An Insight into Efflux-Mediated Arsenic Resistance and Biotransformation Potential of Enterobacter Cloacae RSC3 from Arsenic Polluted Area.

Indiscriminate discharge of heavy metals/metalloids from different sources into the sustainable agro-ecosystem is a major global concern for food security and human health. Arsenic (As), categorized as group one human carcinogen is a quintessential toxic metalloid that alters the microbial compositions and functions, induce physiological and metabolic changes in plants and contaminate surface/ground water. The management of arsenic toxicity, therefore, becomes imminent. Acknowledging the arsenic threat, the study was aimed at identifying arsenic resistant bacteria and evaluating its arsenic removal/detoxification potential. Of the total 118 bacterial isolates recovered from arsenic rich environment, the bacterial strain RSC3 demonstrating highest As tolerance was identified as Enterobacter cloacae by 16S rRNA gene sequence analysis. Enterobacter cloacae tolerated high concentration (6000 ppm) of As and exhibited 0.55 h-1 of specific growth rate as calculated from growth kinetics data. Strain RSC3 also displayed varying level of resistance to other heavy metals and many antibacterial drugs in plate bioassay. The bacterial strain RSC3 possessed gene (arsC) which causes transformation of arsenate to arsenite. The arsenate uptake and efflux of the bacterial cells was revealed by high throughput techniques such as AAS, SEM/TEM and EDX. The simultaneous As reducing ability, and multi metal/multi-antibiotics resistance potentials of E. cloacae provides a promising option in the microbes based remediation of As contaminated environments.

Understanding the phytotoxic impact of Al3+, nano-size, and bulk Al2O3 on growth and physiology of maize (Zea mays L.) in aqueous and soil media.

The release and accumulation of metal-oxide nanoparticles in soils have threatened terrestrial plants. However, limited knowledge is available on the accumulation of nano-Al2O3 (22 nm), bulk-Al2O3 (167 nm), and Al3+ by maize plants and the subsequent impact on its physiology and growth in agar (0.7% w/v), hydroponic (1X), and soil. Maize plants were cultivated with 0.05-2 mg g-1 or ml-1 of three Al types and their biological attributes, oxidative status, Al bioaccumulation, and translocation were measured. The ICP-MS results revealed a dose-dependent increase (P ≤ 0.05 or ≤0.01) in Al content in maize tissues following nano-Al2O3 and Al3+ exposure, however, plants exposed to bulk-Al2O3 showed no significant uptake of Al. Atomic mapping by EDX during SEM analysis and TEM revealed varied distributions of nano-Al2O3 from roots to aerial parts and intracellular transportation. Al deposition in tissues followed the order: Al3+ > nano-Al2O3 > bulk-Al2O3 and therefore, a similar trend of toxicity was observed for seed germination, the emergence of plant organs, length, biomass accumulation, total chlorophyll, phosphorus content, and total soluble protein. Oxidative stress was profoundly induced dose-dependently and was highest at 2 mg ml-1 or g-1 of Al3+ and nano-Al2O3 when superoxide radical formation, proline induction, activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (GPX), and glutathione reductase (GR) and membrane lipid peroxidation were measured. Aluminum toxicity was found higher in hydroponically grown maize compared to soil-grown maize. Forty days exposure in soil showed greater inhibition of maize growth compared to 20 days exposure. This study is significant in understanding the maize response to different Al types in soil and soil-free media.

Bioprospecting Plant Growth Promoting Rhizobacteria for Enhancing the Biological Properties and Phytochemical Composition of Medicinally Important Crops.

Traditionally, medicinal plants have long been used as a natural therapy. Plant-derived extracts or phytochemicals have been exploited as food additives and for curing many health-related ailments. The secondary metabolites produced by many plants have become an integral part of human health and have strengthened the value of plant extracts as herbal medicines. To fulfil the demand of health care systems, food and pharmaceutical industries, interest in the cultivation of precious medicinal plants to harvest bio-active compounds has increased considerably worldwide. To achieve maximum biomass and yield, growers generally apply chemical fertilizers which have detrimental impacts on the growth, development and phytoconstituents of such therapeutically important plants. Application of beneficial rhizosphere microbiota is an alternative strategy to enhance the production of valuable medicinal plants under both conventional and stressed conditions due to its low cost, environmentally friendly behaviour and non-destructive impact on fertility of soil, plants and human health. The microbiological approach improves plant growth by various direct and indirect mechanisms involving the abatement of various abiotic stresses. Given the negative impacts of fertilizers and multiple benefits of microbiological resources, the role of plant growth promoting rhizobacteria (PGPR) in the production of biomass and their impact on the quality of bio-active compounds (phytochemicals) and mitigation of abiotic stress to herbal plants have been described in this review. The PGPR based enhancement in the herbal products has potential for use as a low cost phytomedicine which can be used to improve health care systems.

Psychrophilic Bacterial Phosphate-Biofertilizers: A Novel Extremophile for Sustainable Crop Production under Cold Environment.

Abiotic stresses, including low-temperature environments, adversely affect the structure, composition, and physiological activities of soil microbiomes. Also, low temperatures disturb physiological and metabolic processes, leading to major crop losses worldwide. Extreme cold temperature habitats are, however, an interesting source of psychrophilic and psychrotolerant phosphate solubilizing bacteria (PSB) that can ameliorate the low-temperature conditions while maintaining their physiological activities. The production of antifreeze proteins and expression of stress-induced genes at low temperatures favors the survival of such organisms during cold stress. The ability to facilitate plant growth by supplying a major plant nutrient, phosphorus, in P-deficient soil is one of the novel functional properties of cold-tolerant PSB. By contrast, plants growing under stress conditions require cold-tolerant rhizosphere bacteria to enhance their performance. To this end, the use of psychrophilic PSB formulations has been found effective in yield optimization under temperature-stressed conditions. Most of the research has been done on microbial P biofertilizers impacting plant growth under normal cultivation practices but little attention has been paid to the plant growth-promoting activities of cold-tolerant PSB on crops growing in low-temperature environments. This scientific gap formed the basis of the present manuscript and explains the rationale for the introduction of cold-tolerant PSB in competitive agronomic practices, including the mechanism of solubilization/mineralization, release of biosensor active biomolecules, molecular engineering of PSB for increasing both P solubilizing/mineralizing efficiency, and host range. The impact of extreme cold on the physiological activities of plants and how plants overcome such stresses is discussed briefly. It is time to enlarge the prospects of psychrophilic/psychrotolerant phosphate biofertilizers and take advantage of their precious, fundamental, and economical but enormous plant growth augmenting potential to ameliorate stress and facilitate crop production to satisfy the food demands of frighteningly growing human populations. The production and application of cold-tolerant P-biofertilizers will recuperate sustainable agriculture in cold adaptive agrosystems.

Sorghum-Phosphate Solubilizers Interactions: Crop Nutrition, Biotic Stress Alleviation, and Yield Optimization.

Sweet sorghum [Sorghum bicolor (L.) Moench] is a highly productive, gluten-free cereal crop plant that can be used as an alternative energy resource, human food, and livestock feed or for biofuel-ethanol production. Phosphate fertilization is a common practice to optimize sorghum yield but because of high cost, environmental hazards, and soil fertility reduction, the use of chemical P fertilizer is discouraged. Due to this, the impetus to search for an inexpensive and eco-friendly microbiome as an alternative to chemical P biofertilizer has been increased. Microbial formulations, especially phosphate solubilizing microbiome (PSM) either alone or in synergism with other rhizobacteria, modify the soil nutrient pool and augment the growth, P nutrition, and yield of sorghum. The use of PSM in sorghum disease management reduces the dependence on pesticides employed to control the phytopathogens damage. The role of PSM in the sorghum cultivation system is, however, relatively unresearched. In this manuscript, the diversity and the strategies adopted by PSM to expedite sorghum yield are reviewed, including the nutritional importance of sorghum in human health and the mechanism of P solubilization by PSM. Also, the impact of solo or composite inoculations of biological enhancers (PSM) with nitrogen fixers or arbuscular mycorrhizal fungi is explained. The approaches employed by PSM to control sorghum phytopathogens are highlighted. The simultaneous bio-enhancing and biocontrol activity of the PS microbiome provides better options for the replacement of chemical P fertilizers and pesticide application in sustainable sorghum production practices.

Differential bioaccumulations and ecotoxicological impacts of metal-oxide nanoparticles, bulk materials, and metal-ions in cucumbers grown in sandy clay loam soil.

Expanding applications of metal-oxide nanoparticles (NPs) and increased environmental deposition of NPs followed by their interactions with edible crops threaten yields. This study demonstrates the effects of aging (45 days in soil) of four NPs (ZnO, CuO, Al2O3, TiO2; 3.9-34 nm) and their corresponding metal oxide bulk particles (BPs; 144-586 nm) on cucumbers (Cucumis sativus L.) cultivated in sandy-clay-loam field soil and compares these with the phytotoxic effects of readily soluble metal salts (Zn2+, Cu2+, and Al3+). Data revealed the cell-to-cell translocations of NPs, their attachments to outer and inner cell surfaces, nuclear membranes, and vacuoles, and their upward movements to aerial parts. Metal bioaccumulations in cucumbers were found in the order: (i) ZnO-NPs > ZnO-BPs > Zn2+, (ii) CuO-NPs > CuO-BPs > Cu2+, (iii) Al3+> Al2O3-NPs > Al2O3-BPs and (iv) TiO2-NPs > TiO2-BPs. Aging of NPs in soil for 45 days significantly enhanced metal uptake (P ≤ 0.05), for instance aged ZnO-NPs at 1 g kg-1 increased the uptake by 20.7 % over non-aged ZnO-NPs. Metal uptakes inhibited root (RDW) and shoot (SDW) dry weight accumulations. For Cu species, maximum negative impact (%) was exhibited by Cu2+ (RDW:SDW = 94:65) followed by CuO-NPs (RDW:SDW = 78:34) and CuO-BPs (RDW:SDW = 27:22). Aging of NPs/BPs at 1-4 g kg-1 further enhanced the toxic impact of tested materials on biomass accumulations and chlorophyll formation. NPs also induced membrane damage of root tissues and enhanced levels of antioxidant enzymes. The results of this study suggest that care is required when aged metal-oxide NPs of both essential (Zn and Cu) and non-essential (Al and Ti) metals interact with cucumber plants, especially, when they are used for agricultural purposes.

Differential responses of maize (Zea mays) at the physiological, biomolecular, and nutrient levels when cultivated in the presence of nano or bulk ZnO or CuO or Zn2+ or Cu2+ ions.

Expanding applications of metal-based nanoparticles (NPs) in industry and agriculture have influenced agro-ecosystems. However, relatively little is known about the bioaccumulation, distribution, and phytotoxicity of ZnO-NPs, CuO-NPs, ZnO-bulk, CuO-bulk, Zn2+, or Cu2+ in maize. Plants were exposed to 0.05-2 mg ml-1 or g-1 of six tested materials in agar (7 days) in hydroponic medium (20 days), or sandy-clay-loam soil (20 or 40 days). Seed germination, emergence and lengths of plumules, principal and seminal roots were significantly inhibited by ZnO-NPs, CuO-NPs, Zn2+, and Cu2+. Toxicity was more pronounced in hydroponic culture than in soil, and perceptible alterations in biomolecules were evident. ICP-MS analysis exhibited progressive uptake of metals while morphological, elemental, and surface/deeper scanning showed translocation and distribution of NPs in tissues. Tested materials induced enhanced superoxide radical production, lipid peroxidation, and antioxidant enzymes and proline levels. Exposure significantly reduced P-accumulation, photosynthesis, and protein production. Zn2+ and Cu2+ were found to be more toxic than NPs. Compared to 20 days exposure in soil, toxicity slightly increased after 40 days. ZnO-NPs and CuO-NPs increased apoptotic sub-G1 population by 22.4% and 38%, respectively. These results provide a better understanding of the mechanistic aspects responsible for the nanotoxicities of ZnO- and CuO-NPs in maize.

Impact of metal-oxide nanoparticles on growth, physiology and yield of tomato (Solanum lycopersicum L.) modulated by Azotobacter salinestris strain ASM.

The current study for the first time demonstrates the interference of a free-living, N2-fixing, and nanoparticle (NP) tolerant Azotobacter salinestris strain ASM recovered from metal-polluted soil with tomato plant-metal oxide NPs (ZnO, CuO, Al2O3, and TiO2) interactions in a sandy clay loam soil system with bulk materials as control. Tomato plants were grown till full maturity in soils amended with 20-2000 mg kg-1 of each metal-oxide NP with and without seed biopriming and root-inoculation of A. salinestris. A. salinestris was found metabolically active, producing considerably high amounts of bioactive indole-3-acetic-acid, morphologically unaffected, and with low alteration of cell membrane permeability under 125-1500 µgml-1 of NPs. However, ZnO-NPs slightly alter bacterial membrane permeability. Besides, A. salinestris secreted significantly higher amounts of extracellular polymeric substance (EPS) even under NP exposure, which could entrap the NPs and form metal-EPS complex as revealed and quantified by SEM-EDX. NPs were also found adsorbed on bacterial biomass. EPS stabilized the NPs and provided negative zeta potential to NPs. Following soil application, A. salinestris improved the plant performance and augmented the yield of tomato fruits and lycopene content even in NPs stressed soils. Interestingly, A. salinestris inoculation enhanced photosynthetic pigment formation, flower attributes, plant and fruit biomass, and reduced proline level. Bacterial inoculation also reduced the NP's uptake and accumulation significantly in vegetative organs and fruits. The organ wise order of NP's internalization was roots > shoots > fruits. Conclusively, A. salinestris inoculation could be an alternative to increase the production of tomato in metal-oxide NPs contaminated soils.

Biosorption of heavy metals by dry biomass of metal tolerant bacterial biosorbents: an efficient metal clean-up strategy.

Heavy metals discharge at an unrestrained rate from various industries into the environment pose serious human health problems. Considering this, the present study aimed at exploring the metal biosorbing potentials of bacterial strains recovered from polluted soils. The bacterial strains (CPSB1, BM2 and CAZ3) belonging to genera Pseudomonas, Bacillus and Azotobacter expressing multi-metal tolerance ability were identified to species level as P. aeruginosa, B. subtilis and A. chroococcum, respectively, by 16S rRNA partial gene sequence analysis. The biosorption of cadmium, chromium, copper, nickel, lead and zinc by three dead bacterial genera were studied as a function of metal concentration, variable pH of the medium and reaction (contact) time. The three bacterial strains exhibited a tremendous metal removal ability which continued even at the highest tested concentration of some metals. Later, a decline in the percentage of biosorbed metals was recorded as the metal concentration was increased with the simultaneous generation of a driving force to overcome mass transfer resistance for movement of metal ions between the solution and the surface of adsorbent. Among test bacteria, B. subtilis biosorbed a maximum of 96% chromium at 25 µg mL-1 while the maximum percentage (91%) of biosorbed metals recorded at 400 µg Cd mL-1 was observed for P. aeruginosa. The sorption of metal ions by dead biomass of three bacterial genera at optimum conditions followed the order-(i) B. subtilis BM2: Pb > Cu > Ni > Cd > Cr, (ii) A. chroococcum CAZ3: Cr > Cd > Cu > Ni > Pb and (iii) P. aeruginosa CPSB1: Cd > Cr > Ni > Cu > Pb > Zn. It was found that the optimum pH for metal adsorption ranged between pH 8 and 9 which, however, declined substantially at pH 5.0 for all three bacterial strains. In general, the biosorption of Cd, Cr, Cu, Ni and Pb by B. subtilis and A. chroococcum and such metals along with Zn by P. aeruginosa occurred maximally up to 60 min of bacterial growth. The adsorption data with regard to five metals provide an outstanding fit to the Langmuir and Freundlich isotherms. The biosorptive ability of three bacterial genera correlated strongly (r2 > 0.9) with each metal. The bacteria belonging to two Gram-negative genera Pseudomonas (P. aeruginosa) and Azotobacter (A. chroococcum) and one Gram-positive genus Bacillus (B. subtilis) demonstrated exceptional metal removal efficiency and, hence, provides a comprehensive understanding of metal-bacteria sorption process which in effect paves the way for detoxifying/removing metals from contaminated environment.

Destruction of Cell Topography, Morphology, Membrane, Inhibition of Respiration, Biofilm Formation, and Bioactive Molecule Production by Nanoparticles of Ag, ZnO, CuO, TiO2, and Al2O3 toward Beneficial Soil Bacteria.

The unregulated discharge of nanoparticles (NPs) from various nanotechnology industries into the environment is expected to alter the composition and physiological functions of soil microbiota. Considering this knowledge gap, the impact of five NPs (Ag, ZnO, CuO, Al2O3, and TiO2) differing in size and morphology on growth behavior and physiological activity of Azotobacter chroococcum, Bacillus thuringiensis, Pseudomonas mosselii, and Sinorhizobium meliloti were investigated. Various biochemical and microscopic approaches were adopted. Interestingly, all bacterial strains were found sensitive to Ag-NPs and ZnO-NPs but showed tolerance toward CuO, Al2O3, and TiO2-NPs. The loss of cellular respiration due to NPs was coupled with a reduction in population size. ZnO-NPs at 387.5 µg mL-1 had a maximum inhibitory impact on A. chroococcum and reduced its population by 72%. Under Ag-NP stress, the reduction in IAA secretion by bacterial strains followed the order S. meliloti (74%) > P. mosselii (63%) > A. chroococcum (49%). The surface of bacterial cells had small- or large-sized aggregates of NPs. Also, numerous gaps, pits, fragmented, and disorganized cell envelopes were visible. Additionally, a treated cell surface appeared corrugated with depressions and alteration in cell length and a strong heterogeneity was noticed under atomic force microscopy (AFM). For instance, NPs induced cell roughness for P. mosselii followed the order 12.6 nm (control) > 58 nm (Ag-NPs) > 41 nm (ZnO-NPs). TEM analysis showed aberrant morphology, cracking, and disruption of the cell envelope with extracellular electron-dense materials. Increased permeability of the inner cell membrane caused cell death and lowered EPS production. Ag-NPs and ZnO-NPs also disrupted the surface adhering ability of bacteria, which varied with time and concentration of NPs. Conclusively, a plausible mechanism of NP toxicity to bacteria has been proposed to understand the mechanistic basis of ecological interaction between NPs and resourceful bacteria. These results also emphasize to develop strategies for the safe disposal of NPs.

Heavy metal induced stress on wheat: phytotoxicity and microbiological management.

Among many soil problems, heavy metal accumulation is one of the major agronomic challenges that has seriously threatened food safety. Due to these problems, soil biologists/agronomists in recent times have also raised concerns over heavy metal pollution, which indeed are unpleasantly affecting agro-ecosystems and crop production. The toxic heavy metals once deposited beyond certain permissible limits, obnoxiously affect the density, composition and physiological activities of microbiota, dynamics and fertility of soil leading eventually to reduction in wheat production and via food chain, human and animal health. Therefore, the metal induced phytotoxicity problems warrant urgent and immediate attention so that the physiological activities of microbes, nutrient pool of soils and concurrently the production of wheat are preserved and maintained in a constantly deteriorating environment. To mitigate the magnitude of metal induced changes, certain microorganisms have been identified, especially those belonging to the plant growth promoting rhizobacteria (PGPR) group endowed with the distinctive property of heavy metal tolerance and exhibiting unique plant growth promoting potentials. When applied, such metal-tolerant PGPR have shown variable positive impact on wheat production, even in soils contaminated with metals, by supplying macro and micro nutrients and secreting active biomolecules like EPS, melanins and metallothionein (MTs). Despite some reports here and there, the phytotoxicity of metals to wheat and how wheat production in metal-stressed soil can be enhanced is poorly explained. Thus, an attempt is made in this review to better understand the mechanistic basis of metal toxicity to wheat, and how such phytotoxicity can be mitigated by incorporating microbiological remediation strategies in wheat cultivation practices. The information provided here is likely to benefit wheat growers and consequently optimize wheat production inexpensively under stressed soils.

Effective Inhibition of Phytopathogenic Microbes by Eco-Friendly Leaf Extract Mediated Silver Nanoparticles (AgNPs).

ABSTRACT: This study was aimed at producing the eco-friendly, safe, and inexpensive silver (Ag) nanoparticles (NPs) and assessing its antimicrobial activity. Fungal pathogens isolated from diseased leaves and fruits of brinjal and bacterial pathogen obtained from a culture collection were used in this study. Green synthesis of AgNPs was performed and optimized using Azadirachta indica leaf extract. The newly synthesized AgNPs (λmax = 437 nm) showed isotropism in size (crystal size/diameter: 21/29 ± 5 nm) and morphology under transmission and scanning electron microscopy and energy dispersive X-ray analysis. The fourier transform infrared spectroscopy data suggested the role of various aliphatic/aromatic moieties and proteins in AgNPs stabilization. The AgNPs reduced the growth of Penicillium sp. maximally by 92% after 6 days. The sensitivity of test fungi towards AgNPs followed the order: Penicillium sp. (92%) > Fusarium sp. (89%) > Aspergillus sp. (69%). Exposure of Ralstonia solanacearum to AgNPs (MIC/MBC 200/400 µg ml-1) displayed damaged cellular envelopes, bulging of cells, and pit formation. The nucleic acid discharge showed a progressive increase from 8 to 34% (r2 = 0.97). The cellular metabolic activity and surface adhering ability of R. solanacearum were completely lost at 400 µgAgNPs ml-1. Results suggested that the AgNPs synthesized in this study had enough anti-pathogenic potential and could inexpensively and safely be used as a promising alternative to agrochemicals. Moreover, the findings observed in this study is likely to serve as an important indicator for the development of effective nano-control agents which in effect would help to manage some deadly phyto-pathogens capable of causing heavy losses to agricultural production systems. GRAPHICAL ABSTRACT: Effective inhibition of phytopathogenic microbes by eco-friendly neem leaf extract mediated silver nanoparticles (AgNPs).

Putative Role of Bacterial Biosorbent in Metal Sequestration Revealed by SEM-EDX and FTIR.

Bacterial exopolysaccharides (EPS) play a critical role in sequestration of metals from contaminated environment. Considering these, this study was aimed at extracting EPS from metal tolerant Pseudomonas aeruginosa CPSB1 and Azotobacter chroococcum CAZ3 and to ascertain its role in metal removal. P. aeruginosa CPSB1 and A. chroococcum CAZ3 secreted 1306.7 and 1660 µg mL-1 EPS, respectively in the presence of 200 and 100 µg mL-1 Pb, respectively with glucose as C source. The binding of metal ions to bacterial EPS was validated by SEM and EDX. The functional group involved in metal chelation was revealed by FT-IR. The metal ions were adsorbed onto EPS and hence, EPS could play a crucial role in metal detoxification. Due to this novel trait, P. aeruginosa CPSB1 and A. chroococcum CAZ3 could be developed as bioinoculant to cleanup metal contaminated sites.

Bioreduction of toxicity influenced by bioactive molecules secreted under metal stress by Azotobacter chroococcum.

Heavy metal pollution destruct soil microbial compositions and functions, plant's performance and subsequently human health. Culturable microbes among many metal abatement strategies are considered inexpensive, viable and environmentally safe. In this study, nitrogen fixing bacterial strain CAZ3 recovered from chilli rhizosphere tolerated 100, 1000 and 1200 µg mL-1 of cadmium, chromium and nickel, respectively and was identified as Azotobacter chroococcum by 16S rDNA sequence analysis. Under metal stress, cellular morphology of A. chroococcum observed under SEM was found distorted and shrinkage of cells was noticed when grown with 50 µg mL-1 of Cd (cell size 1.7 µm) and 100 of µg mL-1 Ni (cell size 1.3 µm) compared to untreated control (cell size 1.8 µm). In the presence of 100 µg mL-1 of Cr, cells became elongated and measured 1.9 µm in size. Location of metals inside the cells was revealed by EDX. A dose dependent growth arrest and consequently the death of A. chroococcum cells was revealed under CLSM. A. chroococcum CAZ3 secreted 320, 353 and 133 µg EPS mL-1 when grown with 100 µg mL-1 each of Cd, Cr and Ni, respectively. The EDX revealed the presence of 0.4, 0.07 and 0.24% of Cd, Cr and Ni, respectively within EPS extracted from metal treated cells. Moreover, a dark brown pigment (melanin) secreted by A. chroococcum cells under metal pressure displayed tremendous metal chelating activity. The EDX spectra of melanin extracted from metal treated cells of A. chroococcum CAZ3 displayed 0.53, 0.22 and 0.12% accumulation of Cd, Cr and Ni, respectively. The FT-IR spectra of EPS and melanin demonstrated stretching vibrations and variations in surface functional groups of bacterial cells. The C-H stretching of CH3 in fatty acids and CH2 groups, stretching of N-H bond of proteins and O-H bond of hydroxyl groups caused the shifting of peaks in the EPS spectra. Similar stretching vibrations were recorded in metal treated melanin which involved CHO, alkyl, carboxylate and alkene groups resulting in significant peak shifts. Nuclear magnetic resonance (NMR) spectrum of EPS extracted from A. chroococcum CAZ3 revealed apparent peak signals at 4.717, 9.497, 9.369 and 9.242 ppm. However, 1H NMR peaks were poorly resolved due largely to the impurity/viscosity of the EPS. The entrapment of metals by EPS and melanin was confirmed by EDX. Also, the induction and excretion of variable amounts of metallothioneins (MTs) by A. chroococcum under metal pressure was interesting. Conclusively, the present findings establish- (i) cellular damage due to Cd, Cr and Ni and (ii) role of EPS, melanin and MTs in adsorption/complexation and concurrently the removal of heavy metals. Considering these, A. chroococcum can be promoted as a promising candidate for supplying N efficiently to plants and protecting plants from metal toxicity while growing under metal stressed environment.

Heavy metal mediated phytotoxic impact on winter wheat: oxidative stress and microbial management of toxicity by Bacillus subtilis BM2.

Heavy metals are toxic environmental contaminants, which severely affect microbial composition and functions and, concurrently, crop production. Due to these issues, the present study focussed on the selection of metal tolerant microbes endowed with metal detoxification abilities and their role in the management and remediation of metal contaminated soils. The metal tolerant bacterium BM2, identified as Bacillus subtilis by 16SrRNA gene sequencing, survived well under metal pressure and tolerated 1600 and 2000 µg mL-1 of Ni and Pb, respectively. The inhibitory impact of metals on wheat increased consistently with a progressive increase in metal concentration. Deposition of Ni and Pb within root and leaf and oxidative stress were validated by SEM, EDX and CLSM. The overall growth parameters of wheat grown under metal stress were improved following B. subtilis BM2 colonization. As an example, B. subtilis with 195 mg Pb kg-1 enhanced the length and dry biomass of shoots by 14% and 23%, respectively, over the control. Also, strain BM2 improved the grain yield significantly by 49% at 870 mg Ni kg-1 and by 50% at 585 mg Pb kg-1 compared to uninoculated plants. Moreover, B. subtilis BM2 relieved the metal stress on wheat and caused a significant drop in proline and malondialdehyde content and the activities of antioxidant enzymes, like catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR). This study, therefore, provided solutions to the metal toxicity problems faced by winter wheat and clearly suggests that the metal detoxification potential of B. subtilis BM2 could be greatly useful in the management of metal polluted soils.

Understanding the phyto-interaction of heavy metal oxide bulk and nanoparticles: evaluation of seed germination, growth, bioaccumulation, and metallothionein production.

The fast-growing use of nano-based products without proper care has led to a major public health concern. Nanomaterials contaminating the environment pose serious threat to the productivity of plants and via food chain to human health. Realizing these, four vegetable crops, radish, cucumber, tomato, and alfalfa, were exposed to varying concentrations of heavy metal oxide (TiO2, ZnO, Al2O3 and CuO) submicron or bulk (BPs) and nanoparticles (NPs) to assess their impact on relative seed germination (RSG), seed surface adsorption, root/shoot tolerance index (RTI/STI), bioaccumulation, and metallothioneins (MTs) production. The results revealed a clear inhibition of RSG, RTI, and STI, which, however, varied between species of metal-specific nanoparticles and plants. SEM and EDX analyses showed significant adsorption of MONP agglomerates on seed surfaces. The concentration of metals detected by EDX differed among vegetables. Among the metals, Al, Cu, Ti, and Zn were found maximum in alfalfa (12.46%), tomato (23.2%), cucumber (6.32%) and radish (21.74%). Of the four metal oxides, ZnO was found most inhibitory to all vegetables and was followed by CuO. The absorption/accumulation of undesirable levels of MONPs in seeds and seedlings differed with variation in dose rates, and was found to be maximum (1748-2254 µg g-1 dry weight) in ZnO-NPs application. Among MONPs, the uptake of TiO2 was minimum (2 to 140 µg g-1) in radish seedlings. The concentration of MTs induced by ZnO-NPs, ZnO-BPs, and CuO-NPs ranged between 52 and 136 µ mol MTs g-1 FW in vegetal organs. Conclusively, the present findings indicated that both the nanosize and chemical composition of MONPs are equally dangerous for vegetable production. Hence, the accumulation of MONPs, specifically ZnO and CuO, in edible plant organs in reasonable amounts poses a potential environmental risk which, however, requires urgent attention to circumvent such toxic problems.

Heavy metal induced oxidative damage and root morphology alterations of maize (Zea mays L.) plants and stress mitigation by metal tolerant nitrogen fixing Azotobacter chroococcum.

Heavy metals are one of the major abiotic stresses that adversely affect the quantity and nutritive value of maize. Microbial management involving the use of plant growth promoting rhizobacteria (PGPR) is a promising inexpensive strategy for metal clean up from polluted soils. Considering these, metal tolerant plant growth promoting nitrogen fixing rhizobacterial strain CAZ3 identified by 16SrRNA gene sequence analysis as Azotobacter chroococcum was recovered from metal polluted chilli rhizosphere. When exposed to varying levels of metals, A. chroococcum survived up to 1400 and 2000 µg mL-1 of Cu and Pb, respectively and expressed numerous plant growth promoting activities even under metal stress. Strain CAZ3 secreted 65.5 and 60.8 µg mL-1 IAA at 400 µg mL-1 each of Cu and Pb, respectively and produced siderophores, ammonia and ACC deaminase under metal pressure. The melanin extracted from A. chroococcum revealed metal chelating ability under EDX. Following application, strain CAZ3 enhanced growth and yield of maize grown both in the presence of Cu and Pb. The dry biomass of roots of inoculated plants grown with 2007 mg Cu kg-1 and 585 mg Pb kg-1 was increased by 28% and 20%, respectively. At 585 mg Pb kg-1, the bioinoculant also increased the kernel attributes. At 2007 mg Cu kg-1 strain CAZ3 enhanced the number, yield and protein of kernels by 10%, 45% and 6%, respectively. Interestingly, strain CAZ3 significantly reduced the levels of proline, malondialdehyde and antioxidant enzymes in foliage. The roots of inoculated plants accumulated greatest amounts of metals compared to other organs. In kernels, the concentration of Pb was more as compared to Cu. The metal concentrations in roots, shoots and kernels, however, declined following CAZ3 inoculation. Copper and lead had substantial distortive impact on root and leaf morphology while cell death were visible under CLSM and SEM. Conclusively, A. chroococcum CAZ3 could be a most suitable and promising option to increase maize production in metal polluted soils despite the soils being contaminated with heavy metals.

Biotoxic impact of heavy metals on growth, oxidative stress and morphological changes in root structure of wheat (Triticum aestivum L.) and stress alleviation by Pseudomonas aeruginosa strain CPSB1.

Rapid industrialization and uncontrolled metal discharge into environment is a global concern for crop production. Metal tolerant bacterium isolated from chilli rhizosphere was identified as Pseudomonas aeruginosa by 16S rDNA sequence analysis. Pseudomonas aeruginosa tolerated high concentrations of Cu (1400 µg ml-1), Cd (1000 µg ml-1) and Cr (1000 µg ml-1). Pseudomonas aeruginosa CPSB1 produced multiple plant growth promoting biomolecules in the presence and absence of metals. Strain CPSB1 solubilized P at 400 µg ml-1 of Cd, Cr and Cu. The strain was positive for indole-3-acetic acid (IAA), siderophores, hydrogen cyanide (HCN), ammonia (NH3) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase when grown with/without metals. The phytotoxic effects on wheat increased with increasing Cd, Cr and Cu rates. The P. aeruginosa CPSB1 inoculated wheat in contrast had better growth and yields under Cu, Cd and Cr stress. The root dry biomass of inoculated plants was enhanced by 44, 28 and 48% at 2007 mg Cu kg-1, 36 mg Cd kg-1 and 204 mg Cr kg-1, respectively. The bioinoculant enhanced number of spikes, grain and straw yields by 25, 17 and 12%, respectively. Pseudomonas aeruginosa CPSB1 significantly declined the levels of catalase (CAT), glutathione reductase (GR) and superoxide dismutase SOD), proline and malondialdehyde (MDA), and reduced metal uptake by wheat. The study demonstrated that P. aeruginosa CPSB1 possessed plant growth promoting potentials, showed metal tolerance capability and had ability to counteract deleterious metal impacts. Due to these, P. aeruginosa CPSB1 could be used as bioinoculant for enhancing wheat production even in metal contaminated soils.