Genetic engineering and new technologies have made progress in integrated pest management (IPM) programs but also face limitations. Technologies like inserting insect-resistant genes from Bacillus thuringiensis into plants or using genetic engineering to optimize the speed at which pathogens kill pests have shown promise. However, producing recombinant pathogens faster-killing hosts results in fewer pathogen bodies produced. Additionally, viruses must be ingested to work and can be deactivated by sunlight or rain. Fungal pathogens are intolerant of low humidity or high heat. While biotechnology has improved crops through herbicide and insect resistance, developing transgenic methods that are economical at a large scale remains a challenge.

1. Genetic engineering & new technologies : their
progress & limitations in IPM programme
Thimmaiah M
1St Ph.D
Department of Agronomy
UAHS, Shivamogga
1

2. Sequence of Presentation
 Introduction
 Genetic engineering technologies in IPM
 Limitations
 Biotechnological progress in IPM
 Use of GPS & GIS Tools in IPM
 Conclusion
 Future Prospects
2

3. Introduction
 Integrated Pest Management (IPM) is a system approach that
combines a wide array of crop production and protection
practices to minimize the economic losses caused by the pests
(insect pests, diseases, nematodes, weeds, rodents, birds etc.).
It emphasizes on careful monitoring of pests and conservation
of their natural enemies.
 Insect pathogens have demonstrated to be environmentally
safe and economical alternative for the control of wide range of
arthropod pests.
 But at present, less than 1% of the insecticides used
worldwide for pest control are based on insect pathogens.
 Those used most widely are different subspecies of the
bacterium, Bacillus thuringiensis (Bt), which constitute
approximately 80% of the pathogens used as insecticides.
3

4. IPM strategies / tools
 Mechanical control
 Cultural control
 Chemical control
 Regulatory control
 Biological control
 Host resistance
 Behavioral control
4

5. IPM
Biological
Host Plant
Resistance
Regulatory
Mechanical
Chemical
Cultural
GIS and GPS
Genetic
engineering
5

7. Genetic engineering: Changes in the genetic constitution
of cells by introduction or elimination of specific genes
using molecular biology techniques.
Herbicide tolerant crop
Virus resistant crop
Insect resistant crop
Transgenic micro-organism
7

8. Incorporation of Insect resistance in plants
 Genes of plant origin: Cloning of the Mi-1 gene from wild
tomato (Lycopersicon peruvianum) has given an opportunity
to control root-knot nematode and potato peach aphid
 Crystalline protein (Cry) Bacillus thuringiensis (Bt) produce
Crystalline protein during sporulation. These are highly
insecticidal at low concentration.
 Vegetative insecticidal proteins (Vip) These proteins are
produced during vegetative growth of cells and are secreted
into the growth medium.
8

10. Advent of genetic Engineering
• The virulence and pathogenicity of pathogen is determined
by the microbial genome as a result of coordinated
expression of a concert of genes.
• The acquisition of these domains or pathogenicity islands,
may be sufficient to develop a transgenic virulent
pathogen.
• The advent of recombinant DNA techniques—in essence,
genetic engineering—has provided a myriad of
opportunities to enhance the efficacy and thus cost-
effectiveness of the insect pathogens as their control
agents.
10

11. 1. Isolating a gene to be inserted
2. Inserting the gene in a Vector(Agent used to carry foreign gene)
3. Inserting Vector into the host.
4. Multiplication of host cells by cloning.
5. Extraction of desired product.
11

12. Baculoviruses
• These are arthropod
specific viruses that
infect species.
• The two genera
– Nucleopolyhedrovirus
(NPV)( Multiple virions
occluded in polyhedra
– Granulovirus (GV:
single virions occluded
in granules) .
12

13. Genetic engineering strategies:
1. Genetic Engineering to Optimize Speed of
Kill
2. Genetic Engineering for Increased Virulence
and modify host range.
13

14. 1.Genetic Engineering to Optimize
Speed of Kill
A. Gene Deletion
B. Gene Insertion
14

15. A.Gene Deletion
• EGT gene (Auxillary gene)
• Ecdysteroid UDP-glucosyl transferase (EGT),
renders the ecdysteroids inactive, blocks
molting of the host insect, thereby prolonging
the actively feeding larval stage.
15

16. Deletion of egt from the Autographa californica
multiple nucleopolyhedrovirus (AcMNPV) genome
resulted in more rapid death and an approximately
40% reduction in feeding damage caused by infected
larvae of Trichoplusia ni and Spodoptera frugiperda
compared to those infected with wild type AcMNPV.
(O,Reilly and Miller, 1991)
16

17. (Han et al., 2015) 17

18. • Deletion of the gene encoding the polyhedral
envelope protein that surrounds the AcMNPV
resulted in a 6-fold increase in infectivity
against first instar Trichoplusia ni compared to
that of wild type virus.
18

19. B.Gene Insertion
 Insertion of a gene encoding a toxin, hormone or enzyme into
the baculovirus genome.
 Several recombinant baculoviruses have been constructed for
over expression of the host insect’s own hormones or enzymes
such as diuretic hormone, eclosion hormone,
prothoracicotrophic hormone and juvenile hormone esterase.
 A wide range of genes encoding insect-specific toxins isolated
from various venomous creatures such as scorpions, spiders,
parasitic wasps and sea anemones have been inserted into
baculovirus genomes.
19

20. • Insertion of Diuretic hormone gene from
Manduca sexta resulted in 20% increase in the
insecticidal activity of a recombinant Bombyx
mori NPV. (Maeda, 1989)
• The insect selective toxin(LqhIT2) from yellow
Israeli scorpion Leiurus quinquestriatus was
inserted in HzSNPV for the control of Helicoverpa
zea. (DuPont, 1996)
• The toxin from scorpion Androctonus australis
was inserted in AcMNPV for the control of
Helicoverpa zea. (Black et. Al., 1997)
20

21. • Another paralytic toxin that holds promise is
the TxP‐I toxin, a component of the venom of
the predatory straw itch mite Pyemotes tritici.
• Korth and Levings (1993), inserted a toxin URF
13 from maize to AcMNPV. When the larvae of
Trichoplusia ni were injected with this virus, all
died by 60h of post injection.
21

22. • Two insect selective toxins ASII and Sh 1 from the
sea anemones Anemonia sulcata and
Stichadactyla helianthus resulted in 38% and 36%
improvements in speed of kill in Trichoplusia ni and
Spodoptera frugiperda larvae. ( Hughes et al.,
1997)
• The expression of insect selective spider toxins µ-
Aga-IV from Agelenopsis sperta and DTX9.2 and
Ta1TX-1 from the spiders Diguetia canities and
Tegenaria agrestis resulted in improved speeds of
kill. (Prikhodko et al.,1996 , Hughes et al., 1997)
22

23. • Targeting basement membrane:
Expression of Cathepsin L protease from flesh flies
resulted in significant decrease in the survival time in
the larvae of Autographa californica infected with
AcMNPV. (Harrison and Bonning, 2012)
• One of the common factors associated with genetic
optimization for increased speed of kill, is that the
faster the virus kills the host insect, the fewer OB are
produced . Hence, large scale production of these
recombinant baculoviruses in vivo becomes a challenge
23

24. 2. Genetic Engineering for Increased
Virulence
 There are several examples of baculoviruses that have
been genetically engineered to reduce the amount of
virus required for a fatal infection of the targeted insect
pest. Enhancin is a metalloprotease commonly
expressed by baculoviruses that degrades insect
intestinal mucin in the peritrophic membrane.
 Insertion of the enhancin gene derived from
Trichoplusia ni GV enhanced AcMNPV virulence by 2 to
14-fold in various insect species.
 Conversely, deletion of two enhancin genes from
Lymantria dispar MNPV reduced viral potency 12-fold
compared to wild type virus.
24

25. • AcMNPV expressing an algal virus pyrimidine
dimer-specifi c glycosylase, cv-PDG, is less
susceptible to UV inactivation, signifi cantly
increased the virulence to kill Spodoptera
frugiperda larvae by 16-fold.
25

26. Bacteria
• Bacillus thuringiensis (Bt) has been the most
successful commercial microbial insecticide
and also has been the subject of the
overwhelming majority of genetic engineering
studies to improve efficacy.
• Bacillus thuringiensis is characterized by the
production of a parasporal body during
sporulation that contains one or more
protein endotoxins in a crystalline form
26

27. The immediate challenge for genetic
engineering of bacteria is to:
1. increase the potency of the toxin(s),
2. broaden the activity spectrum,
3. improve the persistence under field
conditions, and
4. reduce the production costs.
27

28. • The cryIAc gene from Bacillus
thuringiensis was integrated
into Pseudomonas fluorescens
P303-1 by electroporation and
the engineered bacteria were
highly insecticidal to cotton
bollworm, H. armigera.
(Duan et al., 2002),
28

29.  A toxin gene from B. thuringiensis
subsp. israelensis inserted into
Bradyrhizobium species that fix
nitrogen in nodules of pigeonpea.
 Experiments in a greenhouse
indicated that this provided
protection against root nodule
damage by larvae of Rivellia
angulata
Nambiar, Ma, and Iyer (1990)
29

30. Bacillus thuringiensis subsp
israelensis expressing the
binary toxin gene from B.
sphaericus showed high
toxicity against different
species of mosquitoes.
(Yuan et al.,1999)
30

31. The mosquitocidal proteins from three
different species; Bin from Bacillus sphaericus
2362, Cry11B—a protein B. thuringiensis
subsp. jegathesan and Cyt1A from Bt subspp
israelensis.
The resulting recombinant B. thuringiensis
produced three distinct crystals and was 3 to 5
times as toxic to Culex species as either Bti IPS-
82 or Bs 2362
31

32. IPS-82 strain of Bti, which produces
the complement of toxins
characteristic of this species, was
transformed with pPHSP-1, the
pcyt1A/STAB plasmid that
produces a high level of Bs Bin
toxin. This recombinant was more
than ten-fold more toxic than
either of the parental strains to
larvae of Cx. quinquefasciatus and
Cx. tarsalis.
32

33. The B. thuringiensis crystal genes have been
introduced into E. coli, B. subtilis, B.
megaterium, and P. fluorescens and form
biopesticide formulations consisting of
encapsulated Cry inclusions.
These encapsulated forms of the Cry
proteins have shown improved persistence
in the environment.
(Gawron-Burke and Baum, 1991)
33

34. Cry1Aa gene from B thuringiensis
subspp kurstaki HD1 was inserted
into maize root colonizer
Pseudomonas flourescence.
Recombinant strains were stable
under environmental conditions
and gave 100% mortality against
Manduca sexta.
(Obukowicz et al., 1986)
34

35.  The cry1Aa1 gene encoding
insecticidal crystal protein (ICP) was
transferred into three isolates (Eh4,
Eh5, and Eh6) of, Erwinia herbicola
(Lohnis).
 The transformed E. herbicola strains
expressed the toxin protein and
conferred insecticidal activity and
resulted in 94 to 100% mortality of
diamondback moth, P. xylostella.
Lin et al. (2002)
35

36. Entomopathogenic fungi
Insect pathogenic fungi are key regulatory factors
in insect pest populations.
Most attention has focused on the ascomycetes
Metarhizium anisopliae and Beauveria bassiana.
The major drawbacks associated with fungal
pesticides include relative instability, requirement
for moist conditions for spore germination,
invasion, and growth, and slow rates of mortality.
36

37. • Paecilomyces fumosoroseus and P.
lilacinus have been transformed
using a Benomyl-resistant b-
tubulin gene from Neurospora
crassa .
• Benomyl-resistant transformants
of P. lilacinus were obtained that
could tolerate greater than 30
µg/ml benomyl and P.
fumosoroseus transformants were
obtained that could tolerate 20
µg/ml benomyl.
(Inglis et al., 1999)
37

38.  Bernier et al. (1989) introduced
benomyl resistance (beta-
tubulin) gene from Neurospora
crassa (encoding resistance to
benomyl) into M. anisopliae.
The transformants were
mitotically stable when
subcultured on nonselective
agar and retained the ability to
infect and kill larvae of M.
sexta.
38

39.  A hybrid chitinase containing the
chitin binding domain from the
silkworm Bombyx mori chitinase
fused to the B. bassiana chitinase
showed the greatest ability to bind
to chitin.
 Constitutive expression of this
hybrid chitinase gene by B.
bassiana reduced time to death of
insects by 23% compared to the
wild-type fungus.
Fan et al. (2007)
39

40. Limitations
Although all of these products are effective when used properly, they have distinct
drawbacks which limit user acceptability.
 The bacterial and viral agents must be ingested to be active, and their killing
action, especially the viruses, is slower than conventional chemicalinsecticides.
 These agents are also subject to rapid inactivation by exposure to sunlight andare
readily washed off the foliage by rain.
 Viral products are expensive to produce since current methods require propagation
in living insect larvae.
 Fungi are very intolerant of low humidity conditions or high temperature, and thus
are generally used only in greenhouses or in coolclimates.
40

41. Biotechnology in Agriculture?
that usesAny technique
substances from these organisms, to make
living organisms or
or
modify a product, to improve plants or animals or
to develop substance for specific uses.
41

42. How is Agricultural Biotechnology used?
Genetic Engineering
Molecular markers
Molecular diagnostics
Vaccines
Tissue culture
42

43. Timeline of Biotechnology in
Agriculture
1938 1962 1990 1994 1995
Sporeine from
France, first
commercial
product
kurstaki,
isolated as
highly potent
strain in France.
Chymosin -1st
product of rDNA
in food supply.
first experiment
on transgenic
plant in field
First commercial
Transgenic crop –
Virus resistance
tobacco by China
FlavrSavr® tomato-
1st genetically
modified crop in
USA and France
1996
Field releaseof
Bt cotton
commercial
cultivation of Bt
cotton in India
2002
43

44. 1. Crop improvement: Improved oil quality in Soybean and
Canola
2. Herbicide resistance: Cotton, Corn, Soybean and Rice
3. Insect Resistance: Cotton, Corn, Rice, Tomato and Potato
4. Virus resistance: Papaya, Squash and Potato
5. Slow-ripening and softening: tomato and melon
6. Male sterility: Canola and Corn.
Application of Biotechnology in Agriculture
44

45. Gene transfer in plants
45

46. Development of transgenic crops expressing insecticidal genes
 Cry toxins Bt: Cry 1 Ab, Cry 1 Ac, Cry IIa, Cry 9c, Cry IIB, Vip I, Vip II etc.
 Plant metabolites : Flavonoids, alkaloids, terpenoids
 Enzyme inhibitors : SBTI, CpTi
 Enzymes : Chitinase, Lipoxigenase
 Plant Lectins : GNA, ACAL, WAA
 Toxins from predators : Scorpion, spiders
 Insect harmones : Neuropeptides and peptide hormones
Pyramidine genes: Engineering transgenic crops with more than
one gene to get multimechanistic resistance.
Insecticidal genes
from sources other
than Bacillus
thuringiensis
46

47. Bt cotton
 1961- Bt was registered as pesticide
 2002: Bt cotton was introduced in India
 India has the largest hectarage of cotton and one
third of the total cotton are planted in the world
 Cotton yield increased from 308 Kg/ha in 2001-02
to 500 kg/ha in 2011-12.
47

48. Major transgenic crops expressing Bt genes for Insect
Resistance
Transgenic Crop
Plants
Foreign Gene Target insect pests
Cotton Cry1A(b), Cry1A(c) H. armigera, H. zea,
Heliothis virescens,
Pectinophora gossypiella.
S. exigua
Maize Cry1A(b), Cry1A(c), Cry9C Chilo partellus, H. zea
Tomato Cry1A(c) Manduca sexta
Tomato Bt(k) M. sexta, H. zea
Rice Cry1A(b), Cry1A(c), CryII(a) Scirpophaga incertulas,
Cnaphalocrosis medinalis
Potato Cry 1A(b), Cry1A(b)6,
CryIII A, CryIII B
Phthorimaea operculella,
Leptinotarsa sp.
Tobacco Cry1A(c ) H. virescens, M. sexta,
Brinjal CryI AC Leucinodes orbonalis
48

49. James (2012)49

50. Requirements identified while producing
transgenic plants
• Resistance should be controlled by single gene.
• Expression of transferred gene should occur in the desired
tissue at the appropriate time.
• Safe for consumption
• Inheritance of the gene in the successive generations should
be very stable.
50

51. Plant derived genes
51

52. Protease inhibitors
 Antimetabolic proteins which interferes with the
process of digestion in insects- strategy by plants.
 Dietary protease inhibitors – detrimental to the
growth and development of insects
 Ex: Helicoverpa, Spodoptera
52

53. α – Amylase inhibitors
• Inhibit the digest enzymes of mammals and insects.
• Seeds of several varieties of common bean, Phaseolus
vulgaris (BAAI) – exhibit resistance to bruchid beetles,
Callasobruchus spp.
• Transgenic
from
tobacco
wheat
plants
(wheat
expressing amylase inhibitors
α-amylase inhibitor, WAAI)
increase the mortality of lepidopteran larvae by 30-40 per
cent.
53

54. Lectins
Plant derived proteins that bind to oligo and polysaccharides
Causes agglutination and cell agrgregation.
Carbohydrate binding lectin protein (including chitin) called
phytohemagglutinin (PHA) found in seeds of common bean.
It binds the chitin in peritrophic membrane of midgut thus
interfere with nutrient uptake.
Wheat (wheat germ agglutinin, WGA) and
snowdrop (Galanthus nivalis agglutinin,GNA)
Alternative to Bt delta endotoxins.
Inhibitory to
homopteran pests-
aphids, plant hoppers
and leaf hoppers
54

55. 55

56. Sl.
No.
Technique Application Examples
1
Agrobacterium-based
plant transformation
Ti- plasmid –to carry novel
DNA into plants
Bt insect resistant
crop plants
2 Particle acceleration
DNA coated gold particles
fired into growing tissue
Transgenic soybean
3 Electroporation
Electric current used to alter
protoplast membranes
permitting DNA uptake
Transgenic rice
4 Microinjection
DNA injected into the nucleus
or cytoplasm of a protoplast
Transgenic tomato
5 RNA interference
Blockage of gene function by
inserting short sequences of
RNA
Potential for
protecting cotton,
rice and maize
against insect pests
Biotechnological methods employed for crop improvement
Atwal and Dhaliwal, 2013 56

57. Genetic engineering of Predator and
Parasitoids
 Transgenic strain of Metaseilus occidentalis
Predator of spider mite.
 Maternal microinjection.
 Transgenic strain can be used routinely in applied
pest management programme.
(Hoy,2000)
57

58. Genetic improvement of predators & Parasitoids
Resistance to pathogens
Resistance to pesticides
Adaptation to different
environmental conditions
High fecundity
Improved host seeking ability
58

59. Potentials of biotechnology in IPM
 Low toxicity of protease inhibitors and Bt δ- endotoxin as
compared to conventional insecticide.
 Expression of toxins in all plant parts - No need of
continuous monitoring of pest.
 Provide protection to those plant parts which are difficult to
be treated with insecticides.
 There is no drift problem and ground water contamination.
 Safe to non target species and human beings.
 Eliminate the problem of shelf life and field stability faced by
pesticide formulation.
 Inbuilt resistance to various insects.
59

60. Risk associated with Biotechnological
approaches
• Human and animal health: Toxicity, food quality, allergenicity
• Risk for agriculture: loss of biodiversity, alternation in nutritional
level, development of resistance.
• Risk for environment: persistence of gene, unpredictable gene
expression, impact on non target organisms.
• Risk for horizontal transfer: interaction among different
genetically modified organisms, genetic pollution through pollen
or seed dispersal, transfer of gene to microorganism.
60

61. RNA interference
 Method of blocking gene function by inserting short sequences of
double stranded ribonucleic acid (dsRNA) that match part of the
target mRNA sequence, thus no proteins are produced.
 Knock down the expression of genes.
61

62. Major Indian centres in transgenic research &
application
• Seven such centres were set up initially at various
Universities/Institutions namely,
• Jawaharlal Nehru University (New Delhi),
• Madurai Kamaraj University (Madurai),
• Tamil Nadu Agricultural University (Coimbatore),
• Osmania University (Hyderabad),
• National Botanical Research Institute (Lucknow) and
• Bose Institute (Kolkata).
• University of Delhi South Campus in 1997. 62

63. Incorporation of herbicide resistant
In 2013, herbicide tolerant crops occupied 99.4 million hectares or 57% of the
175.2 million hectares of biotech crops planted globally.
tolerance selection
genetic engineering techniques
Variety Herbicide
LibertyLink corn, GR corn,
LibertyLink soybean
Liberty (glufosinate)
herbicide.
Roundup Ready corn,
Roundup Ready soybean
Roundup and some other
glyphosate products
63

64. Mechanism of herbicide tolerance
Producing a new protein that detoxifies the
herbicide
 Modifying the herbicide’s target protein
 Producing physical or physiological barriers
preventing the entry of the herbicide into the
plant.
64

65. GPS & GIS

66. GPS stands for Global Positioning System
GIS stands for Geographic Information System
66

67. Uses
Scouting monitoring pest
population
Predicting pest outbreak and
movement
Identifying and categorizing
pattern of damage
Assessing the success
Refining the control tactis
Extent of weed infestation
Insect and pest population
67

68. Sampling has to be done in field level
Digital mapping of the location of
sample site (with GPS)
resulting GIS layer can than be used to
interpolated
estimate a pest population/ crop damage
Making control decision on the basis of
pest population estimates
Scouting monitoring pest population
68

69. Precision application of agrochemicals
Weeds mapping and using pre-emergent herbicide the
following year
Pest population and crop yield are mapped for particular
fields and appropriate agrochemicals can be applied only on the
spot that require them
GIS software is linked to the application equipment and is
used to activate and stop the sprat nozzles. A computer that has
environment sensors can also be used to be more precise e.g.
Decrease overspray due to drift from wind
69

70. Limitations
The effects of transgenic on the natural regulation of
pests and biodiversity are often negative.
By cross pollination herbicide resistant genes can
enter weedy relatives.
Widespread use of transgenic plant can render them
susceptible or accelerate evolution in pest.
IPM favors minimized use of chemical whereas the
availability of herbicide resistant crop promotes the use
of more chemical
70

71. Many lectins are toxic/allergenic to mammals
Use of GIS and GPS is not economical for small area
Technical support is required to use such
techniques
Use of GIS to apply insecticide is possible but less effective
as insects are much vagile
Integrated farming positively affects natural
control agents while yield reductions are low and
economic returns are stable or even increase.
71

72. Conclusion
• Biotechnological approaches play important role in insect-
pest management.
• The efficacy of bio-control agents can be increased through
rDNA technology
• DNA barcoding can help in quick and accurate identification.
• DNA fingerprinting helps for identification of biotypes and
genetic changes in insect-pest.
72

73. Future prospects
•Biotechnological approaches should be shaped within
context of sustainable agriculture system
•Insect population trends can be demonstrated and
thus can be used to develop predictive models
73

74. 74