CHAPTER 2 MATERIALS AND METHODS - …shodhganga.inflibnet.ac.in/.../10603/40705/7/07_chapter2.pdf40 CHAPTER 2 MATERIALS AND METHODS In this chapter, the materials and methods used

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CHAPTER 2

MATERIALS AND METHODS

In this chapter, the materials and methods used for the synthesis

and characterisation of silica nanoparticles are described in detail. In

addition, appropriate methodologies and strategies used to explore the effect

of nano silica and micro silica on soil bacterial toxicity, cytotoxicity in cell

lines and maize seed stability, its growth and physiological characteristics,

fungal resistance and biocontrol activity are specified in detail.

2.1 MATERIALS

All the chemicals used in the present investigation were of

analytical grade purchased from Merck, Mumbai, India. High purity,

analytical grade substrate and reagents used for different biochemical

estimation studies were procured from Sigma-Aldrich, USA. Silicic acid

(H4SiO4, MW 96.11), micro silica (SiO2, MW 60.08, 40 150 mesh), and

sodium silicate (Na2SiO3, MW 284.20) were used as conventional silica

sources for soil and seed treatments. All the chemicals used in this study were

used as received without any further purification. Sterile distilled water

collected from Ultrapure water purification system (Arium 611 Ultrafilter,

Sartorius AG, Ge after double distillation

process was used throughout the experiments. Rice husk for the extraction of

silica nanoparticles were obtained from local rice mill (Tiruchengode,

Namakkal District, Tamil Nadu, India). Maize hybrid seeds (Zea mays L., TIP

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TOP) were collected from Rasi hybrid seeds (Attur, India) for entire

analytical studies using silica sources.

2.2 INDIGENOUS HIGH TEMPERATURE TUBULAR

FURNACE REACTOR

High temperature tubular muffle furnace reactor set-up was

indigenously designed in this study to produce silica nanoparticles from rice

husk at large scale. The block diagram and the photographic image of the

indigenous set-up are given in Figures 2.1 and 2.2, respectively.

Figure 2.1 Block diagram of the indigenous high temperature tubular muffle furnace reactor

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2.2.1 Components of the Indigenous Furnace Reactor Set-up

High temperature tubular muffle furnace reactor set-up was

constructed with two major compartments explained in 2.2.1 A and 2.2.1 B.

A) High Temperature Tubular Rotating Muffle Furnace

Tubular furnace set-up was designed in connection with a geared

motor to facilitate slow circular rotation with uniform burning of the rice

husk. It was made with a total working area of 99000 cm3 (30 × 35 cm) and a

heating zone using Kanthal fibrothal heating elements backed by ceramic

fibre blankets for insulation where maximum heating temperature of 1273 K

was achieved using Autonix Digital controller. The maximum heating rate of

6–8 K per minute exists in this furnace. One of the salient features of this

reactor set-up is the heavy weight rod that was fitted at the bottom of the

furnace to tilt the furnace in a horizontal axis by dumble action hand. It is a

cost effective way to harvest the burnt ash in collector vessel/glass reactor.

B) Glass Reactor

The transparent cylindrical glass vessel is devised with a total water

holding capacity of 20 L and a wall thickness of 10 mm to process the burnt

ash harvested from the furnace (A). Glass vessel acts as a collecting drum

where silica nanoparticles were extracted from the rice husk ash. Glass

container is built-in with the acid-proof teflon blades connected to a motor to

achieve homogeneous stirring of the solution. Acids and bases were

simultaneously added to rice husk ash for extraction through the side upper

nozzle of the glass container. The required heat to the glass container is

provided by an external hot plate.

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2.2.2 Working Principle

Large quantity of rice husk (5 kg) was fed into tubular furnace and

burnt at 1123 K for 3 h. After 3 h of burning, the heat regulator was switched

off with a continued rotation until the temperature decreases to room

temperature. Then, the furnace was tilted by releasing the rod fitted below the

furnace to harvest burnt ash into the collecting drum to extract nano silica

from rice husk. Of the total rice husk biomass, 60% was harvested as nano

silica powder in 7 h of processing time. The automated reactor set-up aids in

the mass production of silica nanoparticles from rice husk.

Figure 2.2 Indigenous high temperature tubular muffle furnace reactor set-up

2.3 SYNTHESIS OF NANO SILICA

Nano silica was extracted from natural raw material, rice husk

using acid precipitation method followed by alkali extraction

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(Kalapathy et al 2000 and Yuvakkumar et al 2014). Rice husk was burnt at

1023 K at the indigenous high temperature tubular muffle furnace for 3 h. The

obtained rice husk ash (RHA) was treated with 6 N HCl under stirring at 343

K for 30 min to leach out impurities under acidic conditions. Then, the ash

was washed with distilled water thrice to attain the pH value 7.0 and added

2.5 N of sodium hydroxide solutions under stirring for 2 h at 353 K to extract

silicate from ash. Supernatant containing the extracted sodium silicate

(Na2SiO3) were gradually added with concentrated sulphuric acid drop wise

until the pH reaches the value of 2.5 at which the solution becomes

transparent white silica sol. The pH was adjusted in such a way to attain the

precipitate of silica sol and then it was thoroughly washed with double

distilled water to remove the sodium interference and consequently washed

with ethanol (99.7% purity). Finally, the pure silica powder was collected

after calcination at 723 K for 2 h. The chemical reaction which occurred

during the extraction of nano silica is given as follows:

Scheme 2.1 Schematic representation for the extraction pathways of nano silica from rice husk ash

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2.4 CHARACTERISATION

Synthesised silica nanoparticles were subjected to different

comprehensive characterisation studies which are described briefly in

Sections 2.4.1 to 2.4.6.

2.4.1 X-Ray Diffraction

The phase and crystalline nature of the prepared samples were

analysed by X-ray diffractometer (XRD) (X’ Pert PRO, PANalytical, the

Netherlands) using CuK Å) as a radiation source. The

diffractometer was scanned over the 2 range of 10º – 80º at 293 K. The

observed peak positions and the relative intensities of the powder pattern were

identified in comparison with the reference diffraction data. The average

crystallite size of the samples was calculated using the following Scherrer’s

formula (Ramamoorthy et al 1999):

kDcos

(2.1)

where D is the crystal size, k the Scherrer’s constant (k=50.9), the

wavelength of the X-ray, the full-peak width at half of the maximum

intensity after correction, and the peak position.

2.4.2 Fourier Transform Infrared Spectroscopy

The peaks of silica functional groups from Fourier transform

infrared spectra (FTIR) were obtained in the wave number region of 4000 –

400 cm–1 at a resolution of 1 cm-1 using a FTIR spectrophotometer (Spectrum

100, PerkinElmer, Waltham, MA, USA). The pellet was prepared by mixing 2

mg of the synthesised silica particles with 200 mg KBr (95% purity) using an

agate mortar. To obtain a translucent pellet, the mixture was then pressed at a

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high pressure of 125 kg cm–2 in a 13 mm-diameter stainless steel container

using the high pressure pellet maker (Niulab, India).

2.4.3 Scanning Electron Microscopy-Energy Dispersive X-Ray

Analysis

The surface morphology and elemental composition of nano silica

were viewed under scanning electron microscopy coupled with energy

dispersive X-ray analysis (SEM-EDXS) (JEOL JSM-6390LV, Japan) at 20

kV with a

scattered over the surface of the sample. The scattered electron from the

sample was then fed to the detector and then to a cathode ray tube through an

amplifier, where the images were formed, which gives the information about

the surface of the sample. The atomic percentage or chemical composition of

the sample under scanning is semi-quantitatively determined as a function of

energy (keV) released by the scattered electrons through EDXS analysis.

2.4.4 Particle Size Distribution Study

Particle size distribution (PSD) of the prepared silica particles was

measured using particle size analyser (Nanophox, Sympatec, Germany) based

on the dynamic light scattering (DLS) technique. The PSD curve of all the

samples was obtained in the range of 1–1000 nm at a scattering angle of 90°.

The He–Ne laser with 10 mW maximum intensity was used as a light source

at a wavelength of 632.8 nm. The three-dimensional photon cross-correlation

technique was used for the simultaneous measurement of PSD and stability.

Prior to measurement, the silica particles were dispersed in an aqueous

solution under sonication (VC 505, Sonics, USA) at 30 kHz for 20 min to

obtain the colloidal solution as a function of density distribution (q).

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2.4.5 Transmission Electron Microscopy

Dispersed silica nanoparticles were examined for their size,

structure, and topography using transmission electron microscopy (TEM, CM

200, Philips, Hillsboro, OR, USA). For the TEM analysis, the dispersed

samples were loaded onto the copper grids operating at 120 kV and then,

dried under infrared (IR) lamp. The selected area electron diffraction (SAED)

pattern was observed to predict the crystalline nature of the samples. Then,

the average particle size was calculated manually by taking minimum and

maximum particle size marked in the image.

2.4.6 Surface Area Analysis

The specific surface area of the prepared silica nanoparticles was

determined with Brunauer–Emmett–Teller (BET) method using surface area

analyser (Autosorb AS-1MP, Quantachrome, Boynton Beach, FL). The

samples were degassed under vacuum at 563 K for 2 h to remove the

physisorbed moisture. The physisorption analysis was done with

N2 adsorption–desorption measurements at liquid nitrogen temperature

(77 K). The total pore volume and average pore diameter were calculated

according to the Barret–Joyner–Halenda method (Barrett et al 1951).

2.5 CYTOTOXICITY STUDY

The silica nanoparticles used for soil and plant application studies

are ultimately consumed by humans; hence, they were screened for

cytotoxicity assay against osteoblast-like MG-63 cell line. The MG-63 cell

line was purchased from National Centre for Cell Sciences, Pune, India. The

cells were cultured in RPMI (Roswell park memorial institute)-1640 medium

supplemented with 10% heat-inactivated foetal bovine serum, 3%

L-glutamine, 100 U mL–1 –1 streptomycin grown

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at 310 K in a humidified atmosphere of 5% CO2 in air. When the cells attain

80–90% confluence, they were seeded into 96-well microtitre plate at a

density of 1×103 cells per well for 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay (Ghosh et al 2010). After 24 h of

incubation, filter-sterilised nano silica at different concentrations from 1 to –1 were loaded in wells, and again incubated at 310 K for 48 h.

The cell morphology was observed in each well using inverted –1 MTT solutions were

added into each well and incubated for 4 h. At the end of incubation, 1 mL

dimethyl sulfoxide was added to reduce the formazan crystals into pink

colour. Then, the optical density (OD) of the test samples was read at 570 nm

spectrophotometrically (U-2900, Hitachi, Japan) with 630 nm as the control.

2.6 SOIL TOXICITY ANALYSIS

2.6.1 Amendment of Soil with Silica Sources and Maize Culture

The fine red soil was collected from the agricultural land of

Tiruchengode, Tamil Nadu, India. The experiment was set in a split-plot

design with silica source as main plot factor. Silica treatments in soil were

done in two sets. In the first set, soil was mixed with only silica sources such

as nano silica, sodium silicate, micro silica and silicic acid at a concentration

of 0.5 g kg–1. In the second set, maize seed was sown in the silica-amended

soils separately to differentiate the changes in microbial population and silica

content from pure soil. No organic fertilizers were added to the pots. The

experiment was performed in triplicates. Then, the maize hybrid seeds were

sown as 3–4 seeds per pot after 48 h of seed incubation in dark condition and

maintained under a photoperiod of 12 h at room temperature.

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The soil without silica sources served as a control. Soil mixed with

silica sources were kept as reference samples to compare the differences in

utilisation of silica by maize. The maize pot without silica sources was

considered as plant control. Similarly, seeds-sown pots amended with silica

sources were considered as tests. The denotations of soil samples for the test

under different treatments are hereafter given in the name of respective silica

sources used. Plants were watered with tap water (pH 7.7–7.9) twice a week.

The soil treatment combinations are mentioned in Table 2.1. Germination

percentage (GP) of seeds subjected to silica treatments were calculated by

considering the average of triplicate samples four days after sowing.

Cumulative germination of triplicate samples is taken from the average of

three GP of samples.

Number of seedsger min atedGer min ation (%) x100Number of seedsin dish

(2.2)

Table 2.1 Soil treatment combinations of different silica sources

Concentration of

silica sources

(g kg-1 of soil)

Soil treatment combinations

Pure soil Maize rhizosphere soil

Nil Control Plant control

0.5 Nano silica Nano silica

0.5 Sodium silicate Sodium silicate

0.5 Micro silica Micro silica

0.5 Silicic acid Silicic acid

2.6.2 Estimation of Microbial Population

After 10 days, soil samples were collected from the pots amended

with silica sources and maize rhizosphere to enumerate the population of

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nitrogen fixers (Azotobacter sp.), phosphate solubilisers, and silicate

solubilising bacteria using Waksman No.77 media (Allen 1959), calcium

phosphate media (Sperber 1958) and silicate solubilising media (Bunt and

Rovira 1955), respectively. The collected soil samples were subjected to serial

dilution and spread plate technique. Cultures of each triplicate samples were

incubated at 301 K for 3–5 days. Characteristic and discrete colonies of

grown cultures were counted and their population was expressed in terms of

colony forming units (CFU g-1). The variations in soil nutrient content such as

soil texture, pH and macronutrients (N, P and K) due to the application of the

silica sources were studied using standard soil testing methods such as

mechanical analysis (Piper 1966), potentiometry (Liang & Karamanos 1993),

conductometry (Sharifi et al 2009), and colorimetric studies

(Srinivasarao et al 2007).

2.6.3 Microbial Biomass Carbon and Nitrogen Content

The soil microbial biomass was estimated using modified

fumigation and extraction method (Carter 1991). Ninhydrin reactive

N-released during the fumigation of soil was determined using ninhydrin

reagent and was used as a measure of microbial biomass. Each soil sample

was divided into two sets. One set of soil sample (10 g soil on oven-dry

weight basis) was fumigated with ethanol-free chloroform for 6 days, dried in

an oven at 313 K for 12 h to remove the solvent and extracted with 2.5 M

potassium chloride. Similarly, non-fumigated soil sample was extracted.

Freshly prepared ninhydrin reagent (4 mL) was added and the mixture was

boiled in a water bath for 20 min. The contents were allowed to cool and the

volume was made up to 10 mL using a 1:1 mixture of methoxy ethanol and

distilled water. The intensity of the above solution was measured

spectrophotometrically.

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1 (Reactive Nin fumigatedsoil Reactive Nin unfumigatedsoil)MBCg of soil x 24Weight of soil

(2.3)

1 (Reactive Nin fumigatedsoil Reactive Nin unfumigatedsoil)MBNg of soil x 2.8Weight of soil

(2.4)

2.6.4 Silica Uptake

Estimation of total silica was performed using modified

silicomolybdenum yellow method (Xia et al 2000) in maize rhizosphere soil

and pure silica amended soil. 5 g of soil sample was weighed and dissolved in

water. Soil-bound silica was decomposed with 0.25 mol L–1 sodium carbonate

in water bath for evaporation and the final volume was made to 50 mL with

double distilled water. To this solution, 2.5 mL of 10% ammonium molybdate

was added and shaken for 15 min. 1 mL of dilute hydrochloric acid (2:1) and

2 mL of masking agent (10% oxalic acid) were added to avoid phosphorous

interference and incubated for 15 min to develop yellow colour. Then, the

absorbance was measured at 410 nm using spectrophotometer. The

concentration of silica was expressed as mg mL–1 of sample extract.

Total silica content in maize leaves was analysed using modified

method proposed by Wei-min et al (2005). After 10 days of maize growth,

young leaves under different silica treatments were collected from each pot

and dried in an oven at 352 K for 48 h. For this estimation, 100 mg samples

were taken. The absorbance of silica extracted samples was read at 650 nm

using spectrophotometer. The concentration of silica was expressed as mg

mL–1 of sample extract.

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2.7 HYDROPONIC INCUBATION OF SEEDS

2.7.1 Preparation of Silica Suspension

Silica sources such as nano silica, micro silica, sodium silicate, and

silicic acid were dispersed at an optimal concentration of 1.0 wt%

(Yuvakkumar et al 2011) in de-ionised water using the sonication method at

amplitude of 30 kHz for 15 min. The maize seeds were surface sterilised by

rinsing them in 60% (v/v) of commercial sodium hypochlorite (NaClO) for

10 min and then with sterile distilled water. Hydroponic solutions containing

0.01× Hoagland solution were prepared and supplemented with the prepared

silica suspensions (1%) containing four silicon sources separately in plastic

pots. The hydroponic treatments of maize with different silica sources are

depicted in Figure 2.3. The entire experiment was conducted in a laboratory

and arranged as completely randomised block design triplicates inclusive of

25 seeds each. Maize seeds were imbibed in a flask containing 100 mL silica-

supplemented hydroponic solution and shaken for 100 rpm at 300 K under

light intensity of 3000 lux with a humidity of 60 %. The solution without

silica was kept as control.

Figure 2.3 Hydroponic experiments for the absorption of different silica sources by maize

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After 16 h of soaking in hydroponic solution, 3–4 seeds from each

treatments flask were taken and sown in pots containing coir pith compost

(Coirpith/sandy loam soil at the ratio of 1:2). The GP and mean germination

time (MGT) of maize seeds were evaluated for 5 days and cultivation was

continued for an additional 15 days at a photoperiod of 12 h at room

temperature. The remaining seeds in the flask were rotated under the same

conditions for 7 days to evaluate Si absorption in all the seeds.

2.7.2 Seed Testing

Seeds collected from the hydroponic solution after 7 days of

incubation were observed for seed stability through morphological

observations. With an aim to study the deposition of SiO2 into maize seed

coats, another experiment was conducted using seed ash. Seeds were dried in

a hot air oven at 353 K for 48 h and then burnt at 1173 K in a muffle furnace

for 3 h to obtain ash for further analysis. Total dry weight percentage was

calculated from the obtained seed ash. Moreover, the SiO2 deposition in seed

ash was quantified using FTIR and X-ray fluorescence (XRF) spectrometry

(EDX-720, Shimadzu, Japan). Qualitative and quantitative elemental analyses

of leaf, root and seed ash of maize samples were performed using XRF. The

powder samples were analysed directly at 10 mm/5mm of focussing on Mylar

thin film without any sample preparation and destruction of the sample

composition.

MGT = No. of seeds germinated

+ No. of seeds germinated

(2.5)No. of seeds in dish No. of remaining seeds

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2.7.3 Analysis of Hydroponic Solution

The effect of silica sources on pH, conductivity and silica content

in the hydroponic solutions was analyzed before and after seed incubation.

The above measurements were carried out using pH and conductivity

electrodes connected to a pH meter (Orion 720A, 3 Star, Thermo Scientific,

USA). The variations in SiO2 absorption of seeds from the hydroponic

solution were determined by the formation of a silicomolybdenum blue

complex as proposed by Wei-min et al (2005). A measure of 100 mg of each

ash samples was taken for the above mentioned study. The absorbance of

silica extracted samples was read at 650 nm using a spectrophotometer.

2.7.4 Root Study

Root samples were carefully removed from pots after 15 days of

maize culture treated with silica sources. These root samples were measured

for root length and number of roots, after which they were allowed to dry in a

hot air oven at 343 K for 36 h. Dried roots were burnt at 1073 K for 5 h in a

high temperature muffle furnace. The effect of silica sources on silica

accumulation and occurrence of silica in roots were studied from root ash

samples by XRF and FTIR analysis.

2.8 FIELD STUDY

Nano silica was compared with micro silica for the growth

enhancement of maize crop under field conditions. The required green house

field was selected from the agricultural land at Tiruchengode, Namakkal

District, Tamil Nadu, India in the area of 15 × 10 ft of each plots containing

sandy loam soil (pH 7.0 ± 0.5). Experimental plots were prepared in

triplicates by adding nano silica at the concentrations of 5, 10, 15 and 20 kg

ha 1 (hereafter termed, respectively, as N5, N10, N15 and N20), micro silica

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at 15 and 20 kg ha 1 (hereafter termed respectively, as B15 and B20) and

control (without silica source). The detailed representation of treatments in

the field is given in Figure 2.4. The plots were then thoroughly ploughed to

spread the particles uniformly. Maize seeds were washed twice with distilled

water. Each plot was sown with 25 maize seeds at a seed distance of 30 cm

and irrigated regularly. The growth stages of the maize were monitored in all

the treatment plots.

Figure 2.4 Block diagram of treatment plot details of maize field

2.8.1 Growth Characteristics

After the seedlings were grown in the four regimes of nano and

micro silica amended fields, the changes in physiological parameters were

regularly observed. During this study, the seed GP of each treatment plot was

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evaluated. Further, the growth characteristics were measured in terms of stem

height, number of shoots and roots, stem diameter, root length and leaf area

(third leaf) after 20 and 40 days of maize growth by collecting samples in a

randomised block design.

2.8.2 Determination of Protein, Chlorophyll and Phenol

The total protein content of 20-day-old maize leaves was

determined according to Bradford method (Bradford 1976) using bovine

serum albumin (BSA) as the standard. The OD of the samples was measured

at 660 nm using spectrophotometer.

Chlorophyll was extracted with 80% acetone and estimated using

UV-Vis spectrophotometer at 650 and 665 nm. The ratio of chlorophyll a to

chlorophyll b in the leaf samples was also determined from the OD of the

chlorophyll extract (Arnon 1949). The variation in total chlorophyll content

was calculated using the following formula:

-1) = (2.55×10-2×OD650) + (0.4×10-2×OD665) (2.6)

Chlorophyll a = (OD665 × 12.7) – (OD645 × 269) (2.7)

Chlorophyll b = (OD645 × 22.9) – (OD665 × 4.68) (2.8)

The total phenolic contents from 3-day-old maize leaves at a

sample weight of 2 g were extracted using methanol. To the leaf extract,

f Folin–Ciocalteau reagent (1 N) was added to the leaf extract and

incubated at 298 K for 15 min to develop blue colour. The absorbance of the

samples was measured at 725 nm spectrophotometrically according to the

method described by Wakabayashi et al (1997).

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2.8.3 Analysis of Elemental Compositions

After 20 days of maize growth, root and leaf samples were

carefully collected and dried in a hot air oven at 373 K for 48 h. Then, the

samples were burnt in high-temperature muffle furnace at 1073 K for 4 h. The

ash thus obtained was further used to estimate the total dry weight percentage,

silica content (Wei-min et al 2005), and elemental analysis using XRF.

2.8.4 Gas Chromatography–Mass Spectroscopy Analysis

The shade-dried maize leaves (20 days old) were extracted by

methanolic extraction and analysed by gas chromatography–mass

spectroscopy (GC-MS, Thermo GC-Trace Ultra Ver. 5.0; Thermo Scientific

MS DSQ II). The GC silica column dimension was 30 m × 0.25 mm at a flow

rate of 1 mL min 1 at 353 K and then, the temperature was raised to 513 K.

The volatile organic compounds present in the control, nano silica and micro

silica treated samples were identified by comparing with the mass spectrum

matched with the inbuilt library (Wiley 9).

2.8.5 Microscopy and X-ray Analysis

High-resolution scanning electron microscope (HRSEM) equipped

with EDXS (FEI Quanta FEG 200; The Netherlands) was used for Si

accumulation studies. Third leaf of maize was cut to 15 mm in length and leaf

blades were examined at an accelerating voltage of 30 kV in a 9 mm working

distance and live detection period of 100 s. For quantification of all samples,

700× magnifications were chosen and the elemental weight percentage was

calculated using ZAF program of the EDXS system. In root anatomical

studies, samples of 15 days old maize roots about 1mm long were cut at a

distance of 10 cm from the apex and the segments were immediately fixed in

a formaldehyde solution (40%) for 24 h. Root sections were cut as thin layer

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transversely and stained with safranin solution for 1 min and then, mounted

on 50% glycerine. Internal root structures were observed under optical

microscope (200×) for the detection of anatomical variations.

2.9 FUNGAL RESISTANCE IN MAIZE

2.9.1 Treatment of Maize with Fungal Pathogens

Silica treatments were carried out in four sets for defense

compound analysis separately against Fusarium oxysporum and Aspergillus

niger. The collected soil was mixed with nano silica at different

concentrations, namely 5, 10, and 15 kg ha 1 (hereafter termed as N5, N10,

and N15), and with micro silica at a concentration of 15 kg ha 1 (hereafter

termed as B15). The maize grown in the soil without silica sources were kept

as control. As the foliar application of silica makes the plant absorb lesser

quantity of silica sources, an experimentation of soil amendment was carried

out. The experiments were performed in triplicates. Maize seeds were sown in

pots and maintained under appropriate growth conditions. Then, the plants

were irrigated with tap water (pH 7.7–7.9) twice a week. Silica accumulation

in leaf amended with nano silica and micro silica sources was detected using

XRF analysis.

2.9.2 Contact Angle Measurement

The contact angle (hydrophobic potential) of maize leaf blade

amended with nano silica and micro silica sources was measured using

contact angle meter (DCAT 11EC; DataPhysics, Germany). The leaf blades in

the size of 15 × 7 mm were kept in contact with water for 10 min under room

temperature. The obtained average contact angle is taken as the hydrophobic

potential of the leaf samples.

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2.9.3 Inoculation of Pathogens

Cultures of F. oxysporum and A. niger were maintained in potato

dextrose agar slants at 277 K. Then, a needle of culture was inoculated in

Erlenmeyer flasks (250 mL) containing 100 mL potato dextrose broth for 72 h

at 301 ± 2 K. Spores were collected after centrifugation at 3000 rpm for

10 min. Pellet was re-suspended in a small amount of sterile distilled water

and then diluted to obtain a fungal suspension of 105 spores per millilitre. The

spore concentration was determined using a haemocytometer. On the fourth

day of seed germination, the spore suspension (105 spores mL–1) was sprayed

on the surface of young maize leaves separately in each set. Then, the plants

were wrapped in porous polythene bag to avoid spreading fungal spores to air.

2.9.4 Examination of Disease Symptoms in Maize

After 72 h of fungal inoculation on maize leaves, disease symptoms

were noticed and evaluated. Symptoms such as leaf spots and lesions were

noted as disease index from each treatment. Then, the plant leaves and roots

were collected and cleansed with sterile distilled water. One gram of each

maize treatment sample was taken for each assay. The estimation of defense

enzymes such as phenylalanine ammonia lyase (PAL), peroxidase, and

polyphenol oxidase (PPO) was carried out after 72 h of inoculation in plant

roots and leaves. Total phenolic contents in maize leaves were estimated

according to the method described by Wakabayashi et al (1997).

2.9.5 Estimation of Enzyme Activity

Root and leaf samples (1 g) of three silica source treatments were

collected and were homogenised in 3 mL of ice-cold 0.1 M sodium borate

buffer, pH 7.0 containing 1.4 mM of 2-mercaptoethanol and 0.1 g of insoluble

polyvinylpyrrolidone. The extract was filtered and the filtrate was centrifuged

60

at 10,000 g for 15 min. The supernatant was used as an enzyme source.

Samples containing 0.4 mL of enzyme extract were incubated with 0.5 mL of

0.1 M borate buffer (pH 8.8) and 0.5 mL of 12 mM l-phenylalanine in the

same buffer for 30 min at 303 K. PAL activities of the enzyme extract were

determined at 290 nm (Ramamoorthy et al 2013).

Root and leaf samples (1 g) of three silica source treatments were

collected and was homogenised in 2 mL of 0.1M phosphate buffer, pH 7.0 at

277 K for the assay of peroxidases. The homogenate was centrifuged at

10,000 g at 277 K for 15 min and the supernatant was used as enzyme source.

The reaction mixture was consisted of 1.5 ml of 0.05 M pyrogallol, 0.5 mL

enzyme extract and 0.5 mL of 1% H2O2 and was incubated at room

temperature (301 K). The changes in absorbance at 420 nm were recorded at

an interval of 30 s for 3 min (Hammerschmidt et al 1982).

Root and leaf samples (1 g) of three silica source treatments were

collected and were homogenised in 2 mL of 0.1M sodium phosphate buffer

(pH 6.5) and ground mixture was centrifuged at 12,000 g for 15 min at 277 K.

The supernatant was used as enzyme source. The reaction mixture consisted

of 200 µL enzyme extract and 1.5 mL of 0.1M sodium phosphate buffer (pH

6.5). To start the reaction, 200 µL of 0.01 M catechol was added in the above

mixture and allowed for 3 min. PPO activity was read at 495 nm using

spectrophotometer (Ramamoorthy et al 2013).

2.10 BIOCONTROL ACTIVITY

2.10.1 Nanomechanical Testing

Mechanical properties of maize leaf blades were determined to

explore the physical strength and roughness attained by silica sources.

Topographical (2D) and hardness measurements were carried out to explore

the surface morphology and roughness of the maize leaves treated with silica

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nanoparticles and microparticles. Maize leaf blade treated with nano and

micro silica sources in the size of 15 × 8 mm were used for the analysis. The

measurement was carried out using Ubi 1 Scanning Quasistatic

nanoindentation (TI-

used to create an indentation with a Berkovich pyramidal indenter. Both the

loading and unloading times were kept constant as 5 s at a rate of 200 nm s 1.

2.10.2 Soil Amendment with Silica and Biocontrol Agent

The soil was mixed with nano silica at different concentrations

(i.e., N5, N10, and N15) and with micro silica (i.e., B15). The above soil

mixtures were further used for the treatment of biocontrol agent,

Pseudomonas fluorescens. A loopful of stock fluorescent pseudomonad

cultures was inoculated and grown in Erlenmeyer flasks (250 mL) containing

100 mL nutrient broth for 24 h on a rotary shaker (100 rpm) at 301 ± 2 K.

Supernatant was removed after centrifugation at 8000 rpm for 10 min at

277 K and then, the pellet was diluted with sterile distilled water to obtain a

bacterial suspension of 108 CFU mL 1. The culture was mixed aseptically

with the soils amended with silica particles. Maize seeds incubated for 24 h at

dark condition were sown in pots. The maize seeds grown in the soil without

silica sources and biocontrol agent were kept as a control.

2.10.3 Estimation of Pseudomonas sp. in Soil

During 10 days of maize growth, roots were grown enough to

collect soil from rhizosphere. The rhizosphere soil supplemented with silica

sources and P. fluorescens was collected from the pots and was subjected to

serial dilution followed by spread plate technique. The total population of

P. fluorescens was enumerated from the soil using King’s B selective media.

Cultures of each triplicate samples were incubated at 301 ± 2 K for 48 h.

Growth of discrete colonies were determined in terms of CFU.

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2.10.4 Examination of Stress Tolerance

After 10 days of Pseudomonas sp. treatment, maize roots were

collected and washed with sterile distilled water. The buffer extraction and

estimation were carried out for plant defense enzymes such as PAL,

peroxidase, and PPO using spectrophotometer. The enzyme activity was

expressed in terms of the changes in absorbance per minute. The total phenols

from maize leaves of different treatments were extracted using methanol and

estimated spectrophotometrically as the method described in Section 2.8.2.

2.11 STATISTICAL ANALYSIS

The differences in the various parameters of silica source treatment

and control in soil and plant samples were calculated using one-way analysis

of variance (ANOVA), followed by multiple comparison tests among the

groups using Tukey’s post hoc test and Duncans’ test. All the statistical tests

were performed using Statistical Package for Social Sciences software

(version 16.0; SPSS, IL, USA). Significant differences from the obtained

mean values of triplicate samples were identified at 5% significant level

(p<0.05). The significance, standard errors and standard deviations of the

obtained mean values were calculated and represented in the respective

figures and tables.

In this chapter, the mass production of silica nanoparticles from rice

husk biomass and their characterisation using appropriate methods and tools

were discussed respectively in detail. In addition, different methodologies

used to assess the role of nano silica on its cytotoxicity, soil toxicity, maize

seed stability, maize growth and physiological characteristics, fungal

resistance, and development of biocontrol action are described in detail by

citing necessary references.