) during the period of standard wind regimes without transcontinental outbreaks, a synchronous concerted turnover of bacterial communities across distant locations of the same island, accompanied as mentioned by a parallel rise in bacterial diversity and community evenness, appears to have occurred; (c) changes in wind provenance could transiently change community composition in the locality placed on the coast facing the incoming wind but not in the one located at the opposite side of the island, and for this reason the community changes brought from dust outbreaks of African origin are observed only in the sampling station exposed to the south; (d) the same winds, once proceeding over land, appear to uplift bacteria belonging to a common core already present over the region, which dilute or replace those that were associated with the air coming from the sea or conveyed by the dust particulates, explaining the two prior points; and (e) the hierarchy of the variables tested in determining bacterial assemblages composition results is as follows: sampling period≫ongoing meteorological events>sampling location within the island.

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.

An NGS-based taxonomic analysis was carried out on airborne bacteria sampled at ground level in two periods (May and September) and two opposite localities on the North-South axis of the Sardinia Island. Located in a central position of the Mediterranean basin, Sardinia constitutes a suitable outpost to reveal possible immigration of bacterial taxa during transcontinental particle discharge between Africa and Europe. With the aim of verifying relative effects of dust outbreaks, sampling period and sampling site, on the airborne bacterial community composition, we compared air collected during dust-carrying meteorological events to that coming from wind regimes not associated to long-distance particle lifting. Results indicated that: (a) a higher microbial diversity (118 orders vs 65) and increased community evenness were observed in the campaign carried out in September in comparison to the one in May, irrespective of the place of collection and of the presence or absence of dust outbreaks. (b) During the period of standard wind regimes without transcontinental outbreaks a synchronous, concerted succession of bacterial communities across distant locations of the same island, accompanied as mentioned by a parallel rise in bacterial diversity and community evenness appears to have occurred. (c) changes in wind provenance could transiently change community composition in the locality placed on the coast facing the incoming wind, but not in the one located at the opposite side of the island; for this reason the community changes brought from dust outbreaks of African origin are observed only in the sampling station exposed to south; (d) the same winds, once proceeding over land appear to uplift bacteria belonging to a common core already present over the region, which dilute or replace those that were associated with the air coming from the sea or conveyed by the dust particulate, explaining the two prior points. (e) the hierarchy of the variables tested in determining bacterial assemblages composition results: sampling period >> ongoing meteorological events > sampling location within the island.

Endophytism within Vitis represents a topic of critical relevance due to the multiple standpoints from which it can be approached and considered. From the biological and botanical perspectives, the interaction between microorganisms and perennial woody plants falls within the category of stable relationships from which the plants can benefit in multiple ways. The life cycle of the host ensures persistence in all seasons, repeated chances of contact, and consequent microbiota accumulation over time, leading to potentially high diversity compared with that of herbaceous short-lived plants. Furthermore, grapevines are agriculturally exploited, highly selected germplasms where a profound man-driven footprint has indirectly and unconsciously shaped the inner microbiota through centuries of cultivation and breeding. Moreover, since endophyte metabolism can contribute to that of the plant host and its fruits’ biochemical composition, the nature of grapevine endophytic taxa identities, ecological attitudes, potential toxicity, and clinical relevance are aspects worthy of a thorough investigation. Can endophytic taxa efficiently defend grapevines by acting against pests or confer enough fitness to the plants to endure attacks? What are the underlying mechanisms that translate into this or other advantages in the hosting plant? Can endophytes partially redirect plant metabolism, and to what extent do they act by releasing active products? Is the inner microbial colonization necessary priming for a cascade of actions? Are there defined environmental conditions that can trigger the unleashing of key microbial phenotypes? What is the environmental role in providing the ground biodiversity by which the plant can recruit microsymbionts? How much and by what practices and strategies can these symbioses be managed, applied, and directed to achieve the goal of a better sustainable viticulture? By thoroughly reviewing the available literature in the field and critically examining the data and perspectives, the above issues are discussed.

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 IS123^T 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.

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.

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.