nanotechnology and its applications in crop protection

1. SHEETAL MEHLA
2018BS13D
Molecular Biology, Biotecnnology And Bioinformatics
CCS Haryana Agricultural University, Hisar

2. Nanotechnology
• Nanotechnology is the design, characterization , production and
application of structures, devices and systems by manipulating
matter on atomic scale.
• Nanotechnology is the collaboration of physics, chemistry, biology,
computer and material sciences integrating with engineering
entering the nanoscale.
• NNI (govt.) says: “Nanotechnology involves ... creating and using
structures, devices and systems that have novel properties and
functions because of their small and/or intermediate size”

3. Terminology
• From the Greek nanos meaning "dwarf”, this prefix is used in
the metric system to mean 10⁻⁹ or 1/1,000,000,000.
• Technology is the making, usage, and knowledge of tools,
machines and techniques in order to solve a problem or
perform a specific function.
• Nanoparticles are the set of particles or sustances where
atleast one dimention is less than approx. 100nm.

4. Premodern Nanotechnology
• 4th Century: The Lycurgus Cup (Rome) is an example of dichroic glass; colloidal gold and
silver in the glass allow it to look opaque green when lit from outside but translucent
red when light shines through the inside.
• 6th-15th Centuries: Vibrant stained glass windows in European cathedrals owed their
rich colors to nanoparticles of gold chloride and other metal oxides.
• 13th-18th Centuries: “Damascus” saber blades contained carbon nanotubes and
cementite nanowires—an ultrahigh-carbon steel formulation that gave them strength.
• 1857: Michael Faraday discovered colloidal “ruby” gold, demonstrating that
nanostructured gold under certain lighting conditions produces different-colored
solutions.
Official website of the United States National Nanotechnology Initiative

6. Nanotechnology
• 1959: Richard Feynman of the California Institute of Technology gave what is considered
to be the first lecture on technology and engineering at the atomic scale, "
There's Plenty of Room at the Bottom" at an American Physical Society meeting at
Caltech.
• 1974: Tokyo Science University Professor Norio Taniguchi coined the term
nanotechnology to describe precision machining of materials to within atomic-scale
dimensional tolerances.
• 1981: Gerd Binnig and Heinrich Rohrer at IBM’s Zurich lab invented the scanning
tunneling microscope.
• 1985: Rice University researchers Harold Kroto, Sean O’Brien, Robert Curl, and Richard
Smalley discovered the Buckminsterfullerene (C60).
• 1989: Don Eigler and Erhard
Schweizer at IBM's Almaden
Research Center manipulated
35 individual xenon atoms
to spell out the IBM logo.

7. • 1991: Sumio Iijima of NEC is credited with discovering the carbon nanotube (CNT).
• 1999–early 2000’s: Consumer products making use of nanotechnology began
appearing in the marketplace, including lightweight nanotechnology-enabled
automobile bumpers that resist denting and scratching,
• golf balls that fly straighter,
• tennis rackets that are stiffer (therefore, the ball rebounds faster),
• nano-silver antibacterial socks,
• clear sunscreens,
• wrinkle- and stain-resistant clothing,
• deep-penetrating therapeutic cosmetics, scratch-resistant glass coatings,

8. Classification of nanostructured
materials• Zero dimention structures (confined in 3 dimentions):
nanoparticles, nanoshells, nanocapsules, fullerens, nanorings,
nanoporous silicon.
• One dimention structures (confined in 2 dimentions): nanorods,
nanotubes, nanowires, quantum wires.
• Two dimention structures (confined in 1 dimention): discs,
platelets, ultrathin films, quantum wells.
• Three dimention structures : These materials are thus
characterized by having three arbitrarily dimensions above 100 nm.

9. STRUCTURE
S
DEVICES
SYSTEMS
NANOTECHNOLOGY

10. Approaches to build something
so small…
• Top down
• Bottom up

14. Aspects of Nanotechnology
• Nanotechnology has two major aspects, the first aspect is
synthesis of nano-size materials and second, the use or
application of nano materials for the desired objectives.
• The synthesis deals with the conversion of the matter of
macro size into the particles of nano size, 1-100 nm. The
synthesis of nanoparticles is not a simple process and requires
specific skill and facilities depending on the method of
synthesis.
• After the synthesis, characterisation of nanoparticles is
another important step which ascertains attainment of the
required particle size of the material and their relative
uniformity.

15. Unique physical properties :
• Smaller size, larger surface area
• Increase in surface area to volume ratio
• Nano sized particles can even pass through the cell wall in plants and animals.
• Nanotechnologists use this process to deliver at cellular level which is more
effective then the conventional method. Dominance of Electromagnetic
forces Encapsulated control for smart delivery system
• Slow release
• Quick release
• Specific release
• Moisture release
• Heat release
• pH release
• Ultra sound
• Magnetic release

16. Characterization of Nanomaterials
• Characterization refers to the study of materials features such
as its composition, structure, size and various properties like
physical, chemical and magnetic characters etc
1.Particle size analysis : Size of nanoparticles
2.UV- Visible spectroscopy analysis : Quantum effects
3.The zeta potential Measurement : Net charge of nanoparticles
4.Microscopic analysis _ surface morphology
• Atomic Force Microscopy(AFM)
• Scanning Electron Microscopy( SEM)
• Transmission Electron Microscopy(TEM)

17. Things behave differently in
nano-world
• Carbon in the form of Graphite (i.e. pencil
lead) is soft, at the nano-scale, can be
stronger than steel and is six times lighter.
• Nano-scale copper is a highly elastic metal at
room temperature, stretching to 50 times its
original length without breaking.
• Shiny orange yellow Gold changes its colour to
brownish black on reducing the size.

18. Tools of nanotechnology

19. Applications of Nanotechnology
in Agriculture
• Analysis of gene expression and Regulation
• Soil management
• Plant disease diagnostics
• Efficient pesticides and fertilizers
• Water management
• Bioprocessing
• Post Harvest Technology
• Monitoring the identity and quality of agricultural produce
• Precision agriculture

20. Nanotechnology In Crop
Protection
• Potential applications of nanotechnology in
crop protection include controlled release of
encapsulated pesticide, fertilizer and other
agrochemicals in protection against pests and
pathogens, early detection of plant disease
and pollutants including pesticide residues by
using nanosensors and generation of
nanotechnology mediated insect resistant
plants.

23. Nanopesticides
“Nano-scale either active ingredients or inert
ingredients with a particle size of 100 nm or
less”
Formulation of a pesticide
• Nano emulsion
• Nano suspension
• Nano encapsulation
• Nano particles

25. Nanoemulsions
• Consist of lipid or polymeric vesicles or
particles
• Size 20-200 nm
• Larger surface area, slower release rate
• Non sedimentation or creaming

26. Nano suspensions
• Submicron colloidal dispersions of pure active
compounds typically range from 50–500 nm
• Solvent-diffusion methods
• Improvement of efficacy due to higher surface area
• Higher solubility, higher mobility
• Induction of systemic activity due to smaller particle
size

27. Nano encapsulation
• Encapsulation -packaging the nanoscale active
ingredient within a kind of tiny 'envelope' or
'shell”.

29. Few strategy
• Slow release – the capsule releases over a longer period of time
• Quick-release – breaks upon contact with a surface (e.g. when pesticide
hits a leaf )
• Moisture release – releases contents in the presence of water (e.g., in soil)
• Heat-release – releases when the environment warms above a certain
temperature
• pH release –Releases only in specific pH (e.g., in the stomach or inside a
cell)
• Ultrasound release – ruptured by an external ultrasound Frequency
• DNANano capsule – smuggles a short strand of foreign DNAinto a living
cell

30. • Agricultural chemical companies such as Monsanto, Syngenta and BASF;
have ventured in developing nanoparticle pesticides.
• The world's leading chemical company already sells a number of pesticide
emulsions containing nanoparticles.
• The positive side of nanoparticle pesticides is that far less need to be
applied and reducing cost and environmental damage.
• Syngenta have obtained a patent for ‘GUTBUSTER’ microcapsule will break
open in alkaline environments, including the stomach of certain insects
(ETC Group, 2004). Syngenta’s US Patent No. 6,544,540
• To date, none of these agrochemicals are currently labeled
as containing nano particles.

31. Metallic Nanoparticles
• Several metallic nanoparticles such as silver and copper have the property of
antimicrobial activity.
• Metallic nanoparticles possess unique chemical and physical properties, small size,
huge surface to volume ratio, structural stability and strong affinity to their targets
(Kumar et al., 2010).
• Polymer-based copper nano-compounds have been investigated with the antifungal
activity against plant infecting fungi (Cioffi et al., 2004).
• Efficacy of silica-silver nanoparticles was studied by Park et al., (2006) against the
plant infecting fungi Rhizoctonia solani, Pythium ultimum, Botrytis cinerea,
Magnaporthe grisea and Colletotrichum gloeosporioides for their control. In recent
times, nano-herbicides, nano-fungicides and nano-pesticides are in use in the
agriculture.
• Sharma, J., Singh, V. K., Kumar, A., Shankarayan, R., & Mallubhotla, S. (2018). Role of
Silver Nanoparticles in Treatment of Plant Diseases. In Microbial Biotechnology (pp.
435-454). Springer, Singapore.
• Singh, A., Singh, N. B., Afzal, S., Singh, T., & Hussain, I. (2018). Zinc oxide
nanoparticles: a review of their biological synthesis, antimicrobial activity, uptake,
translocation and biotransformation in plants. Journal of Materials Science, 53(1),
185-201.

32. Nanoparticles in post-harvest
disease management
• Nanotechnology has been effectively applied in agricultural and
horticultural products by increasing shelf life, controlling growth of
microorganisms by nanofilms and coatings, controlling influence of gases
and the harmful rays (UV), using Nano biosensors for detection of quality
and spoilage (Yadollahi et al., 2009). Nanotechnology can be applied in
postharvest operations such as drying, storage and preservation of
agricultural products. Chitosan, a deacetylated derivative of chitin, is
found to be very effective in reducing postharvest decay of fruit and
vegetables (Liu et al., 2007).
• Shi et al. (2013) studied the novel chitosan/nanosilica hybrid film and its
effect on preservation quality of longan fruits under ambient
temperature. Coating extended shelf life, reduced browning index,
retarded weight loss and inhibited the increase of malondialdehyde
amount and polyphenoloxidase activity in fresh longan fruit.

33. DNA Nanostructures Coordinate
Gene Silencing
• Plant bioengineering will be necessary to sustain plant biology
and agriculture, where the delivery of biomolecules such as DNA,
RNA, or proteins to plant cells is at the crux of plant
biotechnology. DNA nanostructures can passively internalize into
plant cells and deliver small interfering RNA (siRNA) to mature
plant tissues.
• DNA origami refers to an assembly technique that folds single-
stranded DNA template molecules into target structures. This is
done by annealing templates with hundreds of short ‘staple’ DNA
strands.

34. Duan, C. G., Wang, C. H., & Guo, H. S. (2012). Application of RNA silencing to plant disease resistance. Silence, 3(1),

35. e-Nose
• Operates like human nose
• Identify different types of odors and their
concentrations
• Electronic nose sniffs out plant pests
• Just by sniffing the air, an electronic nose can
tell when cucumber and capsicum pepper
plants are damaged – and even diagnose the
problem.

36. Risks Of Nanopesticides For The
Environment
• Nanomaterials in nanopesticides are deliberately released into the
environment due to the nature of their application. Therefore, benefits and
risks to humans and the environment must be considered carefully.
• The EU Biocidal Products Regulation states that every manufacturer must
prove that a pesticide is safe before admitting a product. Since 2013,
nanomaterials have also been explicitly included in the regulation.
• Despite the many ideas on how nanomaterials can be used to improve
conventional pesticides, there are currently no nanopesticide products on the
market. On the one hand, the development and approval of a pesticide is very
tedious and expensive, and, it is currently unclear how great the savings by
using a nanopesticide compared to a conventional pesticide really is.
• The current understanding is not yet sufficient to reliably assess the benefits
and risks of nanopesticides. Further studies on the fate and risks of
nanopesticides in the environment are thus needed.

37. 1. Efficient Management of Fruit Pests by
Pheromone Nanogels.
2. Clay nanosheets for topical delivery of
RNAi for sustained protection against
plant viruses.

38. Case study

39. • (a) Bactrocera dorsalis (B. dorsalis) (b)infection of the fruit guava by B.dorsalis; (c,d) eggs of
B.dorsalis laid below the skin of the host fruit and the attacked fruit shows the signs of the
ovipositional damage in the form of minute depressions; (e) the affected fruits soften at the site of
infestation which then rot and drop down prematurely and (f) the maggots feed on the pulp of the
fruits.

40. Materials
• Commercial ME was obtained from Sisco Research Laboratories Pvt.Ltd., India and was
used without further purification.
• (a) Molecular structures of the gelator, 1 and methyl eugenol (ME).
• (b) colorless ME

41. Immobilization of methyl eugenol in nanogel
Freeze dried
SEM and TEM
Thermal stability
Heat- cool
cycles
Exposure
to open
orchard
GC- MS
analysis
Slow release of
ME from the
nanogels
Enhanced
shelf- life
Field
trials for
nanogels
Statistical
analysis
AgeRes
software
characterization

42. • TEM and SEM images of the nanogel showing
the existence of nano-fibrillar networks.

43. Thermal stability
The melting temperature of the nanogels, i.e. gel-to-sol transition (Tgel) increased
progressively with increasing concentration of gelater.
• a ‘sol’ after warming 12 mg/mL of 1 in ME at 70˚C; inset shows glass vials holding
0.2 mL of the ‘sol’ and
• a ‘gel’ at room temperature (25˚C) after cooling the previous solution without
any external perturbation; inset shows the inverted vials holding the gel suitable
for the field study.
• At the concentration of 12 mg/mL of 1 in ME the Tgel reached ,63˚C which was
well above the ambient temperature even during peak summer in India. Thus the
thermal stability of the sample should be adequate for the agricultural field
trials even in hot climates around different geographical regions of the world.

44. Slow release of ME from the nanogels
• The release pattern of the volatile pheromone in terms of its relative rates of
evaporation at a particular temperature was determined both for the liquid ME alone
and ME immobilized in nanogel (12 mg/mL) under identical conditions.

45. Enhanced shelf life
The results showed that the fruit flies were attracted specifically to the plate A and to the plate
C throughout the period and not to the plate B which held the gelator1 in toluene (a non-
pheromone solvent). This indicates that the pheromone is biologically active in the nanogel .
The same plates were preserved at room temperature (30˚C) and were exposed again in the same guava
orchard after 21 days. Interestingly this time the fruit flies were attracted to the plate A only which
contained the nanogel and not to the plate B or C for the entire time period of observation. This indicates
that the nanogel laced with ME still retained the pheromone for sustained release and has also retained
significant pest attractant property due to the higher shelf-life of ME.

46. Field Trials
• Sampling process:
• 25 cm long plastic bottle of ,7 cm diameter with a closed screw-cap. This is kept hanging
from the branch of a tree with the help of a hook in an upside down orientation. Two
circular holes(diam.,4.5 cm) have been made on the bottle in such a way that they face up
and down opposite to each other. Two holes are useful for the facile passage of fruit flies
that are attracted to pheromone. Water is introduced into the bottle through the lower
hole and maintained at nearly the same level. The pheromone nanogel sample is kept in a
hanging vial and its opening faces downwards just a few cm above the water level. With
this arrangement, even when it rains, the excess of water gets automatically drained away
from the lower hole allowing it to maintain the water level. As the opening of the nanogel
vial is oriented downwards, rain water cannot enter into it. Also at the end of each day,
the contents from the plastic bottle may be released by opening the screw-cap. This
allows collection of dead flies after the day-long exposure in the orchard. The same bottle
may be reused following the above procedure throughout the season.

47. • The number of catches for the control bottle containing ME alone also attracted pests initially.
However, this showed a progressive decrease in comparison to the bottle containing nanogel and
from the 8th day onward no fly was attracted to this bottle. This indicates that the effectiveness of
ME alone towards the B.dorsalis without the gelator 1 is at best limited to one week in the real
field environment

48. Conclusion
• The present invention provides a simple and effective route to as low delivery
of pheromones from a nanogel. This does not require any use of
environmentally harmful and toxic chemicals such as, cross linkers,
antioxidants, antimicrobials, pesticides, volatile organics etc. While other
existing methods pose risk of human contacts where ME is added to water in
the bait, the present method avoids any direct contact with the fruit crop and
the agricultural workers with pheromone. This keeps the crop absolutely clean
from the chemical contaminations unlike the prevalent practice of spraying
toxic pesticides on the orchard.
• The nanogel of pheromone ME described herein is insoluble in water, which
makes it superior to hydrogels and microcapsules. The nanogel does not
significantly swell and shrink in presence of water, confirming that the humidity
plays no effect whatsoever on this material.
• The flexibility in using the nanogel in any season at any temperature (60˚C) are
feasible due to the oxidative, photochemical and thermal stability of the
nanogel.

50. Preparation of LDH
nanosheets
ds RNA construct
Degradation of LDH and
release of dsRNA
Bioclay
Spray
Local lesion assay Stability of dsRNA
loaded into LDH
Bioclay formation
Uptake of dsRNA into
plant cells and induction
of RNAi
Confocal
microscopy
TEM analysisXRD
Leaf retention Rnase degradation
CHARACTERIZATION
Non aqueous
precipitation
Heat treatment
Purification
Dispersion in
water

51. Characterization of LDH nanosheets and
dsRNA loading into LDH
• A. TEM image, indicates that LDH has a
hexagonal nanosheet morphology, with
a lateral dimension in the range of 20–
80 nm.
• B. XRD pattern of LDH nanosheets,
shows that LDH possessed the typical
lamellar structure, as shown by (003)
and (006) diffraction peaks in the thin
film mode.

52. Loading dsRNA on LDH
• Loading of dsRNA at the dsRNA–LDH
mass ratios of 1:1, 1:2, 1:3, 1:4, 1:5
and 1:10, corresponding to lanes 3, 4,
5, 6, 7 and 8, respectively. M=1 kb+
ladder, dsRNA only (lane 1) and LDH
only (lane 2). The dsRNA loading
includes dsRNA from the control
reaction in the MEGAscript RNAi kit,
Life Technologies (control-dsRNA)
(500 bp), in vitro PMMoVIR54-dsRNA
(977 bp) and E. coli HT115-expressed
CMV2b-dsRNA. LDH-bound dsRNA
does not migrate and can be seen as
fluorescence in the well (indicated by
arrows). Complete loading was
achieved at a dsRNA–LDH mass ratio
of 1:4 (lane 6).

53. Degradation of LDH and release of
dsRNA
• Control 5% CO2 treated
• Enhanced release of CMV2b-dsRNA from LDH in suspension stored under 5% CO2
compared with normal atmospheric conditions. Following a one-week incubation,
residual CMV2b-dsRNA–LDH was pelleted and the amount of dsRNA in the pellet was
assessed by northern blot analysis. Lane 1, residual CMV2b-dsRNA bound to LDH
after incubation under natural atmospheric CO2 levels; lanes 2–5, residual CMV2b-
dsRNA bound to LDH after incubation under 5% CO2. The lower panel shows loading
profile of RNA.

54. Leaf retention to check stability of nanoclay.
• a–d, Confocal microscopy of Arabidopsis leaves 24h post treatment before and after washing: shown
are images for Cy3 only (a); Cy3-LDH (b); CMV2b-dsRNA–Cy3 (c); and CMV2b-dsRNA–Cy3-LDH (d).
Bright-field (BF) image (column 1), Cy3 florescence image (column 2) and merged image of the two
(column 3) are shown.

55. RNase degradation and shelf life
• E. Treatment of control dsRNA and control-dsRNA–LDH with RNase A. The dsRNA of the treated and untreated
dsRNA–LDH samples was released from LDH prior to gel electrophoresis. M=1 kb+ ladder.
• F. CMV2b-dsRNA and CMV2b-dsRNA–LDH sprayed on N. tabacum leaves on day 0 and sampled at various time
points post spray. Northern blot analysis of total RNA resolved with 1% agarose gel and probed with DIG CMV2b-
dsRNA oligonucleotide probe shows that CMV2b-dsRNA is almost completely degraded at 20 days whereas
CMV2b-dsRNA–LDH can still be detected at 30 days. Lane (+) is in vitro-transcribed CMV2b-dsRNA as control. Lane
(–) is RNA extracted from N. tabacum leaves without any treatment.
Increased shelf lifeNo effect of Rnase
degradation

56. •
• BioClay (dsRNA–LDH) spray provides protection against viruses in local lesion assays. a, Local lesions caused by CMV inoculation on
cowpea. Plants at the two-leaf stage were sprayed with LDH, CMV2b-dsRNA and CMV2b-BioClay on day 0 (n=8–16 leaves per
treatment group). Plants were mechanically inoculated with CMV at 1 or 5 days post treatment. Lesions were counted 10 days
pvc. b, Local lesions caused by PMMoV inoculation on N. tabacum cv. Xanthi. Plants were sprayed with either water, LDH,
PMMoVIR54-dsRNA or PMMoVIR54-BioClay on day 0 (n=10–25 leaves per treatment group). Plants were mechanically inoculated
with PMMoV at either 5 or 20 days post treatment and necrotic lesions were counted 10 days post viral challenge. c,d, Images
showing extent of necrotic lesions on N. tabacum cv. Xanthi leaves challenged with PMMoV 5 days post spray treatment (c) and 20
days post spray treatment (d). *P<0.05,**P<0.01and***P<0.001 are significant using the Kruskal–Wallis test with post-hoc Nemenyi
test for multiple comparisons between samples compared with LDH. Data represent mean±s.e.m.

57. Discussion
• The effectiveness of the BioClay platform can be attributed to the elegant design and
functionality of the LDH nanosheets to load large dsRNA , strongly adhere to the leaf
surface even after vigorous rinsing and enhance the stability of dsRNA for a longer
period under environmental conditions. The sustained release of dsRNA is facilitated
through the formation of carbonic acid on the leaf surface from atmospheric CO2 and
humidity, which help degrade the LDH nanosheets using the following reactions.
• The results of the GUS reporter system show that dsRNA is able to enter when applied
to the whole plant, and silence transgene expression by the RNAi pathway. We could
also detect CMV2b-specific RNAs only in new unsprayed leaves in plants that were
sprayed with CMV2b-BioClay.
• The versatilityof the dsRNA–LDH complex or BioClay concerning crop protection has
been proven in both local lesions (Xanthi and cowpea) and systemic hosts (tobacco)
using PMMoV and CMV. Delivering the dsRNA as the BioClay spray instead of naked
dsRNA has extended the virus protection window from 5–7 days15,16 to at least 20
days.
• BioClay has the capacity to change the way we protect plants with the potential of
reducing pesticide usage and overcoming the obstacles faced by genetically modified
crops.

59. Inaugurating the Global Forum On Agricultural Research(GFAR)
Triennial conference –New Delhi 2006 ,President Dr. A.P.J
ABDUL KALAM Focused on the Nanotechnology as the new
technology that must be applied in Agriculture and Food
industry

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• Official website of the United States National Nanotechnology Initiative

61. THANK YOU 
"The Next Big Thing Is Really Small”