Amelioration of phytotoxic impact of biosynthesized zinc oxide nanoparticles: Plant growth promoting rhizobacteria facilitates the growth and biochemical responses of Eggplant (Solanum melongena) under nanoparticles stress.

The consistently increasing use of zinc oxide nanoparticles (ZnONPs) in crop optimization practices and their persistence in agro-environment necessitate expounding their influence on sustainable agro-environment. Attempts have been made to understand nanoparticle-plant beneficial bacteria (PBB)- plant interactions; the knowledge of toxic impact of nanomaterials on soil-PBB-vegetable systems and alleviating nanotoxicity using PBB is scarce and inconsistent. This study aims at bio-fabrication of ZnONPs from Rosa indica petal extracts and investigates the impact of PBB on growth and biochemical responses of biofertilized eggplants exposed to phyto-synthesized nano-ZnO. Microscopic and spectroscopic techniques revealed nanostructure, triangular shape, size 32.5 nm, and different functional groups of ZnONPs and petal extracts. Inoculation of Pseudomonas fluorescens and Azotobacter chroococcum improved germination efficiency by 22% and 18% and vegetative growth of eggplants by 14% and 15% under NPs stress. Bio-inoculation enhanced total chlorophyll content by 36% and 14 %, increasing further with higher ZnONP concentrations. Superoxide dismutase and catalase activity in nano-ZnO and P. fluorescens inoculated eggplant shoots reduced by 15-23% and 9-11%. Moreover, in situ experiment unveiled distortion and accumulation of NPs in roots revealed by scanning electron microscope and confocal laser microscope. The present study highlights the phytotoxicity of biosynthesized ZnONPs to eggplants and demonstrates that PBB improved agronomic traits of eggplants while declining phytochemicals and antioxidant levels. These findings suggest that P. fluorescens and A. chroococcum, with NPs ameliorative activity, can be cost-effective and environment-friendly strategy for alleviating NPs toxicity and promoting eggplant production under abiotic stress, fulfilling vegetable demands.

Phyto-interactive impact of green synthesized iron oxide nanoparticles and Rhizobium pusense on morpho-physiological and yield components of greengram.

The iron oxide nanoparticles (IONPs) prepared by green synthesis method using Syzigium cumini leaf extract was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The XRD confirmed the crystalline structure of green synthesized NPs measuring around 33 nm while SEM revealed its nearly spherical shape. Rhizobium species recovered from greengram nodules, identified by 16s rRNA gene sequencing as Rhizobium pusense produced 30% more exopolysaccharides (EPS) in basal medium treated with 1000 µg IONPs/ml. Compositional variation in EPS was observed by Fourier-transform infrared spectroscopy (FTIR). There was no reduction in rhizobial viability and no damage to bacterial membrane was observed under SEM and confocal laser scanning microscopy (CLSM), respectively. Effects of IONPs and R. pusense, used alone and in combination on the growth and development of greengram plants varied considerably. Plants grown with IONPs and R. pusence, used alone and in combination, showed a significant increase in seed germination rate, length and dry biomass of plant organs and seed components compared to controls. The IONPs in the presence of rhizobial strain further increased seed germination, plant growth, seed protein and pigments. Greater protein content (442 mg/g) was observed in seeds at 250 mg/kg of IONPs compared to control. Plants raised with mixture of IONPs plus R. pusense had maximum chlorophyll content (39.2 mg/g FW) while proline content decreased by 53% relative to controls. This study confirms that the green synthesis of IONPs from S. cumini leaf possess useful plant growth promoting effects and could be developed as a nano-biofertilizer for optimizing legume production.

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.

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.

Comparative in situ ROS mediated killing of bacteria with bulk analogue, Eucalyptus leaf extract (ELE)-capped and bare surface copper oxide nanoparticles.

This study demonstrates a simple one-pot green method for biosynthesis of terpenoids encapsulated copper oxide nanoparticles (CuONPs) using aqueous leaf extract of Eucalyptus globulus (ELE), as reducing, dispersing, and stabilizing agent. Indeed, the greater attachment and internalization of ELE-CuONPs in Gram-positive and -negative biofilm producing clinical bacterial isolates validated the hypothesis that terpenoids encapsulated CuONPs are more stable and effective antibacterial and antibiofilm agent vis-à-vis commercially available nano and micro sized analogues. Gas chromatography-mass spectroscopy (GC-MS) analysis of pristine ELE identified 17 types of terpenoids based on their mass-to-charge (m/z) ratios. Amongst them four bioactive terpenoids viz. terpineols, 2,6-octadienal-3,7-dimethyl, benzamidophenyl-4-benzoate and ß-eudesmol were found associated with the CuONPs as ELE-cap, and most likely involved in the nucleation and stabilization of ELE-CuONPs. Further, the Fourier transformed infrared (FTIR) analysis of ELE-CuONPs also implicated other functional biomolecules like proteins, sugars, alkenes, etc. with ELE terpenoids corona. Flow cytometric (FCM) data exhibited significantly enhanced intracellular uptake propensity of terpenoids encapsulated ELE-CuONPs and accumulation of intracellular reactive oxygen species (ROS), which ensued killing of planktonic cells of extended spectrum ß-lactamases (ESßL) producing Escherichia coli-336 (E. coli-336), Pseudomonas aeruginosa-621 (P. aeruginosa-621) and methicillin-resistant Staphylococcus aureus-1 (MRSA-1) clinical isolates compared to the bare surface commercial nano-CuO and bulk sized CuO. The study for the first-time demonstrated the (i) differential bio-nano interface activities due to ELE surface and varied cell wall composition of test bacterial isolates, (ii) antibacterial effect and biofilm inhibition due to disruption of proteins involved in adhesion and biofilm formation triggered by CuONPs induced intracellular oxidative stress, and (iii) indigenous terpenoids-capped bio-inspired CuONPs are more stable and effective antibacterial and antibiofilm agent as compared with commercially available nano-CuO and bulk-CuO.

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.

Fungicide tolerant Bradyrhizobium japonicum mitigate toxicity and enhance greengram production under hexaconazole stress.

Bacterial strain RV9 recovered from greengram nodules tolerated 2400µg/mL of hexaconazole and was identified by 16S rDNA sequence analysis as Bradyrhizobium japonicum (KY940048). Strain RV9 produced IAA (61.6µg/mL), ACC deaminase (51.7mg/(protein·hr)), solubilized TCP (105µg/mL), secreted 337.6µg/mL EPS, and produced SA (52.2µg/mL) and 2,3-DHBA (28.3µg/mL). Exopolysaccharides produced by strain RV9 was quantified and characterized by SEM, AFM, EDX and FTIR. Beyond tolerance limit, hexaconazole caused cellular impairment and reduced the viability of strain RV9 revealed by SEM and CLSM. Hexaconazole distorted the root tips and altered nodule structure leading thereby to reduction in the performance of greengram. Also, the level of antioxidant enzymes, proline, TBARS, ROS and cell death was increased in hexaconazole treated plants. CLSM images revealed a concentration dependent increase in the characteristic green and blue fluorescence of hexaconazole treated roots. The application of B. japonicum strain RV9 alleviated the fungicide toxicity and improved the measured plant characteristics. Also, rhizobial cells were localized inside tissues as revealed by CLSM. Colonization of B. japonicum strain RV9 decreased the levels of CAT, POD, APX, GPX and TBARS by 80%, 5%, 13%, 13% and 19%, respectively over plants grown at 80µg/(hexaconazole·kg) soil. The ability to detoxify hexaconazole, colonize plant tissues, secrete PGP bioactive molecules even under fungicide pressure and its unique ability to diminish oxidative stress make B. japonicum an attractive choice for remediation of fungicide polluted soils and to concurrently enhance greengram production under 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.

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.

Glyphosate induced toxicity to chickpea plants and stress alleviation by herbicide tolerant phosphate solubilizing Burkholderia cepacia PSBB1 carrying multifarious plant growth promoting activities.

In this study, strain PSBB1 isolated from Vicia faba rhizosphere was identified as Burkholderia cepacia, by 16S rDNA sequence analysis and characterized. Strain PSBB1 tolerated glyphosate up to 3200 µg ml-1 and produced IAA (81.6 µg ml-1), ACC deaminase (69.3 mg-1 protein h-1), SA (39.3 µg ml-1) and 2,3-DHBA (26.6 µg ml-1), solubilized insoluble P (50.8 µg ml-1) and secreted 29.4 µg ml-1 exopolysaccharides, which decreased with increasing concentrations of glyphosate. Cell damage following glyphosate application was visible under SEM and CLSM. The phytotoxicity of glyphosate on chickpea was variable but significant. B. cepacia mitigated toxicity and enhanced the size, dry matter, symbiosis, seed attributes and nutritional contents of chickpea. Further, B. cepacia strain PSBB1 declined the levels of CAT, POD, APX and GPX and MDA contents at 4332 µg kg-1 soil glyphosate. Proline also increased under glyphosate stress but declined in B. cepacia inoculated plants. The ability to tolerate higher concentration of glyphosate, the capacity to secrete plant growth regulators even under herbicide stress and potential to reduce the level of proline and antioxidant enzymes makes B. cepacia as an interesting choice for enhancing chickpea production in soils contaminated even with herbicides.

Toxicity of fungicides to Pisum sativum: a study of oxidative damage, growth suppression, cellular death and morpho-anatomical changes.

Considering the fungicidal threat to the sustainable agro-environment, the toxicological impacts of three fungicides, namely kitazin, hexaconazole and carbendazim, on the biological, chemical and morpho-anatomical changes of peas were assessed. Fungicide applications in general caused a slow but gradual reduction in growth, symbiosis and yields of peas, which, however, varied appreciably among species and concentrations of the three fungicides. Of the three fungicides, carbendazim had the most lethal effect, in which it delayed seed germination and also diminished the overall pea growth. Carbendazim at 3000 µg kg-1 maximally reduced the germination, SVI, size of roots and shoots and total dry matter accumulation in roots, shoots and whole plants distinctly by 40%, 84%, 72%, 73%, 68%, 75% and 73% (p ≤ 0.05), respectively. Hexaconazole at 120 µg kg-1 significantly (p ≤ 0.05) declined total chlorophyll, carotenoids, grain yields, grain protein, root P and shoot N by 19%, 28%, 46%, 69%, 48% and 51%, respectively, over the control. The synthesis of stress biomarkers and oxidative stress were increased with increasing dosage rates of fungicides. Proline content in roots, shoots, leaves and grains, MDA, electrolyte leakage and H2O2 of plants grown in soil treated with 288 µg kg-1 kitazin were increased significantly (p ≤ 0.05) by 73%, 52%, 41%, 24%, 59%, 40% and 27%, respectively, relative to the control. Antioxidant defence enzymes were greater in pea foliage. The SEM and CLSM images revealed an obvious alteration in root tips, enhanced cellular damage and cell death when plants were raised under fungicide stress. Also, morpho-anatomical variations in fungicide-treated foliage were visible in the SEM images. Overall, the present study suggests that a careful and secure strategy should be adopted before fungicides are chosen for enhancing pulse production in different agro-climatic regions.

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.