This is a lead lecture deliverd at BET 2010, CCSHAU, Hisar India on 20th Oct 2010.

1. Botanical Pesticides in Pest Management:
Prospects and Constraints
C. Devakumar
cdevakumar@gmail.com

2. Global Biopesticide Market (in millions of dollars)

3. •The global pesticide market was valued at nearly $43
billion in 2009 and is expected to grow at a compound
annual growth rate (CAGR) of 3.6% to reach $51
billion in 2014.
•Biopesticides segment is expected to grow at a
15.6% compound annual growth rate (CAGR) from
$1.6 billion in 2009 to $3.3 billion in 2014.

4. Registration of safer chemicals
Proportion of pesticide active ingredients that are considered to be safer
(biological chemicals and reduced-risk conventional chemicals) has steadily
increased over the last several years.
Source: EPA, 1999.

5. Cost to develop and time to market of various products

9. BOTANICAL PESTICIDES
Scope Drawback
Indigenous; most suited rural  Action “ayurvedic” not
milieu allopathic
· Biodegradable – Green  Situation / location /target
specific
· Time tested through co-
evolution  Low level & stability
 No volume and sales /
· Least side – effects
turnover - No MNC would
· Complex m.o.a; slow- prefer
resistance development  Low yield / chemo-type,
· Useful lead-templates for genotype, eco-type
potent version variations; plant part, age;
storage conditions critical
Ideally suited to biotechnological
innovations & integration  Crude works better;
synergistic
Biodiversity

10. Push-pull or Stimulo – Deterrent Diversionary Strategy
Push Pull
Away from the crop Traps / trap crops
Masking of host attraction; (1R,5S) Host attractants (Isothiocyanates of
Myrtenal for Aphids fabae); oilseed rape for the seed weevil and
Methyl salicylate against cereal cabbage stem flea beetle
aphids
Repellents, antifeedants, oviposition Aggregation, sex and oviposition
deterrents semiochemicals
Attractants for predators and Visual cues
parasitoids;
e.g. Faba beans produce 6-methy-5-
hepten-2-one, an attractant for aphid
parasitoid

11. CRITERIA OF SELECTION OF PLANT OR PLANT PARTS
Traditional knowledge (Ethno-medical)
Farmers’ observation
Literature reports
Chemotaxonomy
Random Screening
Abundant availability

12. PROBLEMS AND PROSPECTS
Commercialisation of botanical pesticides
Quality of raw material
Product standardization
Quality control
Stringent registration requirement
Problem of pest registration

13. Botanical Pesticides: IPR and Policy Issues
Framing pesticide policy to protect human and
ecosystem health
Patentability of the product should be considered. Prior
publication of pesticidal properties of a compound could
cause patent problem
Patenting synthetic analogues with no mention of the
natural source of the chemical family might be safer than
patenting the natural product
Softening registration requirements for natural products

14. PROSPECTS
Raising the threshold of active ingredients
Selection of germplasm tolerant to abiotic and biotic stress
Survey of plant biodiversity
Tissue and cell culture
On-farm production of botanical pesticides
Improving photo and thermal stability
Scientific investment in terms of money and human resource

15. BIOTECHNOLOGICAL TOOLS IN
DISCOVERY

16. The development through in vivo screening

17. Gene expression profiling, a revolutionary tool in
herbicide discovery
Gene Expression Profiling (GEP) with DNA microarrays (chips) is a
new technology used to measure changes in the entire transcriptome,
i.e. full complement of active genes, of an organism in a single
experiment.
A catalogue of genetic fingerprints of the plant Arabidopsis thaliana,
is created and each fingerprint being characteristic for a single
herbicidal MoA is then used to rapidly classify herbicidal compounds
from Ultra High Throughput Vivo Screening (UHTVS) according to
their MoA.
Besides GEP also provides the opportunity to identify the affected
metabolic pathway which is a first step towards the identification of
novel herbicidal targets. Additionally GEP can identify the MoA of
pro-drugs, which cannot be elucidated by conventional biochemical
methods.
GEP provides insight into the interactions of any herbicidal

18. The Principle of Gene Expression Profiling.

19. The virtual discovery cycle

20. Model organisms for target identification

21. The research screening platform

22. Strategy of identifying new targets

23. Test systems for targets in UHTBS

24. UHTVS - Automated evaluation of activity

25. Unique research platform – Network of complementary
technologies to meet the challenges in compound
discovery

26. STRATEGIRES
CAPACITY BUILDING
NATIONAL / GLOBAL NETWORK
OR
CONSORTIA
PUBLIC PRIVATE PARTNERSHI

27. BIOPROSPECTING OF ANTIFUNGAL
PHYTOCHEMICALS

28. THE MOST EFFECTIVE ANTIFUNGAL PHYTOCHEMICALS
AGAINST SHEATHBLIGHT OF RICE
Seedlings soaked for 4 h in 0.1 % formulations pre-transplanting
Piper betle
Eugenol
Aza concentrate
Garlic bulb extract
Chinaberry
Carbendazim
Terpinyl acetate
Linalyl acetate
Control
45 49 53 57 61 65 69
12/23/2010 Infection (%)
PRE-SRC PRESENTATION

29. MCounts Patchouli oil 5ppm,11-47-04 AM, 4-3-2008.SMS TIC Filtered
1.25 25:250
GC-MS ANALYSIS OF PATCHOULI OIL
Pogostemon cablin
Disease control 76% 26.037 β- Patchoulene 4.35%
1.00 Carbendazim 93.4%
26.816 E-caryophyllene 3.02%
36.041 min
36.060 min
27.148 α- guaiene 13.53%
28.943 min
27.146 min
27.597 seychellene 7.24%
0.75 27.893 α- Patchoulene 5.11%
28.943 azulene 17.12%
36.041 patchouli alcohol 36.86%
0.50
27.591 min
CH3
HO
26.037 min
27.891 min
H3C
26.815 min
H3C
27.977 min
0.25
0.00
12/23/2010 PRE-SRC PRESENTATION
25.0 27.5 30.0 32.5 35.0
minutes

30. MCounts Cedar-Hex-5ppm1.SMS TIC Filtered
40:400
GC-MS ANALYSIS OF CEDARWOOD OIL
1.50
Himalayan Cedar Cedrus deodara (D. Don) G. Don. f. (“Deodar”)
19.208 min
Disease control 67.8 %
1.25 Carbendazim 93.4 %
α-himachalene 11.6%
γ-himachalene 7.5%
1.00 β-himachalene 40.4%
Himachalol, 3.4%
Deodarone 4.9%
0.75
(E)-α-atlantone 2.1%
17.982 min
0.50
18.693 min
+ 19.842 min
0.25
+ 19.445 min
18.813 min
20.078 min
0.00
12/23/2010 PRE-SRC PRESENTATION
15 16 17 18 19 20 21 22 23
minutes

31. Effect of palmarosa oil and neem products on
seed infection and seed vigour index in wheat
Treatment Conc. D. s. A. a. Vigour
infection infection
(%) (%) (%)
index
NEEM 5 EC 0.05 0 8 1104
Mancozeb 0.25 0 5 1069.5
(PR+NP) 0.08 8 10 1056
PR (contact) 0.08 8 10 897
PR-fumigation 0.09 36 16 880
(PR+NO) 0.08 20 18 864
PR+NB 0.08 25 25 836
Control 52 24 630
(NP) 0.50% 45 22 559
Neem oil (NO) 0.3 40 20 558

32. Effect of botanicals on seed infection and
seed germination of wheat
Mancozeb NEEM 5 EC
PR+NP Eugenol
PR (contact) Inoculated control
Mancozeb NEEM 5 EC
PR+NP Eugenol
PR (contact) Inoculated control
60 100
45
seed germination (%)
seed infection (%)
30 80
15
0 60
30 60 90 120
Storage period in days after application

33. ANTIFUNGAL SPECTRUM OF NEEM PRODUCTS
Neem products C. M. D. P. sojae F. solani
graminicola phaseolina sorokiniana
Neem oil 90EC 20.5 (68.46) 40.0 (50.0) 28.0 (46.15) 50.5 (40.58) 38.0 (5.0)
NIM-76 20.5 (68.46) 31.0 (61.25) 15.1 (70.96) 50.0 (41.17) 27.0 (32.5)
Neem meliacins 20.0 (69.23) 20.0 (75.0) 20.0 (61.53) 45.0 (47.05) 37.5 (6.25)
Neem 5 EC 0 (100) 6.5 (91.87) 0 (100) 10.0 (88.23) 30.0 (25.0)
Azadirachtin (10%) 32.0 (50.76) 15.0 (81.25) 20.0 (61.53) 38.0 (55.29) 33.5 (16.25)
Mancozeb (0.25%) 0 (100) 0 (100) 0 (100) 0 (100) 6.5(83.75)
Control 65 (0) 80 (0) 52 (0) 85 (0) 40 (0)

34. Essential Oils:
Minimum inhibitory concentration (ppm)
P. F.
D. sorokiniana C. graminicola M. phaseolina
Essential oils sojae solani
Palmarosa 439.8 474.6 481.3 502.9 527.0
Lemon
grass 524.1 523.5 526.7 510.9 578.0
Jamrosa 600.0 588.0 597.3 568.3 622.6
citronella 640.2 640.8 630.9 667.1 686.9

35. Comparison of the best treatments on
fungal infection and vigour index in wheat
1200 55
Fungal infection (% )
44
Vigour indices
1000
33
22
800
11
600 0
(P ct)
ug )
on l
l
(L P)
co )
(P O)
(L B)
(c ct)
5
(P eb
o
ro
P
B
M EC
en
+N
+N
+N
+N
+N
ta
ta
nt
z
co
on
M
G
R
G
R
R
an
E
E
(c
E
R
LG
N
P
Treatments
vigour index D.s infection ( % )
Alternaria infection ( % ) D.s infection ( % )

36. Cyclotides are a structurally unique family of small disulfide-rich proteins
embedded with highly stable structural motif and showing a broad range
of roles in plant defense such as proteinase inhibition, anti-bacterial, anti-
viral effects and insecticidal activities.
(Peligrini et al. 2007 Chen et al. 2005; Daly et al. 2004; Ireland et al. 2008; Jennings
et al. 2001; 2005; Lindholm et al. 2002; Svangard et al. 2004).

37. Bioprospecting of cyclotides in Viola species
Viola betonicifolia and V.
canescene
Frozen in liquid N2 &
grounded
Dried root powder (7 g)
CH2Cl2:MeOH (1:1) left overnigh
Extract partitioned with CH2Cl2 and
then water
Concentrated under vacuum and
then freeze dried
Dried material (100 mg)
Dissolved 33mg in 1 ml of 20% ACN:H2O with 0.1% HCOO
Subjected to reverse phase
chromatography
Eluted with 90% ACN:H2O with
Freeze dried to get cyclotide enriched fraction LC-MS analysis
0.1% HCOOH

38. Total Ion Chromatogram by Flow Injection Analysis (FIA)
VC
VB

39. TIC of VB sample through HPLC
TIC
XIC at Rt 5.37

40. XIC of VB sample through HPLC
TIC
XIC at Rt4.46

41. TIC and XIC of VB sample through HPLC
TIC
XIC at Rt 5.37

42. TIC and XIC of VC sample thr. HPLC
XIC at Rt. 23.30

43. MADHUCA INDICA (MAHUA)
(Family Sapotaceae)
• Mahua valued for its oil bearing seeds and flowers
• Traditionally utilized for alcoholic beverage production
• Seeds kernel constitute over 50% non-drying oil (66% total
unsaturated and 33% total saturated)
• Mahua seed kernel contains 2.5% toxic saponin content which
must be removed for utilizing mahua cake as animal feed
• Efforts have been made to separate saponins from defatted seed
cake for possible use as pest control agents

44. Mahua based Phytoadjuvants: Isolation and derivatization
Madhuca Seed/kernel powder Insecticidal Saponins,
prosapogenins, sapogenin,
Hexane their esters and mahua
oil
Defatted seed extract
O
Methanol C
HO O
OH
Saponin rich material Arab
Glu Glu Glu O
Xyl
Partitioning HOH2C OH
Arab
Water: n-BuOH
Api
Saponin concentrate
Prosapogenins
Precipitation
Acetone
Saponin mixture
Madhuca saponins (MI-I , MI-II and MI-III)
Sapogenin

45. Analytical / prep-LC method for separation of
Madhuca saponins
Analytical LC Prep LC
Detector PDA-213 nm PDA-213 nm
Column RP-18 254 x 4mm (5 µm) 250 x 50 mm (10 µm)
Mobile phase Methanol -Water : 60:40 Methanol -Water : 60:40
Flow rate 0.4 ml min-1 10 ml min-1
MI-III ( M +.1535.9) MI-I (M +.1241)
O
C O
HO O C
OH
Arab HO O
Glu Glu Glu O OH
Xyl Arab
HOH2C OH Glu Glu O
Arab Xyl
HOH2C OH
Api Arab

46. Photo-activated xanthotoxins from Ammi majus seeds
OCH3 O O O
O O O
OCH3
8-methoxypsoralen 5-methoxypsoralen
Xanthotoxins are known for the treatment of vitiligo, leucoderma asthma,
angina and digestive system disorders
The plant is widely cultivated in India for these bioactive furanocoumarins
which are used in the treatment of vitiligo and psoriasis
Like other many photo-activated compounds these furacoumarins may find use
in pest control

47. Isolation and LC analysis of xanthotoxins from
Ammi majus seeds
The seed contains furanocoumarins (5-, and 8-methoxypsoralens), which
stimulate pigment production in skin that is exposed to bright sunlight
Process has been developed and standardized for the efficient extraction of
furanocoumarin from seeds using fluorinated solvents such as phytosols.
Extractions were completed within three cycles
LC method has been standardized for the analysis of furanocoumarins using UV
detector (230 nm), RP-18 column and acetonitrile as solvent system

48. CURCUMA LONGA (Turmeric)
Turmeric oil Pest control (stored grains)
Turmerone Pest control
Curcumins Antifeedant/antifungal
antioxidant/anticancer
Reduced curcumins Anti-oxidant/anti-ageing/cosmetic
Turmerin Anti-oxidant

49. CURCUMINS: Isolation and LC analysis
Curcumin I
Curcumin II
Curcumin III
RP 18 column 250mm x 4 mm
Injection 20 ul
Solvent system Acetonitrile
Curcumin ethers
Flow rate 0.5
ml/min
UV detector 254 nm
Retention time (Rt, min)
Curcumin I 5.89
Curcumin II 5.57
Curcumin III 5.39

50. CURCUMA LONGA : REDUCED CURCUMINS
HO OH
HO OH
H2/Pd/C
H3CO OCH3
H3CO OCH3
O O
O O
Curcumin I Tetrahydro curcumin I
(Keto)
HO OH HO OH
H2/Pd/C
H3CO OCH3 H3CO OCH3
O OH O OH
Hexahydro curcumin I Tetrahydro curcumin I
(enol) (enol)
HO OH HO OH
H2/Pd/C
H3CO OCH3 H3CO OCH3
OH OH OH OH
Hexahydro curcumin I Octahydro curcumin I
(Keto)

51. Turmerin: Isolation and analysis of
water soluble peptide
Turmeric powder
Suspended in boiling distilled water
Filter/centrifuge/decolorize supernatant
Lyophilization
Turmerin (0.1%)
 It is stable to heat and light radiations
 It is a 5-KDa peptide containing 40 AAs
 Turmerone is an effective antioxidant/DNA
protectant/antimutagen