Search Results (37)

Vegetal sources are a continuous research field and different types of extracts have been obtained over time. The most challenging part is compounding them in a pharmaceutical product. This study aimed to integrate a mixture (EX) of four extracts (SE-Sophorae flos, GE-Ginkgo bilobae folium, ME-Meliloti herba, CE-Calendulae flos) in formulations with polymers (polyhydroxybutyrate, polylactic-co-glycolic acid) and their physicochemical profiling. The resulting samples consist of particle suspensions, which were subjected to Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy analysis. When compared to single-extract formulations spectra, they revealed band changes, depending on the complex interactions. Using X-ray Diffractometry, the partially crystalline phase was highlighted for EX-PLGA, while the others were amorphous. Moreover, Atomic Force Microscopy pointed out the nanoscale particles and the topography of the samples, and the outstanding roughness belonging to EX-PHB-PLGA. A 30 min period of immersion was enough for the formulations to spread on the surface of the compression stockings material (CS) and after drying, it became a polymeric film. TGA analysis was performed, which evaluated the impregnated content: 5.9% CS-EX-PHB, 6.4% CS-EX-PLGA, and 7.5% CS-EX-PHB-PLGA. In conclusion, the extract’s phytochemicals and the interactions established with the polymers or with the other extracts from the mixture have a significant impact on the physicochemical properties of the obtained formulations, which are particularly important in pharmaceutical product development. Full article

To investigate the precise and efficient symbiosis between Sinorhizobium meliloti LL2 and different alfalfa varieties, we conducted experiments using eight alfalfa varieties along with the S. meliloti LL2. Our objective was to identify highly effective symbiotic combinations by analyzing differences in nodulation, nitrogen fixation, and biomass accumulation. The results revealed that Gannong NO.9 had higher values for single effective root nodule weight (1.30 mg) and the number of infected cells in root nodules (2795) compared to other varieties (p < 0.05). Additionally, Gannong NO.9 exhibited the highest nitrogenase activity (0.91 μmol·g⁻¹·h⁻¹), nitrogen fixation percentage (67.16%), and amount of nitrogen fixation (18.80 mg/pot). Moreover, there was a significant 26.50% increase in aboveground tissue nitrogen accumulation compared to the control check (CK) (p < 0.05). Furthermore, underground tissue showed excellent values for nitrogen accumulation (35.68 mg/plant) and crude protein content (17.75%) when compared with other treatments. The growth of plants was demonstrated by the combined impact of nodulation and nitrogen fixation. The distribution of biomass after nitrogen fixation was compared to the control group (p < 0.05) to investigate accumulation. The eight combinations of symbiotic nitrogen fixation (SNF) were classified into six distinct types based on their significantly different biomass growth rates compared to CK. ① Aboveground accumulation type: Gannong NO.9 (there was a 24.31% increase in aboveground dry weight); ② aboveground and underground accumulation type: Qingshui (the aboveground dry weight increased by 135.94%, while the underground dry weight grew by 35.26%); ③ aboveground accumulation, underground depletion type: Gannong NO.5 ( ); ④ zero-growth type (there was no significant difference in dry weights, both above and below ground, compared to CK): WL168HQ, WL319HQ and Longzhong; ⑤ aboveground and underground depletion type: WL298HQ (the aboveground dry weight decreased by 29.29%, while the underground dry weight decreased by 20.23%); ⑥ underground depletion type: Gannong NO.3 (the underground dry weight showed a decrease of 34.49%); no type with aboveground consumption and underground accumulation was found. The study clarified the optimal combination of LL2 and Gannong NO.9, finding that biomass accumulation after symbiotic nitrogen fixation is variety-dependent. Full article

The western corn rootworm, Diabrotica virgifera virgifera, (Coleoptera: Chrysomelidae) is a serious pest of maize in the USA and Europe. Microbial plant biostimulants such as bacteria, fungi, and algae are designed to stimulate plant nutrition and growth, with some hypothesized to also possess insecticidal properties. We tested 10 biostimulants (four bacteria, five fungi, and one alga) under laboratory and greenhouse conditions. Most biostimulants did not affect the eggs, larvae, or adults of D.v. virgifera. However, in the laboratory, 10% of biostimulants improved egg hatching, and 40% killed some larvae, including the fungi Beauveria bassiana, Rhizophagus irregularis, and Trichoderma asperellum, and the bacterium Ensifer meliloti. Under potted-plant conditions in the greenhouse, these insecticidal effects were not detectable. However, several biostimulants slightly increased height and shoot length of uninfested maize plants, but reduced volume and length of their roots as well as above-ground biomass. Interestingly, 30% of the biostimulants enhanced the plant’s defence against larvae, for example, Bacillus amyloliquefaciens, B. subtilis, and E. meliloti. These may warrant further research into their modes of action as well as field trials to better understand and optimize their potential use in sustainable and integrated pest management. Full article

The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were “hot spots” of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration “hot spots”, not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial–phage coevolution. Full article

Biological nitrogen fixation by rhizobia-nodulated legumes reduces the dependence on synthetic nitrogen fertilizers. Identification of locally adapted rhizobia may uncover economically valuable strains for sustainable agriculture. This study investigated the diversity and symbiotic potential of rhizobia associated with three Medicago species from Eastern Romania’s ecosystems. Phenotypic screening ensured that only rhizobial species were retained for molecular characterization. 16S rDNA sequencing clustered the isolates into four distinct groups: Sinorhizobium meliloti, Sinorhizobium medicae, Rhizobium leguminosarum, and Mesorhizobium spp. The chromosomal genes (atpD, glnII, recA) and nifH phylogenies were congruent, while the nodA phylogeny grouped the Mesorhizobium spp. isolates with R. leguminosarum. Medicago sativa was the most sampled plant species, and only S. meliloti and R. leguminosarum were found in its nodules, while Medicago falcata nodules hosted S. meliloti and Mesorhizobium spp. Medicago lupulina was the only species that hosted all four identified rhizobial groups, including S. medicae. This study provides the first report on the Mesorhizobium spp. associated with M. falcata nodules. Additionally, R. leguminosarum and two Mesorhizobium genospecies were identified as novel symbionts for Medicago spp. Comparative analysis of Medicago-associated rhizobia from other studies revealed that differences in 16S rDNA sequence type composition were influenced by Medicago species identity rather than geographic region. Full article

Fatty acid and central carbon metabolism are crucial energy metabolism reactions. However, to date, few studies have examined their distribution characteristics within the alfalfa–rhizobia symbiotic system. To clarify the distributional differences and accumulation rates of fatty acids and central carbon with this system, we measured the plant phenotype, nodule formation, nitrogen fixation capacity, and key nitrogen metabolism enzyme activities of Medicago sativa ‘Gannong No. 9’ 35 days post-inoculation (dpi) with Sinorhizobia meliloti LL11. Additionally, we employed targeted metabolomics to analyze central carbon and fatty acid metabolites in various tissue samples of symbiotic and control (C.K.) plants, as well as in S. meliloti LL11. We found that plant height; root length; aboveground fresh and dry weights; underground fresh and dry weights; and nitrate reductase, nitrogen reductase, glutamine synthetase, and glutamate synthase activities were significantly higher in the leaves and roots of symbiotic plants than in those of C.K. plants. Compared to symbiotic plants, C.K. plants exhibited higher total central carbon and fatty acid metabolite content, accounting for 38.61% and 48.17% of C.K. plants, respectively. We detected 32 central carbon and 40 fatty acid metabolites in S. meliloti LL11, with succinate (343,180.8603 ng·mL⁻¹) and hexadecanoic acid (4889.7783 ng·mL⁻¹) being the most. In both symbiotic and C.K. plants, central carbon metabolite was considerably higher than the fatty acid metabolite central. Moreover, the carbon metabolites found in symbiotic plants were primarily distributed in pink nodule roots (PNRs), with malate exhibiting the highest content (4,800,612.3450 ng·g⁻¹), accounting for 53.09% of total central carbon metabolite content. Fatty acid metabolites were mainly found in pink root nodules (P.N.s), which are sites of nitrogen fixation. Trans-10-nonadecenoic acid and hexadecanoic acid exhibited the highest contents, comprising >15% of the total fatty acid metabolite content. We found that petroselaidic acid is only present in P.N., which seems to be closely related to the nitrogen fixation reaction in P.N. In general, symbiotic plants transfer central carbon metabolites to nodules via PNRs to drive nitrogen fixation. However, in P.N.s, these metabolites are limited, leading to accumulation in PNRs. Fatty acid metabolites, crucial for nitrogen fixation, are prevalent in P.N.s. Conversely, C.K. plants without nitrogen fixation distribute these metabolites primarily to the stems, emphasizing growth. This study provides new insights into the energy metabolism of symbiotic nitrogen fixation. Full article

Type IVc Pili (T4cP), also known as Tad or Flp pili, are long thin microbial filaments that are made up of small-sized pilins. These appendages serve different functions in bacteria, including attachment, biofilm formation, surface sensing, motility, and host colonization. Despite their relevant role in diverse microbial lifestyles, knowledge about T4cP in bacteria that establish symbiosis with legumes, collectively referred to as rhizobia, is still limited. Sinorhizobium meliloti contains two clusters of T4cP-related genes: flp-1 and flp-2, which are located on the chromosome and the pSymA megaplasmid, respectively. Bundle-forming pili associated with flp-1 are involved in the competitive nodulation of alfalfa plants, but the role of flp-2 remains elusive. In this work, we have performed a comprehensive bioinformatic analysis of T4cP genes in the highly competitive S. meliloti GR4 strain and investigated the role of its flp clusters in pilus biogenesis, motility, and in the interaction with alfalfa. Single and double flp-cluster mutants were constructed on the wild-type genetic background as well as in a flagellaless derivative strain. Our data demonstrate that both chromosomal and pSymA flp clusters are functional in pili biogenesis and contribute to surface translocation and nodule formation efficiency in GR4. In this strain, the presence of flp-1 in the absence of flp-2 reduces the competitiveness for nodule occupation. Full article

Commercial bacterial exopolysaccharide (EPS) applications have been gaining interest; therefore, strains that provide higher yields are required for industrial-scale processes. Succinoglycan (SG) is a type of bacterial anionic exopolysaccharide produced by Rhizobium, Agrobacterium, and other soil bacterial species. SG has been widely used as a pharmaceutical, cosmetic, and food additive based on its properties as a thickener, texture enhancer, emulsifier, stabilizer, and gelling agent. An SG-overproducing mutant strain (SMC1) was developed from Sinorhizobium meliloti 1021 through N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutation, and the physicochemical and rheological properties of SMC1-SG were analyzed. SMC1 produced (22.3 g/L) 3.65-fold more SG than did the wild type. Succinoglycan (SMC1-SG) overproduced by SMC1 was structurally characterized by FT-IR and ¹H NMR spectroscopy. The molecular weights of SG and SMC1-SG were 4.20 × 10⁵ and 4.80 × 10⁵ Da, respectively, as determined by GPC. Based on DSC and TGA, SMC1-SG exhibited a higher endothermic peak (90.9 °C) than that of SG (77.2 °C). Storage modulus (G′) and loss modulus (G″) measurements during heating and cooling showed that SMC1-SG had improved thermal behavior compared to that of SG, with intersections at 74.9 °C and 72.0 °C, respectively. The SMC1-SG′s viscosity reduction pattern was maintained even at high temperatures (65 °C). Gelation by metal cations was observed in Fe³⁺ and Cr³⁺ solutions for both SG and SMC1-SG. Antibacterial activities of SG and SMC1-SG against Escherichia coli and Staphylococcus aureus were also observed. Therefore, like SG, SMC1-SG may be a potential biomaterial for pharmaceutical, cosmetic, and food industries. Full article

Sustainable-forage production is globally increasing, especially in marginal areas where the edaphic conditions for plant growth are not optimal. Soil salinization influences the symbiotic interaction between alfalfa and rhizobia. The efficiency of different symbiotic pairs (Sinorhizobium meliloti—Medicago sativa) was evaluated in relation to NaCl application (100 mM) on two different alfalfa cultivars (Marina and Etrusca) and 21 S. meliloti strains isolated in Algeria. At 100 mM NaCl, it was observed that there was a higher variability of plant dry weight compared to the control. The strains able to improve plant growth at 100 mM NaCl were different and specific for each alfalfa cultivar, highlighting that (symbiont) G × (host) G interaction is magnified under stressed (saline) conditions (E). Three strains were then identified as candidate inoculants for M. sativa cv Marina and used for an in-field experiment with induced stress (no irrigation), together with S. meliloti GR4 (a highly competitive strain). In-field experiments, showed a high variability, and a significant difference of plant biomass was observed only for those inoculated with S. meliloti GR4. Obtained results suggest that multiple traits should be considered for inoculant-strain selection, and for an efficient translation from lab to field, it requires extensive comprehension of the mechanisms driving G × G × E interaction. Full article

Legume–rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant’s immune response to maintain numerous rhizobial endosymbiosis in nodules. Full article

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria. Full article

Sinorhizobium meliloti 1021 bacteria secretes a considerable amount of flavins (FLs) and can form a nitrogen-fixing symbiosis with legumes. This strain is also associated with non-legume plants. However, its role in plant growth promotion (PGP) of non-legumes is not well understood. The present study evaluated the growth and development of lettuce (Lactuca sativa) and kale (Brassica oleracea var. acephala) plants inoculated with S. meliloti 1021 (FL⁺) and its mutant 1021ΔribBA, with a limited ability to secrete FLs (FL⁻). The results from this study indicated that inoculation with 1021 significantly (p < 0.05) increased the lengths and surface areas of the roots and hypocotyls of the seedlings compared to 1021ΔribBA. The kale and lettuce seedlings recorded 19% and 14% increases in total root length, respectively, following inoculation with 1021 compared to 1021ΔribBA. A greenhouse study showed that plant growth, photosynthetic rate, and yield were improved by 1021 inoculation. Moreover, chlorophylls a and b, and total carotenoids were more significantly (p < 0.05) increased in kale plants associated with 1021 than non-inoculated plants. In kale, total phenolics and flavonoids were significantly (p < 0.05) increased by 6% and 23%, respectively, and in lettuce, the increments were 102% and 57%, respectively, following 1021 inoculation. Overall, bacterial-derived FLs enhanced kale and lettuce plant growth, physiological indices, and yield. Future investigation will use proteomic approaches combined with plant physiological responses to better understand host-plant responses to bacteria-derived FLs. Full article

Soil Sinorhizobium phage AP-16-3, a strain phylogenetically close to Rhizobium phage 16-3, was isolated in a mountainous region of Dagestan, belonging to the origin of cultivated plants in the Caucasus, according to Vavilov N.I. The genome of phage AP-16-3 is 61 kbp in size and contains 62 ORFs, of which 42 ORFs have homologues in the genome of Rhizobium phage 16-3, which was studied in the 1960s–1980s. A search for Rhizobium phage 16-3-related sequences was performed in the genomes of modern strains of root nodule bacteria belonging to different species, genera, and families. A total of 43 prophages of interest were identified out of 437 prophages found in the genomes of 42 strains, of which 31 belonged to Sinorhizobium meliloti species. However, almost all of the mentioned prophages contained single ORFs, and only two prophages contained 51 and 39 ORFs homologous to phages related to 16-3. These prophages were detected in S. meliloti NV1.1.1 and Rh. leguminosarum OyaliB strains belonging to different genera; however, the similarity level of these two prophages did not exceed 14.7%. Analysis of the orphan genes in these prophages showed that they encoded predominantly virion structural elements, but also enzymes and an extensive group of hypothetical proteins belonging to the L, S, and E regions of viral genes of phage 16-3. The data obtained indicate that temperate phages related to 16-3 had high infectivity against nodule bacteria and participated in intragenomic recombination events involving other phages, and in horizontal gene transfer between rhizobia of different genera. According to the data obtained, it is assumed that the repetitive lysogenic cycle of temperate bacteriophages promotes the dissolution of the phage genetic material in the host bacterial genome, and radical updating of phage and host bacterial genomes takes place. Full article

Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide–based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB. Full article

Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5 transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti. Full article