Rhizobial strain involvement in symbiosis efficiency of chickpea–rhizobia under drought stress: plant growth,nitrogen fixation and antioxidant enzyme activities

Chickpea plants were inoculated with two strains of Mesorhizobium ciceri: local strain (C-15) and non-local strain (CP-36) in order to evaluate plant growth parameters, activities of nitrogenase and antioxidant enzymes under drought stress as well as control condition within 15 days of imposition of drought stress. Biomass production, nodulation, nitrogen fixation and antioxidant enzyme activities under drought condition were compared. Under control condition, symbiotic efficiency in symbiosis formed by C-15 was higher than that in symbiosis derived by CP-36. Although drought stress decreased shoot dry weight, root dry weight, nodule dry weight and nitrogen fixation in both symbioses, the rate of decline in plants inoculated with CP-36 was higher than that in symbiosis chickpea with C-15. Therefore, symbioses showed different tolerance level under drought condition which was essentially correlated with symbiotic performance at non-stressful conditions. Under drought stress, nodular peroxidase (POX) activity increased in both symbioses but was higher in nodules produced by C-15. Ascorbate peroxidase (APX) increased significantly in nodules of symbiosis of chickpea with C-15. Catalase (CAT) and glutation reductase (GR) declined in both symbioses which decline extent in symbiosis with C-15 was lower than that in the nodules of CP-36. These results suggested contribution of rhizobial partner in enhancing the tolerance of symbioses to drought stress, which was related with the increase of antioxidant enzyme activities (APX and POX) under drought conditions.     

Tolerance of Mesorhizobium type strains to different environmental stresses

The symbiosis between rhizobia and legumes is affected by different environmental conditions. Our aims were to evaluate stress tolerance of Mesorhizobium species and investigate species-specific stress response mechanisms. Tolerance of Mesorhizobium type strains to temperature, salt and pH stress was evaluated. Mesorhizobium thiogangeticum showed highest growth with 1.5% NaCl and Mesorhizobium ciceri at pH 5. Mesorhizobium plurifarium showed higher growth at 37°C. SDS-PAGE analysis revealed changes in the protein profiles, namely the overexpression of a 60 kDa protein, following heat stress. Under salt stress, five overexpressed proteins were identified in M. plurifarium and M. thiogangeticum. Northern analysis revealed an increase in groEL expression in Mesorhizobium huakuii and Mesorhizobium septentrionale after heat shock; by contrast, a decrease was detected in Mesorhizobium albiziae and M. thiogangeticum, upon salt shock. A high diversity in tolerance to temperature, salt and pH stress was detected among Mesorhizobium species. M. thiogangeticum and M. ciceri are moderately halophilic and acidophilic, respectively. Several proteins, overproduced in different strains, may be involved in stress tolerance. groEL expression increased upon heat and decreased upon salt shock. To our knowledge, this is the first study focusing tolerance to temperature, salt and pH stress, as well as groEL expression, in Mesorhizobium type strains.     

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.     

Effects of salinity on protein and lipopolysaccharide pattern in a salt-tolerant strain of Mesorhizobium ciceri

AIMS: To characterize the physiological and metabolic responses of Mesorhizobium ciceri strain ch-191 to salt stress, investigating the changes induced by salinity in protein and lipopolysaccharide profiles, as well as determining the accumulation of amino acids, glutamate and proline. METHODS AND RESULTS: Strain ch-191 of M. ciceri was grown with different NaCl concentrations. Protein and lipopolysaccharide patterns were determined by electrophoresis. The strain ch-191 tolerated up to 200 mmol l-1 NaCl, although higher salt dosages limited its growth and induced changes in the protein profile. The most noteworthy change in the LPS-I pattern was the decrease in the slowest band and the appearance of an intermediate mobility band. The accumulation of proline in response to salt stress surpassed that of glutamate. CONCLUSION: The protein profile showed major alterations at salinity levels which inhibited growth. However, the alterations in the LPS profile and accumulation of compatible solutes were evident from the lowest levels, suggesting that these changes may constitute adaptative responses to salt, allowing normal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection and characterization of salt-tolerant strains, which also show efficient symbiotic performance under salinity, may constitute a strategy for improving Cicer arietinum-Mesorhizobium ciceri symbiosis in adverse environments.     

Characterization of a symbiotically defective serine auxotroph of Mesorhizobium ciceri

A Tn5-induced mutant strain (TL68) of Mesorhizobium ciceri unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Unlike its wild-type parent (strain TAL620), the mutant had an absolute dependence on serine to grow. Cloning of the DNA region containing Tn5 and sequence analysis showed that Tn5 was inserted into the gene coding for 3-phosphoglycerate dehydrogenase, which catalyses the first step in the serine biosynthetic pathway. The role of serine biosynthesis of M. ciceri in the establishment of nitrogen-fixing symbiosis with chickpea (Cicer arietinum L) was investigated using the mutant TL68. The serA(-) mutant (TL68) was unable to elicit the development of efficient nodules on the roots of Cicer arietinum L. The addition of serine to the plant-growth medium restored the ability of the mutant to nodulate Cicer arietinum, and the nodules were able to fix nitrogen.     

Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India

In a combined approach of phenotypic and genotypic characterization, 28 indigenous rhizobial isolates obtained from different chickpea growing regions in peninsular and northern India were analyzed for diversity. The field isolates were compared to two reference strains TAL620 and UPM-Ca142 representing M. ciceri and M. mediterraneum respectively. Phenotypic markers such as resistance to antibiotics, tolerance to salinity, temperature, pH, phosphate solubilization ability, growth rate and also symbiotic efficiency showed considerable diversity among rhizobial isolates. Their phenotypic patterns showed adaptations of rhizobial isolates to abiotic stresses such as heat and salinity. Two salt tolerant strains (1.5% NaCl by T1 and T4) with relatively high symbiotic efficiency and two P-solubilising strains (66.7 and 71 microg/ml by T2 and T5) were identified as potential bioinoculants. Molecular profiling by 16S ribosomal DNA Restriction Fragment Length Polymorphism (RFLP) revealed three clusters at 67% similarity level. Further, the isolates were differentiated at intraspecific level by 16S rRNA gene phylogeny. Results assigned all the chickpea rhizobial field isolates to belong to three different species of Mesorhizobium genus. 46% of the isolates grouped with Mesorhizobium loti and the rest were identified as M. ciceri and M. mediterraneum, the two species which have been formerly described as specific chickpea symbionts. This is the first report on characterization of chickpea nodulating rhizobia covering soils of both northern and peninsular India. The collection of isolates, diverse in terms of species and symbiotic effectiveness holds a vast pool of genetic material which can be effectively used to yield superior inoculant strains.     

Modulation of symbiotic efficiency and nodular antioxidant enzyme activities in two <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> genotypes under salinity

To analyse nodular expression of antioxidant enzymes depending on plant genotype and salinity, two Phaseolus vulgaris genotypes, tolerant BAT477 and sensitive COCOT, were inoculated with the reference strain Rhizobium tropici CIAT899 and grown under 25 and 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity measured by the acetylene reducing activity (ARA) as an indicator of nitrogenase (E.C. 1.7.9.92) activity were more affected by salt concentrations in COCOT than in BAT477, particularly with 50 mM NaCl. Electrophoresis analysis of antioxidant enzymes in nodules, roots and free-living rhizobia showed that only catalase (CAT E.C. 1.11.1.6) isoenzymes varied with genotype. The sensitive genotype showed lower antioxidant enzyme activities than tolerant genotype and it was more affected by salinity. In the tolerant genotype catalase and ascorbate peroxidase (APX, E.C. 1.11.1.11) were inhibited by salt stress, whereas superoxide dismutase (SOD, E.C. 1.15.1.1) and peroxidase (POX, E.C. 1.11.1.7) were activated by salinity. Statistical analysis allowed suggesting that tolerance to salinity is associated with a differential regulation of distinct superoxide dismutase and peroxidase activities.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

Role of Arbuscular Mycorrhizal Symbiosis in Proline Biosynthesis and Metabolism of Cicer arietinum L. (Chickpea) Genotypes Under Salt Stress

Salinity causes osmotic stress and negatively impacts plant growth and productivity. Proline is one of the most important osmoprotectants synthesized under stressed conditions. Accumulation of free proline occurs due to enhanced biosynthesis and repressed degradation, and both processes are controlled by feedback regulatory mechanisms. Arbuscular mycorrhizal (AM) fungi are considered to be bioameliorators of salinity stress due to their wide-ranging presence in contaminated soils and their role in modulation of biochemical processes. Chickpea is considered sensitive to salinity. However, reports on AM-induced osmoprotection through regulation of proline biosynthesis in chickpea genotypes are scant. The present study investigated the influence of AM symbiosis on proline metabolism in two chickpea (Cicer arietinum L.) genotypes (PBG-5 and CSG-9505) under salt stress and correlated the same with sodium (Na⁺) ion uptake. Salinity reduced plant biomass (roots and shoots), with roots being more negatively affected than shoots. Mycorrhizal colonization with Glomus mosseae was much stronger in PBG-5 and was correlated with reduced Na⁺ ion uptake and higher growth when compared with CSG-9505 under stressed and unstressed conditions. Mycorrhizal symbiosis with chickpea roots boosted proline biosynthesis by significantly increasing pyrroline-5-carboxylate synthetase (P-5-CS) and glutamate dehydrogenase (GDH) activities with a concomitant decline in proline dehydrogenase (ProDH) activity under salt stress. The enhancement of the activity of these enzymes was higher in PBG-5 than in CSG-9505 and could be directly correlated with the percent mycorrhizal colonization and Na⁺ uptake. The study indicated a strong role of AM symbiosis in enhancing stress tolerance in chickpea by significantly modulating proline metabolism and Na⁺ uptake.     

Genomic diversity of chickpea-nodulating rhizobia in Ningxia (north central China) and gene flow within symbiotic Mesorhizobium muleiense populations

Diversity and taxonomic affiliation of chickpea rhizobia were investigated from Ningxia in north central China and their genomic relationships were compared with those from northwestern adjacent regions (Gansu and Xinjiang). Rhizobia were isolated from root-nodules after trapping by chickpea grown in soils from a single site of Ningxia and typed by IGS PCR-RFLP. Representative strains were phylogenetically analyzed on the basis of the 16S rRNA, housekeeping (atpD, recA and glnII) and symbiosis (nodC and nifH) genes. Genetic differentiation and gene flow were estimated among the chickpea microsymbionts from Ningxia, Gansu and Xinjiang. Fifty chickpea rhizobial isolates were obtained and identified as Mesorhizobium muleiense. Their symbiosis genes nodC and nifH were highly similar (98.4 to 100%) to those of other chickpea microsymbionts, except for one representative strain (NG24) that showed low nifH similarities with all the defined Mesorhizobium species. The rhizobial population from Ningxia was genetically similar to that from Gansu, but different from that in Xinjiang as shown by high chromosomal gene flow/low differentiation with the Gansu population but the reverse with the Xinjiang population. This reveals a biogeographic pattern with two main populations in M. muleiense, the Xinjiang population being chromosomally differentiated from Ningxia-Gansu one. M. muleiense was found as the sole main chickpea-nodulating rhizobial symbiont of Ningxia and it was also found in Gansu sharing alkaline-saline soils with Ningxia. Introduction of chickpea in recently cultivated areas in China seems to select from alkaline-saline soils of M. muleiense that acquired symbiotic genes from symbiovar ciceri.     

Salinity tolerant Trichoderma harzianum reinforces NaCl tolerance and reduces population dynamics of Fusarium oxysporum f.sp. ciceri in chickpea (Cicer arietinum L.) under salt stress conditions

The antagonistic potential of salinity tolerant (ST) Trichoderma (Th) isolates against Fusarium oxysporum f.sp. ciceri (foc) was tested, along with their capability to induce relative salt stress tolerance in chickpea with the aim to exploit their use as biological agents in reducing deleterious effects of salinity and controlling Fusarium wilt of chickpea under saline soil conditions. Under laboratory conditions, salt stress was created by supplementing nutrient medium with different concentrations of NaCl viz. 0, 70, 150 and 240?mM NaCl and a pot experiment was conducted using natural saline soil (EC – 6.6 dS?m^?1). Out of 45 Th isolates studied, only five isolates viz. Th-13, Th-14, Th-19, Th-33 and Th-50 were selected to be ST as these were able to grow and sporulate in growth medium containing up to 240?mM NaCl. In saline medium, ST isolates greatly surpassed salinity sensitive (SS) isolate with respect to growth rate, mycelial dry weight, sporulation and biological proficiency against foc. Out of five ST isolates that retained their tolerance to different salt stress levels, Th-14 and Th-19 showed maximum antagonism against foc. Under greenhouse conditions, chickpea plants obtained from seeds bioprimed with Th-14 and Th-19 performed well both at germination and seedling stage in comparison to control in saline soil. As compared to untreated plants, characterisation of Th treated plants confirmed that they had reinforced contents of proline along with relatively higher levels of total phenols, membrane stability index and superoxide dismutase activity while lower accumulation of hydrogen peroxide and malondealdehyde contents. ST isolates, Th-14 and Th-19 significantly reduced foc-induced wilt disease incidence in chickpea plants. The population density of both the Th isolates in rhizosphere far exceeded that of foc under both saline and non-saline soils. However, Th-14 was found more efficient in increasing relative salt stress tolerance in chickpea and reducing the foc growth in rhizosphere under present materials and conditions. These findings provide a novel paradigm for developing alternative, environmentally safe strategy to alleviate salt stress and manage fungal diseases such as foc that aggravates under saline soils.     

Compatible rhizosphere microbes mediated alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid activities

The study was conducted to examine efficacy of a rhizospheric microbial consortium comprising of a fluorescent Pseudomonas (PHU094), Trichoderma (THU0816) and Rhizobium (RL091) strain on activation of physiological defense responses in chickpea against biotic stress caused by the collar rot pathogen Sclerotium rolfsii. Results of individual microbes were compared with dual and triple strain mixture treatments with reduced microbial load (1/2 and 1/3rd, respectively, of individual microbial load compared to single microbe application) in the mixtures. Periodical studies revealed maximum activities of phenylalanine ammonia lyase [E.C. 4.1.3.5] and polyphenol oxidase [E.C. 1.14.18.1] and accumulation of total phenol content in chickpea in the triple microbe consortium treated plants challenged with the pathogen compared to the single microbe and dual microbial consortia. Similarly, the expression of the antioxidant enzymes superoxide dismutase [E.C.1.15.1.1] and peroxidase [E.C.1.11.1.7] was also highest in the triple microbial consortium which was correlated with lesser lipid peroxidation in chickpea under the biotic stress. Histochemical staining clearly showed maximum and uniform lignification in vascular bundles of chickpea stem sections treated with the triple microbes. The physiological responses were directly correlated with the mortality rate as least plant mortality was recorded in the triple microbe consortium treated plants. The results thus suggest an augmented elicitation of stress response in chickpea under S. rolfsii stress by the triple microbial consortium in a synergistic manner under reduced microbial load.     

Comparative study of nitrogen fixation and carbon metabolism in two chick-pea (Cicer arietinum L.) cultivars under salt stress

Two cultivars of Cicer arietinum with differential tolerance to salinity have been compared by analysing growth, photosynthesis, nodulation, nitrogenase activity, and carbon metabolism in the nodule cytosol. The aim was to help elucidate the relationships between, on the one hand, sucrose and malate metabolism in nodules and, on the other, the inhibition of nitrogen fixation under salt stress. Chick-pea cultivars Pedrosillano (sensitive) and ILC1919 (tolerant) inoculated with Mesorhizobium ciceri strain Ch-191 were grown in a controlled environmental chamber and were treated with salt (0, 50, 75, and 100 mM NaCl) from sowing to harvest time (28 d). Plant growth and photosynthesis were more affected by salt in Pedrosillano than in ILC1919. Also the effect of salt on nodulation and nitrogen fixation was much more pronounced in Pedrosillano. The increase in nodular mass in ILC1919 can partially counteract the inhibition of nitrogenase activity. The enzymes of sucrose breakdown were inhibited by NaCl, but in ILC1919 a rise in alkaline invertase was observed with salinity, which could compensate for the lack of the sucrose synthase hydrolytic activity. The activity of PEPC was stimulated by salt in ILC1919. Also, this cultivar showed higher malate concentrations in root nodules.     

Characterization of an rpoN mutant of Mesorhizobium ciceri

AIMS: To study the genetic basis of C(4)-dicarboxylate transport (Dct) in relation to symbiotic nitrogen fixation in Mesorhizobium ciceri. METHODS AND RESULTS: A Tn5-induced mutant strain (TL16) of M. ciceri, unable to grow on C(4)-dicarboxylates, was isolated from the wild-type strain TAL 620. The mutant lacked activities of the enzymes, which use C(4)-dicarboxylates as substrate. The sequencing of the 3.2kb EcoRI fragment, which was the site of Tn5 insertion, revealed three complete and two partial open reading frames. In the mutant, Tn5 interrupted the rpoN gene, of which only one copy was there. Complementation and biochemical studies suggest that the M. ciceri rpoN activity is required for C(4)-Dct, maturation of bacteroids and symbiotic nitrogen fixation. The fine structure of the ineffective nodules produced by TL16 on Cicer arietinum L changed in comparison with those produced by the wild type. CONCLUSIONS: The mutant strain TL16 suffered a disruption in the rpoN gene. Only one copy of rpoN gene is present in M. ciceri. The mutation abolishes Dct activity. It additionally abolishes the symbiotic nitrogen fixation activity of the bacteroids in the nodules. SIGNIFICANCE AND IMPACT OF THE STUDY: This first document in M. ciceri shows that a functional rpoN gene is essential for the transport of dicarboxylic acids and symbiotic nitrogen fixation.     

Enzymes of malate metabolism in Mesorhizobium ciceri CC 1192

Electrophoretic studies were performed on enzymes concerned with the oxidation of malate in free-living and bacteroid cells of Mesorhizobium ciceri CC 1192, which forms nitrogen-fixing symbioses with chickpea (Cicer arietinum L.) plants. Two malate dehydrogenases were detected in extracts from both types of cells in native polyacrylamide electrophoresis gels that were stained for enzyme activity. One band of malate dehydrogenase activity was stained only in the presence of NADP+, whereas the other band was revealed with NAD+ but not NADP+. Further evidence for the occurrence of separate NAD- and NADP-dependent malate dehydrogenases was obtained from preliminary enzyme kinetic studies with crude extracts from free-living M. ciceri CC 1192 cells. Activity staining of electrophoretic gels also indicated the presence of two malic enzymes in free-living and bacteroid cells of M. ciceri CC 1192. One malic enzyme was active with both NAD+ and NADP+, whereas the other was specific for NADP+. Possible roles of the multiple forms of malate dehydrogenase and malic enzyme in nitrogen-fixing symbioses are discussed.     

Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress

To analyse nodular antioxidant enzyme expression in response to salt stress, Phaseolus vulgaris genotype BAT477 was inoculated with reference strain CIAT899, and treated with 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity were analysed. Results showed that: (1) all parameters, particularly in nodules, were affected by salt treatments, and (2) confirmed preferential growth allocation to roots. The ARA was significantly decreased by salt treatments. Protein dosage confirmed that nodules were more affected by salt treatment than were roots. We analysed superoxide dismutase, catalase, ascorbate peroxidase and peroxidase in nodules, roots and a free rhizobial strain. Our results indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD expressed the same CuZn, Fe and Mn SOD isoforms in nodules and roots, whereas in free rhizobia we found only one Fe and Mn SOD. APX and POX nodule and root profiles had only root origins, as no rhizobial band was detected. Under salt stress, plant growth, nitrogen fixation and activities of antioxidant defense enzymes in nodules were affected. Thus, these enzymes appear to preserve symbiosis from stress turned out that NaCl salinity lead to a differential regulation of distinct SOD and POX isoenzyme. So their levels in nodules appeared to be consistent with a symbiotic nitrogen fixing efficiency hypothesis, and they seem to function as the molecular mechanisms underlying the nodule response to salinity.     

Identification of genes involved in salt tolerance and symbiotic nitrogen fixation in chickpea rhizobium Mesorhizobium ciceri Ca181

In an attempt to find the genes involved in salt tolerance of the highly adaptable chickpea rhizobium strain, Mesorhizobium ciceri Ca181, a Tn5 transposon insertion library was generated and screened to identify five mutants with inability to survive in the presence of 0.1 M NaCl. The genes disrupted in these mutants due to insertion of the transposon were identified by sequencing of Tn5 flanking sequences after inverse PCR. One of the mutants had a disruption in diguanylate cyclase gene which is involved in bacterial biofilm formation and persistence. The second mutant had a disruption in an ABC transporter membrane protein gene, which is involved in the uptake of nutrients and cellular osmoprotection. The third mutant had a disruption in a gene showing homology with rhamnulose 1-phosphate aldolase which has an important role in the central metabolism of L-rhamnulose. The fourth mutant had a disruption in a capsule synthesis gene and the fifth mutant had an insertion in an oxidoreductase gene. When these mutants were inoculated into the host chickpea plant under normal non-saline conditions, they formed symbiotic nodules but with severely reduced nitrogenase activity. Hence, it appears that bacterial ability to adapt to hyper-osmotic salt stress conditions is also important for its nitrogen fixing ability in the chickpea root nodules. Allele mining for variant forms of the identified genes in the germplasm resources of M. ciceri may help in the development of highly adaptive and efficient nitrogen fixing strains of the chickpea rhizobium.     

Interactive effects of silicon and arbuscular mycorrhiza in modulating ascorbate-glutathione cycle and antioxidant scavenging capacity in differentially salt-tolerant <Emphasis Type="Italic">Cicer arietinum</Emphasis> L. genotypes subjected to long-term salinity

Salinity is the major environmental constraint that affects legume productivity by inducing oxidative stress. Individually, both silicon (Si) nutrition and mycorrhization have been reported to alleviate salt stress. However, the mechanisms adopted by both in mediating stress responses are poorly understood. Thus, pot trials were undertaken to evaluate comparative as well as interactive effects of Si and/or arbuscular mycorrhiza (AM) in alleviating NaCl toxicity in modulating oxidative stress and antioxidant defence mechanisms in two Cicer arietinum L. (chickpea) genotypes—HC 3 (salt-tolerant) and CSG 9505 (salt-sensitive). Plants subjected to different NaCl concentrations (0–100 mM) recorded a substantial increase in the rate of superoxide radical (O₂ ^·?), H₂O₂, lipoxygenase (LOX) activity and malondialdehyde (MDA) content, which induced leakage of ions and disturbed Ca²⁺/Na⁺ ratio in roots and leaves. Individually, Si and AM reduced oxidative burst by strengthening antioxidant enzymatic activities (superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPOX)). Si was relatively more efficient in reducing accumulation of stress metabolites, while mycorrhization significantly up-regulated antioxidant machinery and modulated ascorbate-glutathione (ASA-GSH) cycle. Combined applications of Si and AM complemented each other in reducing reactive oxygen species (ROS) build-up by further enhancing the antioxidant defence responses. Magnitude of ROS-mediated oxidative burden was lower in HC 3 which correlated strongly with more effective AM symbiosis, better capacity to accumulate Si and stronger defence response when compared with CSG 9505. Study indicated that Si and/or AM fungal amendments upgraded salt tolerance through a dynamic shift from oxidative destruction towards favourable antioxidant defence system in stressed chickpea plants.