SCREENING ON PLANT

EXTRACTS FOR OVICIDAL

ACTIVITY AGAINST

Deroceras reticulatum EGGS

University of Santiago de Compostela

Faculty of Biology

Spain

Autores:, José CASTILLEJO, Javier IGLESIAS,

* Investigación financiada por:

• la Unión Europea. Nº de Proyecto: FAIR5-PL97-3355.

Título: “Novel technologies for integrated control of slug damage in key horticultural crops”

• la Xunta de Galicia. Nº de Proyecto: PGIDT00AGR20001PR

Título: “Alternativas a los molusquicidas comerciales actuales”

Objectives

PLANTS

PLANT EXTRACTS

ACTIVE PRODUCT

SYNTHETIC

PRODUCT

® PATENT

COMERCIALIZATION

HOW and

WHEN to

apply it ?

AGAINST EGGS

AGAINST ADULTS

Preparing the extracts for testing:

Plants were collected and identified

Different parts of the plants were separated:

Rhizomes Leaves Fruits

After dry, were powdered with a domestic coffee grinder

They were stored in bottles

Seeds Flowers Roots

Preliminary tests

Water extracts

Acetone/water extracts (7:3)

Water extracts

The powder of the plant was allowed to macerate

during 24 hours

The extract was filtered and applied to the dishes

Control dishes receives only water

Then were dried at environment temperature

Acetone / Water extracts (7:3)

After macerate, the extract was filtered

Acetone was removed in the rotavapour

Control dishes received the mixture acetone/water

after remove the acetone

Testing the plants:

5 doses of the extract and one control. The doses were arranged in a ten-fold geometric series

3 replicates per dose

3 to 5 eggs per replicate

1 ml of extract was applied to each dish

After dry were remoistened with 1 ml of water

Digitalis purpurea L.

Common name: Foxglove (España:

dedalera)

Constituents:

1. glucosides : digitoxin, digitalin,

digitalein, digiton

2. volatile oil

3. fatty matter

4. gum

5. sugar

FOXGLOVE: any Eurasian scrophulariaceous plant of the

genus Digitalis, esp. D. purpurea, having spikes of purple or

white thimble-like flowers. The soft wrinkled leaves are a

source of digitalis

Digitalis’ extract tested

LEAVES

FRESH

Fp

WATER

(ppm)

80,000

40,000

8,000

ACETONE

/ WATER

(ppm)

100,000

Fp = Filter paper

Ss = Standard soil

Eucalyptus globulus Labill.

Common name: Tasmanian blue gum.

Constituents: Monoterpenes: a-pinene, limonene, b-pinene, p-cymene, terpinene, aromadendrene....

Sesquiterpenes, Monoterpene alchohols, sesquiterpenols, aldehydes.

Oxides: cineole

BLUE GUM: a tall fast-growing widely cultivated

Australian myrtaceous tree, Eucalyptus globulus,

having aromatic leaves containing a medicinal oil, bark

that peels off in shreds, and hard timber. The juvenile

leaves are bluish in colour

Eucalyptus’ extracts tested

LEAVES STEAMS

FRESH DRY FRESH DRY

Fp Fp Fp Fp

WATER

(ppm)

80,000

40,000

8,000

ACETONE/

WATER

(ppm)

100,000

Fp = Filter paper

Ss = Standard soil

Euphorbia helioscopia L.

Common name: Sun spurge (Euforbia)

Constituents: euphorbone; 12-deoxyphorbol; wax; resin; lignin; basorrin; volatile oils; euphorbo-resene; euphorbic acid; calcium malate

SPURGE: Any of various euphorbiaceous plants of the

genus Euphorbia that have milky sap and small flowers

typically surrounded by conspicuous bracts. Some species

have purgative properties

Euphorbia’s extracts tested LEAVES STEAMS ROOTS FRUIT

FRESH DRY FRESH DRY FRESH DRY FRESH DRY

Fp Fp Fp Fp Fp Fp Fp Fp

W

A

T

E

R (ppm)

100,000

80,000

50,000

40,000

8,000

ACENTO

NE /

WATER

(ppm)

100,000

Foeniculum vulgare Miller.

Common name: Fennel (Hinojo)

Constituents: Monoterpenes: limonene; Monoterpenols: fenchol; Phenols: methyl chavicol; Aldehydes; Cetones: camphre; Oxides: cineole; Coumarines; Furocumarines. FENNEL: a strong-smelling yellow-flowered umbelliferous

plant, Foeniculum vulgare, whose seeds and feathery leaves

are used to season and flavour food

Foeniculum’s

extracts tested

LEAVES STEAMS

FRESH DRY

Fp Fp

WATER

(ppm)

80,000

40,000

8,000

ACETONE /

WATER

(ppm)

100,000

Laurus nobilis L.

Common name: Bay,

laurel.

Constituents: volatile

oils (cineole, pinene,

sabinene, eugenol acetate,

terpenoids...), tannic acid,

bitter principles.

LAUREL: a small Mediterranean lauraceous

evergreen tree, Laurus nobilis, with glossy

aromatic leaves, used for flavouring in cooking,

and small blackish berries

LEAVES

FRESH DRY

Fp Fp

W

A

T

E

R

(ppm)

80,000

40,000

8,000

Laurus’ extracts tested

Rosmarinus officinalis L.

Common name:

Rosemary (romero)

Constituents: 1. volatile oils: borneol, linalol,

esters, cineol, d-pinene,

camphene

2. Acids: phenolic, triterpenic,

rosmaricine, isorosmaricine

3. Flavonoids: diosmin

ROSEMARY: an aromatic European shrub,

Rosmarinus officinalis, widely cultivated for its

grey-green evergreen leaves, which are used in

cookery for flavouring and yield a fragrant oil

used in the manufacture of perfumes: family

Labiatae (labiates). It is the traditional flower of

remembrance

LEAVES

FRESH DRY

Fp Fp

WATER

(ppm)

80,000

40,000

8,000

ACETONE

/ WATER

(ppm)

100,000

Rosmarinus’ extracts tested

Rubia peregrina L.

Common name: Wild

madder.

Constituents: rubian,

rubiadin, ruberythric acid,

purpurin, tannin, sugar, alizarin,

pseudopurpurin,

xanthopurpurin.

MADDER: any of several rubiaceous plants of

the genus Rubia, esp. the Eurasian R. tinctoria,

which has small yellow flowers and a red fleshy

root.

Rubia’s extracts tested

LEAVES

FRESH

Fp

WATER

(ppm)

80,000

40,000

8,000

Ruta graveolens L.

Common name:

Rue (Ruda)

Constituents:

furocouamarines, rutin, volatile

oils, alkaloids, coumarines.

RUE: any rutaceous plant of the genus Ruta,

esp. R. graveolens, an aromatic Eurasian shrub

with small yellow flowers and evergreen leaves

which yield an acrid volatile oil, formerly used

medicinally as a narcotic and stimulant. Archaic

name: herb of grace

Ruta’s extracts tested LEAVES STEAMS FLOWERS

FRESH DRY FRESH DRY FRESH DRY

Fp Ss Fp Ss Fp Ss Fp Ss Fp Ss Fp Ss

W

A

T

E

R (ppm)

100,000

80,000

50,000

40,000

10,000

8,000

1,000

100

A

C

E

T

O

N

E

/

W

A

T

E

R

(ppm)

100,000

80,000

50,000

40,000

10,000

8,000

1,000

100

Fp = Filter paper

Ss = Standard soil

Cistus ladanifer L.

Common name: Rock

rose (Heliantemo)

Constituents:

Monoterpenes: pinene,

camphene; Monoterpene

alcohols; Esters: linalyle

acetates; Phenols: eugenol,

thymol; Aldehydes; Cetones.

ROCKROSE: any of various cistaceous shrubs or

herbaceous plants of the Mediterranean genera

Helianthemum, Tuberaria, and Cistus, cultivated

for their yellow-white or reddish roselike flowers.

Cistus’ extracts tested

LEAVES

DRY

Fp

W

A

T

E

R

(ppm)

100,000

50,000

25,000

12,500

6,250

DEFINITIVE Ruta’S EXPERIMENTS ON

FILTER PAPER

LEAVES

DRY

Fp

W

A

T

E

R

(ppm)

80,000

40,000

20,000

10,000

5,000

ACETONE /

WATER

(ppm)

80,000

40,000

20,000

10,000

5,000

Ruta’s dry leaves

water extracts

Mortality over time

0

20

40

60

80

100

0 5 10 15 20

Time in days

% m

ort

ali

ty

80000 ppm 40000 ppm 20000 ppm

Dose-response curve/ LD50

22939

18455

32707

0

20

40

60

80

100

1000 10000 100000

Dose in ppm (log scale)

% m

ort

ali

ty

5 days 7 days 10 days LD50

Hatches

0

20

40

60

80

100

0 5 10 15 20 25

Time in days

% h

atc

h

Control 10000 ppm 5000 ppm

Ruta’s dry leaves

acetone / water (7:3) extracts

Mortality over time

0

20

40

60

80

100

0 5 10 15 20 25

Time in days

% m

ort

ali

ty

80000 ppm 40000 ppm 20000 ppm

10000 ppm 5000 ppm

Dose-response curve

27841786216907

0

20

40

60

80

100

1000 10000 100000

Dose in ppm (log scale)

% m

ort

ali

ty

2 days 3 days 5 days LD50

Hatches

0

20

40

60

80

100

0 5 10 15 20 25

Time in days

% h

atc

h

Control 5000 ppm 2500 ppm 1250 ppm

Asphodelus album Mill.

Common name: Asphodel

Constituents: alcohol, inuline, volatile oil

ASPHODEL: any of various S European liliaceous

plants of the genera Asphodelus and Asphodeline,

having clusters of white or yellow flowers. Compare

bog asphodel.

Asphodelus’ extracts tested

FRUITS

DRY

Fp

W

A

T

E

R

(ppm)

100,000

50,000

25,000

12,500

6,250

Asphodelus’ dry fruits

water extracts

Mortality over time

0

20

40

60

80

100

0 5 10 15 20

Time in days

% m

ort

ali

ty

100000 ppm 50000 ppm

Dose-response curve

53938

0

20

40

60

80

100

10000 100000

Dose in ppm (log scale)

% m

ort

ality

3 days LD50

Hatches

0

20

40

60

80

100

0 5 10 15 20 25

Time in days

% h

atc

h

Control 50000 ppm 25000 ppm

12500 ppm 6250 ppm

CONCLUSIONS

Up to now, Ruta and Euphorbia extracts showed the highest ovicidal activity on paper at the lowest doses and showed some ovicidal activity on soil.

From now on, although we shall continue the tests with new plants, we shall concentrate the research on these two plants.

We have already started to do a number of

new extracts of both Ruta and Euphorbia.

Using a variety of solvents in order to locate

and if possible, to identify the active

constituents responsible of the ovicidal

activity.

Also, for this year we plan to test some of

the extracts on natural soil under semi-

natural conditions.

FRACTIONAL

EXTRACTION OF

SOLVENTS

PLANT EXTRACTS

The process has 4 steps:

Maceration 3 times with MeOH

Concentration and test

Division of solvents

Concentration and test

First and second step: maceration

with MeOH, concentration and test.

Dry powder plant was put in an Erlenmeyer flask.

The solvent was put on it. After 10 hours the solution was passed through a filter paper.

The solvent was concentrated in a rotavapour. Over the plant was placed more MeOH.

This process was repeated 3 times.

The solvent was accumulated in the same glass ball. Then it was concentrated to vacuum in the rotavapour.

Part of the extract obtained was used in the tests, the rest of them was used in the extraction process.

Powder of the plant with MeOH

ROTAVAPOUR

Third and fourth step: division of

solvents, concentration and test.

The solvents used were:

o Cl4C

o CH2Cl2

o Hexane

o BuOH

SUMMARY

METHANOLIC EXTRACT

Hexanic extract

Cl4C extract

CH2Cl2 extract

n-BuOH extract Water extract

MeoH:H2O 8:2

Cl4C

MeOH:H2O 9:1

Hexane (x2)

MeOH:H2O 6:4

CH2Cl2

1º) remove MeOH

2º) n-BuOH (x4)

CONTROL DE LAS PLAGAS DE BABOSAS EN CULTIVOS

HORTÍCOLAS. EFECTOS COLATERALES DE LOS

MOLUSQUICIDAS SOBRE LA FAUNA EDÁFICA*.

1.- Indica los tipos de disolventes empleados para extraer los principios activos

empleados como ovicidas

2.- Indica 2 plantas cuyos extractos tienen efecto ovicida

3.- Dibuja un huevo de la babosa Deroceras reticulatum y señala sus partes

4.- Indica las partes de las plantas donde se encuentran los principios activos

con acción ovicida y señala los procesos previos que se realizan antes de

proceder a la extracción de estos

5.- Indica las causas por las que los extractos de Ruda tiene efecto ovidica sobre

papel de filtro, pero no sobre suelo estándar (artificial)

6.- Factores exógenos que afectan al ciclo biológico de las babosas

* En el respeto al equilibrio está el éxito de nuestra lucha particular

Fin de la Comunicación

*