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.

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.

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.

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.

In situ phytostabilisation capacity of three legumes and their associated Plant Growth Promoting Bacteria (PGPBs) in mine tailings of northern Tunisia.

PGPBs-legumes associations represent an alternative procedure for phytostabilisation of heavy metals polluted soils mainly generated by industrial and agricultural practices. In this study we evaluated the capacity of Vicia faba, Lens culinaris and Sulla coronaria, inoculated in situ by specific heavy metals resistant inocula, for the phytostabilisation of copper, lead and cadmium respectively. The experimentation was performed in mine tailings of northern Tunisia. Results proved that inoculation enhanced roots and shoots biomass production of faba bean by 14% and 12%, respectively, and significantly improved pods yield by 91%. In lentil, the inoculation ameliorated shoot biomass up to 27%. The highest nitrogen fixation was recorded by Sulla coronaria. The three symbioses accumulated heavy metals essentially in roots, and poorly in shoots. In addition, cadmium accumulation in roots of inoculated sulla was enhanced by 39%. Furthermore, inoculations decreased heavy metals availability in the soil up to -10% of Cu and -47% of Pb respectively in roots of faba bean and lentil. Our results suggested a positive effect of co-inoculation of legumes by appropriate heavy metals resistant PGPBs for the phytostabilisation of mine tailings. Elsewhere, the enhancement in the antioxidant enzymes activities demonstrated the role of the three inocula to alleviate the heavy metals induced stress.

Impact of dual inoculation with Rhizobium and PGPR on growth and antioxidant status of Vicia faba L. under copper stress.

Plant-growth-promoting bacteria (PGPR) may help reduce the toxicity of heavy metals in plants in polluted environments. In this work, the effects of dual inoculation with Rhizobium and PGPR strains on the growth of Vicia faba grown under copper stress were assessed during hydroponic culture. Growth parameters, copper (Cu) accumulation and antioxidant enzyme activities were studied. Copper concentrations above 1mM damaged plant growth, but co-inoculation reduced its harmful effect. Co-inoculation of plants treated with 1mM Cu and 2mM Cu increased the dry weights as compared with Cu-treated and uninoculated plants. However, it decreased copper uptake up to 80% in the roots of 1-mM-Cu-treated plants as compared to non-inoculated control. Copper tolerance in Vicia faba is linked to the activity of antioxidant systems that are modulated by metal concentrations: both superoxide dismutase (SOD) and catalase (CAT) were higher in the presence of Cu; a lower Cu dose of 0.5mM stimulated ascorbate peroxidase (APX) and/or peroxidase (POX) activities in shoots and roots; however in nodules CAT appeared to be the main peroxidase in H2O2 scavenging. The 1mM Cu treatment enhanced SOD, CAT and APX activities in roots and only SOD and POX were activated in shoots. All enzyme activities were inhibited by inoculation of 2mM Cu. The effect of inoculation with copper-accumulating PGPRs and the status of the antioxidant enzyme system were linked to changes in the copper tolerance status of Vicia faba. Our results suggested that Vicia faba inoculation with Rhizobium and PGPR Enterobacter clocae and Pseudomonas sp. could help to alleviate copper stress under hydroponic conditions. This result should be tested under field conditions for soil fertilization and phytostabilisation purposes.

Inoculation of Lens culinaris with Pb-resistant bacteria shows potential for phytostabilization.

Phytoremediation comprises a set of plant and microbe-based technologies for remediation of soil heavy metal contamination. In this work, four Pb-resistant bacteria (Agrobacterium tumefaciens, Rahnella aquatilis, and two Pseudomonas sp.) were selected among a collection of isolates from root nodule of Lens culinaris. They had a high degree of bioaccumulation ability in nutrient medium containing 2 mM Pb, and the maximum Pb accumulation of whole cell was found after 48-h incubation. These Pb-resistant bacteria synthesized plant growth promoting substances such as indole acetic acid and siderophore. The presence of the Pb resistance genes (pbrA) in these bacteria has been confirmed by PCR. L. culinaris cultivated in two experimental soils with different levels of contamination showed that Pb contamination affected plant growth; therefore, it's co-inoculation with the consortium of Pb-resistant bacteria improved plant biomass. The present study demonstrated that lentil accumulated Pb primarily in their roots and poorly in their shoots; in addition, it's co-inoculation in moderately Pb-contaminated soil induced a reduction in Pb accumulation in roots and shoots by 22 and 80 %, respectively. Whereas in highly Pb-contaminated soil, we registered a diminution in concentration of Pb in shoots (66 %) and an augmentation in roots (21 %). The contamination of soil by Pb caused an oxidative stress in lentil plant, inducing modulation in antioxidant enzymes activities, essentially in superoxide dismutase (SOD) and peroxidase (GPOX) activities which were more pronounced in lentil cultivated in highly Pb-contaminated soil, in addition, co-inoculation enhanced these activities, suggesting the protective role of enzymatic antioxidant against Pb-induced plant stress.Thus, the present study demonstrated that co-inoculation of lentil with A. tumefaciens, R. aquatilis, and Pseudomonas sp. formed a symbiotic system useful for phytostabilization of highly and moderately Pb-contaminated soils.

Phytostabilization of moderate copper contaminated soils using co-inoculation of Vicia faba with plant growth promoting bacteria.

There is a need to conduct research on the selection of microbial isolates from rhizosphere of plants growing on heavy metal contaminated soils for specific restoration programs. This article suggest a consortium of bacteria combining Rhizobium sp. CCNWSX0481, Rhizobium leguminosarum bv. viciae, Enterobacter cloacae and Pseudomonas sp. 2(2010) that was examined for the ability to promote Vicia faba.L. growth when cultivated on the vineyard of soil moderately contaminated with copper. Data showed that inoculation was significant in nodulation; it increases the number and the weight of nodules of 50%. Co-inoculation was also found to positively influence growth and seed yield, through increasing fresh shoot and fresh root weights by 33 and 26%, respectively, and through rising numbers of seed per pod and pods per plant. In contrast, co-inoculation produced a significant reduction of accumulated copper in roots attending 35%, however, the treatment revealed no significant effects on the copper contents in pods and seeds. The tested inoculum could be an option to promote V. faba growth and to enhance soil fertilization in moderate copper contaminated soils. Further studies on the influence of co-inoculation practices on copper migration in soil-plant systems are recommended to acquire more information for evaluation of this legume safety.

Identification of effective Pb resistant bacteria isolated from Lens culinaris growing in lead contaminated soils.

Soil bacteria are a new phytoremediation system for the removal of heavy metals from soils. In this study, fifteen soil bacteria were isolated from root nodules of lentil growing in heavy metals contaminated soils, particularly by lead. Molecular characterization of the collection showed a large diversity, including Agrobacterium tumefaciens, Rahnella aquatilis, Pseudomonas, and Rhizobium sp. These soil bacteria had a wide range of tolerance to heavy metals. Among them, strains of A. tumefaciens and R. aquatilis tolerated up to 3.35 mM Pb; whereas Pseudomonas tolerated up to 3.24 mM Pb. The inoculation of lentil grown hydroponically with inoculums formed by these efficient and Pb resistant bacteria enhanced plant biomass. The treatment of this symbiosis by 1 mM Pb for 10 days or by 2 mM Pb for 3 days demonstrated that lentil had Pb accumulation capacity and can be considered a Pb accumulator plant, elsewhere, roots accumulated more Pb than shoots, and the inoculation decreased the Pb up take by the plants, suggesting that this symbiosis should be investigated for use in phytostabilization of Pb-contaminated soils. At the same time, a modulation in the antioxidant enzyme activity and a specific duration was required for the induction of the superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX) response and to adapt to Pb stress. These results suggested that these enzymes may be involved in the main mechanism of antioxidative defense in lentil exposed to Pb oxidative stress.