Grape seed extract prevents chlorpyrifos-induced toxicity in rat liver through the modulation of phase I detoxification pathway.

Chlorpyrifos (CPF) poisoning is a public health problem for which there is not currently any effective prophylaxis. In this study, we investigated the protective effect of grape seed extract (GSE) against CPF-induced hepatotoxicity. Rats were daily treated either with CPF (2 mg/kg) or CPF and GSE (20 mg/kg) for 1 week, sacrificed, and their livers dissected for biochemical, molecular, and histopathological analyses. CPF generated liver dysfunction by altering carbohydrate, lipid, amino acid, ammonia and urea metabolism, and provoked mitochondrial impairment through disturbing tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and mitochondrial viability. CPF also induced cholinergic excitotoxicity along with oxidative stress and histopathological alterations. Interestingly, treatment with GSE prevented all the detrimental effects of CPF through the regulation of cytochrome P450 (CYP450) gene expression. Molecular docking analysis indicated that GSE-containing polyphenols acted as epigenetic modulators through inhibiting DNA (cytosine-5)-methyltransferase 1 (DNMT1), thus favoring the CYP2C6 detoxification pathway. Thereby, GSE might be a promising strategy in the protection of the liver against CPF toxicity.

Tetraclinis articulata essential oil emulsion use as alternative to chemical fungicide to control tomato grey mould disease.

Tetraclinis articulata essential oil proved to be effective in controlling tomato grey mould, so we would investigate its effect on some tomato defense mechanisms. The pretreatment of Botrytis cinerea infected tomato plants with TAEO emulsion enhanced the activity of antioxidant enzymes activity SOD, CAT, APX, and GPX, and total polyphenols content and it decreased IC50 of free radical-scavenging activity and H2O2 content. Results showed amelioration in antioxidant status in TAEO emulsion treated and B. cinerea infected plants indicating that treatment decreased infection in tomato plants. The qRT-PCR analysis of defense genes expression Chitinase SlChi, transcription factors SlWRKY and SlAP2/ERF, Lipoxygenase SlLOX, and Thioredoxin SlTRX showed that they were up-regulated as early as 12 hpi sustained with a second increase at 48 hpi in TAEO emulsion pretreated and infected plants. These results suggest the potential use of TAEO emulsion as natural product to induce tomato antioxidant status and activate defense genes.

Date Palm Waste Compost Application Increases Soil Microbial Community Diversity in a Cropping Barley (Hordeum vulgare L.) Field.

Application of date palm waste compost is quite beneficial in improving soil properties and crop growth. However, the effect of its application on soil microbial communities is less understood. High-throughput sequencing and quantitative real-time PCR (qPCR) were used to evaluate the effect of compost application on the soil microbial composition in a barley field during the tillering, booting and ripening stages. The results showed that compost treatment had the highest bacterial and fungal abundance, and its application significantly altered the richness (Chao1 index) and α-diversity (Shannon index) of fungal and bacterial communities. The dominant bacterial phyla found in the samples were Proteobacteria and Actinobacteria while the dominant fungal orders were Ascomycota and Mortierellomycota. Interestingly, compost enriched the relative abundance of beneficial microorganisms such as Chaetomium, Actinobacteriota, Talaromyces and Mortierella and reduced those of harmful microorganisms such as Alternaria, Aspergillus and Neocosmospora. Functional prediction based on Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) showed that amplicon sequence variant (ASV) sequences related to energy metabolism, amino acid metabolism and carbohydrate metabolism were associated with compost-treated soil. Based on Fungi Functional Guild (FUNGuild), identified fungi community metabolic functions such as wood saprotroph, pathotroph, symbiotroph and endophyte were associated with compost-treated soil. Overall, compost addition could be considered as a sustainable practice for establishing a healthy soil microbiome and subsequently improving the soil quality and barley crop production.

Effects of Date Palm Waste Compost Application on Root Proteome Changes of Barley (Hordeum vulgare L.).

Proteomic analysis was performed to investigate the differentially abundant proteins (DAPs) in barley roots during the tillering stage. Bioinformatic tools were used to interpret the biological function, the pathway analysis and the visualisation of the network amongst the identified proteins. A total of 72 DAPs (33 upregulated and 39 downregulated) among a total of 2580 proteins were identified in response to compost treatment, suggesting multiple pathways of primary and secondary metabolism, such as carbohydrates and energy metabolism, phenylpropanoid pathway, glycolysis pathway, protein synthesis and degradation, redox homeostasis, RNA processing, stress response, cytoskeleton organisation, and phytohormone metabolic pathways. The expression of DAPs was further validated by qRT-PCR. The effects on barley plant development, such as the promotion of root growth and biomass increase, were associated with a change in energy metabolism and protein synthesis. The activation of enzymes involved in redox homeostasis and the regulation of stress response proteins suggest a protective effect of compost, consequently improving barley growth and stress acclimation through the reduction of the environmental impact of productive agriculture. Overall, these results may facilitate a better understanding of the molecular mechanism of compost-promoted plant growth and provide valuable information for the identification of critical genes/proteins in barley as potential targets of compost.

Antioxidant, antibacterial, and antileishmanial potential of Micromeria nervosa extracts and molecular mechanism of action of the bioactive compound.

AIMS: This study aimed to determine the antibacterial and antileishmanial potential of Micromeria nervosa extracts. The identification of the antileishmanial compound and the study of its molecular mechanism of action have also been undertaken. METHODS AND RESULTS: Ethanol extract showed high polyphenol content and diethyl ether extract exhibited high DPPH scavenging and low beta-carotene bleaching activity (IC50 = 13.04 ± 0.99 and 200.18 ± 3.32 µg mL-1, respectively). However, diethyl ether extract displayed high antibacterial activity against Gram-positive strains including methicillin-resistant Staphylococcus aureus (MIC = 31.25 µg mL-1), Staph. aureus ATCC6538 (MIC = 62.5 µg mL-1), and Listeria monocytogenes ATCC 19115 (MIC = 125 µg mL-1), as well as high antileishmanial activity against the promastigote forms of L. infantum and L. major (IC50 = 11.45 and 14.53 µg mL-1, respectively). The active compound was purified using bioassay-guided fractionation and thin layer chromatography, and identified as ursolic acid using high-performance liquid chromatography coupled with a photodiode array and mass spectrometry. The purified compound was strongly inhibitory against the promastigote and amastigote forms of L. infantum and L. major (IC50 = 5.87 and 6.95 µg mL-1 versus 9.56 and 10. 68 µg mL-1, respectively) without overt cytotoxicity against Raw 264.7 macrophage cells (SI = 13.53 and 11.43, respectively). The commercial compound (ursolic acid) showed similar activity against amastigotes and promastigotes forms of L. infantum and L. major. Moreover, its molecular mode of action against leishmaniasis seems to involve the expression of the ODC and SPS genes involved in thiol pathway. CONCLUSION: Extracts of M. nervosa can be considered as a potential alternative to antimicrobial and antileishmanial drugs.

Plant growth promoting rhizobacteria modulates the antioxidant defense and the expression of stress-responsive genes providing Pb accumulation and tolerance of grass pea.

To ensure the success of phytoremediation, it is important to consider the appropriate combination of plants and microorganisms. This study was conducted to get a better insight into the underlying molecular and biochemical mechanism of grass pea (Lathyrus sativus L.) induced by plant growth promoting rhizobacteria (PGPR), when exposed for 3, 6, 9, and 14 days to 1 mM Pb in a hydroponic system. The significant positive effect of bacterial inoculation was reproduced in various parameters. Results indicated that inoculation of PGPR significantly increased the accumulation of Pb by 20%, 66%, 43%, and 36% in roots and by 46%, 55%, 37%, and 46% in shoots, respectively after 3, 6, 9, and 14 days of metal exposure compared to the uninoculated plants. The metal accumulation in grass pea plants triggered a significant elevation in the synthesis of non-protein thiols (NPT), particularly in inoculated plant leaves where it was about 3 and 2-fold higher than the uninoculated set on the 6th and the 9th day. Nevertheless, Pb treatment significantly increased oxidative stress and membrane damage in leaves with the highest hydrogen peroxide (H2O2) production and tissue malondialdehyde (MDA) concentration recorded in uninoculated plants. Furthermore, the PGPR inoculation alleviated the oxidative stress, improved significantly plant tolerance, and modulated the activities of antioxidant enzymes (SOD, CAT, APX, GR, DHAR, and MDHAR). Similarly, the expression patterns of LsPCS, LsGCN, LsCNGC, LsGR, and LsGST through qRT-PCR demonstrated that bacterial inoculation significantly induced gene expression levels in leaves 6 days after Pb treatment, indicating that PGPR act as regulators of stress-responsive genes. The findings suggest the key role of PGPR (R. leguminosarum (M5) + Pseudomonas fluorescens (K23) + Luteibacter sp. + Variovorax sp.) in enhancing Pb accumulation, reducing metal toxicity, strengthening of the antioxidant system, and conferring higher Pb tolerance to grass pea plants. Hence, the association Lathyrus sativus-PGPR is an effective tool to achieve the goal of remediation of Pb contaminated sites.

Proteomic Analysis of Barley (Hordeum vulgare L.) Leaves in Response to Date Palm Waste Compost Application.

Composts are an emerging biofertilizers used in agronomy that can improve crop performance, but much less is known regarding their modes of action. The current study aimed to investigate the differentially abundant proteins (DAPs) in barley leaves associated with growth promotion induced by application of date palm waste compost. Morphophysiological measurements revealed that compost induced a significant increase in plant height, chlorophyll content, gas exchange parameters and plant biomass. LC-MS/MS analyses indicate that compost induced global changes in the proteome of barley leaves. A total of 62 DAPs (26 upregulated and 36 downregulated) among a total of 2233 proteins were identified in response to compost application. The expression of DAPs was further validated based on qRT-PCR. Compost application showed altered abundance of several proteins related to abiotic stress, plant defense, redox homeostasis, transport, tricarboxylic acid cycle, carbohydrate, amino acid, energy and protein metabolism. Furthermore, proteins related to metabolic processes of phytohormone, DNA methylation and secondary metabolites were induced. These results indicate that barley responds to compost application by complex metabolism pathways and may result in a positive alteration in a physiological and metabolic barley plant state which consequently could lead to improved growth and stress adaptation observed in compost-treated plants.

Effects of Salt Stress on Transcriptional and Physiological Responses in Barley Leaves with Contrasting Salt Tolerance.

Salt stress negatively impacts crop production worldwide. Genetic diversity among barley (Hordeum vulgare) landraces adapted to adverse conditions should provide a valuable reservoir of tolerance genes for breeding programs. To identify molecular and biochemical differences between barley genotypes, transcriptomic and antioxidant enzyme profiles along with several morpho-physiological features were compared between salt-tolerant (Boulifa) and salt-sensitive (Testour) genotypes subjected to salt stress. Decreases in biomass, photosynthetic parameters, and relative water content were low in Boulifa compared to Testour. Boulifa had better antioxidant protection against salt stress than Testour, with greater antioxidant enzymes activities including catalase, superoxide dismutase, and guaiacol peroxidase. Transcriptome assembly for both genotypes revealed greater accumulation of differentially expressed transcripts in Testour compared to Boulifa, emphasizing the elevated transcriptional response in Testour following salt exposure. Various salt-responsive genes, including the antioxidant catalase 3, the osmoprotectant betaine aldehyde dehydrogenase 2, and the transcription factors MYB20 and MYB41, were induced only in Boulifa. By contrast, several genes associated with photosystems I and II, and light receptor chlorophylls A and B, were more repressed in Testour. Co-expression network analysis identified specific gene modules correlating with differences in genotypes and morpho-physiological traits. Overall, salinity-induced differential transcript accumulation underlies the differential morpho-physiological response in both genotypes and could be important for breeding salt tolerance in barley.

Exogenous application of spermidine mitigates the adverse effects of drought stress in faba bean (Vicia faba L.).

In Tunisia, drought stress is a major environmental factor limiting crop production and causing relatively low and unstable faba bean yields. In the present study, we explored the putative role of spermidine (0.5, 1, 1.5 and 2mM) in ameliorating the effects of drought stress induced by polyethylene glycol (PEG-6000, -0.58MPa) in faba bean seedlings. Drought stress reduced photosynthetic performance, chlorophyll and relative water content in leaves of faba bean variety Badii. Moreover, drought increased proline, electrolyte leakage and malondialdehyde content by inducing reactive oxygen species (hydrogen peroxide) generation in leaves. However, applying spermidine increased the activities of catalase, superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase. The results show that the application of spermidine especially at a rate of 1.5mM effectively reduces oxidative damage and alleviates negative effects caused by drought stress. In addition, exogenous spermidine increased the expression of polyamine biosynthetic enzymes' genes (VfADC , VfSAMDC and VfSPDS ), and reduced the expression of VfSPMS suggesting that exogenous spermidine can regulate polyamines' metabolic status under drought challenge, and consequently may enhance drought stress tolerance in faba bean. Real-time quantitative polymerase chain reaction analysis revealed that some drought responsive genes (VfNAC , VfHSP , VfNCED , VfLEA , VfCAT , VfAPX , VfRD22 , VfMYB , VfDHN , VfERF , VfSOD and VfWRKY ) from various metabolic pathways were differentially expressed under drought stress. Overall, these genes were more abundantly transcribed in the spermidine-treated plants compared to untreated suggesting an important role of spermidine in modulating faba bean drought stress response and tolerance.

Antibacterial and antibiofilm activity of Peganum harmala seed extract against multidrug-resistant Pseudomonas aeruginosa pathogenic isolates and molecular mechanism of action.

Biofilm formation of the opportunistic pathogen Pseudomonas (P). aeruginosa is one of the major global challenges to control nosocomial infections due to their high resistance to antimicrobials and host defense mechanisms. The present study aimed to assess the antibacterial and the antibiofilm activities of Peganum (P). harmala seed extract against multidrug-resistant P. aeruginosa isolates. Chemical identification of the active compound and determination of its molecular mechanism of action were also investigated. Results showed that P. harmala n-butanol "n-BuOH" extract exhibited antibacterial activity against multidrug-resistant P. aeruginosa isolates. This extract was even more active than conventional antibiotics cefazolin and vaamox when tested against three P. aeruginosa multidrug-resistant isolates. In addition, P. harmala n-BuOH extract exhibited potent bactericidal activity against PAO1 strain at MIC value corresponding to 500 µg/mL and attained 100% killing effect at 24 h of incubation. Furthermore, P. harmala n-BuOH extract showed an antibiofilm activity against P. aeruginosa PAO1 and exhibited 80.43% inhibition at sub-inhibitory concentration. The extract also eradicated 83.99% of the biofilm-forming bacteria. The active compound was identified by gas chromatography-mass spectrometry as an indole alkaloid harmaline. Transcriptomic analysis showed complete inhibition of the biofilm-related gene pilA when PAO1 cells were treated with harmaline. Our results revealed that P. harmala seed extract and its active compound harmaline could be considered as a candidate for a new treatment of multidrug-resistant P. aeruginosa pathogens-associated biofilm infections.

Transcriptomic Analysis of Salt-Stress-Responsive Genes in Barley Roots and Leaves.

Barley is characterized by a rich genetic diversity, making it an important model for studies of salinity response with great potential for crop improvement. Moreover, salt stress severely affects barley growth and development, leading to substantial yield loss. Leaf and root transcriptomes of a salt-tolerant Tunisian landrace (Boulifa) exposed to 2, 8, and 24 h salt stress were compared with pre-exposure plants to identify candidate genes and pathways underlying barley's response. Expression of 3585 genes was upregulated and 5586 downregulated in leaves, while expression of 13,200 genes was upregulated and 10,575 downregulated in roots. Regulation of gene expression was severely impacted in roots, highlighting the complexity of salt stress response mechanisms in this tissue. Functional analyses in both tissues indicated that response to salt stress is mainly achieved through sensing and signaling pathways, strong transcriptional reprograming, hormone osmolyte and ion homeostasis stabilization, increased reactive oxygen scavenging, and activation of transport and photosynthesis systems. A number of candidate genes involved in hormone and kinase signaling pathways, as well as several transcription factor families and transporters, were identified. This study provides valuable information on early salt-stress-responsive genes in roots and leaves of barley and identifies several important players in salt tolerance.

Evaluating the contribution of osmotic and oxidative stress components on barley growth under salt stress.

Salt stress is considered one of the most devastating environmental stresses, affecting barley growth and leading to significant yield loss. Hence, there is considerable interest in investigating the most effective traits that determine barley growth under salt stress. The objective of this study was to elucidate the contribution of osmotic and oxidative stress components in leaves and roots growth under salt stress. Two distinct barley (Hordeum vulgare) salt-stress tolerant genotypes, Barrage Malleg (BM, tolerant) and Saouef (Sf, sensitive), were subjected to 200 mM NaCl at early vegetative stages. Stressed and control leaves and roots tissue were assessed for several growth traits, including fresh and dry weight and plant length, as well as the content of osmoprotectants proline and soluble sugars. In addition, malondialdehyde content and activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as their corresponding gene expression patterns, were investigated. The results showed better performance of BM over Sf for leaf dry weight (LDW), root dry weight (RDW) and root length (RL). The salt-tolerant genotype (BM) had better osmoprotection against salt stress compared with the salt-sensitive genotype (Sf), with a higher accumulation of proline and soluble sugars in leaves and roots and a stronger antioxidant system as evidenced by higher activities of SOD, CAT and APX and more abundant Cu/Zn-SOD transcripts, especially in roots. Stepwise regression analysis indicated that under salt stress the most predominant trait of barley growth was Cu/Zn-SOD gene expression level, suggesting that alleviating oxidative stress and providing cell homeostasis is the first priority.

Physiological responses and expression of sugar associated genes in faba bean (Vicia faba L.) exposed to osmotic stress.

Faba bean (Vicia faba L.) is the major food legume crop in Tunisia. However, its growth and yield is strongly affected by water-limited environments. In this study, osmotic stress exhibited a negative effect on Bachar and Badii cultivar. Nevertheless, the deteriorating effects of osmotic stress were relatively low on studied parameters of Bachar due to its better efficiency to reduce oxidative damage by increasing enzymatic activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), accumulation of total chlorophyll (Chlt), soluble sugars and leaf relative water content (RWC). GC-MS analysis determined a total of 11 soluble carbohydrates induced by osmotic stress and differentially accumulated in the both cultivars. Bachar showed elevated levels of mannose, glucose, galactose, ribose, rhamnose and myo-inositol which might help to maintain osmotic adjustment, membranes and proteins protection from the damaging effect of reactive oxygen species. Sugar metabolism related genes (VfNINV3, VfPHS2, VfFRK4, VfHXK1, VfGPI1, VfSTP1.1, VfpGlcT1.1, VfSTP5.1, VfpGlcT1.2, VfSWEET2.1, VfVINV2, VfSUS1, VfPGM1, VfSUT1.1, VfGPT1, VfSPS1, VfSPP1, VfPHS1, VfSUT4.1 and VfTMT1.1) were differentially expressed in both cultivars demonstrating their important roles in sugar accumulation. Most of these genes were upregulated in the leaves of Bachar under moderate and severe stress, which could lead to increase glycolysis and tricarboxylic acid cycle in order to accelerate energy production, necessary to increase osmotic regulation and consequently enhancing the osmotic stress tolerance in that cultivar. Overall, sugars accumulation ability can be used as a useful indicator for the osmotic stress tolerant potential in faba bean breeding programs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s12298-021-00935-1).

Alleviation of drought stress in faba bean (Vicia faba L.) by exogenous application of ß-aminobutyric acid (BABA).

Drought stress is one of the most prevalent environmental factors limiting faba bean (Vicia faba L.) crop productivity. ß-aminobutyric acid (BABA) is a non-protein amino acid that may be involved in the regulation of plant adaptation to drought stress. The effect of exogenous BABA application on physiological, biochemical and molecular responses of faba bean plants grown under 18% PEG-induced drought stress were investigated. The results showed that the application of 1 mM of BABA improved the drought tolerance of faba bean. The application of BABA increased the leaf relative water content, leaf photosynthesis rate (A), transpiration rate (E), and stomatal conductance (gs), thereby decreased the water use efficiency. Furthermore, exogenous application of BABA decreased production of hydrogen peroxide (H2O2), malondialdehyde and electrolyte leakage levels, leading to less cell membrane damage due to oxidative stress. Regarding osmoprotectants, BABA application enhanced the accumulation of proline, and soluble sugars, which could improve the osmotic adjustment ability of faba bean under drought challenge. Interestingly, mended antioxidant enzyme activities like catalase, guaiacol peroxidase, ascorbate peroxidase and superoxide dismutase and their transcript levels may lead to counteract the damaging effects of oxidative stress and reducing the accumulation of harmful substances in BABA-treated faba bean plants. In addition, exogenous BABA significantly induced the accumulation of drought tolerance-related genes like VfMYB, VfDHN, VfLEA, VfERF, VfNCED, VfWRKY, VfHSP and VfNAC in leaves and roots, suggesting that BABA might act as a signal molecule to regulate the expression of drought tolerance-related genes.

In situ effects of Lathyrus sativus- PGPR to remediate and restore quality and fertility of Pb and Cd polluted soils.

Rehabilitation of heavy metals contaminated soils using association between legumes and beneficial rhizospheric microorganisms such as plant growth-promoting bacteria (PGPR) is a major challenge in agronomy. The present study focuses on assessing the impact of field inoculation with I1 (Rhizobium leguminosarum (M5) + Bacillus simplex + Luteibacter sp. + Variovorax sp.) and I5 (R. leguminosarum (M5) + Pseudomonas fluorescens (K23) + Luteibacter sp. + Variovorax sp.) on growth and phytoremediation potential of Lathyrus sativus plants as well as soil quality and fertility. The experimentation was carried out in mine tailings of northern Tunisia. Obtained Results indicated that the in situ inoculation with I1 and I5 significantly increased the shoots (47% and 22%) and roots dry weights (22% and 29%), as well as nodules number (48% and 31%), respectively, compared to uninoculated plants. The maximum Pb accumulation in the above-ground tissue was recorded in plants inoculated with I5 (1180.85 mg kg-1 DW). At the same time, we noticed a reduction in total Pb and Cd in the rhizosphere of inoculated plots mainly in those inoculated with I5 reaching 46% and 61%, respectively, compared to uninoculated plots. Likewise, I5 inoculum significantly enhanced soil total nitrogen (35%) and available phosphorus (100%), as well as ß-glucosidase (16%), urease (32%) and alkaline phosphatase (12%) activities. Here we demonstrate the usefulness of L. sativus inoculated with I5 inoculum formed by mixing efficient and heavy metals resistant PGPR to boost an efficient reclamation of Cd and Pb contaminated soils and, ultimately, to improve their quality and fertility.

Identification of two pesticide-tolerant bacteria isolated from Medicago sativa nodule useful for organic soil phytostabilization

Plant-microbe interactions such as rhizobacteria legumes are interesting in organic farming that has undergone significant expansion in the world. The organic agriculture is as an environment-friendly technique and a sustainable alternative to intensive agricultural system. Three types of soil were chosen, organic (ORG), conventional (CON), and fallow land (NA) to isolate soil bacteria-nodulating Medicago sativa, in order to develop microbial inoculants for use in agricultural sustainable system. Soil analysis revealed significant higher amounts of total nitrogen, organic carbon, total phosphorus, and matter detected in ORG. As for heavy metals, ORG showed high Cu content due to the authorized chemical use in organic farming. A sample of 130 bacteria was isolated from Medicago sativa nodule, genetically characterized by PCR/RFLP of ribosomal 16S RNAs, and a great dominance of Sinorhizobium meliloti (88.4%, 73.8%, and 55.5%) is obtained among NA-, CON-, and ORG-managed soils, respectively. The ORG showed the high bacterial diversity with 13.3% of non-identified strains. The resistance against five pesticides (Prosper, Cuivox, Fungastop, Nimbecidine, and Maneb) revealed a maximum of inhibitory concentration about 10 mg l−1 of Prosper, 12 mg l−1 of Cuivox, 6 ml l−1 of Fungastop, 7.5 ml l−1of Nimbecidine, and 25 ml l−1 of Maneb. The analysis of the symbiotic properties and plant growth-promoting potential revealed two efficient strains significantly increased alfalfa dry weight through producing siderophores, phosphorus, and indole acetic acid (13.6 mg ml−1 and 19.9 mg ml−1 respectively). Hence, we identify two tolerant and efficient strains, Achromobacter spanium and Serratia plymuthica, isolated from Medicago sativa nodule with valuable potential able to phytostabilize pesticide-contaminated soils

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Identification of two pesticide-tolerant bacteria isolated from Medicago sativa nodule useful for organic soil phytostabilization.

Plant-microbe interactions such as rhizobacteria legumes are interesting in organic farming that has undergone significant expansion in the world. The organic agriculture is as an environment-friendly technique and a sustainable alternative to intensive agricultural system. Three types of soil were chosen, organic (ORG), conventional (CON), and fallow land (NA) to isolate soil bacteria-nodulating Medicago sativa, in order to develop microbial inoculants for use in agricultural sustainable system. Soil analysis revealed significant higher amounts of total nitrogen, organic carbon, total phosphorus, and matter detected in ORG. As for heavy metals, ORG showed high Cu content due to the authorized chemical use in organic farming. A sample of 130 bacteria was isolated from Medicago sativa nodule, genetically characterized by PCR/RFLP of ribosomal 16S RNAs, and a great dominance of Sinorhizobium meliloti (88.4%, 73.8%, and 55.5%) is obtained among NA-, CON-, and ORG-managed soils, respectively. The ORG showed the high bacterial diversity with 13.3% of non-identified strains. The resistance against five pesticides (Prosper, Cuivox, Fungastop, Nimbecidine, and Maneb) revealed a maximum of inhibitory concentration about 10 mg l-1 of Prosper, 12 mg l-1 of Cuivox, 6 ml l-1 of Fungastop, 7.5 ml l-1of Nimbecidine, and 25 ml l-1 of Maneb. The analysis of the symbiotic properties and plant growth-promoting potential revealed two efficient strains significantly increased alfalfa dry weight through producing siderophores, phosphorus, and indole acetic acid (13.6 mg ml-1 and 19.9 mg ml-1 respectively). Hence, we identify two tolerant and efficient strains, Achromobacter spanium and Serratia plymuthica, isolated from Medicago sativa nodule with valuable potential able to phytostabilize pesticide-contaminated soils.

Heavy metal accumulation in Lathyrus sativus growing in contaminated soils and identification of symbiotic resistant bacteria.

In this study, two populations of leguminous plants Lathyrus sativus were grown in four soils that were collected from sites differently contaminated by heavy metals. Evaluations included basic soil properties, concentrations of major nutrients and four metals (copper, zinc, lead and cadmium) in these soils. Investigation of Lathyrus sativus response to contamination showed that the increase of heavy metal concentration in soils affected biomass of plant, number of nodules and plant metal uptake. Heavy metal tolerance of 46 isolated bacteria from the root nodules was evaluated and demonstrated that the maximum concentration of Cd, Pb, Cu and Zn tolerated by strains were 0.8, 2.5, 0.2, and 0.5 mM, respectively. Twenty-two isolates were tested for their effects on plant biomass production and nodule formation and showed that only R. leguminosarum nodulated Lathyrus sativus, while some bacteria improved the shoot and root dry biomass. Sequences of their 16S rDNA gene fragments were also obtained and evaluated for tentative identification of the isolates which revealed different bacterial genera represented by Rhizobium sp, Rhizobium leguminosarum, Sinorhizobium meliloti, Pseudomonas sp, Pseudomonas fluorescens, Luteibacter sp, Variovorax sp, Bacillus simplex and Bacillus megaterium. The existence of Pb- and Cd-resistant genes (PbrA and CadA) in these bacteria was determined by PCR, and it showed high homology with PbrA and CadA genes from other bacteria. The tested resistant population was able to accumulate high concentrations of Pb and Cd in all plant parts and, therefore, can be classified as a strong metal accumulator with suitable potential for phytoremediation of Pb and Cd polluted sites. Heavy metal resistant and efficient bacteria isolated from root nodules were chosen with Lathyrus sativus to form symbiotic associations for eventual bioremediation program, which could be tested to remove pollutants from contaminated sites.

Effect of Pb-resistant plant growth-promoting rhizobacteria inoculation on growth and lead uptake by Lathyrus sativus.

In search of efficient and resistant plant growth-promoting rhizobacteria (PGPR) strains with multiple activities, a total of twelve bacterial belonging to R. leguminosarum, S. meliloti, Pseudomonas sp., P. fluorescens, Luteibacter sp., Variovorax sp., B. simplex, and B. megaterium were isolated from root nodules of grass pea (Lathyrus sativus L.) grown in contaminated soils. Upon screening, all test strains were able to synthesize indoleacetic acid; more than 90% were siderophore producers and 75% showed varying levels of phosphate solubilizing ability. The gaseous metabolite biosynthesis showed that 42% of strains were cyanogenic. The lead (Pb) bioaccumulation differs with incubation times between cell wall and cytoplasm. Indeed, the most part of Pb was adsorbed to cell surface. A pot experiment was conducted for investigating the capability of combined bacteria to promote plant growth of Lathyrus sativus under controlled conditions. Subsequently, the performance of symbiosis Lathyrus sativus-PGPR (I4: R. leguminosarum (M5) + B. simplex + Luteibacter sp. + Variovorax sp.) was investigated under lead stress using hydroponic culture to elucidate the effect of bacterial inoculation on Pb uptake as well as plant growth. Results showed that under 0.5 mM Pb, inoculation with I4 significantly increased shoots and roots biomass by 59% and 56%, respectively, and improved Pb uptake in both shoots and roots by 39% and 47%, respectively, as compared to uninoculated plants. The inoculation of Lathyrus sativus with efficient and Pb resistant PGPR is a promising symbiosis that having significant potential to improve phytoremediation of Pb-polluted soils.

Genetic variability in Tunisian populations of faba bean (Vicia faba L. var. major) assessed by morphological and SSR markers.

The genetic diversity of 21 faba bean populations was examined using morphological and molecular markers. DNA was extracted from 189 individuals and 8 microsatellite markers were genotyped individually in these 21 populations. A total of 53 alleles were obtained in all populations, with an average of 6.62 alleles per locus. The expected and observed heterozygosity was 0.38 and 0.62 respectively. The average polymorphism index content of SSR markers was 0.61, ranging from 0.31 to 0.81. The unweighted pair group method with arithmetic mean dendrogram clustered all the populations into two groups, each for them subdivided into 3 sub-groups according to geographical origin. Morphological variation showed that the populations were not grouped according to their geographical origin. Therefore, patterns of differentiation of morphological traits did not coincide with molecular differentiation, indicating that morphological variation does not reflect genetic subdivision in studied faba bean populations. Analysis of molecular variance revealed high levels of genetic variation (83%) within population and provides a good base for designing genetic improvement programs. The result of Principal Component Analysis (PCA) revealed that three dimensional principal components (PC1, PC2 and PC3) contributed 40.56% of the total variability and accounted with values of 20.64, 11.22 and 8.70%, respectively. Cluster analysis based on PCA indicated three separate groups of populations. The genetic relationships found between the 21 populations samples were the same in both the PCA and STRUCTURE analysis which support the results observed. These data may serve as a foundation for the development of faba bean breeding programs.