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.

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.

Comprehensive identification, evolutionary patterns and the divergent response of PRX genes in Phaseolus vulgaris under biotic and abiotic interactions.

Peroxiredoxins (Prxs) are novel cysteine-based peroxidases which are involved in protecting cells from oxidative damage by catalyzing the reduction of different peroxides. The present study addressed, for the first time, genome-wide identification, evolutionary patterns and expression dynamics of Phaseolus vulgaris Prx gene family (PvPrx). Nine Prx proteins were identified in P. vulgaris based on homology searches. The phylogeny analysis of Prxs from seven plant species revealed that Prx proteins can be clustered into four groups (1C-Prx, 2C-Prxs, PrxQ and type II Prxs). Both tandem and segmental duplication contributed to PvPrx gene family expansion. Intragenic reorganizations including gain/loss of exon/intron and insertions/deletions have also contributed to PvPrx gene diversification. The collinearity analysis revealed the presence of some orthologous Prx gene pairs between A. thaliana and P. vulgaris genomes. The Ka/Ks ratio indicated that two of the three PvPrx duplicated gene pairs have undergone a purifying selection. Redundant stress-related cis-acting elements were also found in the promoters of most PvPrx genes. RT q-PCR analysis revealed an upregulation of key PvPrx members in response to symbiosis and different abiotic factors. The upregulation of targeted PvPrx members, particularly in leaves exposed to salinity or drought, was accompanied by an accumulation of hydrogen peroxide (H2O2). When exogenously applied, H2O2 modulated almost all PvPrx genes, suggesting a potential H2O2-scavenging role for these proteins. Collectively, our analysis provided valuable information for further functional analysis of key PvPrx members to improve common bean stress tolerance and/or its symbiotic performance. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03246-8.

Genome-wide identification, characterization and expression analysis of glutaredoxin gene family (Grxs) in Phaseolus vulgaris.

Glutaredoxins (Grxs) are ubiquitous oxidoreductase proteins implicated in development and abiotic stress response mainly through maintaining redox homoeostasis. Here, we conducted the first systematic analysis of the Grx gene family (PvGrx) in the most popular legume Phaseolus vulgaris (common bean). A total of 50 PvGrx genes were identified, and divided into four classes (CC-type, CGFS-type, CPYC-type and Grl-type) based on the phylogenetic analysis. The different classes have different introns-exons structures and conserved motifs, indicating functional divergence in the PvGrx family. Both tandem and segmental duplications were found to be involved in the expansion of PvGrx family that underwent a purifying selection by excluding the deleterious loss-of-function mutations. Cis-acting regulatory elements and gene ontology analyses predicted their role of distinctive members in abiotic stress response and hormonal signalling. RNA-seq based expression analysis revealed their differential expression pattern during plant development. On the other hand, RT q-PCR analysis revealed that target PvGrx isoforms were associated with nodule organogenesis and symbiosis based on their expression profiles. In addition, a battery of PvGrx candidates were markedly upregulated by different abiotic stressors suggesting their broad spectrum of functions. These findings serve as a reference for functional analysis and genetic improvement in P. vulgaris and related legume species.

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.

Genome-wide analysis and expression profiling of H-type Trx family in Phaseolus vulgaris revealed distinctive isoforms associated with symbiotic N2-fixing performance and abiotic stress response.

Thioredoxins (Trxs) are implicated in plant development and stress tolerance through redox regulation of target proteins. Trxs of Type h (Trxhs) constitute the largest and the most complicated cluster in the Trx family because of their unknown individual functions. Here, we identified and characterized the Phaseolus vulgaris Trxh family during development, mutualistic interactions and in response to abiotic stress. P. vulgaris (common bean) Trxh gene family (PvTrxh) encompasses 12 isoforms (PvTrxh1-h12), subdivided into 3 groups according to their amino acid sequence features. In silico RNA-seq -based expression analysis showed a differential expression of PvTrxh genes during development. RT-qPCR analysis of PvTrxh genes during nodule organogenesis revealed their highest expression in the nodule primordium (NP). Interestingly, in response to symbiosis, specific PvTrxh isoforms (PvTrxh3 and h5) were found to be highly upregulated compared to mock-inoculated plants. In addition, their expression patterns in the NP positively correlated with the symbiotic N2-fixing efficiency of the Rhizobium strain, as revealed by a number of symbiotic efficiency parameters (ARA, leghemoglobin content, biomass, and total soluble proteins), concomitantly with increased amounts of hydrogen peroxide (H2O2). On the other hand, distinctive PvTrxh isoforms were found to be upregulated in plant leaves, where H2O2 amounts were elevated, in response to both salt and drought constraints. When exogenously applied, H2O2 upregulated specific PvTrxh isoforms in plant leaves and roots. These findings point to a specific, rather than redundant, function for Trxh proteins in common bean beside the association of distinctive Trxh isoforms with symbiosis and abiotic stress response.

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.

Effect of Vicia faba L. var. minor and Sulla coronaria (L.) Medik associated with plant growth-promoting bacteria on lettuce cropping system and heavy metal phytoremediation under field conditions.

Researches involving the use of association between legumes and PGPBs (plant growth-promoting bacteria) in heavy metal phytoremediation process were mainly performed for soils highly contaminated. However, even in agriculture soils, with moderate or low contamination levels, plants can accumulate high rates of heavy metals. So, food chain contamination by these metals presents a real threat to animal and human health. This work aimed to evaluate the use of two legumes/PGPB symbioses; Vicia faba var. minor and Sulla coronaria have been inoculated with specific heavy metal-resistant inocula in a crop rotation system with Lactuca sativa as a following crop, in order to assess their effects on soil fertility, lettuce yield, and heavy metal content. Our results showed that legume inoculation significantly enhanced their biomass production, nitrogen and phosphorus content. The use of our symbioses as green manure before lettuce cultivation, as a rotation cropping system, affected positively soil fertility. In fact, we recorded a higher organic matter content, with rapid decomposition in the soil of inoculated plots. Besides, results demonstrated a greater nitrogen and phosphorus content in this soil, especially in the plot cultivated with inoculated V. faba var. minor. The improvement of soil fertility enhanced lettuce yield and its nitrogen and phosphorus content. Moreover, inoculated legumes extracted and accumulated more heavy metals than non-inoculated legumes. Our symbioses play the role of organic trap for heavy metals, making them unavailable for following crops. These facts were supported by lettuce heavy metal content, showing a significant decrease in metal accumulation, mainly zinc and cadmium, in edible parts. Results showed the usefulness of the studied symbioses, as a main part of a rotation system with lettuce. Our symbioses can be suggested for agriculture soil phytoremediation, aiming to enhance non-legume crop yield and limit heavy metal translocation to food chain.

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.

Antioxidant systems responses and the compatible solutes as contributing factors to lead accumulation and tolerance in Lathyrus sativus inoculated by plant growth promoting rhizobacteria.

Short-term lead (Pb) uptake by plants is important to better understand the mechanisms of metal uptake, plant tolerance and detoxification strategy. Thus we examined the response of Lathyrus sativus to 1 mM Pb application in hydroponic sorption kinetics at 24, 48 and 72 h, and we investigated the contribution of two inocula I1 (R. leguminosarum (M5) + B. simplex + Luteibacter sp + Variovorax sp) and I5 (R. leguminosarum (M5) + P. fluorescens (K23) + Luteibacter sp + Variovorax sp) in plant mechanisms responses. Pb application induced its immediate uptake by L. sativus with highest concentrations, which increased gradually mostly for inoculated plants. The control plant shoots accumulated the highest concentration of lead at 24 h. However, at 48 and 72 h this potential uptake was significantly enhanced in plants inoculated with I5. Moreover, inoculation increased significantly root Pb-uptake with the maximum reached at 72 h. We observed a progressive decline in chlorophyll contents after Pb exposure in control plants that was higher than in PGPR-treated plants and the greatest improvement (152%) was recorded in I5 inoculated leaves. The PGPR also promoted significant elevation in the carotenoid content with the highest increases (188%) in plants inoculated with I5 at 72 h. Data illustrated remarkable augmentation in malondialdehyde, ion leakage level and decrease in membrane stability. Whereas, inoculation enhanced significantly cellular membrane integrity through increases in membrane stability index as compared to the control plants. In response to Pb, proline biosynthesis, as well as total soluble sugars concentration, immediately increased and the stimulatory effect was more pronounced in inoculated plants at 72 h. Lead considerably altered the activities of SOD, GPOX, CAT and APX enzymes in leaves and roots in a time- and inoculation- dependent manner. It is concluded that antioxidant enzymes, carotenoids, soluble sugars and proline were involved in the main defense mechanism and tolerance of Lathyrus sativus to Pb oxidative stress, as well lead accumulation, and are likely to operate in combination.

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.

Physiological responses and antioxidant enzyme changes in Sulla coronaria inoculated by cadmium resistant bacteria.

Plant growth promoting bacteria (PGPB) may help to reduce the toxicity of heavy metals on plants growing in polluted soils. In this work, Sulla coronaria inoculated with four Cd resistant bacteria (two Pseudomonas spp. and two Rhizobium sullae) were cultivated in hydroponic conditions treated by Cd; long time treatment 50 µM CdCl2 for 30 days and short time treatment; 100 µM CdCl2 for 7 days. Results showed that inoculation with Cd resistant PGPB enhanced plant biomass, thus shoot and root dry weights of control plants were enhanced by 148 and 35% respectively after 7 days. Co-inoculation of plants treated with 50 and 100 µM Cd increased plant biomasses as compared to Cd-treated and uninoculated plants. Cadmium treatment induced lipid peroxidation in plant tissues measured through MDA content in short 7 days 100 µM treatment. Antioxidant enzyme studies showed that inoculation of control plants enhanced APX, SOD and CAT activities after 30 days in shoots and SOD, APX, SOD, GPOX in roots. Application of 50 µM CdCl2 stimulated all enzymes in shoots and decreased SOD and CAT activities in roots. Moreover, 100 µM of CdCl2 increased SOD, APX, CAT and GPOX activities in shoots and increased significantly CAT activity in roots. Metal accumulation depended on Cd concentration, plant organ and time of treatment. Furthermore, the inoculation enhanced Cd uptake in roots by 20% in all treatments. The cultivation of this symbiosis in Cd contaminated soil or in heavy metal hydroponically treated medium, showed that inoculation improved plant biomass and increased Cd uptake especially in roots. Therefore, the present study established that co-inoculation of S. coronaria by a specific consortium of heavy metal resistant PGPB formed a symbiotic system useful for soil phytostabilization.

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.

Characterization of efficient plant-growth-promoting bacteria isolated from Sulla coronaria resistant to cadmium and to other heavy metals.

The inoculation of plants with plant-growth-promoting rhizobacteria has become a priority in the phytoremediation of heavy-metal-contaminated soils. A total of 82 bacteria were isolated from Sulla coronaria root nodules cultivated on four soil samples differently contaminated by heavy metals. The phenotypic characterization of these isolates demonstrated an increased tolerance to cadmium reaching 4.1mM, and to other metals, including Zn, Cu and Ni. Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) analysis showed a large diversity represented by genera related to Agrobacterium sp., R. leguminosarum, Sinorhizobium sp., Pseudomonas sp., and Rhizobium sp. Their symbiotic effectiveness was evaluated by nodulation tests. Taking into consideration efficiency and cadmium tolerance, four isolates were chosen; their 16SrRNA gene sequence showed that they belonged to Pseudomonas sp. and the Rhizobium sullae. The selected consortium of soil bacteria had the ability to produce plant-growth-promoting substances such as indole acetic acid and siderophore. The intracellular Cd accumulation was enhanced by increasing the time of incubation of the four soil bacteria cultivated in a medium supplemented with 0.1mM Cd. The existence of a cadmium-resistant gene was confirmed by PCR. These results suggested that Sulla coronaria in symbiosis with the consortium of plant-growth-promoting rhizobacteria (PGPR) could be useful in the phytoremediation of cadmium-contaminated soils.