Enhancing the Mobilization of Native Phosphorus in the Mung Bean

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Enhancing the mobilization of native phosphorous in mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi Ramesh Raliya, Jagadish Chandra Tarafdar, and Pratim Biswas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05224 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016

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Journal of Agricultural and Food Chemistry

Enhancing the mobilization of native phosphorous in mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi

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Ramesh Raliya , Jagadish Chandra Tarafdar and Pratim Biswas

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Washington University in St. Louis, St. Louis, MO-63130, USA ǁ

Central Arid Zone Research Institute, Jodhpur – 342003, India

Manuscript ID jf-2015-05224n.R1 Submitted to Journal of Agricultural and Food Chemistry March 13, 2016

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*Corresponding Author Phone: +1 314-936-5548. Fax: 314-935-5464. Email: [email protected]

ACS Paragon Plus Environment


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Enhancing the mobilization of native phosphorous in mung bean rhizosphere using ZnO nanoparticles synthesized by soil fungi

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ABSTRACT: Phosphorous (P) is a limiting factor to plant growth and productivity in almost

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half of the world’s arable soil, and its uptake in plants is often constrained due to low solubility

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in the soil. To avoid repeated and large quantity application of rock phosphate as a P fertilizer

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and enhance the availability of native P acquisition by the plant root surface, in this study

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biosynthesized ZnO nanoparticle was used. Zn acts as a cofactor for P-solubilizing enzymes

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such as phosphatase and phytase and nano ZnO increased their activity between 84 and 108%.

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Resultant P uptake in mung bean increased by 10.8%. In addition, biosynthesized ZnO also

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improves plants phenology such as stem height, root volume, biochemical indicators such as leaf

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protein and chlorophyll contents. In the rhizosphere, increased chlorophyll and root volume

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attract microbial populations that maintain soil biological health. ICP-MS results exhibited ZnO

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nanoparticles were distributed in all plant parts including seeds. However, the concentration of

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Zn was within the limit of the dietary recommendation. To the authors’ knowledge, this is the

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first holistic study focusing on native P mobilization using ZnO nanoparticles in the life cycle of

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mung bean plants.

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KEYWORDS: native P mobilization, ZnO nanoparticle, phosphatase, phytase, mung bean

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Low utilization of native-P by crops and high fixation of applied P had been a major concern in

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crop production for over three decades.1 Different approaches viz. phosphorus solubilizing

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bacteria (PSB), phosphatase and phytase producing fungi (PPF), organic acid producing

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microorganisms, and Vesicular-arbuscular mycorrhiza (VAM) have been tried in past to address

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these problems.2-4 But success of these phosphorus mobilizing micro-organisms (PMM) had

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been limited for two reasons (a) Low Soil Organic Carbon (SOC) which limit the energy supply

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to PMM (b) High evaporation from surface soils which adversely affect the survivability of

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PMM. Plants exude about 25 to 30% of the photosynthates through roots. These exudates consist

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of low molecular weight amino acids, amino sugars, organic acids and polysaccharides,

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enzymes, and can provide energy to PMM and C skeleton for synthesis of nutrient mobilization

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and uptake.5-7 Any attempt to increase the proportion of root exudates would logically result in

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more symbiosis with microbial community, and enhance the activity of soil nutrient mobilizing

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enzymes. Therefore, in this study, we aim to enhance P mobilization enzyme activity, soil

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microbial population as a result of photosynthesis in mung bean plants. In this study, we select

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mung bean because of its nutritional value for the essential nutrients. It is well established that

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increased activity of phosphatase and phytase enzyme play a role in native P mobilization and

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enhance acquisition by root surface.8 To enhance P availability and uptake, various attempts has

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been developed such as direct application of phosphate rock,9 P fertilization10 and foliar

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application.11, 12

INTRODUCTION

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In the recent years, many applications of nanotechnology have been explored across the

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whole spectrum of economic sectors, including agriculture, food, forest, and the environment,

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with demonstrated societal benefits.13 Scientific community agrees that nanoscale technology is



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promising for food and agriculture, the development must be done in a sustainable manner.14 In

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the context of P mobilization, best of our knowledge, there are only two reports, one from our

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group in which, we reported ZnO effect on P-mobilizing enzyme secretion and gum contents in

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clusterbean15 and recently by Zahra et al.,16 reported that the application of TiO2 and Fe3O4

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nanoparticles increased phyto-availability of P in Lactuca sativa. In the present study, we used

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ZnO nanoparticles for native P mobilization in the rhizosphere and acquisition by mung bean.

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Zinc is the structural component, a cofactor of P mobilizing enzymes (phosphatases and

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phytase) and microbial enzyme dehydrogenase.17 They are known to stabilize the enzyme

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complexes in plants. We aim on utilizing nanoscale property of zinc for enhancing native P

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mobilization by increasing P mobilizing enzyme activity in soil, and boosting plant metabolism

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that would most likely lead to enhance photosynthesis and higher root exudation which in turn

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would increase the energy supply and supply of C skeleton compounds to PMM. This approach

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would enable in breaking of existing barriers to utilization of native P and reduce dependence on

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conventional P fertilizers, expensive and limited natural mineral resources all over the world.

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To synthesize zinc (in the form of ZnO) nanoparticles, a variety of chemical methods

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are available.18, 19 However, to minimize the risk and use of harmful chemicals, we used ZnO

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nanoparticles synthesized from the rhizospheric fungi with a fungal protein coating,15,

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making this an environmentally benign process. Nanoparticles were applied on the foliage part

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of mung bean to avoid direct contact with the soil ecosystem. Wang et al.,21 developed an

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approach that could be used to deliver nanoparticles precisely by an aerosol route. The aim of

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this study was to explore the use of biologically synthesized ZnO nanoparticles to increase

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rhizosphere soil P availability for plant acquisition by minimizing the risk of nanoparticle


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toxicity. Physiological response, phenological impact and quantification of ZnO nanoparticle

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biodistribution in mung bean was investigated.

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Synthesis and Characterization of ZnO Nanoparticles. The ZnO nanoparticles were

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synthesized from the soil fungus, Aspergillus fumigatus TFR-8 by following the method reported

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earlier.15 In brief, Aspergillus fumigatus TFR-8 mycelial fungal balls (NCBI GenBank Accession

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No. JQ675291) were grown up in broth medium (composed of 0.3 % malt extract, 1 % sucrose,

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0.3 % yeast extract, and 0.5 % peptone). After incubation, the cell-free filtrate was obtained by

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separating the fungal biomass using 0.45-microsize membrane filter. Using cell-free filtrate, salt

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solution of zinc nitrate was prepared with final concentration of 0.1 mM. The biotransformed

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product was collected periodically for characterization of particle size. Biosynthesized ZnO

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nanoparticles were characterized by UV-Vis spectroscopy, Dynamic Light Scattering (DLS)

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analysis (Zetasizer Nano ZS) to determine the hydrodynamic particle size that is useful to predict

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particle size distribution in the solvent. Transmission Electron Microscopy (TEM, JEM-2100F)

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measurements were carried out at 200 kV accelerating voltage to confirm the size and

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morphology, and selective area electron diffraction (SAED) pattern of the synthesized ZnO

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nanoparticles. Elemental analysis was carried out using X-Ray Photoelectron Spectrometer

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(Physical Electronics® 5000 VersaProbe II Scanning ESCA (XPS) Microprobe). Crystal nature

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of the particles were characterized by X-Ray Diffraction (Bruker D8).

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Characterization of Experimental Soil and Plant Growth. The experimental soil was

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collected from farm field of Central Arid Zone Research Institute. The soil was air dried and

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sieved through a 2 mm mesh prior to experimental use. The soil type was classified as sandy-

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loam soil, in which percentage of sand, slit and clay of 85.6%, 6.9% and 7.5% respectively. The

MATERIAL AND METHODS



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organic matter of the soil was 0.3%. Therefore, organic farm manure was mixed with soil (1:10)

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before use. Resulting soil properties were: pH 8.1±0.2, Zinc 0.6 µg/g, nitrogen 462±18.5 mg/kg,

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P 733±23.5 mg/kg and potassium 695±21.9 mg/kg. Bacteria, fungi and actinomycetes counts

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(colony forming unit; CFU/ml) of 12×10-6, 8×10-4 and 6×10-5 correspondingly.

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Five seeds of mung bean were sown in each pot, was thinned to three at a later stage of

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germination. The pots were placed (in a random block design and exchanged their position every

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alternate day to avoide environmental impact) in a greenhouse with 16h photoperiod, 34/25 ºC

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day/night temperature, 65% relative humidity and 360 µmol m-2S-1 light intensity. Each

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treatment including control and bulk ZnO was set in five replicates. No additional fertilizer and

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pesticides were used through the life cycle. Alternate day, watering was carried out.

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Nanoparticles Exposure to Mung bean Foliage. Two weeks old mung bean plants’ foliage was

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exposed with ZnO nanoparticles and bulk ZnO. Concentration of ZnO suspension was 10 mg/L,

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found an optimum in screening experiment. The solution was homogenized by sonication for 45

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minutes before use. A total of 25 mL suspension, each of bulk and nano ZnO was sprayed on

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each plant by an atomizer, generate 250-300 µ size droplets. Samples were collected after 14

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days of treatment for phenotypic analyzes, plant – soil, biochemical analyzes and rhizosphere

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microbial population count.

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Plant-Rhizosphere Response to ZnO Nanoparticles.

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Phenotype. Plant phenotype is a comprehensive assessment of growth and development. In this

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study, plant height, root length, root area, root diameter and number of root nodules were

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recorded. After 28 days, plants of each treatment was uprooted, stems were cut at the soil

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surface, and height were measured using meter scale. Roots were carefully shaken to remove

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excess soil, and clumps of soil trapped between roots were taken out. The roots were transferred


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to a beaker and cleaned by gentle shaking of the beaker after adding 0.2 mM CaCl2 solution. The

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roots were taken out of the soil extract and processed further for root length, area, and diameter

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measurement using Delta T-Scan Software (Delta-T Devices, UK).

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Representative leaves were used further for protein and chlorophyll measurement.

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Rhizospheric soil sample of each treatment was taken out (100 g) from the bulk soil of the pots

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in which plants were grown, as well as from absolute control pot in which no plants were grown

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and frozen at 4 °C for further analysis.

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Rhizosphere Soil Enzyme. All the studied enzyme activities were calculated based on the

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changes of absorbance and Beer’s Law. In this study, P mobilizing and microbial activity

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enzymes, as an indicator of biological activity in rhizospheric soil were investigated.

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Acid and Alkaline Phosphates Activity. The acid and alkaline phosphatase was assayed by the

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method reported earlier.22 In short, 1 g of sieved soil (< 2 mm) mixed in 0.2 mL of toluene, 4 mL

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of p-nitrophenyl phosphate (PNP) solution prepared in acetate buffer (pH 4.5, for acid

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phosphatase) and borax-NaOH buffer (pH 9.2, for alkaline phosphatase). Further, samples were

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incubated at 30 °C in the dark for 1 h, followed by 1 mL of 0.5 M CaCl2 and 4 mL of 0.5 M

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NaOH were added. Swirled the test tube for a few seconds, and filtered the soil suspension

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through a Whatman no 42 filter paper. The yellow color intensity of the filtrate was measured at

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420 nm wavelength. The p-nitrophenol content of the filtrate was calculated from the standard

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curve obtained with standards containing 0, 10, 20, 30, 40 and 50 µg of p-nitrophenol.

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Phytase Activity. Phytase activity was assayed by Ames’ method.23 Two gram of sieved soil

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sample mixed with 4 mL of 100 M sodium acetate buffer (pH 4.5), and 1 mL of 1µM sodium

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phytate. The sample was incubated at 37° C for 1 h in the dark. After 1 h incubation, the reaction



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was terminated by the addition of 0.5 mL of 10% TCA. Liberated inorganic P was analyzed by

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spectrophotometer analyzes at 660 nm using chlorostannous reduced molybdophosphoric

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hydrochloric acid. One unit of phytase activity was defined as the amount of enzyme, which

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liberated 1 µM Pi (inorganic P) per second.

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Dehydrogenase Activity. Dehydrogenase activity was measured in soils immediately after soil

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sampling that was assayed by the method of Tabatabai.24 In summary, 1 g soil mixed with, 0.2

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mL of 3% 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) and 0.5 mL of 1% glucose. After a

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gentle shake, the sample was incubated for 24 h at 37 °C in dark, followed by addition of 10 mL

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of methanol. The sample was further incubated at 4 °C for 3 h. The production of

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triphenylformazan (TPF) was determined by measuring absorbance at 485 nm.

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Microbial Population Count. Microorganisms play an essential role in maintaining biological

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soil health. In this study, foliar application of ZnO nanoparticle effect on rhizospheric bacteria,

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fungi and actinomycetes populations were assessed. Serial dilution technique25 was used to

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count microbial CFU.

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Plant P. Grinded and sieved (2 mm size) plant samples were digested using a tri-acid mixture

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(HNO3: H2SO4: HClO4; 9:2:1) as described by Jackson.26 To estimate 10 mL of digested plant

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material was diluted with 25 mL DI water and add 15 mL added HNO3, ammonium molybdate

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(5%) and ammonium metavanadate (0.25%) in equal proportions (1:1:1). Phosphorous was

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quantified by measuring absorption at 485 nm and calculated according to the formula described

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elsewhere.27

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Soluble Leaf Protein. Total soluble proteins were quantified using the method suggested by

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Lowry et al.28 with certain modification. In short, fresh leaves were crushed in 5 mL of 10%


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trichloroacetic acid to precipitate the total soluble proteins and purified using centrifugation at

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8×103 g for 10 minutes at 4 °C. In this method, folin reagent (a mixture of sodium tungstate,

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molybdatye and phosphate) together with a copper sulfate mixed with isolated protein. The

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resultant, blue-purple color produced due to the reduction of phosphomolybdotungstate to

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hetero-polymolybdenum blue by the copper catalyzed the oxidation of amino acids, was

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quantified by absorbance at 660 nm using a spectrophotometer.

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Chlorophyll Pigment Content. To measure the chlorophyll content, plant leaves were collected

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on the 28th day and washed with tap water followed by DI water. One gram of fresh plant leaves

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were cut into small pieces and dipped in absolute acetone for 12h under dark. After incubation,

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the extracted chlorophyll was recorded at 661.6 nm, 664.8 nm, and 470 nm wavelength. Total

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chlorophyll content was calculated according to the formula described elsewhere.29

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Biodistribution and Transport of ZnO Nanoparticles. To investigate nanoparticles movement in

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mung bean plant, quantitative estimation was carried out by ICP-MS by following the sample

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digestion method describing elsewhere.21 Anatomical study of root, stem, leaf and mung bean

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seed was carried out by TEM. Root, stem, and leaf were collected on the 28th day, whereas, seeds

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were collected at 78th day. Small pieces of the tissue was immersed in 2.5% phosphate buffered

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glutaraldehyde for 4 h followed by rinsed with 0.1 M phosphate buffer and subjected to

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secondary fixation using 2% osmium tetroxide. Dehydration was carried out in a series (30 –

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100%) of five ethanol washes. The tissues were then infiltrated with a mixture of epoxy resin

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and propylene oxide as a transitional solvent, followed by complete infiltration with resin.

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Finally, the resin was polymerized in the oven at 60 ⁰C and the of plant tissues were ultra-

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sectioned using a microtome, followed by staining of sections with 4% uranyl acetate before

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imaging by TEM at 120 KV.



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Statistical analysis.

The data obtained from pot and field experiments were subjected to

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Statistical analysis by the analysis of variance method.30 The significance of different sources of

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variance was tested by “Error Mean Square” method of Fisher’s at probability level of 0.05 for

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appropriate degree of freedom. The standard of means (SEM ±) and the value of least

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significance difference (LSD) were calculated as described by Gomez and Gomez.31

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Physico-chemical Characterization of ZnO Nanoparticles. The morphology of ZnO

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nanoparticles was investigated by TEM and HR-TEM analyzes. Nearly spherical ZnO

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nanoparticles of 22.4±1.8 nm were more or less homogenously distributed as revealed by TEM

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micrograph (Figure. 1 A).

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agreement with a hydrodynamic diameter (inset of Figure. 1A) of the particles dispersed in DI

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water. Aqueous dispersion of ZnO nanoparticles was fairly stable up to 24 h under experimental

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condition. The stability of particles is possible due to in situ corona formation by fungal

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extracellular protein used in synthesis procedure.15 Negative surface zeta potential (-31.4 mV) of

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the particles play a role in nano-biointeraction with plant cell and keep them radially dispersed

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due to Coulomb repulsion.32 Crystal property of synthesized ZnO nanoparticles can be revealed

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by plane structure observed in HR-TEM micrograph, which is further supported by SAED

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pattern (Figure. 1B). In addition, crystal nature of particles were confirmed by X-Ray diffraction

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peaks (Fig. S1), match with characteristic zincite crystal plane.33 The chemical composition of

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the nanoparticles was determined by XPS analyzes (Figure. 2), where characteristic peaks for Zn

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(2p1 and 2p3) and O (1s) were observed. From the higher resolution XPS spectra of Zn 2p1 and

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2p3, it can be seen that the photoelectron peak of the sample appears at the binding energy of

RESULTS AND DISCUSSION

The physical diameter of synthesized nanoparticles is in well


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1043.6 eV and 1020.5 eV. Dominated peak of Zn 2p3 at 1020.5 and oxygen at 529.2 is

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associated with the binding of Zn2+ in ZnO zincite structure.34 Low-intensity carbon peak

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observed in the XPS survey spectrum is due to substrate used to mount sample powder for the

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XPS analyzes.

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The impact of ZnO Nanoparticles on the Physiological Growth. Zinc is a known as an

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essential micronutrient for all plants.35 The bulk counterpart either zinc sulfate or zinc oxide has

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been extensively applied in the field to abate Zn deficiency.36 To avoid the sulfate interference,

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in this study, we used bulk ZnO to compare the effect of ZnO nanoparticles. The concentration

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of applied nanoparticles were 10 mg/L, an optimal concentration of any micronutrient element

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for plant growth.37 Above the concentration limit, it imparts toxicity to the plants. Both, bulk

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and nano ZnO were tested for mung bean plants and several growth parameters were checked

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thereafter. Figure 3 shows that both bulk and nano ZnO had a significant positive effect in terms

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of stem height and root length. However, no significant difference were observed in root

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diameter and nodule with respect to control. Stem height of plants treated with ZnO

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nanoparticles was observed 32.8% higher than control. Similarly, root length, area, and nodule

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was increased by 29.9%, 53.3% and 58.9%, respectively. Plants phenotypic characteristic is

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crucial to determine plant biomass and extensive effect on root directly co-relate with water and

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nutrient absorption by plants. An important feature of legume root, is nodulation, a site for

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nitrogen fixation bacterial species.38

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The response of Zno Nanoparticles Mung bean Plants to Soil Enzyme of Native P

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Mobilization in Rhizosphere and Soil Biological Health. A large portion of the P in soil can

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exist in an organic form, must be converted to inorganic P before it can be utilized by plants.39

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The cycling of organic P and externally applied P fertilizer has a large impact on P availability in



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agricultural ecosystem.40 Phosphorous is very stable element in soils because they are very

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reactive with other compounds and bound as ester phosphate such as Ca-P, Al-P or Fe-P, become

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increasingly insoluble in the soil.1 Resultant P is unavailable to plants either due to runoff with

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excess rainfall or due to complex structure of P with other compounds. Phosphorous is taken up

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by plants wither in the form of H2PO4- or HPO42-. The mineralization of organic P is mediated by

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phosphatase and phytase enzyme, also known as P mobilizing enzymes that hydrolyzes C-O-P

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ester bonds.6, 40 Plants roots and rhizospheric microbes, produces or secrets phosphatases and

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phytase.41 Phosphatase and phytase enzyme play an important role in acquisition of P by plant

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roots and a significant factor of nutritional efficiency.5,

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enzymes including phytase and phosphatase that assist in performing reaction to mobilize native

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P.17,

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alkaline) and phytase as a result of ZnO nanoparticle exposure to plants is well complement with

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the previous studies as discussed above. ZnO nanoparticles of 23 nm having an improved

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catalytic property than bulk counterpart, significantly increased acid phosphatase (98.07%),

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alkaline phosphatase (93.02%), phytase (108%) and dehydrogenase (84.21%) activity in

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rhizosphere soil with respect to control (Figure 4). Although bulk ZnO also increased the

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enzyme activity but almost half of the nanoparticle efficiency, because of several reason such as

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(a) availability/internalization (b) surface zeta potential (c) surface area to volume size ratio and

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(d) intra cellular transport and reach out to various plants parts and subsequently to rhizospheric

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zone. Increase phosphatase and phytase activity is also complement with an increased in P

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acquisition in plants by 10.85% in nano ZnO treated plants and 2.04% in bulk ZnO treated plants

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(Figure. 5). Results of increased availability in soil and acquisition of P by plants due to nano

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ZnO can also be verified from increased rhizospheric zone by significantly increased root

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Zinc is the cofactor for various

In this study, experimental results of increased activity in soil phosphatase (acid and


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volume (Figure. 3) and soil microbial (fungi, bacteria and actinomycetes) population (Figure. 6)

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and dehydrogenase enzyme activity (Figure. 5), an indicator of microbial activity in soil.

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Enhanced microbial population help to maintain soil structure and soil biological health, an

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important phenomenon for various nutrient cycle.

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Biodistribution and Phytoaccumulation of Zn in Exposed Plant. ICP-MS data showed the

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presence of Zn in leaf, stem, root and shoot after foliar treatment with 10 mg/kg ZnO

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nanoparticles and its bulk counterpart (Figure. 7). The concentration of Zn in mung bean tissues

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followed the sequence leaf > stem > seed > root. Trends of Zn accumulation was similar for nano

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and bulk ZnO. The presence of Zn in mung bean seed indicate the possibility of introducing Zn

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ions/ZnO nanoparticles into the food chain. However, the amount of Zn (

Enhancing the Mobilization of Native Phosphorus in the Mung Bean

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