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?

Callosobruchus maculatus

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 javanicapp j

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

Lippia javanicapp j

linalool

perilaldehyde

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

Implicationsp

Elit t i l f ti id l l t d tElite materials of pesticidal plants need to be promoted for pest control and p ppropagation

Caution required when recommending use of pesticidal plants for pest control without chemotyping or validating in trialswithout chemotyping or validating in trials