Effect of Wastewater on the Composition of Bacterial Microbiota of Phragmites australis Used in Constructed Wetlands for Phytodepuration.

Phytodepuration occurs in the plant-mediated remediation processes exploited to remove pollutants from wastewater, and Phragmites australis is one of the most used plants. This goal is achieved using constructed wetlands (CW), which are engineered systems designed to mimic the natural processes of pollutants removal. The aim of this work was to characterize the bacterial communities associated to P. australis, soils, and permeates of the CW of Calice (Prato, Italy), to evaluate the possible effect of wastewaters on the CW bacterial communities, through a next-generation sequencing-based approach. A total of 122 samples were collected from different tissues of P. australis (i.e., roots, aerial parts, and stem), soil (i.e., rhizospheric and bulk soil), and permeates, and analyzed. All samples were collected during five sampling campaigns, with the first one performed before the activation of the plant. Obtained results highlighted a specific microbiota of P. australis, conserved among the different plant tissues and during time, showing a lower alpha diversity than the other samples and not influenced by the more complex and variable environmental (soils and permeates) bacterial communities. These data suggest that P. australis is able to select and maintain a defined microbiota, a capacity that could allow the plant to survive in hostile environments, such as that of CW.

Effect of Site and Phenological Status on the Potato Bacterial Rhizomicrobiota.

The potato is the fourth major food crop in the world. Its cultivation can encounter problems, resulting in poor growth and reduced yield. Plant microbiota has shown an ability to increase growth and resistance. However, in the development of effective microbiota manipulation strategies, it is essential to know the effect of environmental variables on microbiota composition and function. Here, we aimed to identify the differential impact of the site of cultivation and plant growth stage on potato rhizosphere microbiota. We performed a 16S rRNA gene amplicon sequencing analysis of rhizospheric soil collected from potato plants grown at four sites in central Italy during two phenological stages. Rhizomicrobiota was mainly composed of members of phyla Acidobacteriota, Actinobacteriota, Chloroflexi, and Proteobacteria and was affected by both the site of cultivation and the plant stages. However, cultivation sites overcome the effect of plant phenological stages. The PiCRUST analysis suggested a high abundance of functions related to the biosynthesis of the siderophore enterobactin. The presence of site-specific taxa and functional profiling of the microbiota could be further exploited in long-term studies to evaluate the possibility of developing biomarkers for traceability of the products and to exploit plant growth-promoting abilities in the native potato microbiota.

Synthetic plant microbiota challenges in nonmodel species.

Plant-associated microbiota are becoming central in the development of ways to improve plant productivity and health. However, most research has focussed mainly on a few model plant species. It is essential to translate discoveries to the many nonmodel crops, allowing the design and application of effective synthetic microbiota.

Differential Response of Wheat Rhizosphere Bacterial Community to Plant Variety and Fertilization.

The taxonomic assemblage and functions of the plant bacterial community are strongly influenced by soil and host plant genotype. Crop breeding, especially after the massive use of nitrogen fertilizers which led to varieties responding better to nitrogen fertilization, has implicitly modified the ability of the plant root to recruit an effective bacterial community. Among the priorities for harnessing the plant bacterial community, plant genotype-by-microbiome interactions are stirring attention. Here, we analyzed the effect of plant variety and fertilization on the rhizosphere bacterial community. In particular, we clarified the presence in the bacterial community of a varietal effect of N and P fertilization treatment. 16S rRNA gene amplicon sequence analysis of rhizospheric soil, collected from four wheat varieties grown under four N-P fertilization regimes, and quantification of functional bacterial genes involved in the nitrogen cycle (nifH; amoA; nirK and nosZ) were performed. Results showed that variety played the most important role and that treatments did not affect either bacterial community diversity or bacterial phyla abundance. Variety-specific response of rhizosphere bacterial community was detected, both in relation to taxa (Nitrospira) and metabolic functions. In particular, the changes related to amino acid and aerobic metabolism and abundance of genes involved in the nitrogen cycle (amoA and nosZ), suggested that plant variety may lead to functional changes in the cycling of the plant-assimilable nitrogen.

Scent of a Symbiont: The Personalized Genetic Relationships of Rhizobium-Plant Interaction.

Many molecular signals are exchanged between rhizobia and host legume plants, some of which are crucial for symbiosis to take place, while others are modifiers of the interaction, which have great importance in the competition with the soil microbiota and in the genotype-specific perception of host plants. Here, we review recent findings on strain-specific and host genotype-specific interactions between rhizobia and legumes, discussing the molecular actors (genes, gene products and metabolites) which play a role in the establishment of symbiosis, and highlighting the need for research including the other components of the soil (micro)biota, which could be crucial in developing rational-based strategies for bioinoculants and synthetic communities' assemblage.

DNA Methylation in Ensifer Species during Free-Living Growth and during Nitrogen-Fixing Symbiosis with Medicago spp.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Here, we explored DNA methylation in bacteria of the genus Ensifer, including its potential role in regulating terminal differentiation during nitrogen-fixing symbiosis with legumes. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cell cultures, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent of methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. Based on these observations, we suggest that CcrM activity is dysregulated and constitutive during terminal differentiation, which we hypothesize is a driving factor for endoreduplication of terminally differentiated bacteroids. IMPORTANCE Nitrogen fixation by rhizobia in symbiosis with legumes is economically and ecologically important. The symbiosis can involve a complex bacterial transformation-terminal differentiation-that includes major shifts in the transcriptome and cell cycle. Epigenetic regulation is an important regulatory mechanism in diverse bacteria; however, the roles of DNA methylation in rhizobia and symbiotic nitrogen fixation have been poorly investigated. We show that aside from cell cycle regulation, DNA methyltransferases are unlikely to have conserved roles in the biology of bacteria of the genus Ensifer. However, we present evidence consistent with an interpretation that the cell cycle methyltransferase CcrM is dysregulated during symbiosis, which we hypothesize may be a key factor driving the cell cycle switch in terminal differentiation required for effective symbioses.

Crossing Bacterial Genomic Features and Methylation Patterns with MeStudio: An Epigenomic Analysis Tool.

DNA methylation is one of the most observed epigenetic modifications. It is present in eukaryotes and prokaryotes and is related to several biological phenomena, including gene flow and adaptation to environmental conditions. The widespread use of third-generation sequencing technologies allows direct and easy detection of genome-wide methylation profiles, offering increasing opportunities to understand and exploit the epigenomic landscape of individuals and populations. Here, we present a pipeline named MeStudio, with the aim of analyzing and combining genome-wide methylation profiles with genomic features. Outputs report the presence of DNA methylation in coding sequences (CDSs) and noncoding sequences, including both intergenic sequences and sequences upstream of the CDS. We apply this novel tool, showing the usage and performance of MeStudio, on a set of single-molecule real-time sequencing outputs from strains of the bacterial species Sinorhizobium meliloti.

Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

Endophytes from African Rice (Oryza glaberrima L.) Efficiently Colonize Asian Rice (Oryza sativa L.) Stimulating the Activity of Its Antioxidant Enzymes and Increasing the Content of Nitrogen, Carbon, and Chlorophyll.

Bacterial endophytes support the adaptation of host plants to harsh environments. In this study, culturable bacterial endophytes were isolated from the African rice Oryza glaberrima L., which is well-adapted to grow with poor external inputs in the tropical region of Mali. Among these, six N-fixer strains were used to inoculate O. glaberrima RAM133 and the Asian rice O. sativa L. cv. Baldo, selected for growth in temperate climates. The colonization efficiency and the N-fixing activity were evaluated and compared for the two rice varieties. Oryza sativa-inoculated plants showed a fairly good colonization efficiency and nitrogenase activity. The inoculation of Oryza sativa with the strains Klebsiella pasteurii BDA134-6 and Phytobacter diazotrophicus BDA59-3 led to the highest nitrogenase activity. In addition, the inoculation of 'Baldo' plants with the strain P. diazotrophicus BDA59-3 led to a significant increase in nitrogen, carbon and chlorophyll content. Finally, 'Baldo' plants inoculated with Kl. pasteurii BDA134-6 showed the induction of antioxidant enzymes activity and the maintenance of nitrogen-fixation under salt stress as compared to the unstressed controls. As these endophytes efficiently colonize high-yielding crop varieties grown in cold temperate climates, they become good candidates to promote their growth under unfavorable conditions.

Nonadditive Transcriptomic Signatures of Genotype-by-Genotype Interactions during the Initiation of Plant-Rhizobium Symbiosis.

Rhizobia are ecologically important, facultative plant-symbiotic microbes. In nature, there is a large variability in the association of rhizobial strains and host plants of the same species. Here, we evaluated whether plant and rhizobial genotypes influence the initial transcriptional response of rhizobium following perception of a host plant. RNA sequencing of the model rhizobium Sinorhizobium meliloti exposed to root exudates or luteolin (an inducer of nod genes, involved in the early steps of symbiotic interaction) was performed on a combination of three S. meliloti strains and three alfalfa varieties as host plants. The response to root exudates involved hundreds of changes in the rhizobium transcriptome. Of the differentially expressed genes, 35% were influenced by the strain genotype, 16% were influenced by the plant genotype, and 29% were influenced by strain-by-host plant genotype interactions. We also examined the response of a hybrid S. meliloti strain in which the symbiotic megaplasmid (∼20% of the genome) was mobilized between two of the above-mentioned strains. Dozens of genes were upregulated in the hybrid strain, indicative of nonadditive variation in the transcriptome. In conclusion, this study demonstrated that transcriptional responses of rhizobia upon perception of legumes are influenced by the genotypes of both symbiotic partners and their interaction, suggesting a wide spectrum of genetic determinants involved in the phenotypic variation of plant-rhizobium symbiosis.IMPORTANCE A sustainable way for meeting the need of an increased global food demand should be based on a holobiont perspective, viewing crop plants as intimately associated with their microbiome, which helps improve plant nutrition, tolerance to pests, and adverse climate conditions. However, the genetic repertoire needed for efficient association with plants by the microbial symbionts is still poorly understood. The rhizobia are an exemplary model of facultative plant symbiotic microbes. Here, we evaluated whether genotype-by-genotype interactions could be identified in the initial transcriptional response of rhizobium perception of a host plant. We performed an RNA sequencing study to analyze the transcriptomes of different rhizobial strains elicited by root exudates of three alfalfa varieties as a proxy of an early step of the symbiotic interaction. The results indicated strain- and plant variety-dependent variability in the observed transcriptional changes, providing fundamentally novel insights into the genetic basis of rhizobium-plant interactions. Our results provide genetic insights and perspective to aid in the exploitation of natural rhizobium variation for improvement of legume growth in agricultural ecosystems.

Symbiotic and Nonsymbiotic Members of the Genus Ensifer (syn. Sinorhizobium) Are Separated into Two Clades Based on Comparative Genomics and High-Throughput Phenotyping.

Rhizobium-legume symbioses serve as paradigmatic examples for the study of mutualism evolution. The genus Ensifer (syn. Sinorhizobium) contains diverse plant-associated bacteria, a subset of which can fix nitrogen in symbiosis with legumes. To gain insights into the evolution of symbiotic nitrogen fixation (SNF), and interkingdom mutualisms more generally, we performed extensive phenotypic, genomic, and phylogenetic analyses of the genus Ensifer. The data suggest that SNF likely emerged several times within the genus Ensifer through independent horizontal gene transfer events. Yet, the majority (105 of 106) of the Ensifer strains with the nodABC and nifHDK nodulation and nitrogen fixation genes were found within a single, monophyletic clade. Comparative genomics highlighted several differences between the "symbiotic" and "nonsymbiotic" clades, including divergences in their pangenome content. Additionally, strains of the symbiotic clade carried 325 fewer genes, on average, and appeared to have fewer rRNA operons than strains of the nonsymbiotic clade. Initial characterization of a subset of ten Ensifer strains identified several putative phenotypic differences between the clades. Tested strains of the nonsymbiotic clade could catabolize 25% more carbon sources, on average, than strains of the symbiotic clade, and they were better able to grow in LB medium and tolerate alkaline conditions. On the other hand, the tested strains of the symbiotic clade were better able to tolerate heat stress and acidic conditions. We suggest that these data support the division of the genus Ensifer into two main subgroups, as well as the hypothesis that pre-existing genetic features are required to facilitate the evolution of SNF in bacteria.