Moez Jebara

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.

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 82xa0bacteria 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.xa0leguminosarum, 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.

Genetic Relationship and Diversity Analysis of Faba Bean (Vicia Faba L. var. Minor) Genetic Resources Using Morphological and Microsatellite Molecular Markers

Assessment of genetic diversity is an essential component in germplasm characterization and utilization. Molecular markers serve as a valuable tool to assess the genetic variation and germplasm identification, which play a key role for faba (Vicia faba L.) bean breeding. In this study, we analyzed the genetic diversity of faba bean accessions based on simple sequence repeats (SSRs) and morphological traits. Forty-six faba bean accessions, originating from different countries and from the ICARDA breeding program, were evaluated by using 15 morphological and agronomic traits and 17 simple sequence repeat (SSR) loci. Significant differences among accessions for the 15 morphological descriptors were observed. Analysis by SSR markers showed a high genetic diversity among the accessions: All SSRs showed polymorphism, and 101 alleles were revealed for all accessions. The number of alleles at each locus ranged from 2 to 10, with an average of 5.94 alleles per marker, and the polymorphic information content (PIC) values ranged from 0.38 to 0.84 with a mean of 0.69. Expected heterozygosity (He), observed heterozygosity (Ho), unbiased expected heterozygosity (UHe), and Shannon’s information index (I) showed existence of high genetic variation between accessions from different pedigree. Analyses of genetic distance of the accessions separated the accessions into two groups and seven and five subgroups according to morphological and SSR analysis, respectively. Principal component analysis (PCA) of the SSR markers showed that the first two principal components (PCs) explained a total of 43.90xa0% of the genetic variation and allowed to distinguish three groups of accessions. Regardless of the method of analysis, Tunisian cultivars are grouped together. For the rest of the accessions, the geographical origin did not seem to be the main factor for structuring the variability of the studied accessions. Elite accessions from the ICARDA faba bean program differed from others and clustered together. The results obtained suggested that the faba bean microsatellite markers can be used to efficiently distinguish faba bean genotypes and to estimate their genetic diversity.

Identification and characterization of drought stress responsive genes in faba bean (Vicia faba L.) by suppression subtractive hybridization

Faba bean (Vicia faba L.) ranks fourth in food legume crop production in the world. However, drought is a potential major constraint to faba bean production and improved faba bean cultivars and development of drought-resistant varieties play a key role in enhancing faba bean crop production. In this study, suppression subtractive hybridization (SSH) technique was used to study differential expression in response to water stress and to identify genes involved in molecular mechanism of drought tolerance. A forward subtractive cDNA library induced by water deficit conditions was constructed used Hara faba bean cultivar grown in pots and treated with either well-watered (WW) or water-stressed (WS). A total of 28 clones were identified as drought stress induced. After sequencing, ten unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis which showed homology to known drought responsive genes including heat shock protein (HSP), late embryogenic abundant (LEA), zinc finger protein transcription factors (ZFP), lipid transfer protein (LTP), chlorophyll a/b-binding protein (ChlBP), thioredoxin h (Trx h), and ATP synthase as well as some functionally unknown transcripts. Their expression was characterized in Leaf, root, flower, cotyledon, and stem tissue. Quantitative RT-PCR analysis revealed that eight genes were consistently up-regulated in Hara compared to Giza3 cultivar, known as drought-tolerant and sensitive respectively under water deficit treatment. The expression of six genes was differentially expressed in different stages of water stress faba bean plant. Drought responsive genes showed changed expression patterns, indicating that they may play important roles in faba bean water stress response. Furthermore, these results indicate that drought-induced genes are related to metabolic pathways and genetic regulation of stress and development and can serve as a foundation for future studies to elucidate drought stress mechanisms of faba bean.

Effect of drought stress on chlorophyll fluorescence, antioxidant enzyme activities and gene expression patterns in faba bean (Vicia faba L.)

ABSTRACT Drought is a major environmental stress that limits faba bean growth, leading to considerable reduction in their productivity. Two genotypes (Giza 3 and Hara) were evaluated under three water treatments (90%, 60% and 30% field capacity). The results showed that water stress affects negatively chlorophyll fluorescence parameters at different levels of water deficit and the variations of Fv/Fm, ΦPSII and electron transport rate in Giza 3 were largely higher than those in Hara. Drought preferentially enhanced the activities of superoxide dismutase, catalase and guaiacol peroxidase whereas it did not affect ascorbate peroxidase activity. Real-time reverse transcriptase-polymerase chain reaction results demonstrated that the expression of aquaporin (PIP), myeloblastosis (MYB), nine-cis-epoxycarotenoid dioxygenase, late embryogenesis abundant, dehydrins (Dhn), heat shock protein (HSP) and APETALA2/ethylene-responsive factor was modified in response to drought conditions and that all genes were differentially expressed in Hara and Giza 3. The observed expression patterns of these genes might be due to water deficit intensity according to cultivar’s tolerance to drought. Hara with the highest level of antioxidant enzyme activities, the accumulation of proline content and the expression level of Dhn, HSP and MYB gene throughout all conditions tested is reported more drought tolerant than Giza 3. This study should contribute to explaining the physiological, biochemical and molecular mechanism tolerance drought stress and improving breeding program in faba bean.

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 50xa0µM CdCl2 for 30xa0days and short time treatment; 100xa0µM CdCl2 for 7xa0days. 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 7xa0days. Co-inoculation of plants treated with 50 and 100xa0µM 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 7xa0days 100xa0µM treatment. Antioxidant enzyme studies showed that inoculation of control plants enhanced APX, SOD and CAT activities after 30xa0days in shoots and SOD, APX, SOD, GPOX in roots. Application of 50xa0µM CdCl2 stimulated all enzymes in shoots and decreased SOD and CAT activities in roots. Moreover, 100xa0µM 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.

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)u2009+u2009B. simplexu2009+u2009Luteibacter sp.u2009+u2009Variovorax 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 0.5u2009mM 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 variabilitly 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.

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 10xa0mgxa0l−1 of Prosper, 12xa0mgxa0l−1 of Cuivox, 6xa0mlxa0l−1 of Fungastop, 7.5xa0mlxa0l−1of Nimbecidine, and 25xa0mlxa0l−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 (13.6xa0mgxa0ml−1 and 19.9xa0mgxa0ml−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.

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 1u202fmM Pb application in hydroponic sorption kinetics at 24, 48 and 72u202fh, 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 24u202fh. However, at 48 and 72u202fh this potential uptake was significantly enhanced in plants inoculated with I5. Moreover, inoculation increased significantly root Pb-uptake with the maximum reached at 72u202fh. 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 72u202fh. 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 72u202fh. 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.