3 optimising pesticidal plant use and getting research ... · farmer surveys ‐priority species...
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O ti i i P ti id l Pl t UOptimising Pesticidal Plant Use and getting research into useand getting research into use
Philip C Stevenson, Steve Belmain, p , ,David Hall, Dudley Farman, Stephen Nyirenda John Kamanula BrightonNyirenda, John Kamanula, Brighton Mvumi, Phosiso Sola, Gudeta Sileshi, G il L i Ni l V it h F li F tGwil Lewis, Nigel Veitch, Felix Forest.
Farmer surveys ‐ Priority species list Farmer Surveys, Database & Lit. > 100 plant species of known potential value
y y p
> 100 plant species of known potential valuePriority indigenous species listAloe feroxBobgunnia madagascariensisg gDolichos kilimandscharicusEuphorbia tirucaliLippia javanicaNeorautanenia mitisNeorautanenia mitisSolanum panduriformeSecuridaca longepedunculataStrychnos spinosaTephrosia vogeliiTephrosia vogeliiVernonia spp.Zanha africanaChenopodium amboensisFagara heitziiNon‐indigenous speciesTithonia diversifolia, Azadirachta indica – Neem.Azadirachta indica Neem. Tagetes minutaCymbopogon spp. Nyirenda, et al. (2011) African Journal of
Agricultural Research 6: 1525‐1537.
Tephrosia vogeliiTephrosia vogelii
Plants reportedly used (%) in Malawi for pest control (n=167)
Kamanula et al., (2011) International Journal of Pest Management. 57: 41‐49.
Is Tephrosia vogelii an effective lt ti t th ti ?alternative to synthetics?
• Validation of Tephrosia spp. –
• Literature filled with assumptions• confusion about role of rotenoids & details of chemistry/bioactivity overlooked.
• Can we optimise use for farmers?
Bruchid bioassayy
Treatments need to beneed to be small to enable small quantities f l tof plant
chemical to be testedbe tested.
Effect of crude plant extracts (2%w/v) applied to cowpea on the mortality of adult bruchidscowpea on the mortality of adult bruchids
8090100
y
24 hrs
48 hrs
72 hrs
P d b40506070
nt m
ortality
Pod borer
10203040
Percen
0
Effect of crude plant extracts (2%w/v) applied to cowpea on the mortality of adult bruchidscowpea on the mortality of adult bruchids
Ento‐toxicity of Tephrosia and Neorautanenia
8090100
y
24 hrs
48 hrs
72 hrs
Ento toxicity of Tephrosia and Neorautanenia
P d b40506070
nt m
ortality
Pod borer
10203040
Percen
0
Rotenoids from Tephrosia vogelii leavesp gMain components in leaves
H OO
OH
OO
O
O OMeR
O
O OMeR
D li R HO
OMe
OMeOMeDeguelin R=H
Tephrosin R = OHRotenone R=H 12α‐hydroxyrotenone R = OH
OO
O OO
O
OHO
OMe
OMe O OMeOH
OMeSarcolobine Toxicarol
Effect on C. maculatus of cowpea seeds treated withT vogelii leaf leaf extract & rotenoids after 48 hT. vogelii leaf, leaf extract & rotenoids after 48 h
80
90
100
60
70
80
umbe
r (%)
Dead
30
40
50
Insect nu
Sick
Healthy
10
20
30
02% leaf AcO extract
2%Deguelin 1000ppm
Deguelin 100ppm
Tephrosin 100ppm
Control pp pp pp
Sick insects are alive but incapacitated i.e., can not lay eggs.
Chemical variation within Tephrosia?Chemical variation within Tephrosia?
HO
OO
O
OMe
OMeR
OMe
Deguelin R=H Tephrosin R = OH
Best species ‐ T. vogelii or T. candida ?( i t I t ti l I tit t i Af i )(growing at International Institute in Africa)
Tephrosia vogeliiHighly toxic to bruchidsbruchids
Tephrosia candida?Tephrosia candida?Promoted for soil
improvement (N fi i
1 2
(N2 fixing, green mulch) and
(assumed) pest 1 2control
Is T. candida also effective at controlling bruchids?
Both are in fact T. vogelii
Tephrosia vogeliiHighly toxic to bruchidsbruchids
Tephrosia vogeliiTephrosia vogeliiPromoted for soil
improvement (N fi i
1 2
(N2 fixing, green mulch) and
(assumed) pest 1 2control
Plastid Ltrn region, ITS nuclear DNA sequences & morphology indicate both to be T. vogelii
LC‐MS chromatograms of T. vogeliih t 1 & 2
PhilSAPP3 x 150 mm Phenomenex Luna C18(2), 95:0:5(0 min) 0:95:5(20 min) 0:95:5(25 min), water : MeOH : ACN +1% formic acidTvogelii herb specimen 34mg/ml
PhilSAPP22-Oct-2009
Tvogelii herb specimen 1 3: Diode Array Range: 4.337e+219.40
Fig 1a HPLC chromatogram of T. vogelii chemotype 1PhilSAPP3 x 150 mm Phenomenex Luna C18(2), 95:0:5(0 min) 0:95:5(20 min) 0:95:5(25 min), water : MeOH : ACN +1% formic acidTvogelii herb specimen 34mg/ml
PhilSAPP22-Oct-2009
Tvogelii herb specimen 1 3: Diode Array Range: 4.337e+219.40
PhilSAPP3 x 150 mm Phenomenex Luna C18(2), 95:0:5(0 min) 0:95:5(20 min) 0:95:5(25 min), water : MeOH : ACN +1% formic acidTvogelii herb specimen 34mg/ml
PhilSAPP22-Oct-2009
Tvogelii herb specimen 1 3: Diode Array Range: 4.337e+219.40
Chemotype 1
chemotypes 1 & 2AU
2.5e+2
3.0e+2
3.5e+2
12.03
deguelin
AU
2.5e+2
3.0e+2
3.5e+2
12.03
deguelin
AU
2.5e+2
3.0e+2
3.5e+2
12.03
deguelin
Chemotype 1
A
5.0e+1
1.0e+2
1.5e+2
2.0e+212.62
18.75
15 12 20.35
tephrosinrotenone
toxicarolsarcolobine
A
5.0e+1
1.0e+2
1.5e+2
2.0e+212.62
18.75
15 12 20.35
tephrosinrotenone
toxicarolsarcolobine
A
5.0e+1
1.0e+2
1.5e+2
2.0e+212.62
18.75
15 12 20.35
tephrosinrotenone
toxicarolsarcolobine
5.0e+1
11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
15.12
Tcandida BI19309 3: Diode Array Range: 6.261e+120.68
Fig 1b HPLC chromatogram of T. vogelii chemotype 2Ob i 5 h l h
5.0e+1
11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
15.12
Tcandida BI19309 3: Diode Array Range: 6.261e+120.68
Ob i 5 h l h5.0e+1
11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
15.12
Tcandida BI19309 3: Diode Array Range: 6.261e+120.68
Obovatin 5‐methyletherChemotype 2
AU
2 0e+1
3.0e+1
4.0e+1
12.00 12.58
Z‐tephrostachin 4 & 5
Obovatin 5‐methylether
AU
2 0e+1
3.0e+1
4.0e+1
12.00 12.58
Z‐tephrostachin 4 & 5
Obovatin 5‐methylether
AU
2 0e+1
3.0e+1
4.0e+1
12.00 12.58
Z‐tephrostachin 4 & 5
Obovatin 5 methyletherChemotype 2
Time
0.0
1.0e+1
2.0e 1
19.93
15.1213.40 20.37
31 & 2 6
Time
0.0
1.0e+1
2.0e 1
19.93
15.1213.40 20.37
31 & 2 6
Time
0.0
1.0e+1
2.0e 1
19.93
15.1213.40 20.37
31 & 2 6
11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.0011.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.0011.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
Compound IDs based on 700MHz NMR and HR‐EI MassSpec.Compounds 1 – 6 are novel
Effect on C. maculatus of cowpea treated with acetone extracts of T vogelii chemotypes after 48 hoursextracts of T. vogelii chemotypes after 48 hours
80
90
100
) Dead
50
60
70
insects (%
)
Sick
Healthy
30
40
50
Num
ber o
f
0
10
20
N
02% 0.20% 0.02% 2% 0.20% 0.02% AcO
Chemotype 1 Chemotype 2 Control
Sick insects are alive but entirely incapacitated
Concentration dependent mortality of C. maculatuson cowpea treated with compounds from T vogeliion cowpea treated with compounds from T. vogelii.
90
100
m)
60
70
80
ality
(±sem
Rotenone
40
50
60
ent m
orta Deguelin
Tephrosin
10
20
30
Mean pe
rc
p
Obovatin 5 Me
0
10
0 100 200 300 400 500 600
M
Dose (ppm, generalised logit linear model)
Securidaca longepedunculataSecuridaca longepedunculatalongepedunculata
Effective stored d t t t t
Securidaca longepedunculata
product protectant Ghana & Zambia.
Root bark pounded & mixed with graing
inefficient
Maize damage (%) by natural infestation of S. zeamais 20 wks after treatment with plants (2%zeamais 20 wks after treatment with plants (2% w/w) reported to be pesticidal by farmers –Choma, Zambia.
45
6 Farmers rankings100
120
4 6 Farmers rankings
80
100
enta
ge
340
60
mag
e pe
rce
12
20
40
Da
0Untreated Bobgunnia Cassia Cissus Securidaca Actellic SD
Securidaca longepedunculataMethyl salicylate
Securidaca longepedunculata
• Methylsalicylate
• Saponins
deterrent and toxic to Sitophilus zeamais but volatilevolatile
Saponins also active &Saponins also active & occur in stem bark
Can stem bark be used instead of root?instead of root?
• More sustainable
St t l 2009 J A F d Ch 57 8860Stevenson et al., 2009 J Ag Food Chem, 57, 8860.Jayasekera, Stevenson, Belmain and Hall, 2002 J. Mass Spectrom, 37, 577Jayasekera, Stevenson, Belmain and Hall, 2005 J. Chem. Ecol., 31, 303.
LC-MS chromatograms of 5 specimens of S.longepedunculata from different locationsh i i i i i h i
60
80
1000
T l Gh Eff ti2
showing variation in saponin chemistry
80
1000
20
40 Tamale, Ghana. Effective 1 2
1000
20
40
60
Bolgatanga, Ghana. Effective
1000
20
40
60
80
Choma, Zambia. Effective
20
40
60
80
100
Root bark Nchenachena, Malawi, Effective
20
40
60
80
1000
Stem bark Nchenachena Malawi Ineffective in field trials
24 26 28 30 32 34 36 38Time (min)
0
20
Securidaca longepedunculataWater extracts saponins
M ffi i t f l t
Securidaca longepedunculata
•More efficient use of plant •Every grain coated
Submersion of grain in soapy extract for treatment &Submersion of grain in soapy extract for treatment & solarisation kills pre-storage infestation.
Farmers tried this
–50% less plant material used
Extracting Securidaca and treating grain
Current field trial of water extract
treated grain – Kasisi Zambiatreated grain Kasisi, Zambia
Extract requires less than half as much plant material as powdered but unpopular – too much effort!
But it works!!!!!
Bobgunnia madagascariensis g g(Desv.) J. H. Kirkbr. & Wiersema (Leguminosae)
Syn Swartzia madagascariensisSyn. Swartzia madagascariensis
(Widespread across African semi arid woodlands except Madagascar)
Kamanula et al., (2011) Intl. J. Pest Manage, 57, 41-49
woodlands except Madagascar).
Flavonoid pentaglycosides from B madagascariensisB. madagascariensis
3'
α-Rha I
3 11 7
3
4'
21'
9
α-Rha IV 1
3
2
1
3 15 410
Rha IIRha III6
α-Rha IIα-Rha III1
1a R 1 = R 2 = OH, R 3 = H 1b R 1 = R 3 = OH, R 2 = H 2a R 1 = R 3 = H, R 2 = OH
Between 5 and 20% by weight of dry pod!
Stevenson, Nyirenda & Veitch. (2010) Tetrahedron Letters, 51, 4727–4730.
2b R 1 = R 2 = H, R 3 = OH
Saponins from B d i i
28COOR2
B.madagascariensis
3
OO
2
HOOC R
32
OO
O
OR3
HOOC R1
1
OOO
O
3 R 1 = CH 3, R 2 = β-Glc, R 3 = β-Xyl 4 R 1 = CH 3, R 2 = β-Glc, R 3 = H5 R 1 = CH 3, R 2 = R 3= HMost biologically active ,
6 R 1 = CHO, R 2 = R 3 = H 7 R 1 = CH 3, R 2 = H, R 3 = β-Glc 8 R 1 = CHO, R 2 = H, R 3 = β-Glc9 R CH R R β Glc
Most biologically active
Polarity?
9 R 1 = CH 3, R 2 = R 3 = β-Glc
Marston et al., (1994) Stevenson et al.(2010)
LC-MS chromatograms of B. madagascariensisat equiv conc from 4 locations showing
1000
9 3
at equiv. conc. from 4 locations showing qualitative and quantitative variation in saponins
0
50 4568
9 Luhomero, Malawi. Effective in field trials
7 56 7
50
1000
Choma, Zambia Ineffective in field trials
50
1000
Rhumphi Malawi
1000
50 Rhumphi, Malawi
0
50 Muzarabani, Zimbabwe Effective in field trials
6 8 10 12 14 1Time (min)
Sarasan, Kite, Sileshi and Stevenson 2011, Plant Cell Reports
Lippia javanicaEffect on tick count / animal of treating cattle with 5%
pp j
Lippia javanica 120
80
100
anim
al
40
60
No.
tick
s/a
0
20
N
Tickbuster L. javanica Untreated®
Madzimure et al., Trop Anim Health Prod (2011) 43:481–489
Contact toxicity of Lippia oil, perillaldehyde, linalool and perillaldehyde+ linalool (1:1) against S zeamais at 24hr
Lippia oil
perillaldehyde+ linalool (1:1) against S. zeamais at 24hr
100
120
Lippia oilPerillaldehydeLinalool
80
100
%)
Perillaldehyde+Linalool
40
60
orta
lity
(%
0
20Mo
-20
00 0.05 0.5 1 2.5 5 10
Concentration (mg/mL)Concentration (mg/mL)
Limonene, linalool, citronellal and perillaldehyde in Nchenachena Lippia leaf oil changes over timeNchenachena Lippia leaf oil changes over time
800
700
800mg/
g oil)
500
600
Lipp
ia oil (m
i
300
400
poun
ds in
L Limonene
Linalool
Citronellal
200
300
nt of c
omp Citronellal
Peril laldehyde
0
100
Amou
October December February April June
Harvesting period
Some volatile compounds identified in Lippia oils from different locations in Malawioils from different locations in Malawi
Location
Compound Nchenachena Chikangawa Jendap g
Perillaldehyde 44 % 0 0.55 %
Li 24 % 0 13 %Limonene 24 % 0 13 %
Ipsdienone 1 % 52 % 31 %
Piperitone 2 % 0 22 %
Germacrene D 4 % 5 % 5 %
(+)-Carvone 3 % 0 0
Summary y
T h i lii d th i idTephrosia vogelii and other species provide highly effective bruchid control but efficacy is variable
• Activity associated with presence of specific• Activity associated with presence of specific plant chemicals
• Occurrence of chemcials is variable between chemotypes but predictablec e otypes but p ed ctab e