Arbuscular mycorrhizal fungi and nitric oxide alleviate cadmium phytotoxicity by improving internal detoxification mechanisms of corn plants.

Plants develop several external and internal mechanisms to increase their tolerance to heavy metals (HMs) toxicity including cadmium (Cd). Symbiosis with arbuscular mycorrhizae fungi (AMF) is one of the plants' strategies to tolerate HMs toxicity. Nitric oxide (NO), as a signaling molecule, is also involved in physiological responses of plants to various stresses. The present work was conducted as a factorial completely randomized design with three replications to study the effects of Funneliformis mosseae fungi and Sodium nitroprusside (SNP, 100 mM) as a donor of NO alone, in combination (AMF + SNP) on corn plant growth, and internal detoxification mechanisms of Cd toxicity in a Cd-contaminated calcareous soil (0, 25, 50, and 100 mg Cd kg-1). The results showed that under Cd stress, AMF inoculation and/or foliar application of SNP significantly increased plant growth (32% to 103% for shoot and 44% to 84% for root) by decreasing Cd concentration in corn plant tissues (23% to 46% for shoot and 19% to 40% for root). Cd-induced oxidative stress was mitigated by AMF and/or SNP by enhancing the activities of antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), and concentration of non-enzymatic antioxidants such as glutathione (GSH) and phytochelatin (PC). Increasing the tolerance index (TI) and decreasing the transfer factor (TF) in the corn plants treated with AMF and/or SNP, confirm the efficient role of SNP and AMF in stimulating the detoxification mechanisms of Cd within the plant cells, which was more pronounced at the lowest Cd level (25 mg Cd kg-1). In conclusion, symbiotic associations of corn plants with AMF alone or in combination with SNP mitigated the detrimental effect of Cd toxicity in corn grown in Cd-contaminated calcareous soil. The corn's internal detoxification mechanisms lowered the Cd concentration in plant tissue which resulted in the improvement of the corn's growth parameters.

Co-application of ACC deaminase-producing rhizobial bacteria and melatonin improves salt tolerance in common bean (Phaseolus vulgaris L.) through ion homeostasis.

A comprehensive body of scientific evidence indicates that rhizobial bacteria and melatonin enhance salt tolerance of crop plants. The overall goal of this research was to evaluate the ability of Rhizobium leguminoserum bv phaseoli to suppress salinity stress impacts in common bean treated with melatonin. Treatments included bacterial inoculations (inoculated (RI) and non-inoculated (NI)), different salinity levels (non-saline (NS), 4 (S1) and 8 (S2) dS m-1 of NaCl) and priming (dry (PD), melatonin (PM100) and hydro (PH) priming) with six replications in growing media containing sterile sand and perlite (1:1). The results showed that the bacterial strain had the ability to produce indole acetic acid (IAA), ACC deaminase and siderophore. Plants exposed to salinity stress indicated a significant decline in growth, yield, yield components, nitrogen fixation and selective transport (ST), while showed a significant increase in sodium uptake. However, the combination of PM100 and RI treatments by improving growth, photosynthesis rate and nitrogen fixation positively influenced plant performance in saline conditions. The combined treatment declined the negative impacts of salinity by improving the potassium translocation, potassium to sodium ratio in the shoot and root and ST. In conclusion, the combination of melatonin and ACC deaminase producing rhizobium mitigated the negative effects of salinity. This result is attributed to the increased ST and decreased sodium uptake, which significantly reduced the accumulation of sodium ions in shoot.

A comparison between the function of Serendipita indica and Sinorhizobium meliloti in modulating the toxicity of zinc oxide nanoparticles in alfalfa (Medicago sativa L.).

Zinc oxide nanoparticles (ZnO-NPs) are among the most commonly used nano-fertilizers (NF). However, elevated levels of ZnO-NPs in soil may affect plant growth and development due to its potential toxicity when accumulated in large amounts in plant tissues. This research was conducted using an in situ rhizobox system with the aims of evaluating zinc uptake from nano-zinc oxide amended rhizosphere soil by alfalfa plant and the effect of plant growth-promoting microorganisms on alleviating the phytotoxicity of ZnO-NPs. Treatments included microbial inoculations (Sinorhizobium meliloti, Serendipita indica) and different ZnO-NP concentrations (0, 400, and 800 mg kg-1) with three replications. The results indicated that S. indica minimized the phytotoxicity of ZnO-NPs to alfalfa by enhancing growth rate and decreasing zinc (Zn) translocation from root to shoot. Compared with plants inoculated with S. meliloti, co-inoculation with S. indica increased the shoot dry weight by 18.33% and 8.05% at 400 and 800 mg kg-1 ZnO-NPs, respectively. However, at the highest level of ZnO-NPs (800 mg kg-1), root inoculation of S. indica and S. indica + S. meliloti decreased Zn translocation factor by 60.2% and 44.3% compared to S. meliloti, respectively. Furthermore, a distinct relation between tolerance of S. indica-colonized plant to ZnO-NPs and the ability of S. indica in inhibiting or retarding degradation of polyunsaturated lipids through prevention of excess reactive oxygen species formation was observed. Malondialdehyde content of inoculated plants with S. indica either alone or in combination with S. meliloti was significantly lower than non-inoculated plants (p< 0.01). Zn-induced oxidative stress was mitigated by S. indica through enhanced activities of catalase and peroxidase enzymes. The findings of the present study indicate the potential use of endophytes fungus S. indica for ensuring food safety and security, and human health in heavy metal-polluted soil by reducing the phytoavailability of heavy metals in the aerial parts of the host plants.

Combination of Siderophore-Producing Bacteria and Piriformospora indica Provides an Efficient Approach to Improve Cadmium Tolerance in Alfalfa.

Application of siderophore-producing microorganisms (SPMs), as an environmentally friendly approach, facilitates plant growth and survival under heavy metals toxicity. This study evaluated the effectiveness of SPMs, belonging to the bacterial genera Rhizobium and Pseudomonas and a root endophytic fungus (Piriformospora indica) to improve the fitness of alfalfa under cadmium (Cd) stress. A greenhouse experiment was performed as a randomized design with factorial arrangement of treatments. Treatments included microbial inoculations (Sinorhizobium meliloti, Pseudomonas fluorescence, and P. indica) and different Cd concentrations (0, 2, 5, 10 mg/kg) with three replications in potting media containing sand and sterile perlite (v/v, 2:1). The effect of Cd on plant growth and development, antioxidant enzymes activities, and accumulation of Cd and nutrients in alfalfa plant was investigated. Alfalfa inoculated with SPMs showed significantly higher biomass and nutrients uptake under both normal and Cd stress conditions than the controls. Under the highest Cd concentration (10 mg/kg), alfalfa plants inoculated with P. fluorescens and P. indica, either alone or in combination, showed the highest shoot dry weights. Cd-induced oxidative stress was mitigated by SPMs through enhanced antioxidant enzyme activities of catalase, ascorbate peroxidase, and guaiacol peroxidase. We showed that P. indica either alone or in combination with the siderophore producing bacteria (SPB) minimized the toxicity of Cd by enhanced growth rate and the lower Cd concentration in the shoots. In conclusion, metal-resistant SPMs could assist alfalfa to survive in Cd-contaminated soil by enhancing plant growth and development. Application of plant-associated microbes is an efficient, environmentally friendly approach to surmount the adverse effects of heavy metals toxicity on plants, animals, and humans. Graphical abstract.

Chemical- and microbial-enhanced phytoremediation of cadmium-contaminated calcareous soil by maize.

Phytoremediation is an appropriate technology used to remove pollutants from environment components. A greenhouse trial was conducted to test the hypothesis that application of surfactant levels and inoculation with Pseudomonas fluorescens bacterium and/or Piriformospora indica fungus enhances the phytoremediation of cadmium (Cd). Maize seeds were sown in Cd-polluted soil, and after 2 months Cd status in plant tissues and Cd phytoremediation criteria was determined. Results showed that application of surfactant increased root and shoot dry weight. Mean Cd uptake in roots and shoots increased following the application of 2 and 4 mmol kg-1 Tween 80, respectively. Application of 2 mmol kg-1 Tween 80 increased mean Cd uptake efficiency, while application of 4 mmol kg-1 Tween 80 increased phytoextraction and translocation efficiencies. Inoculation with P. indica and P. fluorescens was mostly effective in increasing Cd uptake and Cd phytoextraction efficiency, respectively. Co-inoculation with P. indica and P. fluorescens had no superiority to application of each inoculant alone. Since most of the Cd remained in roots, phytostabilization is probably the main mechanism controlling Cd phytoremediation by maize. According to the results, application of Tween 80 and inoculation with P. indica and P. fluorescens effectively enhanced phytoremediation of Cd-contaminated soil by maize.

Salinity-associated microRNAs and their potential roles in mediating salt tolerance in rice colonized by the endophytic root fungus Piriformospora indica.

Piriformospora indica (P. indica), an endophytic root fungus, supports the growth and enhanced tolerance of plants to biotic and abiotic stresses. Several recent studies showed the significant role of small RNA (sRNA) molecules including microRNAs (miRNAs) in plant adaption to environmental stress, but little is known concerning the symbiosis-mediated salt stress tolerance regulated at miRNAs level. The overarching goal of this research is to elucidate the impact of miRNAs in regulating the P. indica-mediated salt tolerance in rice. Applying sRNA-seq analysis led to identify a set of 547 differentially abundant miRNAs in response to P. indica inoculation and salt stress. These included 206 rice-specific and 341 previously known miRNAs from other plant species. In silico analysis of miRNAs predictions of the differentially abundant miRNAs led to identifying of 193 putatively target genes, most of which were encoded either genes or transcription factors involved in nutrient uptake, sodium ion transporters, growth regulators, and auxin- responsive proteins. The rice-specific miRNAs targeted the transcription factors involved in the import of potassium ions into the root cells, the export of sodium ions, and plant growth and development. Interestingly, P. indica affected the differential abundance of miRNAs regulated genes and transcription factors linked to salt stress tolerance. Our data helps to understand the molecular basis of salt stress tolerance mediated by symbionts in plant and the potential impact of miRNAs for genetic improvement of rice varieties for tolerance to salt stress.

Maize water status and physiological traits as affected by root endophytic fungus Piriformospora indica under combined drought and mechanical stresses.

MAIN CONCLUSION: Under combined drought and mechanical stresses, mechanical stress primarily controlled physiological responses of maize. Piriformospora indica mitigated the adverse effects of stresses, and inoculated maize experienced less oxidative damage and had better adaptation to stressful conditions. The objective of this study was to investigate the effect of maize root colonization by an endophytic fungus P. indica on plant water status, physiological traits and root morphology under combined drought and mechanical stresses. Seedlings of inoculated and non-inoculated maize (Zea mays L., cv. single cross 704) were cultivated in growth chambers filled with moistened siliceous sand at a matric suction of 20 hPa. Drought stress was induced using PEG 6000 solution with osmotic potentials of 0, - 0.3 and - 0.5 MPa. Mechanical stress (i.e., penetration resistances of 1.05, 4.23 and 6.34 MPa) was exerted by placing weights on the surface of the sand medium. After 30 days, leaf water potential (LWP) and relative water content (RWC), root and shoot fresh weights, root volume (RV) and diameter (RD), leaf proline content, leaf area (LA) and catalase (CAT) and ascorbate peroxidase (APX) activities were measured. The results show that exposure to individual drought and mechanical stresses led to higher RD and proline content and lower plant biomass, RV and LA. Moreover, increasing drought and mechanical stress severity increased APX activity by about 1.9- and 3.1-fold compared with the control. When plants were exposed to combined stresses, mechanical stress played the dominant role in controlling plant responses. P. indica-inoculated plants are better adapted to individual and combined stresses. The inoculated plants had greater RV, LA, RWC, LWP and proline content under stressful conditions. In comparison with non-inoculated plants, inoculated plants showed lower CAT and APX activities which means that they experienced less oxidative stress induced by stressful conditions.

The effect of Piriformospora indica on the root development of maize (Zea mays L.) and remediation of petroleum contaminated soil.

As the depth of soil petroleum contamination can vary substantially under field conditions, a rhizotron experiment was performed to investigate the influence of endophyte, P. indica, on maize growth and degradation of petroleum components in a shallow and a deep-reaching subsurface layer of a soil. For control, a treatment without soil contamination was also included. The degree in contamination and the depth to which it extended had a strong effect on the growth of the plant roots. Contaminated soil layers severely inhibited root growth thus many roots preferred to bypass the shallow contaminated layer and grow in the uncontaminated soil. While the length and branching pattern of these roots were similar to those of uncontaminated treatment. Inoculation of maize with P. indica could improve root distribution and root and shoot growth in all three contamination treatments. This inoculation also enhanced petroleum degradation in soil, especially in the treatment with deep-reaching contamination, consequently the accumulation of petroleum hydrocarbons (PAHs) in the plant tissues were increased.

Proteomics study reveals the molecular mechanisms underlying water stress tolerance induced by Piriformospora indica in barley.

Piriformospora indica is a mutualistic root endophytic fungus, which transfers several benefits to hosts including enhance plant growth and increase yield under both normal and stress conditions. It has been shown that P. indica root-colonization enhances water stress tolerance based on general and non-specific plant-species mechanism. To better understand the molecular mechanism of P. indica-mediated drought stress tolerance, we designed a set of comparative experiments to study the impact of P. indica on barely plants cultivar "Golden Promise" grown under different drought levels [Filed capacity (F.C.) and 25% F.C.]. P. indica enhanced root and shoot biomass of colonized plants under both well-watered and water-deficit conditions. Proteome analysis of P. indica-colonized barley leaves under well-treated and water-deficit conditions resulted in detection of 726 reproducibly protein spots. Mass spectrometry analysis resulted in the identification of 45 differentially accumulated proteins involved in photosynthesis, reactive oxygen scavenging, metabolisms, signal transduction, and plant defense responses. Interestingly, P. indica increased the level of proteins involved in photosynthesis, antioxidative defense system and energy transport. We propose that P. indica-mediated drought stress tolerance in barely is through photosynthesis stimulation, energy releasing and enhanced antioxidative capacity in colonized plants. BIOLOGICAL SIGNIFICANCE: Plant mutualistic symbionts offer long-term abiotic stress tolerance through the host adaptation to environmental stress. There have been a few published proteomic studies of plant symbionts to drought, and this is thought to be the first proteomic analysis, demonstrating the impact of endophyte on barley plant under drought stress. For some of identified proteins like TCTP and PCNA, a connection to physiological function in plants is novel, and can be the best candidates for sources of drought tolerance in future studies.

A proteomics approach to study the molecular basis of enhanced salt tolerance in barley (Hordeum vulgare L.) conferred by the root mutualistic fungus Piriformospora indica.

Piriformospora indica is a root-interacting mutualistic fungus capable of enhancing plant growth, increasing plant resistance to a wide variety of pathogens, and improving plant stress tolerance under extreme environmental conditions. Understanding the molecular mechanisms by which P. indica can improve plant tolerance to stresses will pave the way to identifying the major mechanisms underlying plant adaptability to environmental stresses. We conducted greenhouse experiments at three different salt levels (0, 100 and 300 mM NaCl) on barley (Hordeum vulgare L.) cultivar "Pallas" inoculated with P. indica. Based on the analysis of variance, P. indica had a significant impact on the barley growth and shoot biomass under normal and salt stress conditions. P. indica modulated ion accumulation in colonized plants by increasing the foliar potassium (K(+))/sodium (Na(+)) ratio, as it is considered a reliable indicator of salt stress tolerance. P. indica induced calcium (Ca(2+)) accumulation and likely influenced the stress signal transduction. Subsequently, proteomic analysis of the barley leaf sheath using two-dimensional electrophoresis resulted in detection of 968 protein spots. Of these detected spots, the abundance of 72 protein spots changed significantly in response to salt treatment and P. indica-root colonization. Mass spectrometry analysis of responsive proteins led to the identification of 51 proteins. These proteins belonged to different functional categories including photosynthesis, cell antioxidant defense, protein translation and degradation, energy production, signal transduction and cell wall arrangement. Our results showed that P. indica induced a systemic response to salt stress by altering the physiological and proteome responses of the plant host.