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The present work was initiated in order to test whether the growth and pathogenic behavior of fungal isolates infecting Faba bean (FB) are influenced by salt and drought stress. A collection of 108 fungal isolates was recovered from different infected FB plants grown in various bioclimatic zones in Tunisia. Koch’s postulates revealed that 54% of those isolates caused root rots infection by 25% to 100% of the whole root system. Eighteen pathogenic fungal isolates were chosen based on their high incidence on various plant growth parameters using the heat map analysis to undergo the in vitro analysis, where 13 isolates belong to Fusarium spp. The in vitro mycelial growth of the 18 strains in potato-dextrose agar amended with NaCl and PEG6000 was strain dependent, in which F. equiseti (VFF12, VFF16), F. graminearum VFF6, F. brachygibbosum VFF9, Alternaria sp. VFF5, Boeremia exigua VFF4, Rutstroemia sp. VFF7 and Rhizopus oryzae VFF1 showed a significant increase by up to 150% compared to controls. Best linear unbiased predictors (BLUPs) for differences in root and shoot dry weights between control (inoculated unstressed plants) and inoculated under salt and drought stress of the tolerant fungal strains were analyzed. BLUPs of VFF16, VFF6, and VFF7 were significantly increased under both stresses compared to controls. While, BLUBs of VFF12, VFF9, and VFF4 were not significant, which maintained similar pathogenic effect. However, BLUPs of VFF5 and VFF1 were significantly decreased. Consequently, soil salinization and water deficiency - occurring nowadays in many parts of the world - can increase the aggressiveness of phytopathogenic fungi.

Alleviation of cadmium-induced root genotoxicity and cytotoxicity by calcium chloride (CaCl2) in faba bean (Vicia faba L. var. minor) seedlings were studied. Faba bean seeds were treated with H2O or 2% CaCl2 for 6 h before germination. Seeds were then exposed to 0 and 50 µM CdCl2 concentrations for 7 days. Genotoxic damaging effects of Cd was examined through the determination of the mitotic index (MI), chromosomal aberrations (CA) and micronucleus (MN) in the meristem cells of faba bean roots. Similarly, effects of Cd stress on metal accumulation, total membrane lipid contents, total fatty acid composition (TFA), lipid peroxidation as indicated by malondialdehyde production, soluble protein and non-protein thiols (NP-SH) contents, hydrogen peroxide production and the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) were evaluated after 7 days of Cd stress in the seedling roots. Cd stress resulted in the reduction of MI, in addition to MN formation and CA induction in the roots of non-primed seeds (treated with H2O). Moreover, Cd induced lipid peroxidation, H2O2 overproduction and loss of membrane lipid amount and soluble protein content, and changes in the TFA composition in roots of faba bean seedlings. SOD activity declined, but CAT and GPX activities increased. However, seed pre-treatment with CaCl2 attenuated the genotoxic and cytotoxic effects of Cd on Vicia faba roots. The results showed that CaCl2 induced reduction of Cd accumulation, improved cell membrane stability and increased the antioxidant defence systems, thus reducing and alleviating Cd genotoxicity and oxidative damage.

The effect of calcareous soil on growth, photosynthetic gas exchanges, symbiotic nitrogen fixation (SNF), iron nutrition, and antioxidative enzymes activities was investigated in two lines of Medicago truncatula: TN8.20 and Jemalong. The results showed that growth (especially in shoots) was significantly lower on calcareous soil in both lines, but Jemalong was more impacted than TN8.20. Plants cultivated on calcareous soil showed significantly lower values for photosynthesis activity (as net CO2 assimilation and stomatal conductance) and nodule performance (nodulation, nodule growth and SNF), but this trend was more pronounced in the sensitive line (Jemalong). The better performance of TN8.20 as compared to Jemalong could be ascribed due to its aptitude to preferentially allocate iron to nodules, thereby ensuring nodule growth and a higher SNF activity, besides showing higher iron use efficiency for SNF. In both lines, superoxide dismutase (SOD, EC.15.1.1), catalase (EC.1.11.1.6), and peroxidase (POX, EC 1.11.1.7) activities were increased significantly when they were grown on a calcareous soil. Yet, this tendency was more pronounced in the tolerant line, TN8.20. Overall, our results strongly suggest that on calcareous soils, the successful adaptation of TN8.20 would be related to its ability to induce an antioxidant response in order to preserve important metabolic paths such as photosynthetic activity and symbiotic nitrogen fixation.

Faba bean (Vicia faba L.) is an important pulse crop in the Mediterranean region. Salinity is one of the most devastating abiotic stresses that alter the growth and development of plants and affect the crop yield, especially in arid and semi-arid area such as Tunisia. To surmount the damaging effect of salinity, it is important to develop tolerant cultivars of faba bean. In this study, we examined the behavior of four faba bean cultivars in response to salinity stress. Plants were grown in hydroponics at 0, 75 and 150 mM of NaCl, and 16 morphological, photosynthetic and physiological parameters were assessed. Our data showed a significant genotypic variation for salt tolerance in faba bean among the studied cultivars. All measured parameters were affected by salinity at moderate salt concentration (75 mM), while there was a more pronounced reduction of all parameters at severe salt concentration (150 mM). The statistics analysis revealed that photosynthetic and biomass parameters were the least altered in cv. ‘Najeh’. Based on the results, cv. ‘Najeh’ can be considered to be a tolerant cultivar and to attract great interest in future breeding programs for modern cultivar improvement.

The ability of Phoma medicaginis to infect legume nodules and the subsequent effect on their functioning was investigated in Medicago truncatula lines, TN.8.20 and Jemalong6, inoculated with an efficient Sinorhizobium meliloti strain. Colonization of nodules by P. medicaginis strain Pm8 was confirmed by morphological and molecular analyses. P. medicaginis effect was more relevant on the most susceptible line, TN.8.20, where it decreased the nitrogen fixation capacity (NFC) significantly in nodules from 62 days after sowing (das) till the end of nodule life cycle; however in Jemalong6 nodules, the NFC inhibition was significant only at the flowering stage. Analyses of stress indices as structure, lipid peroxidation and leghemoglobin content did not show significant structural or metabolic alterations in infected nodules. Superoxide dismutase (SOD) activity was increased in nodules of both M. truncatula lines up to 62 das. After 78 das, increase of SOD activity continued only in Jemalong6 nodules. Changes in peroxidase (POX) activity were correlated to those revealed on NFC. Indeed, POX was decreased in nodules of TN.8.20 line but remained almost unchanged in Jemalong6. These results, suggest that POX represents a strong component of the defence mechanisms in nodules of resistant M. truncatula lines, where it could be involved in reinforcing the cell wall barrier. The stimulation of SOD activity is also a key mechanism in maintaining nodule integrity via controlling ROS overproduction when counteracting fungal attacks.

The biochemical processes underlying the expression of resistance in the roots of Medicago truncatula against Aphanomyces euteiches infection was investigated, with emphasis on oxidative stress. The levels of H2O2, superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, soluble phenolics and lignin were measured in the roots of two lines, A17 partially resistant and F83005.5 susceptible to A. euteiches at three infection stages; penetration of the epidermis (1 dpi), colonization of the cortex (3 dpi) and invasion of the root stele (6 dpi). A rapid and large decrease of the H2O2 levels in A17 roots occurred. However, in F83005.5 roots, the decrease in H2O2 levels was delayed until 3 dpi. In A17 roots, the activities of ascorbate peroxidase, peroxidase and catalase were induced as early as 1 dpi, whereas a general decrease in the activity of the four antioxidant enzymes was observed in F83005.5 roots. The levels of soluble phenolics and lignin were increased in A17 roots at 3 and 6 dpi, respectively. The H2O2 levels were negatively correlated to ascorbate peroxidase, catalase and lignin production at 1, 3 and 6 dpi, respectively in A17 roots. Physiological concentrations of H2O2 found in M. truncatula infected roots had no detrimental effect on the in vitro growth of this oomycete. Our data suggest that H2O2 does not have a direct antimicrobial effect on M. truncatula resistance to A. euteiches, but is involved in cell wall strengthening around the root stele, preventing pathogen invasion of the vascular tissues.

Sinorhizobium meliloti strain A5, isolated from root nodules of Medicago truncatula growing spontaneously in Tunisian dry lands, showed better growth performance under different sodium chloride concentrations than the standard reference strain RCR2011. Strain A5 has the ability to grow even at 684 mM NaCl. Two-dimensional (2D) electrophoretic analysis of the salt effect on proteome profiles revealed the greatest change on the 4th day of exposure. The halotolerant strain A5 showed more upregulated protein spots than the standard RCR2011 strain (23% of the analyzed protein spots vs only 10% in strain RCR2011) and fewer downregulated protein spots (28% vs 69%). Proteins over-expressed in salty medium as determined by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for both strains were identified as an outer-membrane protein, an l-amino-acid binding periplasmic transporter protein, a 50S ribosomal protein (L25), a heat shock protein, and a putative oxido-reductase protein. Down-regulated proteins were identified as a putative iron-binding periplasmic transporter protein, a putative lipoprotein precursor, a putative spermidine/putrescine-binding periplasmic transporter protein, and a putative amino-acid binding ABC transporter protein. The profile of strain A5 showed higher over-expression of an oxidoreductase and a heat-shock protein, and specifically an induction of a putative oligopeptide ABC transporter ATP-binding protein.

Thirty-six symbiotic associations involving six chickpea cultivars against six rhizobial strains were evaluated for symbiotic performance and responses to osmotic stress applied by mannitol (50 mM) in aerated hydroponic cultures. Analyses in different symbioses were focused on biomass production, nodulation, nitrogen fixation, and their modulation under osmotic stress conditions, as well as expression of nodular antioxidant enzymes. Mesorhizobium ciceri reference (835) and local (CMG6) strains, as well as the local (C11) M. mediterraneum allowed the best symbiotic efficiency for all chickpea cultivars. The osmotic stress induces severe decrease ranging 30–50% in aerial biomass and 50–70% for nitrogen fixation. Nevertheless, plants inoculated with M. ciceri (835) and M. mediterraneum (C11) preserve a relatively high growth (4 g plant−1) with nitrogen-fixing activity (25 μmols h−1 plant−1). The bacterial partner was the most important factor of variance of the analysed parameters in osmotic stress or physiological conditions where it gets to 60–85%. The strains allowing the best competent symbioses were proposed for field assays. Under osmotic stress, nodular peroxidase (POX) and ascorbate peroxidase (APX) activities were significantly enhanced. The increase of POX and APX was inversely correlated with the inhibition of aerial biomass production (p = 0.05) and nitrogen-fixing capacity (p = 0.01), suggesting a protective role of these enzymes in nodules. Superoxide dismutase (SOD) was also activated in stressed nodules. However, the spectacular decrease in catalase (CAT) activity discounts its involvement in osmotic stress response.