Mitigating pb toxicity in Sesbania sesban L. through activated charcoal supplementation: a hydroponic study on enhanced phytoremediation.

BACKGROUND: Soil contamination by heavy metals is a critical environmental challenge, with Pb being of particular concern due to its propensity to be readily absorbed and accumulated by plants, despite its lack of essential biological functions or beneficial roles in cellular metabolism. Within the scope of phytoremediation, the use of plants for the decontamination of various environmental matrices, the present study investigated the potential of activated charcoal (AC) to enhance the tolerance and mitigation capacity of S. sesban seedlings when exposed to Pb. The experiment was conducted as a factorial arrangement in a completely randomized design in hydroponic conditions. The S. sesban seedlings were subjected to a gradient of Pb concentrations (0, 0.02, 0.2, 2, and 10 mg/L) within the nutrient solution, alongside two distinct AC treatments (0 and 1% inclusion in the culture media). The study reached its conclusion after 60 days. RESULTS: The seedlings exposed to Pb without AC supplementation indicated an escalation in peroxidase (POX) activity, reactive oxygen species (ROS), and malondialdehyde (MDA) levels, signaling an increase in oxidative stress. Conversely, the incorporation of AC into the treatment regime markedly bolstered the antioxidative defense system, as evidenced by the significant elevation in antioxidant capacity and a concomitant reduction in the biomarkers of oxidative stress (POX, ROS, and MDA). CONCLUSIONS: With AC application, a notable improvement was observed in the chlorophyll a, total chlorophyll, and plant fresh and dry biomass. These findings illuminate the role of activated charcoal as a viable adjunct in phytoremediation strategies aimed at ameliorating heavy metal stress in plants.

Protein fraction from Sesbania virgata (Cav.) Pers. seeds exhibit antioxidant and antifungal activities.

Sesbania virgata (Cav.) Pers. seeds are protein sources with health and environmental benefits. In this research, proteins with lectin activity were identified in a protein fraction from S. virgata seeds (PFLA), as well its antioxidant and antimicrobial potentials, in addition to cytotoxic effects. To obtain PFLA, seed flour was homogenized in Glycine-NaOH (100 mM; pH 9.0; NaCl 150 mM) and precipitated in ammonium sulfate. PFLA concentrates bioactive lectins (32 HU/mL, 480 HU/gFa, 18.862 HU/mgP) and essential amino acids (13.36 g/100g protein). PFLA exerts antioxidant activity, acting as a promising metal chelating agent (~77% of activity). Analyzes of cell culture assay results suggest that antioxidant activity of PFLA may be associated with the recruitment of essential molecules to prevent the metabolic impairment of cells exposed to oxidative stress. PFLA (256 - 512 µg/mL) also exhibits antifungal activity, inhibiting the growth of Aspergillus flavus, Candida albicans, Candida tropicalis and Penicillium citrinum. Cytotoxic analysis indicates a tendency of low interference in the proliferation of 3T3 and HepG2 cells in the range of PFLA concentrations with biological activity. These findings support the notion that PFLA is a promising adjuvant to be applied in current policies on the management of metal ion chelation and fungal infections.

A Review on Medicinal and Ethnomedicinal Uses, Biological Features, and Phytochemical Constituents of Sesbania sesban L. Merr., A Nitrogen-Fixing Plant Native to the Republic of Chad.

This study reports on a literature review of the leguminous tree Sesbania sesban (L.) Merr which is found in the N'djamena region, the Republic of Chad. The study focused on S. sesban's medicinal and ethnomedicinal uses, biological features, and phytochemical constituents to assist in future evaluations. A literature review was conducted using academic websites, such as Science Direct and Springer, online international plant databases, and data from national herbaria. S. sesban is a perennial shrub or tree that measures 3-4 m in height. This species is becoming rare in N'djamena but can be found in the rainy season, while in winter, it occurs mainly in ponds (called the Chadian dialect "Bouta") and on the shores of the Chari and Logone rivers. The local inhabitants in Chad use the species as medicine, livestock feed, and fuelwood and for improving soil fertility and repelling desert encroachment. Traditional healers use its leaves to treat breast cancer and edema. S. sesban is an essential species native to the Republic of Chad that needs conservation and valorization. Viewing its importance and rarity in N'djamena , a strategy for replanting the species in gardens, homes, and fields around N'djamena and other regions of Chad is recommended.

The Impact of Inoculation of Two Strains of Rhizobacteria on Radionuclide Transfer in Sesbania Grandiflora.

Transfer factors of some naturally-occurring and artificial radionuclides from an agricultural soil to rhizobacteria-treated Sesbania grandiflora, a small leguminous tree, were studied. Two plant growth promoting rhizobacteria (PGPR) strains (SCR17 and PCE2) were used to carry out an agricultural experiment in pots in semi-arid region (Syria). The results showed the bacterial strain (SCR17) increased the transfer and accumulation of 238U and 40K in Sesbania grandiflora, while both bacterial strains showed no effect on the accumulation of 234Th, 226Ra, 210Po and 210Pb in the treated plants. The transfer factor of 137Cs from soil to rhizobacteria-treated Sesbania grandiflora was negligible. The values of the transfer factors of 234Th, 226Ra, 210Po and 40K were found to be within the global values, while the values of the transfer factors of 238U and 210Pb were found to be relatively higher. This study highlights the importance of using Phytoremediation by PGPR strains for radionuclides-contaminated soils. Therefore, this method is a promising technique for the restoration and rehabilitation of contaminated sites with radionuclides, as it is low cost, easy to apply, and environmentally friendly.

Effective inhibition of Sesbania grandiflora bioactive compounds against C-di-GMP phosphodiesterase of Pseudomonas aeruginosa.

Sesbania grandiflora also known as Agasthya has potent antibiofilm activity and its bioactive compounds obtained from the leaves are medicarpin, isoniazid and 4-methyl oxazole. Extra cellular polymeric substances (EPS) created by the bacterium involve the formation of biofilm and this causes the infections such as nosocomial infections, and urinary tract infections. Pseudomonas aeruginosa has been linked with high levels of intracellular Cyclic-di-Guanosine Monophosphate (c-di-GMP; PA4781) in biofilm formation. In this study, Human BLAST analysis of c-di-GMP Phosphodiesterase has been carried out and it shows an insignificant result and it is believed to be a possible drug target for UTI infection caused by P. aeruginosa. Its protein structure was retrieved from PDB database which was subjected to molecular docking against S. grandiflora bioactive compounds and control drug ciprofloxacin. Compounds taken for the study were screened for ADMET properties and drug-likeliness properties. Molecular interaction analysis of c-di-GMP with medicarpin compound shows -6.75 Kcal/mol binding energy with two hydrogen bonds when compared to the control drug with -6.86 kcal/mol binding energy and two hydrogen bonds respectively. Hence, our findings in the current study suggest that medicarpin could be an inhibitor of c-di-GMP and possess anti-biofilm activity, which could be validated experimentally.

Effect of stabilization time and soil chromium concentration on Sesbania virgata growth and metal tolerance.

Sesbania virgata is a pioneer shrub from the Fabaceae family, native to riparian environments in northeast of Argentina, southern of Brazil and Uruguay. In peri-urban riparian soils, metal contamination is a frequent problem, being its bioavailability partly determined by the stabilization time and frequency of contamination events. The effect of time elapsed between chromium (Cr) soil enrichment and plant seeding and Cr doses on S. virgata tolerance and metal absorption were evaluated. Treatments were developed by adding Cr (80-400 ppm) to the soil and allowing two days or fifteen months to elapse before sowing, and a control treatment without Cr addition. After 150 days from seeding, bioaccumulation and translocation factors, growth parameters (dry biomass and its aerial/radical allocation pattern, stem length and its elongation rate), morphological parameters (root volume and leaf area), and physiological parameters (chlorophyll content) of the specimens were determined. The emergence of S. virgata was inhibited since 150 ppm when Cr was added to the soil two days before seeding, with Cr accumulation in roots starting at 80 ppm (17.4 ± 2.5 mg kg-1). Under 15 months of metal stabilization, S. virgata plants survived across the entire range of Cr doses tested, with accumulation in roots since 100 ppm (35.5 ± 0.2 mg kg-1) and metal translocation to aerial tissues only under 400 ppm. The results obtained showed that S. virgata did not have high BCF and TF values, suggesting that it cannot be classified as bioaccumulator of Cr under the tested conditions. However, its presence in environments contaminated with Cr can be beneficial, as it helps to stabilize the metal in the soil.

Impacts of soil and water conservation measures on soil physicochemical properties in the Jibgedel Watershed, Ethiopia.

Soil erosion significantly affects agricultural production. Soil and Water Conservation (SWC) measures have been constructed to reduce soil loss. However, the impact of SWC measures on physicochemical soil properties has rarely been investigated in most parts of Ethiopia. Therefore, this study was designed to evaluate the effects of SWC measures on selected soil physicochemical properties in the Jibgedel watershed, West Gojjam zone, Ethiopia. The study also assessed the farmers' perception of the benefits and impacts of SWC measures. Composite and core soil samples were taken at a depth of 0 to 20 cm from four farmlands with SWC measures (soil bund, stone bund, and soil bund with sesbania tree) and without SWC measures in three replications. Results have shown that employing SWC measures in the farmland significantly improved most of the physicochemical properties of the soil compared to farmland without SWC measures. Bulk density from soil bund with and without sesbania trees was significantly lower than stone bund and untreated farmland. Soil organic carbon, total nitrogen, electrical conductivity, and available phosphorus from soil bund with sesbania tree were significantly higher than other treatments. The result also revealed that most farmers perceived that the implemented SWC measures improved soil fertility and crop yield. SWC measures are easier to adopt for integrated watershed management when farmers are well-versed in them.

Synergistic Flame Retardancy of Phosphatized Sesbania Gum/Ammonium Polyphosphate on Polylactic Acid.

Phosphating sesbania gum (DESG) was obtained by modifying sesbania gum (SG) with 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) and endic anhydride (EA). The structure of DESG was determined using Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H-NMR). Flame-retardant polylactic acid (PLA) composites were prepared by melt-blending PLA with DESG, which acted as a carbon source, and ammonium polyphosphate (APP), which acted as an acid source and a gas source. The flame retardancy of the PLA composite was investigated using vertical combustion (UL-94), the limiting oxygen index (LOI) and the cone calorimeter (CONE) test. Thermal properties and morphology were characterized via thermogravimetric analysis (TGA) and field emission scanning electron microscopy (FESEM), respectively. Experimental results indicated that when the mass ratio of DESG/APP was equal to 12/8 the LOI value was 32.2%; a vertical burning test (UL-94) V-0 rating was achieved. Meanwhile, the sample showed a lowest total heat release (THR) value of 52.7 MJ/m2, which is a 32.5% reduction compared to that of neat PLA. Using FESEM, the uniform distribution of DESG and APP in the PLA matrix was observed. The synergistic effect of DESG and APP effectively enhanced the flame retardancy of PLA. Additionally, the synergistic mechanism of DESG and APP in PLA was proposed.

Mechanism and application of Sesbania root-nodulating bacteria: an alternative for chemical fertilizers and sustainable development.

Chemical fertilizers are used in large-scale throughout the globe to satisfy the food and feed requirement of the world. Demanding cropping with the enhanced application of chemical fertilizers, linked with a decline in the recycling of natural or other waste materials, has led to a decrease in the organic carbon levels in soils, impaired soil physical properties and shrinking soil microbial biodiversity. Sustenance and improvement of soil fertility are fundamental for comprehensive food security and ecological sustainability. To feed the large-scale growing population, the role of biofertilizers and their study tends to be an essential aspect globally. In this review, we have emphasized the nitrogen-fixing plants of Sesbania species. It is a plant that is able to accumulate nitrogen-rich biomass and used as a green manure, which help in soil amelioration. Problems of soil infertility due to salinity, alkalinity and waterlogging could be alleviated through the use of biologically fixed nitrogen by Sesbania plants leading to the conversion of futile land into a fertile one. A group of plant growth-promoting rhizobacteria termed as "rhizobia" are able to nodulate a variety of legumes including Sesbania. The host-specific rhizobial strains can be used as potential alternative for nitrogenous fertilizers as they help the host plant in growth and development and enhance their endurance under stressed conditions. The review gives the depth understanding of how the agriculturally important microorganisms can be used for the reduction of broad-scale application of chemical fertilizers with special attention to Sesbania-nodulating rhizobia.

Mechanistic elucidation of germination potential and growth of Sesbania sesban seedlings with Bacillus anthracis PM21 under heavy metals stress: An in vitro study.

Soils contaminated with heavy metals such as Chromium (Cr) and Cadmium (Cd) severely impede plant growth. Several rhizospheric microorganisms support plant growth under heavy metal stress. In this study, Cr and Cd stress was applied to in vitro germinating seedlings of a Legume plant species, Sesbania sesban, and investigated the plant growth potential in presence and absence of Bacillus anthracis PM21 bacterial strain under heavy metal stress. The seedlings were exposed to different concentrations of Cr (25-75 mg/L) and Cd (100-200 mg/L) in Petri plates. Growth curve analysis of B. anthracis PM21 revealed its potential to adapt Cr and Cd stress. The bacteria supported plant growth by exhibiting ACC-deaminase activity (1.57-1.75 µM of α-ketobutyrate/h/mg protein), producing Indole-3-acetic acid (99-119 µM/mL) and exopolysaccharides (2.74-2.98 mg/mL), under heavy metal stress condition. Analysis of variance revealed significant differences in growth parameters between the seedlings with and without bacterial inoculation in metal stress condition. The combined Cr+Cd stress (75 + 200 mg/L) significantly reduced root length (70%), shoot length (24%), dry weight (54%) and fresh weight (57%) as compared to control. Conversely, B. anthracis PM21 inoculation to seedlings significantly increased (p ≤ 0.05) seed germination percentage (5%), root length (31%), shoot length (23%) and photosynthetic pigments (Chlorophyll a: 20%; Chlorophyll b: 16% and total chlorophyll: 18%), as compared to control seedlings without B. anthracis PM21 inoculation. The B. anthracis PM21 inoculation also enhanced activities of antioxidant enzymes, including superoxide dismutase (52%), peroxidase (66%), and catalase (21%), and decreased proline content (56%), electrolyte leakage (50%), and malondialdehyde concentration (46%) in seedlings. The B. anthracis PM21 inoculated seedlings of S. sesban exhibited significantly high (p ≤ 0.05) tissue deposition of Cr (17%) and Cd (16%) as compared to their control counterparts. Findings of the study suggested that B. anthracis PM21 endured metal stress through homeostasis of antioxidant activities, and positively impacted S. sesban growth and biomass. Further experiments in controlled conditions are necessary for investigating phytoremediation potential of S. sesban in metal-contaminated soils in presence of B. anthracis PM21 bacterial strain.

Effects of copper oxide nanoparticles on germination of Sesbania virgata (FABACEAE) plants.

Nanotechnology is a field that, over the years, has been growing in several research areas, such as medicine, agriculture and cosmetics, among others. As a result, there is a continuous increase in the production, use and disposal of these materials in the environment. The behaviour and (bio) activity of these materials in the atmosphere, water and soil are not fully studied. Therefore, it is necessary to carry out an analysis of the risks of contamination, as well as the possible effects and impacts of nanoparticles (NPs) on the ecosystem. In an attempt to investigate these effects on plants, the present study aimed to investigate the impact of copper oxide nanoparticles (CuO NPs) on the seed germination process of Sesbania virgata. For this, the Sesbania virgata seeds were subjected to different concentration of CuO NPs (0, 100, 200, 300 and 400 mgL-1) and their germination and development were monitored by optical analysis (thermography and chlorophyll a fluorescence). The results show that the CuO NPs induced a reduction on the maximum emission of chlorophyll a, which was concentration-dependent. The data also showed that CuO NPs promoted an increase in the energy dissipated by non-photochemical pathways and the surface temperature of the seeds. Additionally, our findings revealed that CuO NPs caused a root growth inhibition. In summary, the present study demonstrates, for the first time, that CuO NPs can negatively affect the physiological status and development of the S. virgata plant, by altering the efficiency of the functioning of photosystem II in its initial developmental stage, depending on the concentration of CuO NPs.

Fungicidal Activity of Recombinant Javanicin against Cryptococcus neoformans Is Associated with Intracellular Target(s) Involved in Carbohydrate and Energy Metabolic Processes.

The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.

Effect of feeding Sesbania sesban and reed grass on growth performance, blood parameters, and meat quality of growing lambs.

This study was conducted to evaluate the effects of green fodders, fresh Sesbania sesban, and reed grass, on growth performance, blood metabolites, and meat quality of growing lambs. Twenty-one male Farafra lambs (19.0 ± 1.87 kg body weight) were randomly distributed into three groups, each with seven animals. The first group was fed on 60% concentrates and 40% fresh alfalfa, and served as a control. The second and third groups were fed also on 60% concentrates plus 40% fresh Sesbania sesban and reed grass, respectively. Lambs were fed the diets until reaching the target slaughter weight of about 50.0 kg, which was obtained after a period of 215 days. Five lambs from each group were randomly selected and slaughtered at the end of the experiment. Slaughter body weight, carcass traits weight, and dressing percentage were estimated. Chemical composition and physical properties of longissimus dorsi muscle, from the animals' right side, were determined. It was found that no significant (P > 0.05) differences among groups in body weight, average daily gain, feed conversion rate, hot carcass, dressing percentage, and carcass components were observed. The forage intake of Sesbania and alfalfa tended to be higher than that of reed forage. There were no significant (P > 0.05) differences in the blood parameters among the treatments, except the urea level which was higher in Sesbania sesban group than the reed one. However, the serum urea concentration in Sesbania and reed groups was similar to that of alfalfa. Sesbania sesban and reed forages decreased (P < 0.05) the eye muscle area and shear force in lambs when compared to the control group. The longissimus dorsi muscle of the Sesbania and reed treatments was found to be numerically higher in the moisture and lower in the fat and collagen contents than the alfalfa group. Most of meat and fat color parameters were improved by feeding of Sesbania and reed forages. In conclusion, Sesbania sesban or reed grass, as unconventional forage sources, can be used as alternatives of the commonly used alfalfa feed. Moreover, these sources could enhance the meat quality of lambs and increasing the tenderness with accepted meat and fat color. So, it can be recommended to use Sesbania or reed grass forages in diets of sheep, to overcome the rising in the price or shortage in the availability of traditional green fodders.

Crude saponin extract from Sesbania grandiflora (L.) Pers pod meal could modulate ruminal fermentation, and protein utilization, as well as mitigate methane production.

The aim of the study was to conduct a basic evaluation of the in vitro effect of crude protein (CP) levels in concentrate and a saponin extract from Sesbania graniflora pods meal (SES) on the kinetics of gas, nutrient digestibility, ruminal fermentation, protein efficiency uses, and methane (CH4) mitigation. Eight treatments were formed according to a 2 × 4 factorial design in a completely randomized design (CRD). The first factor referred to the levels of CP at 14 and 16% on dry matter (DM) basis in the concentrate diet, and the second factor referred to the levels of SES supplementation at 0, 0.2, 0.4, and 0.6% of the total substrate on a DM basis. The results showed that S. graniflora pod meal contained 21.73% CP, 10.87% condensed tannins, and 16.20% crude saponins, respectively. Most kinetics of gas as well as cumulative gas were not influenced by the CP levels or SES addition (P > 0.05) except gas production from immediately soluble fraction (a) was significantly different by CP levels. Ammonia-nitrogen concentration of incubation at 4 h was significantly difference based on the CP levels and SES supplementation (P < 0.05). Increasing SES levels significantly (P < 0.05) decreased protozoal population. In vitro digestibility of DM and organic matter was not changed by CP levels or SES addition. Butyrate and acetate to propionate ration were decreased, and propionate was increased when increasing SES dose (P < 0.05), while CP levels did not change total volatile fatty acids and molar portions. The ruminal CH4 concentration was reduced by 44.12% when 0.6% SES was added after 8 h of incubation. Therefore, SES supplementation could enhance protein utilization and improve rumen fermentation particularly lowering CH4 production.

LuxR-Type Regulator AclR1 of Azorhizobium caulinodans Regulates Cyclic di-GMP and Numerous Phenotypes in Free-Living and Symbiotic States.

LuxR-type regulators play important roles in transcriptional regulation in bacteria and control various biological processes. A genome sequence analysis showed the existence of seven LuxR-type regulators in Azorhizobium caulinodans ORS571, an important nitrogen-fixing bacterium in both its free-living state and in symbiosis with its host, Sesbania rostrata. However, the functional mechanisms of these regulators remain unclear. In this study, we identified a LuxR-type regulator that contains a cheY-homologous receiver (REC) domain in its N terminus and designated it AclR1. Interestingly, phylogenetic analysis revealed that AclR1 exhibited relatively close evolutionary relationships with MalT/GerE/FixJ/NarL family proteins. Functional analysis of an aclR1 deletion mutant (ΔaclR1) in the free-living state showed that AclR1 positively regulated cell motility and flocculation but negatively regulated exopolysaccharide production, biofilm formation, and second messenger cyclic diguanylate (c-di-GMP)-related gene expression. In the symbiotic state, the ΔaclR1 mutant was defective in competitive colonization and nodulation on host plants. These results suggested that AclR1 could provide bacteria with the ability to compete effectively for symbiotic nodulation. Overall, our results show that the REC-LuxR-type regulator AclR1 regulates numerous phenotypes both in the free-living state and during host plant symbiosis.

CRISPR/Cas9 genome editing shows the important role of AZC_2928 gene in nitrogen-fixing bacteria of plants.

AZC_2928 gene (GenBank accession no. BAF88926.1) of Azorhizobium caulinodans ORS571 has sequence homology to 2,3-aminomutases. However, its function is unknown. In this study, we are for the first time to knock out the gene completely in A. caulinodans ORS571 using the current advanced genome editing tool, CRISPR/Cas9. Our results show that the editing efficiency is 34% and AZC_2928 plays an extremely important role in regulating the formation of chemotaxis and biofilm. CRISPR/Cas9 knockout of AZC_2928 (△AZC_2928) significantly enhanced chemotaxis and biofilm formation. Both chemotaxis and biofilm formation play an important role in nitrogen-fixing bacteria and their interaction with their host plants. Interestingly, AZC_2928 did not affect the motility of A. caulinodans ORS571 and the nodulation formation in their natural host plant, Sesbania rostrata. Due to rhizobia needing to form bacteroids for symbiotic nitrogen fixation in mature nodules, AZC_2928 might have a direct influence on nitrogen fixation efficiency rather than the number of nodulations.

CheY1 and CheY2 of Azorhizobium caulinodans ORS571 Regulate Chemotaxis and Competitive Colonization with the Host Plant.

The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions.IMPORTANCEAzorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

Diverse Genomic Backgrounds Vs. Highly Conserved Symbiotic Genes in Sesbania-Nodulating Bacteria: Shaping of the Rhizobial Community by Host and Soil Properties.

Aiming at investigating the overall diversity, biogeography, and symbiosis gene evolutionary history of the Sesbania cannabina-nodulating rhizobia in China, a total of 874 rhizobial isolates originating from the root nodules of this plant grown at different sites were characterized and compared with those of some reference strains. All of the S. cannabina-nodulating rhizobia were classified into 16 (geno) species, including seven novel genospecies in the genera Ensifer, Rhizobium, Neorhizobium, and Agrobacterium, with Ensifer sesbaniae and Neorhizobium huautlense as the dominant and universal species. Ten of these species were found to nodulate other leguminous hosts or to lack nodulating abilities and were defined as symbiovar sesbania. Biogeographic patterns were observed, for which pH, TN, AK, and AP were the main determinants. The effects of pH were opposite to those of TN and AK, while AP presented effects independently of TN, AK, and pH. Symbiotic genes of these rhizobia showed a common origin, but nodA evolved faster than nifH. Point mutation is the main driving force in the evolution of both nodA and nifH, and lateral transfer of symbiotic genes might play an important role in the formation of diverse S. cannabina-nodulating rhizobial species. S. cannabina only nodulates with Sesbania rhizobia, demonstrating its severe selection on rhizobial symbiosis genes. Soil pH and physiochemical characteristics could affect rhizobial survival and competitive nodulation. This study provides insight into the community shifts and evolution of rhizobia in relation to their host and soil environments.

Analysis of host protein interactions in plant viruses: an in silico study using Sesbania mosaic virus.

The dynamics of interactions of viral proteins with their host are pivotal in establishing a successful infection and ensuring systemic spread. To uncover these, an in silico analysis of the interactions between the coat protein (CP) of Sesbania mosaic virus (SeMV), a group IV virus with single-stranded positive-sense RNA genome was carried out with the known crystal structures of proteins belonging to the Fabaceae family, which is its natural host. SeMV is an isometric plant virus which infects Sesbania grandiflora, a member of Fabaceae, and causes mosaic symptoms. Earlier results have indicated that the assembly and disassembly events of SeMV favor the formation of CP dimers. Hence, the ability and strength of interactions of CP dimer with the host proteins were assessed using in silico protein-protein docking approaches. A set of 61 unique crystal structures of native proteins belonging to Fabaceae were downloaded from the Protein Data Bank (PDB) and docked with the CP dimer of SeMV. From the docking scores and interaction analysis, the host proteins were ranked according to their strength and significance of interactions with the CP dimers. The leads that were identified present themselves as strong candidates for developing antivirals against not only SeMV but also other related viruses that infect Fabaceae. The study is a prototype to understand host protein interactions in viruses and hosts.

Azorhizobium caulinodans c-di-GMP phosphodiesterase Chp1 involved in motility, EPS production, and nodulation of the host plant.

Establishment of the rhizobia-legume symbiosis is usually accompanied by hydrogen peroxide (H2O2) production by the legume host at the site of infection, a process detrimental to rhizobia. In Azorhizobium caulinodans ORS571, deletion of chp1, a gene encoding c-di-GMP phosphodiesterase, led to increased resistance against H2O2 and to elevated nodulation efficiency on its legume host Sesbania rostrata. Three domains were identified in the Chp1: a PAS domain, a degenerate GGDEF domain, and an EAL domain. An in vitro enzymatic activity assay showed that the degenerate GGDEF domain of Chp1 did not have diguanylate cyclase activity. The phosphodiesterase activity of Chp1 was attributed to its EAL domain which could hydrolyse c-di-GMP into pGpG. The PAS domain functioned as a regulatory domain by sensing oxygen. Deletion of Chp1 resulted in increased intracellular c-di-GMP level, decreased motility, increased aggregation, and increased EPS (extracellular polysaccharide) production. H2O2-sensitivity assay showed that increased EPS production could provide ORS571 with resistance against H2O2. Thus, the elevated nodulation efficiency of the ∆chp1 mutant could be correlated with a protective role of EPS in the nodulation process. These data suggest that c-di-GMP may modulate the A. caulinodans-S. rostrata nodulation process by regulating the production of EPS which could protect rhizobia against H2O2.