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CHAPTER 3

RESULTS AND DISCUSSION

This chapter describes the results observed from the

characterisation studies of prepared nano silica powders and the effect of

silica nanoparticles against animal cell line, soil beneficial bacteria, soil

nutrients, growth response of maize under hydroponic and field conditions,

and the disease resistance in maize are discussed in detail. This chapter is

classified into five sections, namely Sections 3.1, 3.2, 3.3, 3.4, and 3.5.

3.1 SYNTHESIS, CHARACTERISATION, AND TOXICITY

ANALYSIS OF SILICA NANOPARTICLES

In this section, synthesis, characterisation and toxicological

assessment of silica nanoparticles against animal cells and soil bacteria for

food crop applications are discussed in detail.

3.1.1 Synthesis and Characterisation of Silica Nanoparticles

The extracted white hydrous silica precipitate from rice husk ash is

formed at pH 4. The calcined silica samples are characterised for their

structure, morphology and purity. XRD pattern of the prepared silica

nanoparticles reveals that the particles are amorphous in nature, which is

confirmed by the observed broad peak at 22 (2 ) (Figure 3.1a). The obtained

FTIR spectra (Figure 3.1b) show the presence of characteristic peaks in the

absorption spectra at 1096 cm 1 and 451 cm 1 corresponding to Si O Si and

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Si O functional groups, respectively. From SEM results (Figure 3.1c), the

silica particles are found as aggregates with spherical morphology. The purity

of the prepared silica powders is determined from EDXS. The results

(Figure 3.1d) show 98.2% purity of silica powders with negligible zinc

contamination. From particle size distribution curve and TEM image

(Figure 3.1e and f), it is confirmed that the particle size of silica particles are

found to be in the range from 20 to 40 nm with spherical morphology. The

SAED pattern of the TEM image also confirms amorphous nature of silica.

The obtained results are compared with the earlier studies on the preparation

of silica nanoparticles from rice husk through different processing conditions

(Yuvakkumar et al 2014). The prepared silica particles are found to possess

high surface area of 361 m2 g 1 (Figure 3.1g). The total pore volume and

average pore diameter are observed to be 0.8479 cc g 1 and 9.3 nm,

respectively.

In crystalline SiO2, all the oxygen atoms are bridging whereas

amorphous silica possess random orientation of oxygen atoms or some are

bonded to only one Si atom, i.e., non-bridging atoms. Amorphous nanosilica

is considered safe for humans by (World Health Organisation) WHO. In

addition, amorphous SiO2 nanoparticles show more promising potential for

agricultural applications (Barik et al 2008) than crystalline silica as it exhibits

hazardous effects to humans. Hence, to mimic naturally existing plant,

amorphous nanosilica particles are synthesised for biological applications.

However, morphology and crystalinity does not have any significant role on

uptake but it influences toxicological effects (Ghormade et al 2011).

65

a) XRD Pattern

b) FTIR Spectrum

Figure 3.1 (Continued)

66

c) Particle size distribution curve

d) SEM image

Figure 3.1 (Continued)

67

e) EDX spectra

f) TEM image

Figure 3.1 (Continued)

keV

68

g) BET plot

Figure 3.1 Characterisation of prepared silica powders

3.1.2 Toxicity of Silica Nanoparticles in Animal Cell Lines

The toxicological response of silica nanoparticles on the higher

organisms is studied using osteoblast cell lines at different concentrations.

The variations in the cell morphology with respect to the nano silica

concentrations are shown in Figure 3.2.

361 m2 g-1

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(a) Control (b) -1

(c) -1 (d) -1

Figure 3.2 Morphology of MG-63 cell lines treated with silica nanoparticles

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Figure 3.3 Percentage of cytotoxicity as a function of treatment of silica nanoparticles

Cells are observed to be aggregated with an increase in the

concentration of 1. In the same way, MTT

assay results for the nano silica-treated cells are graphically represented in

Figure 3.4. From the figure, the total cell viability percentage of MG-63 cells

under different concentrations is elucidated against 100% control samples. 1, cell viability tends to decrease significantly. This may be

due to the formation of particle agglomeration in RPMI medium as well as

interference during the cell proliferation which results in decreased cell

viability. Nano silica is biologically inert up to 125 -1 against MG-63

cell line. In addition, the in vitro cytotoxic responses of MG-63 cell line to

silica nanoparticles shows good biocompatibility. Hence, the high-surface

area silica nanoparticles are not showing any toxicological responses to food

crops. The reduction in cell viability with an increase in particles’

concentration is related to the higher accumulation of amorphous particles

into animal cells which may cause osmotysis leading to higher cell death. In

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contrast, micro silica is also not causing any cell death due to the prevention

of particle entry into cells.

3.1.3 Effect of Silica Sources on Seed Germination

The effect of nano silica on the microbial population and

availability of silica to soil and maize rhizosphere is clearly exploited in this

study in comparison with conventional silica sources. The germination

percentage (GP) of maize seeds amended with different sources of silica is

shown in Figure 3.4. Among different silica sources, maize seeds treated with

nano silica show the highest GP (98.5%) which may be due to immediate

uptake of silica nanoparticles by seeds and biochemical induction

(Yuvakkumar et al 2011).

Figure 3.4 Effect of silica sources on maize seed germination

In contrast, GP is reduced under sodium silicate treatment (92.5%)

which is due to the direct contact of seeds with alkaline Na2SiO3 in soil.

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From statistical analysis, it is inferred that there is no significant difference

(p < 0.05) between microsilica and silicic acid treatments. On the other hand,

nano silica significantly influences GP (%) when compared with the control.

3.1.4 Effect of Silica Sources on Bacterial Population

The population of microorganisms such as phosphate-solubilising

bacteria (PSB), nitrogen fixers, and silicate solubilising bacteria (SSB) is

enumerated in all the soil samples mixed with different silica sources, as

shown in Figure 3.5. This is carried out on the groups of beneficial microbes

by taking the characteristic colonies grown on the appropriate media. The

significant variation in total microbial population is confirmed from the

results of ANOVA at 0.05 level (p 0.05). When compared to control, the

silica-treated soils reveal an increase in microbial population. The increase in

the population of PSB due to the effect of micro silica is in line with an earlier

study (Veluchamy et al 2009). Moreover, nano silica treatment induces higher

population of PSB (3.8 × 104 CFU g 1) than that of SSB, which is same as

that of control (Figure 3.5). This observation reveals that addition of silica

may act as a substrate for phosphorus uptake systems in soil as well as in

plants.

Among the amendment of four different silica sources, the

population of nitrogen fixers is considerably high in all the treatments except

sodium silicate (2.2 × 104 CFU g 1). The observed lower population in

Na2SiO3-treated soil (3.4 × 104 CFU g 1) is due to formation of an alkaline

environment (pH 10.0 ± 0.02) in the soil which is not favourable for the

growth of beneficial microbes. In case of silicic acid and micron silica

treatments, a considerable increase of N2 fixers is observed when compared

with control.

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Generally, the population of SSB is less in normal soil that

solubilises sodium silicate. In addition, the presence of silicate is a direct

source for SSB to enhance its population. Changes in SSB among different

treatments are observed to be significantly stimulated (3.2 × 104 CFU g 1) by

sodium silicate. In soil treatment with different silica sources, an increase is

observed in the microbial population in nano silica-amended soils except

SSB. The observed increase is due to the size dependent property of nano

silica, which may act as a readily utilisable silica source for microorganisms.

The uptake of nanoparticles by microorganisms is via direct/active transport

or transfection of nanoparticles (Williams et al 2006) which supports the

present observation.

Figure 3.5 Effect of silica sources on maize seed germination. * indicates the significance obtained by Tukey’s test at 5%level (p < 0.05)

The enumeration of soil microbes in maize rhizosphere soil shown

in Figure 3.6 is to explore the efficiency of nano silica on improving the

fertility of plain soil as well as of rhizosphere soil. While comparing the

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silica-treated soils without seeds, rhizosphere soils show an increase in

microbial population in all treatments. Nano silica-treated soils show the

highest population of PSB (4.4 × 104 CFU g 1) and nitrogen fixers

(4.8 × 104 CFU g 1) except SSB, which are significantly influenced by sodium

silicate treatments (2.0 × 104 CFU g 1).

Figure 3.6 Effect of silica sources on the microbial population in maize rhizosphere. * shows the significance obtained by Tukeys’ test at 5% level (p < 0.05)

In contrast, sodium silicate-treated soils contain the lowest

population of PSB (2.2 × 104 CFU g 1). An increase in the population of PSB

of nano silica-amended soil indicates the enhanced soil fertility and available

nutrients to the plants. These results are in accordance with an earlier report

(Prakash et al 2010) showing a higher interaction of solubilised silica with

other nutrients, phosphorus in particular. Ten days of plant growth increases

the population of nitrogen fixers in rhizosphere, which may be stimulated due

to the added silica sources.

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The highest population of nitrogen fixers in nano silica-treated soil

may provide more nitrogen source to maize through N2 fixation. The

increased population of PSB in nano silica-treated soil is due to the

availability of more phosphorus to plants, as the silicon itself competes for

phosphorus in plant system (Prasetyoko 2006). As both phosphorus and

silicon influence the P content and the population of PSBs, the uptake of

either source increases the population of PSBs (Ma & Takahashi 2002).

Hence, silica may act as a substrate for PSBs that result in an increase of

microbial population and P availability. In contrast, an earlier report shows

that addition of silica sources reduces nitrogen level and raises phosphorus

level in few plants (Matichenkov & Ammosova 1996) but not in maize as

nitrogen fixers is considered. The observed results in maize rhizosphere soils

are consistent with those found in the literature (Aira et al 2010), that is,

inorganic fertilisation modifies the composition of rhizosphere resulting in an

increase in microbial community structure. These types of fertilizers (silica

sources) also determine the soil microbial communities because of the

differences in the soil nutrient contents (Owino-Gerroh & Gascho 2004).

The reduced population of SSB (2.0 × 104 CFU g 1) in nano silica-

treated rhizosphere is due to lack of specific interaction with the bacteria

(Figure 3.6). The soil treated with other silica sources shows an increase in

population than that treated with nano silica, which may be due to weak

interactions of bacteria with soluble silica sources for biological conversions.

These soil treatment studies aid the presence of SSB which in turn enhances

the content of available silica in soil. Among all the treatments, the population

of SSB is not significantly increased by other silica sources except sodium

silicate, which inhibits the population of the other two groups of microbes.

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3.1.5 Effect of Silica Sources on Soil Nutrient Content

The changes in total MBC and MBN contents of silica-treated soils

are shown in Table 3.1.

Table 3.1 Soil microbial biomass carbon and nitrogen contents

Treatment Biomass Carbon

( g g 1 of soil)

Biomass Nitrogen

( g g 1 of soil)

Soil treatment

Control 690 ± 25 81 ± 2.9

Nano silica 1924 ± 114 227 ± 13.3

Sodium silicate 258 ± 52 31 ± 6.1

Micron silica 620 ± 97 72 ± 11.3

Silicic acid 1103 ± 131 130 ± 15.3

Soil after seed sowing

Plant control 584 ± 37 69 ± 4.4

Nano silica 1508 ± 124 178 ± 14.5

Sodium silicate 170 ± 19 20 ± 2.3

Micron silica 547 ± 43 65 ± 5.0

Silicic acid 893 ± 71 106 ± 8.3

The results of total MBC and MBN contents of all the treatments

are in agreement with the present microbial population studies. The observed

results show an increase in carbon and nitrogen content ratio in nano silica-1 respectively).

Thus, the more accurate estimation of soil biomass is obtained from the above

method because it avoids the error occurring in colony-counting experiment.

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The applied concentrations of silica source (0.01, 0.03, 0.05, 0.07

and 0.09%) to the maize crop and their morphological changes have been

analysed in previous reports (Yuvakkumar et al 2011). This study has focused

to explore the significance of nano silica on improving soil fertility in

comparison with other conventional sources in an eco-friendly manner.

Differences in soil silica content because of silica sources are

estimated based on the formation of silicomolybdenum-yellow complex, as

shown in Figure 3.7. Significant difference is observed in soil silica content

among the different treatments. The silica content is high in both nano silica-

and Na2SiO3-treated soils (3.62 and 3.82 mg mL 1). The reduction in silica

content of all the maize rhizosphere samples other than unsown soil shows the

uptake of silica sources by maize roots. The silica contents of rhizosphere soil

samples are in the order of Na2SiO3 > microsilica > silicic acid > nano silica

(Figure 3.7). Higher silica content in Na2SiO3-amended soil is because of the

gradual conversion of silica source into available form to plants. Hence, silica

content is not entirely used by maize roots within 10 days (Youssef et al

2010). The reduction of silica content in nano silica-treated soil and the same

in rhizosphere (2.4 and 2.0 mg mL 1) shows that nano silica particles are

readily used by maize.

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Figure 3.7 Variations in silica concentration of all silica amended soils. * indicates the observed significant difference obtained at 5% level (p < 0.05) by Tukey’s test

The completely utilised silicic acid enters into the plant system in

the form of monosilicic acid (Savant et al 1997), which in turn allows soil

factors to use the applied silicon sources. However, silica solubilisation period

and uptake by plants are found to be long in Na2SiO3- and microsilica-treated

soils. Silica uptake by maize is quantitatively estimated, and the changes in

maize silica content are shown in Figure 3.8. Analysis of maize silica content

is essential to confirm the quantity of the available silica to plants. The

variations in silica content and its accumulation while applying microsilica

sources are studied in other crops such as rice and wheat (Ma & Takahashi

2002, Wei-min et al 2005). They have shown a considerable increase in silica

availability to rhizosphere as well as to plants.

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Figure 3.8 Variations in total silica concentration of maize samples. * indicates the observed significant difference obtained at 5% level (p < 0.05) by Tukey’s test

From the observed results, it is seen that nano silica treatment

shows an increase in silica content (14.75 mg mL 1) in the maize extract

among the other silica sources. Thus, the observed results indicate the

possible physiological role of nano silica on maize growth. Biotic and abiotic

stress tolerance of maize is generally rendered by the deposition of silicon in

plant tissues (Da Cunha & Do Nascimento 2009).

The effect of silica sources on essential soil nutrient contents is

shown in Table 3.2. The soil texture is not changed with silica treatments

whereas the pH is slightly affected by the treatment of micron silica (pH 8.32)

and silicic acid (pH 8.07). Moreover, the pH is highly influenced due to the

presence of Na2SiO3 in soil. From Table 3.2, it is clear that macronutrients are

greatly influenced by nano silica, that is, it enhances phosphorus content to 18

kg ha 1 and simultaneously reduces nitrogen content to 238 kg ha 1. Nano

silica and microsilica treatments alleviate the total macronutrient contents and

soil properties whereas it is not so in silicic acid treatment (Table 3.2).

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Table 3.2 Effect of different silica sources on soil nutrient contents

Test (Average) ControlNano silica

Sodium silicate

Micro silica

Silicic acid

pH 8.42 8.45 8.88 8.32 8.07

Electrical conductivity (dS m-1) 1.13 0.23 1.2 1.16 0.38

Texture SL SL SL SL SL

Lime NC NC NC NC NC

Total available N (kg ha-1) 322 238 227 213 302

Total available P (kg ha-1) 7 18 12 9 16

Total available K (kg ha-1) 353 233 304 326 295Note: SL - Sandy loam; NC - Non-calcareous

The changes in soil inorganic nutrients with respect to silica

fertilisation are due to the production of organic compounds by increased

microbial activity and desorption of inorganic nutrients from soil mineral

compounds (Aziz et al 2010). Although soil microorganisms and total

biomass content respond to application of different silica sources, significant

enhancement for better nutrition for maize is observed from nano silica

treatment. The addition of nano silica in soil also influences macronutrient

level, low-phosphorus stressed maize and other metal tolerance to promote

growth and development of maize seedlings (Yang et al 2008). Moreover, the

influence of nutrient content is found to be better in nano silica treatments

than that by micro silica sources. Hence, the application of nano silica does

not only maintain the soil pH but also alleviate the availability of

macronutrients to maize rhizosphere soil. This study explains the beneficial

effect of biocompatible silica nanoparticles to soil microbial biota as well as

soil fertility improvement.

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3.2 ANALYSIS OF SILICA UPTAKE UNDER HYDROPONIC

CULTIVATION OF MAIZE SEEDS

Germination percentage (GP) and mean germination time (MGT)

of maize seeds treated with different sources of silica are given in Table 3.3.

GP and MGT are observed to be high (95.5%) in silica nanoparticles-treated

pots while it is found to be low (75.77%) in Na2SiO3 treated pots. No

significant difference is observed between micro SiO2 and H4SiO4 treatments.

Seed germination and root elongation are considered as the widely used acute

phytotoxicity tests with susceptibility for unstable materials and sensitivity

(Munzuroglu & Geckil 2002). In the above study, GP (%) is delayed in

Na2SiO3 treatment. Nano silica is directly transported into seeds because of its

smaller size when compared with micro SiO2 and H4SiO4. Generally, the

symptom of chemical phytotoxicity is growth retardation, resulting in the

plants being stunted. Here, no marked negative effect on germination and root

elongation is noticed with respect to the silica treatments, even though silica

nanoparticles continued to be present in the hydroponic solution. The number

of roots and root length that varied upon treatments is presented in Table 3.3.

The roots and root length are not significantly affected by silica treatments.

Table 3.3 Effect of silica sources on maize seed germination and root length variations

Seed treatment

GP (%) MGT Number ofroots*

Root length (mm)

Number of shoots*

Control 93.33ab 2.8 7 ±1 145 ± 2.1 4.2 ± 0.04

Nano SiO2 95.50a 1.73 8 ± 2 168 ± 3.4a 4.8 ± 0.12

Micro SiO2 80.00 3.6 7 ± 2 151 ± 2.6 4.2 ± 0.15

Na2SiO3 75.77 4.2 9 ± 2 150 ± 2.2 4.2 ± 0.32

H4SiO4 90.50ab 1.73 8 ± 2 162 ± 3.8a 4.2 ± 0.27

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a indicates significant at p<0.05 level by Tukey’s test; ab shows subsequent

significance; * shows that values are non-significant

Absorbed silica during hydroponic incubation does not significantly

change the length of maize roots. In contrast, there is a considerable increase

in the root length of plants treated with silica nanoparticles and micro silica.

These results have a significant difference among treatment at p < 0.05 by

Tukey’s test. Bao shan et al (2004) studied in Larix olgensis by treating

chemically synthesised silica nanoparticles and observed the promoted

seedling growth. In another study, a mixture of silica and titania nanoparticles

results in enhanced seed germination and plant enzyme activity

(Lu et al 2002), which coincides with our results of enhanced GP and root

length. Hence, the root elongation of sensitive plant species would have a

silica source-dependent response.

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Figure 3.9 FTIR spectra of root samples showing the effect of silica sources on functional groups in terms of transmittance

Presence of silica functional groups and peak shifts are identified in

root ashes through FTIR spectra, as shown in Figure 3.9. The corresponding

peaks of silica for Si–O–Si, Si–OH, and Si–O are found in the regions of

1057, 790, and 673 and 463 cm–1 respectively. Even though peak intensity

varies with different silica sources, transmittance percentage is same in all the

treatment samples. The nutritional changes in maize root ashes after silica

treatments are given in Table 3.4.

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Table 3.4 Elemental composition of maize root ashes under treatments of different silica sources

Analyte (wt%)

Control Nano SiO2 Micro SiO2 Na2SiO3 H4SiO4

SiO2 33.08 43.50 38.48 20.22 35.03

Fe2O3 16.30 22.29 18.09 15.04 18.69

K2O 18.58 19.81 19.09 36.58 26.64

CaO 12.57 11.09 11.59 16.05 14.75

TiO2 0.67 1.34 0.74 0.77 1.04

P2O5 1.92 1.04 1.87 4.71 2.77

MnO 0.12 0.23 0.12 0.22 0.22

SO3 0.25 0.22 0.15 0.39 0.30

ZnO 0.05 0.16 0.07 0.11 0.19

CuO 0.01 0.03 0.01 0.04 0.04

MoO3 - - - 0.01 -

According to the XRF analysis, silica-treated plant roots show a

marked increase in silica content. Silica nanoparticles-treated roots (43.5%)

show significantly higher silica content with altered nutrient contents when

compared with control (33.1%). These results show that plant metabolic

changes are also influenced by the nanoscale particles (Monica and

Cremonini 2009). Na2SiO3 treated maize root ash contains least quantity of

SiO2 (20.2%). The effect of nanoparticles’ uptake on the metabolism of maize

remains unknown. However, it will be closely related to the chemical

composition, chemical structure, particle size, and surface area of the

nanoparticles. It is well known that roots are the first largest tissues to

encounter excess concentration of pollutants and toxins compared with

shoots; hence, elemental analyses of roots and the presence of Si functional

85

groups in roots are necessary. These analyses confirm that solubility and

uptake of oxide nanoparticles greatly affect the cell culture response

(Bao-shan et al 2004, Lin & Xing 2007) and radicals after penetrating the

seed coats that directly contact the nanoparticles.

3.2.1 Effect of Silica Sources on Seed Stability

Seed incubation for seven days in hydroponic solution

supplemented with Si sources alters the seed stability with respect to the kind

of silica sources (Figure 3.10). During hydroponic incubation of maize seeds,

seed coat rigidity and stability in the external environment are

morphologically observed. When compared to silica nanoparticles, other

silica sources affect the seed coat and make the seeds more fragile.

The increased fragility of H4SiO4-treated seeds makes the seeds

highly prone to fungal attack, which might be due to the poor accumulation of

SiO2. In contrast, silica nanoparticles-treated seeds have good seed viability

(Figure 3.10); hence, it might form complexes and organic compounds in the

cell wall of epidermal cells, thereby increasing their resistance to degradation

by the release of fungi enzymes (Datnoff & Rodrigues 2005). Na2SiO3 causes

discolouration of seeds as a result of alkaline pH. Although Na2SiO3 is

reported to act as a substitute for potassium-deficient maize plants

(Brunner et al 2006), its direct contact with seeds and roots is found to confer

an alkaline environment. However, potassium deficiency reduces mechanical

stability and nutritional quality to pathogens ameliorated by Si. This concept

seen in maize seeds agrees well with the stability of nano SiO2 -treated seed

coats and better emergence of seedlings when compared with other sources

(Jordan-Meille & Pellerin 2008).

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Figure 3.10 Maize seed stability after seven days of hydroponic incubation treated with different silica sources

The variations in the dry weight percentage of seeds treated with

different silica sources are plotted in Figure 3.11. Seed dry weight percentage

(%) increases because of optimum nano silica fertilisation, which ultimately

enhances the Si accumulation in plants. Studies on the dry weight of maize

seeds reveal that dry weight (%) of seed ash is high in silica nanoparticles-

treated seed (6.25 ± 0.22) than in seeds treated with micro silica and Na2SiO3

particles. Reduced dry weight (1.06 ± 0.2) is observed under Na2SiO3

treatment. No ash is found during seed burning after H4SiO4 treatment in any

of the triplicates because of fungal infection. Although silica nanoparticles

treated seeds show lower dissolution rate in a hydroponic solution, its

presence alleviates the uptake of other nutrients by plants (Rus et al 2004).

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Figure 3.11 Dry weight percentage of maize seed ash after treatments with different silica sources

FTIR results (Figure 3.12) show the presence of characteristic

peaks for silica functional groups in seed ash similar to the earlier FTIR

results of roots revealed in Figure 3.9. No peak shifts are identified for

gauging the amount of Si availability in maize. All the Si treatments exhibited

similar percentage of transmittance. However, the peak intensity is

considerably shorter than the peaks observed in roots due to the naturally

accumulated silica in trace quantity in seeds than in roots

(Datnoff & Rodrigues 2005). Determination of silica content in seeds is

necessary to ascertain the Si uptake from the hydroponic solution. However,

in our study, silica nanoparticles render seed erectness when compared with

other silica sources and control seeds.

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Figure 3.12 Transmittance spectra of maize seed ash treated with different silica sources

The XRF results indicate the elemental variations in seed ashes

after 7 days of soaking in silica suspension, as given in Table 3.5.

Table 3.5 Nutrient composition of maize seed ash treated with nano SiO2, micro SiO2 and Na2SiO3

Treatment Macro nutrients (wt%) Micro nutrients (wt%)

K2O P2O5 CaO Fe2O3 SO3 MnO ZnO CuO ZrO2 MoO3 SiO2

Normal seed 69.1 6.84 12.5 1.28 0.2 0.13 0.06 0.14 0.004 - 9.49

Control 51.08 29.53 3.53 5.33 - 0.21 0.36 0.07 - - 9.58

Nano SiO2 42.49 35.43 1.63 1.75 - 0.18 0.11 0.03 - - 18.17

Micro SiO2 47.34 33.57 1.9 1.76 - 0.14 0.28 0.03 - 0.01 14.7

Na2SiO3 68.98 17.58 0.09 2.64 - 0.16 0.03 0.03 - 0.37 9.16

LSD (p<0.05) - - - - - - - - - - 0.002*

*Least significant difference

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Generally, the presence of silica in seeds is very less when

compared with other parts of the plant (Datnoff & Rodrigues 2005). Increased

silica content under silica nanoparticles treatment influences the macro and

micronutrient levels in seeds. Influence of nano SiO2 on alleviating micro and

macro nutrients studied in the present investigations is similar to previous

investigations (Miao et al 2010, Yang et al 2008) and adds an advantage of

nano SiO2 treatment for improving crop yield.

3.2.2 Silica content

The estimation of silica content in the nutrient solution as well as in

seeds helps to understand the silica absorption by maize plants

(Figure 3.13 and 3.14).

Figure 3.13 Silica content of hydroponic solution after the incubation of maize seeds

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Variations in the silica content with respect to the silica source

treatment in seeds and external hydroponic solutions determine the quantity of

Si transport into maize seeds. Silica uptake of seeds is high under nano silica

treatment (12.25 mg mL–1) and is low in hydroponic solution

(10.75 mg mL–1). This reduced silica content in hydroponic solution reflects

the transport of trace quantity of Si into seeds that is significantly higher in

other sources.

Occurrence of SiO2 in control samples may be due to the release of

trace quantity of soluble silica from the seeds into solution. Interestingly, the

external hydroponic solutions of micro SiO2 treatment exhibit increased SiO2

content (32.5 mg mL–1) than its seed ash (10.75 mg mL–1). This observation

explains the poor uptake of micro SiO2 by seeds.

Figure 3.14 Silica content of maize seeds treated with different silica sources after hydroponic incubation

92

The fine particles generally lead to good emergence and early

seedling growth as compared to the larger aggregates and also render better

seed–soil contact. This concept agrees with silica nanoparticles’ application

that finer the aggregates of fertilizer better the uptake by plants

(Braunack 1995).

3.2.3 pH and Conductivity

Table 3.6 shows the effect of silica sources on the changes in pH

and conductivity on the first day and seventh day of hydroponic incubation

imbibed with maize seeds. The experimental pH variations seen in the

hydroponic solution are in the range from 6.5 to 7.5. Measurement of pH and

conductivity in hydroponic solutions is essential as the seeds are observed to

be more vulnerable to pH changes.

Table 3.6 Effect of silica sources on the changes in pH value and conductivity of hydroponic solution

TreatmentpH Conductivity (m cm–1)

1st day 7th day 1st day 7th day

Control 7.03 ±0.02cd 6.72 ± 0.18ab 0.04 ± 0.001a 0.044 ± 0.001a

Nano SiO2 8.57 ±0.15bc 6.79 ± 0.10ab 0.019 ± 0.002bc 0.007 ± 0.00ab

Micro SiO2 9.34 ±0.21ab 6.49 ± 0.24ab 0.083 ± 0.002ab 0.016 ± 0.005ab

Na2SiO3 11.14 ± 0.20a 9.11 ± 0.08a 0.001 ± 0.00cd 0.005 ± 0.00bc

H4SiO4 7.21 ±0.13cd 7.04 ± 0.03ab 0.019 ± 0.003bc 0.017 ± 0.002ab

a, b, c and d indicates the significance of samples at p <0.05 level by Tukey’s

test. Single letter shows high significance and combination shows subsequent

significance.

93

In Na2SiO3 added solution, pH and conductivity studies reveal

toxicity because the solution has an alkaline pH (9.11 ± 0.08). Despite the

salty nature of Na2SiO3 increases the conductivity, its high dissolution rate

reduces the conductivity (0.001 m cm–1). In particular, solutions under nano

and micro SiO2 treatments show less conductivity (0.007 and 0.016 m cm–1)

at seventh day when compared with the first day. The differences may be due

to the uptake of other nutrients by Si induction thereby increasing

conductivity (Miao et al 2010, Rus et al 2004). Even though sodium ions are

essential nutrients for maize as a substitute for K deficiency, these ions

increase both pH and salinity in a hydroponic solution (Jordan-Meille &

Pellerin 2008). Thus, both silica absorption and seed stability are enhanced

when nano silica is added along with nutrient supplements.

3.3 GROWTH AND PHYSIOLOGICAL RESPONSE OF MAIZE

UNDER FIELD CULTIVATION STUDIES

3.3.1 Growth Characteristics

Experimental maize plots containing four regimes of nano silica

and micro silica, including control are analysed for both morphological and

physiological discriminations as evidenced from Figure 3.15. Changes in

growth parameters such as seed germination, number of shoots and roots,

stem diameter, stem height, root length and leaf area of 20 and 40 day old

plants at varying concentrations of nano silica and micro silica, respectively,

are presented in Tables 3.7 and 3.8.

94

i) 6th day ii) 16th day

iii) 20th day iv) 20th day

v) 60th day vi) 60th dayFigure 3.15 Growth of maize under field conditions amended with nano

and micro silica sources at different days

GP of seeds is not affected by any of the plots tested and non-

significant differences during germination among the samples are observed. It

is inferred from the observed results that the growth parameters are enhanced

in maize with the increase in concentration of nano silica up to 20 days. The

gradual increase in growth parameters with respect to nano silica regime

Control N5 N10 N15 N20 Control N5 N10 N15 N20

95

provides enlarged leaf area that promotes photosynthetic activity. However,

number of shoots and roots do not show considerable variations. But lateral

roots and root length are observed to be increased due to nano silica

fertilisation.

Table 3.7 Effect of nano silica on growth parameters in maize after 20 days of growth

Growth

parameter

Sample

Germination (%)

Root (Nos)

Root length(cm)

Shoots(Nos)

Stem height(cm)

Stem diameter

(cm)

Leaf area (cm2

plant 1)

Control 97 11 11 4 13.3 0.8 114.7 ±2.8

N 5 97 20 12 5 14.5 1.1 128 ±4.1

N10 97.3 11 10.5 5 16.5 1.1 162 ±6.4

N15 98 18 18 6 19 1.4 260 ±10.1

N20 98 24 19 7 52 1.6322

±10.26

B 15 97 12 9 5 15.5 1 117.6 ±4.5

B 20 97.5 11 7.5 5 18 1.2 180.4 ±5.9

*LSD0.05 0.0 0.0 0.0 0.0 0.0 0.0 - *Least significant difference (LSD) at 5% level

After 20 days, the growth characteristics of maize remains same in

all the treated plots including micro silica treatments (Table 3.8) and plant

does not show substantial increase in the growth characteristics with

increasing concentration of silica sources, especially stem height and leaf

area. Similarly, the effect of nano silicon on the growth characteristics of

tomato is studied under salinity levels and has delivered promising results in

salt tolerance (Haghighi et al 2012).

96

Table 3.8 Effect of nano silica on growth parameters in maize after 40 days of growth

Growth characteristics

Root (Nos)

Root length(cm)

Shoots (Nos)

StemHeight(cm)

Stemdiameter

(cm)

Leaf area(cm2 plant 1)

Control 20 18 7 61 1.9 164.3±5.4

N5 22 15 7 74 2.4 294.8±11.3

N10 18 18 11 82 2.9 368.8±21.7

N15 20 12 12 81 3.2 360±21.8

N20 16 20 12 79 3.3 375.1±20.1

B15 10 12 7 62 0.9 199.2±5.4

B20 4 12 11 65 2.4 290±11.62

LSD0.05 0.001 0.0 0.0 0.0 0.0 -

3.3.2 Total Soluble Silica and Chlorophyll Content

While estimating the total leaf silica content based on the formation

of silicomolybdenum blue complex, increase in silica content with respect to

nano silica concentration is observed (Table 3.9). N15 and N20 possess

higher silica content (17.83 ± 1.29 and 12.55 ± 1.04 g mL 1, respectively)

than any other concentrations including micro silica (2.50 and 1.85 g mL 1,

respectively). The Si in the plant tissue can be solubilised and indirectly

measured in the extracted solution. Increased silica content in nano silica

treatments among other sources reflects the effective uptake of silica sources

from soil. Leaf samples collected from experimental plots (20 days) reveal the

gradual increase in chlorophyll a and b content according to the concentration

gradient of nano silica (Table 3.9) in contrast to micro counterpart (0.012 and

0.03 g mL 1) of 20 days old samples of maize leaves.

97

Table 3.9 Variations in total silica content and chlorophyll (a and b) content in maize leave samples amended with nano silica

Sample Silica content1)

Chlorophyll a1)

Chlorophyll b 1)

Chlorophyll

a to b ratio

Control 2.00 ± 0.52 0.017 0.01 1.647

N5 3.00 ± 0.41 0.01 0.007 1.581

N10 5.75b ± 0.83 0.024 0.01 2.311

N15 17.83a ±1.29 0.045 0.043 1.049

N20 12.55a ± 1.04 0.047a 0.042 1.121

B15 2.50 ± 0.61 0.012 0.007 1.598

B20 1.85 ± 0.57 0.03 0.02 1.493

LSD0.05 0.006 0.003 0.0

Note: Values within a column followed by single letters (a, b) show

significant varietal difference by Duncan’s test and least significant difference

(LSD) at 5% level

Moreover, higher chlorophyll content is achieved at N15 and N20

(0.045 and 0.047 g mL 1, respectively) than other regimes of silica

treatments. However, the actual action of nano metal/metal oxides on

chloroplasts and phyto compounds in plants is not well ascertained

(Dimkpa et al 2012, Mahmood et al 2005, Kidd et al 2001). It is interesting to

note that nano silica increases Si accumulation and leaf erectness which

stimulate the factors of chlorophyll synthesis at optimal regimes of silica

accumulation. The acceleration may be due to increased leaf area that renders

better light absorption and photosynthetic activity of chlorophyll a and b.

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3.3.3 Dry Weight, Protein and Phenol Determination

Changes in total maize dry weight percentage (%) and essential

plant biochemical components such as content of protein and total phenols in

20 days old maize roots and leaves are presented in Table 3.10. Root weight is

found to be high at N15 (24.29 ± 0.4%) and low at N20 in maize than micro

silica (5.67%) and control (7.73%). The higher dry weight percentage in roots

reflects the increased accumulation of silica in leaf bundle sheath. Shoot dry

weight (27.68 ± 0.34%) at N20 is less among the four concentrations of silica

nanoparticles. In micro silica treatment, reduction in dry weight percentage

(4.78 and 9.49%) is observed. While comparing the total dry weight of shoots

and roots, roots show lesser dry weight. This reduction is correlated with the

observation of Hossain et al (2002) that transition of excess SiO2 to older

leaves is also included in total shoot dry weight where as it is not included

during the root silica absorption.

Influence of silica regimes on the content of total protein and

phenol of all the treatment samples are shown in Table 3.10. Protein content

is found to linearly accelerate according to the concentrations of nano silica

especially at N15 (29.08 mg g-1) with negligible differences in total quantity

among all treatments. The increased protein content with the regimes of nano

silica and micro silica may be attributed to the existence of metabolic balance

between induction of proteins such as cell wall transporters and damping off

stress-responsive enzyme activities as a function of nanoscale fertilisation in

maize.

99

Table 3.10 Effect of nano silica on dry weight percentage and contents of total protein and phenol in maize roots and shoots

Sample Dry weight (%) Total protein

(mg g 1) Total phenol

( g mL 1)

Root Shoot Shoot Shoot

Control 7.73bc ± 0.04 5.05a ±0.03 25.28 0.364

N5 4.4a ± 0.13 6.61 a ±0.18 27.31 0.192

N10 8.77bc ± 0.05 33.96ab ±1.42 27.53 0.202

N15 24.29ab ± 0.4 21.52ab ±0.27 29.08 0.06

N20 5.89a ± 0.01 27.68ab ±0.34 27.09 0.422

B15 5.67 a ± 0.11 4.78a ±0.29 26.77 0.323

B20 11.67ab ± 0.35 9.49ab ±0.38 27.08 0.43

LSD0.05 - - 0.0 0.05

Note: ab, bc - show significant varietal difference and single letters indicates

non significance by Duncan’s test and least significant difference (LSD) at

5% level

Total phenol content of 20 day old maize leaves drastically reduces

at N15 (0.06 g mL 1) even though irregular modulations occur with other

regimes of silica treatments especially higher concentration at B20 (0.43 g

mL 1). Increased phenolic contents in low silica plants are correlated with

previous results obtained on Si-deprived plants that the observed silicification

may be substituted by the production of structural phenolic compounds

(Carver et al 1998). In addition, low phenolic content at N15 (0.06 g mL 1)

may reflect the induction of stress tolerance mechanism in maize.

100

3.3.4 Leaf Nutrient Variations

FTIR spectra of leaf ashes for the occurrence of corresponding

silica functional groups (Si O Si, Si OH, and Si O respectively at 1057,

693, and 463 cm 1) are confirmed in all the samples with varying intensity

(Figure 3.16). This implies the gradient of silica accumulation in plants to

substantiate the uptake of nanoscale silica by plant. It infers that the

occurrence of possible silica functional group is similar in all treatments.

Elemental studies on dry leaf ash on maize reflect the nutritional alleviation

exerted by nano silica in comparison with micro silica sources (Table 3.11).

Figure 3.16 Occurrence of silica functional groups in terms transmittance of treated maize leaf samples

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SiO2 content of the leaves also varies with the increasing silica

regimes. SiO2 accumulation in plants is not significant with the concentration

including control samples except at N15 where SiO2 content is 19.18%

(Table 3.11). XRF spectrometry is a direct method of elemental analysis used

on oven-dried plant matter. Alleviation of K and P contents has occurred due

to increased SiO2 deposition. Role of silica in ameliorating P and K contents

is explained in earlier reports (Miao et al 2010, Yang et al 2008) and the same

is enhanced by nano silica than by micro silica.

Table 3.11 Elemental compositions of the treated leaf ash samples

Analyte

(wt%)Control N5 N10 N15 N20 B15 B20

K2O 90.35 87.10 87.35 77.39 89.05 91.35 91.01

SiO2 4.93 8.42 8.02 19.18 7.66 4.39 1.30

Fe2O3 0.28 0.49 0.31 0.31 0.43 0.16 0.30

SO3 0.22 0.48 0.50 0.31 0.48 0.13 0.46

MnO 0.13 0.30 0.20 0.22 0.19 0.10 0.17

CuO 0.05 0.11 0.08 0.06 0.09 0.03 0.07

P2O5 4.01 3.01 3.52 2.49 2.05 3.34 5.68

ZnO 0.03 0.02 0.04 0.04 0.05 0.02 0.04

Hence, the accumulation of silica and other elements in maize

shoots varies greatly. However, the extent of SiO2 deposition in maize leaves

is found to be maximum at N15. In micro silica sources, the silica content is

the same as that of control sample (4.4%). This may be due to the large

particle size (micron size) that is not utilised efficiently by plants. Other

102

regimes of nano silica reveal no significant differences among treatment while

it greatly varies from that of micro silica (1–4%) as well as control. Therefore,

maize silica uptake seems to be promoted at the concentration of 15 kg ha 1

from soil. These results justify that the corn variety is amenable to SiO2

fertilisation up to N15 (15 kg ha 1) for the cultivation.

3.3.5 Variations in Organic Compounds

The modulations in total organic compounds according to the

treatments of nano silica and micro silica are elucidated from GC-MS results.

A relative abundance of volatile compounds in maize leaf extract with respect

to the retention time is presented in chromatograph (Figure 3.17). Area

percentage (%) of aldehydes, ketones, alkanes, alcohols, acid-esters, and

ethers of the micro and nano silica-treated leave samples is evaluated using

Wiley Library 9 and their abundance is plotted in Figure 3.18. Specific stress-

tolerant mechanisms in the plant are reflected by the abundance of the

phenolic compounds in the leaf extract. Total organic compounds like

phenols, aldehydes, ketones etc., are found to be reduced in maize leaves

grown in nano silica-amended soil. The expression of such compounds is

found to respond more in leaf extract of control and micro silica treatments.

From Figure 3.13, it is noticed that there are no considerable variations in

other organic compounds. These results are correlated with the study on the

protective mechanism of silicon in rice through phenols (Goto et al 2003).

103

Figure 3.17 GC MS chromatogram of maize leaf extract with respect to silica treatments

104

Figure 3.18 Total area percentages of organic compounds in maize leaf extract of silica treatments

3.3.6 Si Accumulation in Leaves and Roots

The difference in the optimal usage of SiO2 by species and organs

is due to differences in the endogenous levels of Si in the plant and

developmental stage (Hodson & Evans 1995). Hence, it is necessary to clarify

the accumulation of silica in roots and leaves through microscopic studies.

HR-SEM results exhibited the accumulation pattern of siliceous compounds

in leaves and EDXS results reveal the total elemental composition of tested

leaves (Figure 3.19). The elemental variations of leaves are shown as a table

insert in appropriate EDXS images. Third leaf of the maize plants is screened

for the variations in the quantity of silica deposition.

105

Figure 3.19 (Continued)

106

Figure 3.19 (Continued)

Interestingly, SEM images at N10 and N15 show wider deposition

of silica, which is confirmed through the reflection of silica and

corresponding balance in total elemental composition (Figure 3.19c and d).

The increased SiO2 accumulation in nano silica (N10 and N15) treatment

helps to avoid water lodging of roots and leaves (Lux et al 1999). Decrease in

silica content during microscopic observation at N20 may be due to the

transition of silica to the older leaf blades when abundant Si is absorbed

(Hossain et al 2002).

107

Figure 3.19 Distribution of silica in maize leaves shown in HR-SEM images coupled with EDXS. Scale bar = 50 m (700×)

This observation is also substantiated from the total dry weight of

plant as discussed earlier in this study. From the histological studies of maize

roots amended with nano and micro silica treatments (Figure 3.20), number of

epidermis cells and cell wall extensibility in roots are not found to influence

with nano silica treatments. However, the cell walls are observed to be thick

in maize roots amended with silica nanoparticles, where Si accumulation is

higher than with micro silica and control.

108

a) Control

b) Nano silica

c) Micro silica

Figure 3.20 Microphotograph of maize roots cross sections (400 ×)

P

X

Si

E

Si

Si

E

E

Si

Si

109

i) Control ii) N5

iii) N10 iv) N15 Figure 3.21 (Continued)

Si is deposited primarily in the epidermal tissues of roots and leaves

in the form of a silica bodies called phytoliths which results in higher

accumulation of silica in maize roots. Hence, nano silica promotes the

induced thickness of epidermal silicon–cellulose layer that supports the

mechanical stability of plants thereby resists lodging (Savant et al 1999).

Similarly, lead anatomical observation of longitudinal sections (Figure 3.21)

are differed in silica deposition with an increase in the regime of nano silica

and considerably high when compared to micro silica treatments.

110

v) N20 vi) B15

vii) B20 Figure 3.21 Microphotograph of maize leaf cross sections (400 ×)

Moreover, the reflectability of SEM images is in line with the

optical microscopic studies. When nano silica enhances the Si uptake into

cells, the dry weight of cells also increases in parallel with the SiO2

concentration. Thus, Si deposition is linked with the cell wall composition

and it can enhance the thickening of the root epidermis than the control plants,

which is in agreement with previous reports of micro silica application studies

(Hossain et al 2002). In contrast, uptake of other nanomaterials like CNTs by

root cells is not significant (Mondal et al 2011). By investigating the

responses of maize growth and physiological components to nano silica

regimes in comparison with micro silica using appropriate methodologies, the

111

concentration of 15 kg ha 1 shows an augmented silica accumulation and

physiological characteristics.

3.3.7 Foliar Spray

Foliar-sprayed maize plots with nano and micro silica are assessed

for their discriminations in phytochemical components. Leaf samples of 20

day- old maize collected from the experimental plots exhibit an increase in

chlorophyll content in nano silica (0.053 g mL 1) (Table 3.12) in contrast to

micro silica (0.027 g mL 1).

Table 3.12 Variations in total chlorophyll (a and b), protein and phenol contents of maize samples amended with nano and micro silica sources

Sample Chlorophyll a1)

Chlorophyll b1)

Protein

(mg g 1)

Phenols

( g mL 1)

Control 0.021±0.005 0.030±0.007 21.43±1.20 0.180±0.040

N15 0.053±0.002 0.048±0.011 25.71±1.06 0.272±0.090

B15 0.027±0.012 0.01±0.00 23.14±0.93 0.214±0.101

LSD*0.05 0.007 0.004 - - *Least significant difference at 5% level

However, the actual action of nano metal/metal oxides on

chloroplasts and phyto compounds in plants is not well ascertained

(Mahmood et al 2005). The acceleration in organic compounds may be due to

increased leaf area that renders better light absorption and photosynthetic

activity of chlorophyll a and b. Changes in total biochemical components

such as content of protein and total phenols in 20 day-old maize leaves are

presented in Table 3.12.

112

Protein content is found to be high at N15 (25.71 mg g 1) and in

B15 (23.14 mg g 1) with negligible differences in total quantity. The increase

in protein content with nano and micro silica may be attributed due to the

existence of metabolic balance between inductions of proteins such as cell

wall transporters. This may also due to damping off stress-responsive enzyme

activities as a function of nanoscale fertilisation in maize. Total phenol

content of leaves is observed to increase at N15 (0.272 g mL 1) than at B15

(0.214 g mL 1). Thus, the observed silicification may be substituted by the

production of structural phenolic compounds (Carver et al 1998). In addition,

the observed high phenol content at N15 may reflect the induction of stress

tolerance mechanism in maize that increases silica uptake.

Table 3.13 Elemental compositions of dry leaf ash of maize

Sample Elements (wt%)

K2O SiO2 Fe2O3 SO3 MnO CuO P2O5 ZnO

Control 90.35 4.93 0.28 0.22 0.13 0.05 4.01 0.03

N15 87.75 9.31 0.43 0.12 0.22 0.06 2.07 0.04

B15 90.91 6.16 0.31 0.1 0.2 0.03 2.26 0.03

Elemental studies on dry leaf ash of maize reflect the nutritional

alleviation exerted by nano and micro silica (Table 3.13). At N15, SiO2

content is found to be 9.3% (Table 3) which is significantly higher than of

micro silica (6.2%) and control (4.4%). Alleviation of potassium (K) and

phosphorous (P) contents occurs due to the increased SiO2 deposition as

reported earlier (Miao et al 2010). The enhancement in K and P content is

found optimum in nano silica than micro silica. Hence, the accumulation of

silica and other elements in maize shoots differs at higher magnitudes. This

may be due to increased particle size (micrometre scale) which is less utilised

by plant. In addition, higher phenol content also enhances the silica deposition

113

rate, which is in line with our observation from nano silica treatment

(Ghanmi et al 2004). Therefore, maize Si uptake seems to be promoted by

nano silica treatment in soil. In the present investigation, it is found that

amorphous silica nanoparticles show enhanced growth parameters when

compared to micro silica particles. As silica being a nutrient ‘anomaly’ in

monocotyledons, it does not confer toxicity to plants even as micro particles.

However, due to the size dependent effect nanosilica particles are easily

absorbed/internalised into plants either via active/passive transport

mechanism.

3.4 EFFECT OF SILICA NANOPARTICLES ON CONFERRING

DISEASE RESISTANCE IN MAIZE

Nano silica is studied for conferring defense response to fungal

infection in maize over micro silica treatment. The focus of this disease

resistance study at initial maize growth stage is essential to prevent pre- and

post harvest infections caused by fungi, which result in yield loss of maize.

3.4.1 Hydrophobic Potential of Silica Treated Maize Leaf

The hydrophobic potential of nano silica- and micro silica-treated

leaves is measured based on the contact angle between the leaf and water. The

hydrophobic potential of nano silica-treated leaves is found to be 86.18°

which is higher than that of the micro-treated leaves (80.11°), as given in

Table 3.14. Generally, an increase in hydrophobic nature of particles may

favour the plant defense action towards biotic and abiotic stresses. Silica

accumulation in maize leaves amended with nano silica and micro silica is

found to be 19.14% and 7.42%, respectively (Table 3.14).

114

Table 3.14 Hydrophobic potential and silica accumulation percentage of maize leaves treated with nano and micro silica particles

SampleContact angle

(°)

Si accumulation

(wt%)

Control 80.00 5.93

Nano SiO2 86.18 19.14

Micro SiO2 80.11 7.42

This is possibly because of the mechanism of increased silica

transport and deposition in roots as well as leaves (Ma and Yamaji 2008,

Fauteux et al 2005). Thus, it is evident that nanoparticles or nanoparticle

aggregates with diameter less than the pore diameter of the cell wall can

easily penetrate and reach plasma membrane. There is an enlargement of

pores or induction of new cell wall pores on interaction with nanoparticles

which in turn enhances the uptake (Navarro et al 2008). In addition,

deposition of amorphous silica in the cell walls leads to leaf erectness and

hence, may prevent the invasion of pathogenic fungi.

3.4.2 Effect of Silica Treatment of Maize Disease Incidence

Fungal inoculation (after 72 h) on the maize amended with different

regimes of nano silica and micro silica particles is evaluated for disease

incidence in terms of number of lesions and diseased spots. As the

concentration of silica nanoparticles increases from 5 to 15 kg ha 1, disease

symptoms on tissues of the leaf decreases. Generally, lesions and spots occur

due to invasion of fungi into the plant leaves on 3 days of infection which is

shown in Table 3.15.

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Table 3.15 Disease incidence in maize amended with nano silica after treatment with fungal pathogens

Treatment

Maize treated withF. oxysporum

Maize treated withA. niger

No. of lesions(cm 2)

Disease spots

No. of lesions (cm 2)

Disease spots

Control 2.67±0.58 30±3.12 4.00±1.00 45±4.01 N5 2.00±0.00 29±2.45 3.00±1.00 34±3.19 N10 1.67±0.58a 21±2.89 2.67±1.15a 28±2.83a

N15 1.00±0.00a 12±3.11a 1.33±0.58a 11±2.46a

B15 2.27±0.58 27±3.02 3.00±1.00 37±1.08 a indicates the significant difference obtained among treatment by Duncan’s’

test at 5% level (p < 0.05)

The degree of resistance is measured as the degree of fungal

susceptibility to the leaves amended with nano silica as well as with micro

silica and control leaves. The disease incidence found in control plant is 2.67

lesions per centimetre, and 2.27 lesions per centimetre in micro silica

inoculated with Fusarium, but lesser in leaves treated with A. niger. Both the

fungi are susceptible to nano silica treatment, especially at N10 and N15.

Fungal incidence is reduced with an increase in concentration of nano silica

where as it is less in micro silica treatment. Leaf samples inoculated with

A. niger show a similar pattern of disease incidence as seen in F. oxysporum.

Both the organisms are more infectious to control and less to micro silica-

treated maize; however, the infection is considerably controlled with nano

silica incorporation in maize plots.

Early studies on micro silica sources show that silicon application

induces resistance in plants (Fauteux et al 2005). But the degree of resistance

conferred by the nanoparticles is found to be higher than that conferred by

micro particles. In our previous study, it is observed that maize seeds are

more stabilised and silica uptake is high in the seeds treated with nano silica

116

when compared with other conventional sources. Similar observations reflect

in this study, that is, the physical stability of plants is found to improve with

nano silica amendment. Micro silica sources are observed to influence biotic

and abiotic stress tolerance mechanisms in monocot plants (Fauteux et al

2005, Fawe 1998). However, nanoparticles play unique role by contributing

direct uptake and accumulation of silica which leads to leaf erectness and

enhances defense response to fungal pathogens. Hence, nano silica may act as

an effective physical barrier against mycelia invasion.

3.4.3 Effect of Nano Silica on Defense Compounds of Infected Maize

Roots

The effects of silica nanoparticles and micro silica on the total

enzymes such as PPOs, peroxidases, and PALs are investigated to evaluate

resistance behaviour of maize roots. An irregular expression of the enzymes is

observed with silica nanoparticles’ treatments as well as with fungal

infections. The PAL activity of the roots amended with different regimes of

nano silica and micro silica individually inoculated with F. oxysporum and

A. niger is given in Table 3.16. The PAL activity of the roots inoculated with

A. niger is found to be low at N15 (97.0 ± 2.41). As the concentration of silica

nanoparticles increases, the PAL activity tends to decrease during the

infection of both fungi.

117

Table 3.16 Effect of nano and micro silica particles on PAL activity of infected maize roots

Treatment

Phenylalanine ammonia lyase activity in roots

( g mL 1)

Control plant F. oxysporum A. niger

Control 107.3±0.57 110.6 ±1.23 113.3±2.14

N5 105.9±1.89 121.7±2.56 108.9±1.87

N10 100.1±5.42a 105.0±1.98 a 103.2±3.56a

N15 91.7±2.36a 127.3±3.58 97.0±2.4 a

B15 112.7±2.11 118.0±4.51a 120.3±3.16 a indicates the significant difference obtained among treatment by Duncan’s’

test at 5% level (p<0.05)

Moreover, micro silica and control samples show similar enzyme

activity. The PAL expression may further reduce because of the lag in fungal

invasion with respect to silica absorption. On comparison of plants inoculated

with F. oxysporum, those inoculated with A. niger are more susceptible to

silica treatments which may be due to the damping-off mechanism in plants

(Liang et al 2003).

The PPO activity of the maize roots after fungal spraying is assayed

against F. oxysporum and A. niger at regular intervals (Figures 3.22 and 3.23).

The intensity of the PPO activity increases in the solution with time which

indicates a gradual interaction of PPOs from plant extract with the freshly

added substrate, i.e., catechol. Among all the treatments, N15 shows the

lowest intensity of the PPO activity against F. oxysporum infection

(Figure 3.22).

118

Figure 3.22 PPO activity of maize treated with Fusarium sp. at different time intervals

However, both N15 and B15 show the least PPO activity during

A. niger inoculation, as shown in Figure 3.23. Generally, the pathogenicity of

A. niger is more protected by nano silica than its micro counterpart. The range

of optical density of samples infected with Fusarium is in the range of

0.05–0.35 min 1, whereas that of the samples infected with Aspergillus sp., it

is 0.03–0.28 min 1. The logical relationship between nano silica and

decreased stress-responsive enzyme activity is due to the deposition of silica

that tranquilises stress-related macromolecules (Fauteux et al 2005). The

result indicates that the prevalence of Aspergillus infection can be reduced by

silica nanoparticles application.

119

Figure 3.23 PPO activity of maize treated with Aspergillus sp. at different time intervals

Figures 3.24 and 3.25 show peroxidase activity of nano silica- and

micro silica amended maize roots inoculated with F. oxysporum and A. niger

respectively, at regular intervals. The exponential reduction in enzyme

activity with respect to time is observed in all the samples. As the time

increases, the freshly added H2O2 is more exposed to oxidation in the

presence of peroxidases in the plant extract as well as to air atmosphere.

Moreover, a decline in enzyme activity with higher concentration of silica

nanoparticles is noticed in both the fungi. The intensity of peroxidase

enzymes in the maize extract inoculated with F.oxysporum (Figure 3.24) and

A.niger (Figure 3.25) are in the range of 2.7–3.4 and 2.8–3.56 OD min 1,

respectively.

120

Figure 3.24 Peroxidase activity of maize samples treated with Fusarium sp. at different time intervals

Figure 3.25 Peroxidase activity of maize samples treated with Aspergillus sp. at different time intervals

Phenol is one of the most stress-responsive plant compounds

(Rodrigues et al 2003). The total phenol activity of collected leaf extracts

shows linear increment with an increase in concentration during the fungal

inoculation. Nevertheless, maize inoculated with F. oxysporum shows higher

121

phenol content (2056 g mL 1) at 15 kg ha 1 than micro silica (1100 g mL 1)

and control samples (Figure 3.26). In contrast, leaves treated with A. niger

contain 743 g mL 1 of phenol which is higher than micro silica

(193 g mL 1) at 15 kg ha 1 (Figure 3.27). The phenol content increases

during fungal infection compared with control, particularly in F. oxysporum.

The increase in phenol content in plants is because either Si induces plant

defense compounds or that the plants are in more stress conditions due to

fungal infection.

However, the plants treated with nano silica are found to be

healthier than control plants which are confirmed through the observed

decrease in disease lesions as compared to others silica sources. In the

presence of silica, rice and wheat are capable of inducing similar biologically

active defence agents, including increased production of glycosylated

phenolics in rice. This observation, that is, an increase in concentration of

phenolics in maize is due to the application of nano silica, is in close

agreement with the one reported for rice (Rodrigues et al 2003). The

protection of fungal infection with an enhanced activity of chitinases,

peroxidases, PPOs, and flavonoid phytoalexins is first explored in cucumbers

by micro silicon application (Liang et al 2003, Currie & Perry 2007). Thus,

the response of plants to nanometal oxides varies with the type of plant

species, their growth stages, and the nature of nanoparticles.

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Figure 3.26 Total phenol content of maize roots after Fusarium sp. Inoculation. * indicates the significant difference among treatments at 5% level by Duncan’s test (p < 0.05)

Figure 3.27 Total phenol content of maize roots after Aspergillus sp. Inoculation. *, ** indicate significant difference at 5% level

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A recent study on nanomaterials in different plants at different

developmental stages reveals contradictory effects (Nair et al 2010). In this

study, the degree of externally induced resistance conferred by the

hydrophobic silica nanoparticles is high at 10 and 15 kg ha 1 when compared

to micro silica. Thus, an intrusion of nano silica via active transport is a

defensible way to avoid cellular damage to pathogens in maize leaves. The

mass production of silica nanoparticles from rice husk biomass requires

simple experimental technique, process methods and instrumentation. Thus,

the production of nano silica from rice husk biomass is cost-effective. This in

turn helps to apply for agricultural applications, disease resistance in

particular.

3.5 ENHANCING THE BIOCONTROL ACTION OF MAIZE

USING SILICA NANOPARTICLES

3.5.1 Physical Strength of Silica Treated Maize Leaf

Essential resistance mechanism in plants other than defense

compounds is the physical strength. The degree of physical strength attained

in leaves by silica nanoparticles can be elucidated from the leaf hardness tests

using nanomechanical testing systems. Figure 3.28 shows the topography of

silica nanoparticles-

observed colour contrast in the images shows that the roughness exists on the

leaf surface at a series of scanning displacement (nm). A thick section of

leaves in nano silica-treated maize is observed for roughness and hardness

(Table 3.17) which are found to be 1640 nm and 26.46 MPa, respectively

(Figure 3.28c).

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a) Control b) Micro silica c) Nano silica

Figure 3.28 Surface topography of maize leaf surface

In contrast, leaves treated with micro silica and control possesses

considerably less roughness and hardness value than nano silica-treated

samples. The displacement of piezoelectric tip as a function of applied force

on the leaf surface is graphically represented in Figure 3.29.

Figure 3.29 Mechanical indentation curves with respect to applied force on the leaf surface

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The depth curve is gradually decreased with an increase in force

from nano, micro and control leaves of maize which is clearly seen in the

graph. Generally, silica nanoparticles are physiosorbed by the cuticular lipids,

disrupting the protective barrier and thereby causing death of insects purely

by physical means (An et al 2010).

Table 3.17 Mechanical properties of maize leaves treated with nano and micro silica sources

Sample Mean height

(nm)

Roughness

(nm)

Hardness

(MPa)

Control -0.001 368.0 1.51

Micro silica - 0.03 383.4 3.25

Nano silica 1.4 1640 26.46

In addition, the surface charge-modified hydrophobic silica

nanoparticles (3–5 nm) are successfully used to control a range of agricultural

insects and animal ectoparasites of veterinary importance. Further, silica

nanoparticles are also applied as thin films on seeds to decrease fungal growth

and boost cereal germination (Karunakaran et al 2013, Fauteux et al 2005).

3.5.2 Effect of Silica Particles on Pseudomonas sp. Population

Pseudomonas sp. population is enumerated after 48 h of incubation

in King’s B medium and is illustrated in Figure 3.30. Higher population

(4.4 × 104 CFU g 1) is induced at N15 when compared to control

(2.6 × 104 CFU g 1) and micro silica (3.2 × 104 CFU g 1) samples. The

efficiency of nano silica in improving the soil biomass due to the treatment

with silica and biocontrol agent is evident in this observation.

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Figure 3.30 Total population of pseudomonads in soil treated with silica sources

In the previous study, enhanced population of beneficial bacteria is

noticed in the sandy loam soil treated with TiO2 and ZrO2 nanoparticles

wherein a higher concentration led to a decrease in population

(Ghanmi et al 2004). On the contrary, our results show that an increase in

nano silica concentration increases the bacterial population. Therefore, it is

inferred that if nano silica enhances the bacterial population, it promotes the

degree of biocontrol action against maize pathogen in soil.

3.5.3 Effect of Pseudomonas sp - Silica Particles Biocomposites on

Maize Defense Compounds

The PPO activity in Figure 3.31 indicates an increase in solution

intensity with an increase in the incubation period after adding the substrate to

the root extract.

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Figure 3.31 PPO activity of maize roots treated with silica sources and Pseudomonas at different time intervals

However, a decrease in enzyme content in the roots is noticed with

an increase in the concentration of nano silica treatment, particularly at N15.

On the other hand, the control and micro silica treated maize roots do not

show any effect on enzyme content. The suppressed enzyme expression may

indirectly convey lesser stress in maize. In addition, peroxidase and

PAL activity of maize roots treated with Pseudomonas and different regimes

of nano silica are shown in Figures 3.32 and 3.33, respectively.

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Figure 3.32 Peroxidase activity of maize roots treated with nanobiocomposites at different time intervals

On comparison of all treatments, N10 and N15 show reduced

expression of peroxidases with a decrease in time interval and an increase in

concentration. However, less enzyme activity is observed in sample B15 than

N5 and control. The above results are in correlation with those of earlier

studies that micro Si treatment induces damping-off mechanisms among

defense compounds (Gamliel & Katan 1993). Hence, silicon-mediated

defense mechanisms in monocotyledons support the application of nano silica

formulated with P.fluorescens to achieve a broad spectrum of biocontrol

activity.

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Figure 3.33 PAL activity of maize roots treated with nanobiocomposites at different time intervals

Total phenol content in a methanolic extract of maize roots is

mL 1) among all treatments 1 1), it

is not differed at 5% significant level (p < 0.05). A sharp rise in phenolic

content at N15 in combination with biocontrol agent is observed even after

three trials, which emphasises the induced resistance. These elevated levels of

phenol may be due to enhancement of fungal resistance compounds released

by P.fluorescens. Although many phenolic compounds in plants may not have

biocontrol activity per se, the oxidation products of pre-existing phenols and

bacterial extracellular enzymes might have biocontrol action

(Gamliel & Katan 1993). This is also because soluble silica easily forms

complex with phenolic compounds, thereby strengthening the mechanical

resistance of plant cells (Ramamoorthy et al 2002).

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Figure 3.34 Total phenol content of maize roots

A similar phenomenon is observed throughout this investigation.

However, the reduction of phenols at N5 and N10 is controversial in this

study which may be due to the insufficient supplement of nano silica to soil.

The defense mechanisms in maize after the treatment of nano silica and

biocontrol agent are presented schematically as follows:

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SiO2 Accumulation of phenolic compounds + physical barrier (i)

P. fluorescens Biocontrol activity (ii)

(iii)

Scheme 3.1 The mechanism of biocontrol action developed in maize treated with silica nanoparticles

Biocontrol agent is also essential for elevated levels of phenols in

plants (Ramamoorthy et al 2002). It is an interesting observation of increasing

phenolic content and improved leaf physical strength to resist fungal stress in

the present study. The crucial colonization level must be reached is estimated

at 105–106 CFU g 1 of root in case of P. fluorescens treatment that protects

plants from G.tritici or Pythium sp. (Barik et al 2008). A potent formulation

can be developed for sustainable cropping from this biological composite

(Silica nanoparticles and P.fluorescens). Hence, nanoscale silica acts as one

of the mediators for defense reactions in planta compared to other

conventional silica sources.

By investigating role of silica nanoparticles in maize through all

possible analyses, the interaction mechanism of silica with soil and maize is

clearly elucidated in a schematic diagram (Figure 3.35).

Active/Passive transport

Application in roots

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Figure 3.35 Summary of uptake and transport mechanism of silica nanoparticles for the beneficial role in maize