NANO MATERIALS FOR THE MANAGEMENT OF PLANT DISEASES

1. Professor Jayashankar Telangana State
Agricultural University
Course in-charge
Dr.B.Rajeswari
Professor
Dept. of Plant Pathology
1
Masters Seminar on
Use of Nano particles in plant disease
management of field crops
Department of Plant Pathology
Presented by
N.Karunakar Reddy
RAM/2017-66

2. 2
through the slides..
 Introduction
 Definition
 Timeline
 Nano materials
 Approaches used
 Applications of Nanotechnology
 Nanotechnology in INDIA
 Case study
 Disadvantages of nanotechnology
 Future prospectives
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3. 3
1. The application of chemical fungicides has caused threat to non-
target organisms and the environment due to their overuse.

 Since the release of xenobiotics results in the increase of
environmental risk, the goal should be to use such compounds
carefully so that they cause least negative impact on the
environment into which they are released.

 Despite good concept IDM is yet to cope up with certain issues
pertaining to practical applications especially in the background
of changing climatic scenario.
INTRODUCTION
•3

4. 4
 To reduce harmful effects on the non-target organisms,
encapsulation of the active ingredient with nano materials
should be attempted
 Nano polymers can allow sensitive ingredients to be physically
enveloped into a protective matrix in order to protect core
materials from adverse reactions due to factors like air or light.
Therefore, nanotechnology would provide green and efficient
alternatives for the management of plant diseases in agriculture
without harming the nature.

5. Hence there is increasing potential for
 Suitable techniques and sensors for precision agriculture, natural resource
management,
 Early detection of pathogens
 Detection of contaminants in food products by harmful pathogens.
 Smart delivery systems for agrochemicals like pesticides.
 Smart systems integration for food processing, packaging and other areas
like monitoring agricultural and food system security.
5

6. HISTORY
• The concepts that seeded
nanotechnology were first discussed in
1959 by renowned physicist Richard
Feynman in his talk There's Plenty of
Room at the Bottom, in which he
described the possibility of synthesis
via direct manipulation of atoms.
• The term "nano-technology" was first
used by Norio Taniguchi in 1974,
though it was not widely known.
6

7. • Inspired by Feynman's concepts, K. Eric
Drexler independently used the term
"nanotechnology" in his book in 1986
Engines of Creation: The Coming Era of
Nanotechnology.
• Also in 1986, Drexler co-founded The
Foresight Institute to help increase public
awareness and understanding of
nanotechnology concepts and implications.
7

8. 8
Definition
The word “nano” comes from the Greek for “dwarf”
 The design, characterization, production and application of structures, devices
and systems by controlling shape and size at the nanoscale
- British Standards Institution (BSI, 2005)
Technology: visualize, characterize, produce and manipulate matter of the size of
1 – 100 nm

9. Nanoscale
9
Nanometers
1 nanometre = one billionth (10ˉ⁹) of metre

10. What Is Unique About Nanotechnology?
 Small size (High surface to volume ratio), therefore requires self
assemblers
 Significantly higher hardness, breaking strength and toughness at low
temperatures and super plasticity at high temperatures
 Additional electronic states, high chemical selectivity of surface sites and
significantly increased surface energy
 New entry ways (high mobility in human body, plants and environment)
10

11. Properties of nano particles
10nm 50nm
Property
Below about 100 nm the rules
that govern the behaviour of the
elements of our known world
start to give way to the rules of
quantum mechanics, and
everything changes
Quantum effects
11

12. Present area of activities in the field of Nanotechnology in India
12
Park et al., 2006

13. ~ 2000 Years
Ago
Sulfide nanocrystals used by Greeks and Romans to dye hair
~ 1000 Years
Ago
Gold nanoparticles of different sizes used to produce different colors in
stained glass panes
1974 “Nanotechnology” - Taniguchi uses the term nanotechnology for the first
time
1981 International Business Machines (IBM) develops Scanning Tunneling
Microscope
1985 “Buckyball” - Scientists at Rice University and University of Sussex
discover C60
1986 • “Engines of Creation” - First book by K. Eric Drexler
1989 IBM logo made with individual atoms
1991 Carbon nanotube discovered by S. Iijima
1999 “Nanomedicine” – 1st nanomedicine book by R. Freitas
2000
2014
“National Nanotechnology Initiative” launched in USA
E. Betzig, W. E. Moerner and Stefan Hell got nobel prize for turning of
optical microscope into nanoscope 13
Timeline of Nanotechnology

14. 14
W. E. Moerner
Stanford University, US
Stefan Hell
Max Planck Institute of Biophysical Chemistry,
Germany
Eric Betzig
Howard Hughes Medical Institute, US
Contribution: Turning an optical microscope into nanoscope
Nobel Prize in Chemistry
2014

15. Dendrimers
Quantumdots
Nanosensors
FullerenesCarbon Nanotubes
Nano Chips
C60 Cadmium selinade
Sequence nanoscale
15
Tools of Nanotechnology
C60

16. 16
How do properties change at nano scale level ?
Increase in surface area to volume ratio
Quantum mechanical effects
Dominance of electromagnetic forces
Random (Brownian) motion
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17. 17
Increase in surface area to volume ratio
Example of Gold Nano particle:
 Sphere of radius 12.5 nm contains total approx. 480,000 atoms.
surface contains approx. 48,000 atoms.
So, approx. 10% atoms are on the surface.
 Sphere of radius 5 nm contains total approx. 32,000 atoms.
surface contains approx. 8000 atoms.
So, approx. 25% atoms are on the surface. 17

18. Optical properties
Colour change
“Bulk” gold looks yellow
12 nanometer gold
particles look red
Quantum mechanical effects
18

19. 3) Dominance of
Electromagnetic Forces
Gravitational force is a function of
mass and distance which is weak
between (low-mass) nanosized
particles.
Electromagnetic force is a function
of charge and distance is not
affected by mass.
4) Random motion:
Random motion is very high for nano scale particles.
19

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METHODS OF NANOPARTICLE PRODUCTION
1. Top down method
2. Bottom up method

21. (Royal Society and Royal Academy of Engineering, 2004)21
METHODS OF NANOPARTICLE PRODUCTION
Pyrolysis,
Sol-gel
process
Physio-chemical
process

22. 22(Mahendra et al., 2012)
Applications in agriculture
Mahendra et al., 2012

23. PLANT PATHOGENS IN BIOSYNTHESIS OF
NANOPARTICLES
The safe method of nanoparticle production is the
biological systems especially microorganisms
Microorganisms offer several advantages like
 Manoeuvrability for desired result using biotechnology
 Ease of handling especially fungi
 Cheapness of production
 Easy scaling up of the process
 High efficiency
 Simplicity
 Nature of greenchemistry or eco-friendliness.
Microorganisms have been regarded as ‘biofactories’ for
production of metallic nanoparticles.

24.  Fungi are relatively recent in their use in synthesis of
nanoparticles.
There has been a shift from bacteria to fungi to be used
as natural ‘nanofactories’ owing to easy downstream
processing, easy handling and their ability to secrete a
large amount of enzymes.
(Mandal et al., 2006)
However, fungi being eukaryotes are less amenable to
genetic manipulation compared to prokaryotes.
FUNGI IN SYNTHESIS…………

27. BACTERIA IN SYNTHESIS…………
Among microbes, prokaryotes have received the most
attention for biosynthesis of nanoparticles.
 Bacteria have been used to biosynthesize mostly
silver, gold, FeS, and magnetite nanoparticles and
quantum dots of cadmiumsulphide (CdS), zinc sulphide
(ZnS) and lead sulphide (PbS).

28. Bacterial based Nanoparticles

29. VIRUS IN SYNTHESIS…………
Plant virus especially spherical/icosahedral viruses represent
the examples of naturally occurring nanomaterials or
nanoparticles.
Plant viruses are made up of single or double stranded
RNA/DNA as genome which is encapsidated by a protein coat.
Their ability to infect, deliver nucleic acid genome to a specific
site in host cell, replicate, package nucleic acid and come out of
host cell precisely in an orderly manner have necessitated them
to be used in nanotechnology.

32. DETECTION AND OTHER USES OF
NANOTECHNOLOGY IN PLANT PATHOLOGY
Nanosized metals as diagnostic probes
Nanoparticles are different from their bulk
counterparts, which, when reduced to nanosize (1-100
nm) achieve certain properties which make them suitable
for development as diagnostic probes.
(Sharon et al., 2010).
Fluorescent silica nanoprobes have potential for rapid
diagnosis of plant diseases. Fluorescent silica
nanoprobes conjugated with the secondary antibody of
goat anti-rabbit IgG was used for detection of a
bacterial plant pathogen Xanthomonas axonopodis pv.
vesicatoria (bacterial spot on solanaceous plants).

33. Proteins like concanavalin A, fibronectin or
immunoglobulin G were surface grafted on micro-
fabricated uncoated as well as gold-coated silicon
cantilevers. These proteins were found to have different
affinities to bind to the molecular structures present on
fungal cell surface.
The biosensors detected the target fungi in the range
of 103 -106 cfu/ml
Nanoscale biosensor/ nanosensors

34. Quantum Dots are few nm in diameter, roughly spherical
(some QDs have rod like structures), fluorescent, crystalline
particles of semiconductors whose excitons are confined in all
the three spatial dimensions.
QDs have emerged as important tool for detection of a
specific biological marker in medical field with extreme
accuracy.
They have been used in cell labelling, cell tracking, in vivo
imaging and DNA detection.
Quantum Dots

35. Carbon nano material as a sensor
Nanofabrication
 Carbon nanomaterials have been developed to act as electrode
for electrochemical analysis . They have the potential to be
developed as electro chemical sensor to detect pesticide
residue in plants.
(Sharon and Sharon, 2008)
 Nanofabrication techniques have been used in creating
artificial plant parts such as stomata and xylem vessel which are
then used to study the infection process and behaviour of
pathogens inside host plant for example Uromyces
appendiculatus (fungus causing rust disease of bean),
Colletotrichum graminicola (fungus causing anthracnose in corn)
and Xylella fastidiosa (xylem limited bacterium causing Pierce’s
disease of grapevine).
(Meng et al., 2005).

36. Nanomaterials for management of plant diseases
Nanosized silver:
Silver (Ag) is known to have antimicrobial activity both in ionic or
nanoparticle forms.
 The powerful antimicrobial effect of silver especially in unicellular
microorganisms is believed to be brought about by enzyme inactivation.
Application of silver in management of plant diseases has been tested with
reference to two fungal pathogens of cereals viz. Bipolaris sorokiniana (spot
blotch of wheat) and Magnaporthe grisea (rice blast). In vitro assays indicated that
silver both in ionic and nanoparticle forms inhibited colony growth of both the
pathogens but M. grisea was comparatively more sensitive to silver application.
Jo et al. (2009
SEM image of
AgNP

37. Nanosized silica-silver:
Silica is well known to enhance stress resistance to plants including plant diseases
promotion of plant physiological activity and growth but it has no direct antimicrobial
effect.
On the other hand silver is known to have excellent antimicrobial effect . Thus a new
composition of nano silica-silver was developed to combat plant diseases.
In vitro test showed higher effectiveness of silica-silver nanoparticles towards fungi at the
dose of 10 ppm causing 100% inhibition of vegetative growth .
 Most of the bacteria tested were inhibited completely with only 100 ppm of silica-silver
nanoparticles. When nanosized silica-silver particles were applied in field condition to
control powdery mildew diseases of cucurbits, 100% control was achieved after 3 weeks.
These nanoparticles were found to be phytotoxic only at a very high dose of 3200 ppm
when tested in cucumber and plants.
Nanosized silica silver inhibited the growth and development of both Gram-positive and
Gram-negative bacteria.
(Park et al., 2006).

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These silica (SiO2 ) nanoparticles are with regularly
arranged pores which increase the surface area of the
nanoparticles.
Targeted delivery of chemicals and DNA can be made by
mesoporous silica nanoparticles .
 It offers the possibility of genetic manipulation of plants,
delivery of chemicals at targeted site in plant, improve
efficiency of used chemical and reduce the chemical residue
problem to the minimum.
Mesoporous silica nanoparticles:

39. Nano-copper was reported to be highly effective in
controlling bacterial diseases viz. bacterial blight of rice
(Xanthomonas oryzae pv.oryzae) and leaf spot of mung
(X. campestris pv. phaseoli)
(Gogoi et al., 2009).
Nano-copper:

40. Movement and behaviour of iron nanoparticles and their curative
affect is being studied more extensively in plants.
 Study to deliver the nanoparticles in the targeted site of a diseased
plant has been acheived.
Application of iron nanoparticles coated with carbon to pumpkin
plants for treating specific plant part that is infected showed positive
results.
Nano-iron

41. Carbon nanotubes have shown growth enhancing effect on tomato
when grown in soil containing carbon nanotubes.
 It is believed that carbon nanotubes entered the germinating tomato
seeds thus facilitating water uptake and plant growth.
(Khodakovsky et al., 2000).
Carbon nanotubes:

42. 42
Keuk-Jun Kim et al. (2009)
Journal of Biometals.
Antifungal activity and mode of action of silver
nanoparticles on Candida albicans

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Flow cytometric analysis for plasma membrane
potential.
Change in the plasma membrane dynamics of
fungal cells.
Intracellular glucose and trehalose release.
Transmission electron microscopic analysis.

44. 44
Flow cytometric analysis for plasma membrane potential

45. Change in the plasma membrane dynamics of fungal cells

46. The concentrations of trehalose and glucose from C.albicans by
nano-Ag and amphotericin B
Amounts of trehalose and glucose
concentrations (µg/mg
Intracellular glucose
and trehalose
Released glucose
and trehalose
Control 7.2 6.8
Nano-Ag 16.1 30.3
Amphotericin B 20.5 27.4

47. (a) control with no nano-Ag
(b) 170µg/ml of nano-Ag
(c) 400 µg/ml of nano-Ag
Transmission electron microscopic analysis

48. Nanomaterials in plant disease management
48

49. Nano formulations - Any formulation that
intentionally includes elements in the nm size range and/or
claim novel properties associated with these small size range.
NANO FORMULATIONS:
The aims of Nanoformulations are generally common to other
pesticide formulations and consist in:
1) Increasing the apparent solubility of poorly soluble active
ingredient
2) Releasing the active ingredient in a slow/targeted manner
and/or protecting the a.i. against premature degradation.
NANO FORMULATIONS
49

50. Nano formulations include:
Nano particles
Nanoemulsion
Nanosuspension
Nanoencapsulation
50

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NANO SUSPENSIONS
Submicron colloidal dispersions of pure active
compounds typically range from 50–500 nm.
 Improvement of efficacy due to higher surface area
 Higher solubility
 Induction of systemic activity due to smaller particle size
 Higher mobility
 Lower toxicity due to elimination of organic solvents
51

52. NANO PARTICLES
Improvement of efficacy- due to higher surface area.
Higher solubility
Systemic activity - due to small particle size.
Higher mobility & low toxicity due to elimination of organic
solvents.
52

53. The nanoparticles used in biopesticides controlled release
formulations (Fig-1) are
1)Nanospheres: Aggregate in which the active compound is
homogeneously distributed into the polymeric matrix.
2)Nanocapsules: Aggregate in which the active compound is
concentrated near the center core, lined by the polymeric
matrix.
3) Nanogels: Hydrophilic (generally cross-linked) polymers
which can absorb high volumes of water.
4)Micelles: Aggregate formed in aqueous solutions by
molecules containing hydrophilic and hydrophobic moieties.

54. 54

55. In vitro efficacy of nanoparticles of CoFe2O4 and NiFe2O4 against
mycelial growth of three different plant pathogenic fungi.
Mycelial growth inhibition (%)
Plant pathogenic fungi 100
ppm
200
ppm
300
ppm
400
ppm
500
ppm
CoFe2O4
Colletotrichum gleosporiodes 39.45 46.39 56.67 77.23 78.91
Dematophora necatrix 39.44 50.00 59.45 75.56 88.90
Fusarium oysporum 41.10 50.28 63.64 75.84 87.62
NiFe2O4
Colletotrichum gleosporiodes 43.25 54.06 61.94 78.06 81.39
Dematophora necatrix 43.61 52.78 61.39 78.89 93.33
Fusarium oysporum 58.06 60.28 68.61 83.33 89.45
Nanomaterial Fungicides: In Vitro and In Vivo Antimycotic Activity
of Cobalt and Nickel Nanoferrites on Phytopathogenic Fungi
Parul sharma et al. (2012)

56. 56
a) Induction of microcycle conidiation
in Colletotrichum gloeosporioides at (1)
500 ppm of nickel nanoparticles
compared to (2) untreated control. b)
Inhibitory effect of (1) nickel
nanoparticles at 500 ppm compared to
(2) untreated control on mycelia
growth of Dematophora necatrix. c)
Inhibitory effect of nickel nanoparticles
at 500 ppm (1) compared to untreated
control (2) on mycelia growth of
Fusarium oxysporum.

57. Evaluation of CoFe2O4 and NiFe2O4 nanoparticles
under pot culture conditions against Fusarium wilt
Ferrite
nanoparticles
Concentration
(ppm)
Disease
incience(%)
Disease
reduction(%)
CoFe2O4 100 ppm 90.47 9.54
200 ppm 76.18 23.83
300 ppm 38.09 61.92
400 ppm 28.57 71.43
500 ppm 9.52 90.49
NiFe2O4
100 ppm 80.95 19.07
200 ppm 57.12 42.88
300 ppm 23.80 76.21
400 ppm 9.52 90.49
500 ppm 0.00 100.00
Control --- 100.00 0.00
Parul sharma et al. (2012)

58. Effect of a) CoFe2O4 and b) NiFe2O4 ferrite
nanoparticles against Fusarium wilt of capsicum annum
under pot culture conditions compared to c) control.

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Days after
infection
Control Chitosan Chitosan NP
Treated
D.I.(%) Lesion
area(mm2)
D.I.(%) Lesion
area(mm2)
D.I.(%) Lesion
area(mm2)
4 20 425 11 245 - -
6 50 1305 16 365 - -
10 100 2175 33 735 - -
Effect of chitosan nanoparticle on suppression
of leaf blast disease on detached rice leaf
Preparation of Chitosan nanoparticles and its effect on
detached rice leaves infected with Pyricularia grisea
Appu Manikandan et al. (2016)

60. Suppression of blast disease on detached leaves of O. sativa (a) Control (10day)
(b) Chitosan (10 day) (c) Chitosan nanoparticle (10 day).

61. a) Ag NPs in solution, b) and c) AgNPs embedded in chitosan matrix and
their Size
An in vitro study of the antifungal activity of
silver/chitosan nanoformulations against important
seed borne pathogens.
Pawan Kaur et al. (2012)

62. Agar well diffusion method
Sample Zone of Inhibition (mm)
A.flavus A.alternata R.solani
Control 0 ± 0 0 ± 0 0 ± 0
Chitosan 10.66 ± 0.76 10 ± 1.73 9.8 ± 1.76
AgNP 10 ± 1 8.3 ± 0.28 8.16 ± 0.76
Ag-ch 19.66 ± 0.28 18.33 ± 0.29 19.60 ± 0.76
Amphotericine-B 19.5 ± 0.5 19.33 ± 1.52 19.66 ± 1.04
Pawan Kaur et al. (2012)

63. Mycelium growth Inhibition
Zone of inhibition of AgNPs (ii, iv, vi) and
Ag/Ch NFs (i, iii, v) against a) A. alternata,
b) R. solani and c) A. flavus.

64. Effects of agar well double diffusion assay
of EON oil treatment at 1 and 2 % on
mycelium growth of FOV after 8 days
incubation (a:
control, b: EON1 %, c: EON 2 %)
Eugenol oil nanoemulsion: antifungal activity against
Fusarium oxysporum f. sp. vasinfectum and
phytotoxicity on cottonseeds
Kamel et al. (2015)

65. Fungal morphology study
a)1 % EON b) untreated macro- and micro-conidiospores c) treated macro- and
micro-conidiospores with 2 % EON

66. SDS PAGE analysis
SDS–PAGE of FOV mats treated with nanoeugenol oil (EON). Lane 1, 3, and 5: (1 % EON).
Lane 2, 4, and 6 (2 % EON). Lane M contains protein marker (66 kDa, Bovine Serum
Albumin), (45 kDa Carbonic Anhydrase), (22 kDa Trypsin Inhibitor). Protein bands in black
line (three stimulated band) and bands in red line (seven reduced band)

67. Treatment (%) DS (%) PEDC (%)
Control 74.00 ± 1.15 0.00 ± 0.00
Bulk Saponin 0.01 71.33 ± 1.76 3.62 ± 0.96
Bulk thymol 0.01 68.00 ± 4.00 13.20 ± 1.37
Thymol nano emulsion
0.01 59.33 ± 4.66 23.06 ± 4.04
0.02 54.00 ± 2.30 27.03 ± 2.72
0.03 33.33 ± 1.76 54.95 ± 2.25
0.04 29.33 ± 0.66 60.34 ± 1.10
0.05 16.66 ± 2.66 77.36 ± 3.96
0.06 3.33 ± 0.66 95.49 ± 0.90
DS= Disease severity
PEDC= Percent efficacy of disease control
Thymol nanoemulsion exhibits potential antibacterial
activity against bacterial pustule disease and growth
promotory efect on soybean.
Sarita et al. (2018)

68. Symptoms of bacterial pustule disease on soybean plants in pot experiments
(a) lesions expanded and merged in control
(b) small yellow to brown lesions in soybean leaf at 0.06%,v/v thymol nanoemulsion.

69. Effect of thymol nanoemulsion on plant growth of soybean. Concentrations of
thymol nanoemulsions ranging from 0.02 to 0.06% v/v, exhibited visual differences
in plant growth.

70. Mode of action of fungicide
Synthesis and characterization of Nano-tricyclazole
-A systemic fungicide.
Venugopala et al. (2016)

71. Performance of Nano-Tricyclazole pesticide
(A) full growth of fungi
(B) after applying nano- pesticide)
Venugopala et al. (2016)

72. • BASF – has applied for patent for “ Nanoparticles
comprising a crop protection agent ”, involves active
ingredient of 10 – 150 nm.
( Chinnamuthu and Boopathi, 2009)
• Bayer Crop Science - has applied for a patent of
agrochemicals in form of emulsion, active ingredient of 10
– 400 nm. (Bayer Crop Science’s US Patent Application
no. 20040132621, “Microemulsion Concentrates.”)
• Syngenta – already sells products containing nanoscale
droplets.
• ‘Primo MAXX-plant growth regulator’
• ‘ Banner MAXX Fungicide’
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Agrochemicals via Encapsulation

73. Potential Risks of Nanotechnology
 Health issues
Nanoparticles could be inhaled, swallowed, absorbed through
skin
They trigger inflammation and weaken the immune system,
and interfere with regulatory mechanisms of enzymes and
proteins
 Environmental issues
Nanoparticles could accumulate in soil, water and plants
 New risk assessment methods are needed
National and international agencies are beginning to study
the risk.
 Results will lead to new regulations.
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74. 74
Conclusion
Nanotechnology has the potential to revolutionize the existing
technologies used in various sectors including agriculture.
Better management and conservation of inputs
Nanotechnology in agriculture – nascent stage.
Promising results - in use of nano materials for delivery of
pesticides & fertilizers.
Applications are near commercialization
Further research is needed to evaluate the risk assessment

75. Conclusion
• Nanotechnology is capable of being used in agricultural
products that protect plants and monitor plant growth and
detect diseases
• Scientists are still seeking new applications of
nanotechnology in agriculture and the food industry
• The agricultural sector and the food industry will indeed see
tremendous changes for the better in the coming years
75

76. Future trends
• The question that whether the coming age of Nanotechnology is the Next
technological revolution ?
• There is great optimism among scientists, politicians and policy makers
who anticipate significant job creation.
• Opportunities for developing new materials and methods that will enhance
our ability to develop faster, more reliable and more sensitive analytical
systems.
• Overall the scenario presents us with the view that nanotechnology is ever
growing in the future
76

77. “…. Nanotechnology is knocking at our
doors .
……We have to launch vertical
missions under an umbrella
organization with the public-private
investment in at least ten nanotechnology
projects in Water, Energy, Agriculture,
Healthcare, Space, Defense.”
Our future lies in
nanotechnology
Dr. A. P. J. Abdul Kalam (2005),
Former President of India 77

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