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Journal of Agricultural and Food Chemistry
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Metallic Nanoparticles (TiO2 and Fe3O4) Application Modify Rhizosphere Phosphorus
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Availability and Uptake by Lactuca sativa
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Zahra Zahra1, Muhammad Arshad1,*, Rafia Rafique1, Arshad Mahmood2, Amir Habib3,
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Ishtiaq A. Qazi1, Saud A. Khan1
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1
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Engineering, National University of Sciences and Technology, Sector H-12, Islamabad, 44000,
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Pakistan
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2
National Institute of Laser and Optronics, Nilore, Islamabad, 45650, Pakistan
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3
School of Chemical and Materials Engineering, National University of Sciences and
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Institute of Environmental Sciences and Engineering, School of Civil and Environmental
Technology, Sector H-12, Islamabad, 44000, Pakistan
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ABSTRACT
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Application of engineered nanoparticles (NPs) with respect to nutrient uptake in plants is
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not yet well understood. The impacts of TiO2 and Fe3O4 NPs on the availability of naturally soil
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bound inorganic phosphorus (Pi) to plants were studied along with relevant parameters. For this
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purpose, Lactuca sativa (Lettuce) was cultivated on the soil amended with TiO2 and Fe3O4 (0,
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50, 100, 150, 200 and 250 mg kg-1) over a period of 90 days. Different techniques like SEM,
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EDX, Raman and FTIR were used to monitor translocation and understand the possible
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mechanisms for phosphorus (P) uptake. The trends for P accumulation were different for roots
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(TiO2>Fe3O4>Control) and shoots (Fe3O4>TiO2>Control). Cystine and Methionine were detected
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in the rhizosphere in Raman spectra. NPs’ affinities to adsorb phosphate ions, modifications in P
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speciation and NPs stress in the rhizosphere had possibly contributed to enhanced root exudation
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and acidification. All these changes led to improved P availability and uptake by the plants.
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These promising results can help to develop an innovative strategy for using NPs for improved
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nutrient management to ensure food security.
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Keywords: Titania, Magnetite, Nanoparticles, Phosphorus Phytoavailability, Lactuca sativa
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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All macro– and micro–nutrients have their own importance and P is one of the key life-
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supporting elements in all the living organisms. It is an essential constituent of adenosine
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triphosphate (ATP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It also facilitates
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phospholipids in forming cell membranes. The P deficiency can affect different plant functions,
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seed development, root structure and ultimately the crop yield.1 Therefore, P is considered to be
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the yield limiting factor in numerous soils.
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The largest challenge in agricultural management of P is its low solubility.2 In soils, high
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concentrations of organic and inorganic phosphates are present, of which about 88 to 99% of
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total inorganic phosphorus (Pi) is bound by Ca+2 and thus unavailable to plants. About 30% of
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the world’s agricultural land needs P fertilizers for good crop production.3 The phosphate
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fertilizers form insoluble complexes in soil and the bioavailability of applied P tends to be less
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due to the immobilization.4 In calcareous and alkaline soils, the added soluble P also gets fixed
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resulting in low crop productivity. So there is growing interest in developing methods to improve
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the accessibility of naturally bound P in soils which could save natural/rock P resources for
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sustainable production of crops. Enhanced root exudates production is an adaptive response to
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acquire poorly mobile soil resources,5 particularly P.
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In the recent years, researchers have incorporated nanotechnology in agriculture and
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worked on the effects of plant growth and possible mechanisms.6,7,8 For the present study, TiO2
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and Fe3O4 NPs were selected for application in soil to grow Lactuca sativa. It is one of the green
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leafy vegetables that are cultivated worldwide. It is a source of mineral nutrients that are
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important for human health and nutrition. It is consumed in raw form due to its high nutritive
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value (i.e., vitamin, fiber, and mineral input to a diet), good taste and low price.9 Moreover, it is
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among the most consumed vegetables, accounting with a mean consumption of 23.4 g per day in
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Europe, which is about 7.2% of the total nutritional intake of vegetables.10 In 2011, its global
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production was about 25 million tons.11
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Among the engineered nanomaterials, TiO2 is the most widely used metal oxide NPs with
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up to 10,000 tons of global production per year.12 In different studies, the effects of TiO2 and
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Fe3O4 NPs have been examined in various plant species
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to nutrient uptake is limited. Both positive and negative effects have been reported upon
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application to higher plants.16 In the soil, microbial ecosystem can also be impacted by the NPs.
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In a study, Fe2O3 and Fe3O4 were reported to have beneficial impacts on the soil bacteria
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community at certain concentrations.17 Ge et al. 18 treated a grassland soil with 0, 500, 1000 and
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2000 mg TiO2 NPs kg-1 soil in microcosms over 60 days. They reported that metal oxide NPs can
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negatively affect the soil bacterial communities. However, these values are very high as
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compared to the treatments used in the present study. There exists a need to explore ENPs effects
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on the rhizophere microbiome
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soils.
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13,14,15
but the information with respect
and their possible role in nutrient management in agricultural
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In plant nutrition, NPs are not often considered as a factor affecting phytoavailable P.
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However a recent study reported increased accumulation of P (3686 mg kg-1) in roots of wheat
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at 250 mg kg-1 of CeO2 NPs treatment as compared to the control (2417 mg kg-1) while CeO2
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NPs at 500 mg kg-1 improved plant growth, shoot biomass and grain yield by 9.0, 12.7, and
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36.6%, respectively.20 In another work, Al2O3 NPs bound P was reported to enhance P uptake by
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plants in aqueous solutions at very low concentration of even 10 mg L–1.21 Pradhan et al.22
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studied the effects of Mn NPs on nitrogen uptake, assimilation, and metabolism in mung bean
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plants. Majority of these studies are short period assays up to two weeks and in rare cases up to
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months. In this context, the objectives of present study were; 1) Effects of TiO2 and Fe3O4 NPs
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application on phytoavailability of P to Lactuca sativa and plant growth, 2) Rhizosphere changes
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in response to NPs applications in soil and, 3) Localization of metallic NPs in plants. To the best
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of our knowledge, this is the first report on P availability trends over an extended contact period
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(90 days) with different kinds of NPs under soil conditions.
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MATERIALS AND METHODS
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Synthesis of TiO2 NPs by Sol-Gel Method
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TiO2 NPs were synthesized using a slightly modified sol–gel method that was developed
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earlier for synthesis of molecularly imprinted Titania.23 Titania precursor was added in 0.5 M
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acidic solution. As the TiCl4 was added, the color of the solution turned yellowish. When the
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solution became transparent, it was neutralized with 0.5M NH4OH till pH became 7.0 followed
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by stirring until the gel like network formed. The gel was allowed to settle. The supernatant was
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discarded and rest of the sol was dried in vacuum–oven at 105 ºC. Dry gel was ground and
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calcined at 400 ºC for 6 h.
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Synthesis of Fe3O4 NPs by Co-precipitation Method
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For preparation of Fe3O4 NPs, FeCl2 (0.1 M) was mixed with FeCl3 (0.2 M) and NaOH
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(0.1 M) was added to the mixture. The solution turned black and placed on heating till the slurry
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rest behind. It was washed several times with distilled water until the pH 7.0 achieved. The
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slurry was dried in an oven at 80 ºC. The dried magnetite clusters were ground to get NPs.24
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Characterization of Synthesized NPs
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The phase composition, crystal structure and crystallite size measurements for the TiO2
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and Fe3O4 NPs were performed by X-ray Diffractometer (XRD, Theta-Theta STOE, Germany) at
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40 keV and 40 mA. Processing of XRD results were done with X'Pert High Score software
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package (PANalytical B.V. Almelo, Netherland). The surface morphology of TiO2 and Fe3O4
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NPs was analyzed using Scanning Electron Microscopy (SEM, Jeol, JSM 6490 A, Japan)
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equipped with Energy Dispersive X-ray Spectroscopy (EDX, Jeol, JED 2300) and ion sputtering
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device (Jeol, JFC 1500). Suspensions of TiO2 and Fe3O4 NPs in ethanol were made-up on quartz
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slides. Sputtering technique was used to evaporate the volatile substances or moisture and
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stabilize the NPs on the substrate to avoid interference or preventing contamination. The
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technique can be used for several conductive materials.25 Then the slides were observed under
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SEM.
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Soil Preparation
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For the present study, sandy-loam soil was selected. The air-dried soil was ground by ball
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mill and passed through mechanical sieve (
