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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.45 n.3 Concepción set. 2000

http://dx.doi.org/10.4067/S0366-16442000000300006 

INSECTICIDAL ACTIVITY OF Plocamium cartilagineum
MONOTERPENES

V. Argandoña 1, T. Del Pozo1, A. San-Martín 2 and J. Rovirosa 2*

Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
1Departamento de Biología, 2 Departamento de Química
(Received: February 23, 2000 - Accepted: May 24, 2000)

ABSTRACT

This work describes the insecticidal activity of two halogenated monoterpenes, (mertensene and violacene) isolated from the red alga Plocamium cartilagineum, and two derivatives (dibromomertensene and dihydromertensene), towards the tomato moth Tuta absolute (Povolny) and the greenbug, Schizaphis graminum, a cereal aphid. The toxicity of violacene to aphids was higher than other compounds causing 92 % aphid mortality after 48h and was similar to insecticides used as aphicides. Violacene, dibromomertensene and dihydromertensene reduced the reproduction index of aphids. Dibromomertensene and violacene protected tomato plants against the tomato moth.

Key Words: Plocamium, halogenated monoterpenes, insecticidal activity

RESUMEN

Este trabajo describe la actividad insecticida de dos monoterpenos halogenados ( mertenseno y violaceno) aislados del alga roja Plocamium cartilagineum, y dos derivados (dibromomertenseno y dihidromertenseno), contra la larva del tomate Tuta absoluta y el pulgón verde, Schizaphis graminum, un áfido de los cereales. La toxicidad de violaceno para los áfidos fue mayor que la de los otros compuestos causando un 92% de mortalidad de los áfidos a las 48h y fue similar a la de los insecticidas usados como aficidas. Violaceno, dibromomertenseno redujeron el índice de reproducción de los áfidos. Dibromomertenseno y violaceno protegieron a las plantas de tomate contra la polilla del tomate.

Palabras claves: Plocamium, monoterpenos halogenados, actividad insecticida.

INTRODUCTION

Seaweeds of the family Plocamiaceae, (order Gigartinales) which are distributed world-wide have been extensively investigated. Each of eight different Plocamium species contain monocyclic or acyclic halogenated monoterpenes. At least three different monoterpenes occur in more than one species, a phenomenon which suggests a common biosynthetic link between them. The largest variety of halogenated monoterpenes has beeen isolated from Plocamium cartilagineum, the most cosmopolitan Plocamium species, with collections ranging from such diverse locations as the Pacific coasts of North America and Australia, the Mediterranean, the Isle of Wight and the inhospitable Janus Island of Antarctica. The taxonomy of P.cartilagineum has been difficult in that two distinct morphological forms seem to exist 1).

Plocamium cartilagineum L.(Dixon) is a marine algae (Rhodophyceae), with a wide geographic distribution. Eight monoterpenes have been detected in samples collected throughout the central coast of Chile. Mertensene 1 and violacene 2 are the most abundant of the monoterpenes identified 2). The relative concentration of these compounds is variable. It has been suggested that this variation could be related to the geographical location where the alga is found 3).

The insecticidal and acaricidal activities of polyhalogenated monoterpenes from P. cartilagineum have been demonstrated against various insects including: Spodoptera frugiperda, Heliothis virescens, Ostrinia nubilalis, (Lepidopterous. Noctuidae), Antronomus grandis, Diabrotica undecimpunctata, Tetranychus urticae and Aphis fabae (Homopterous)2). 2 was the compound that showed the greatest insecticidal activity against the four last organism, while mertensene had a moderate activity only against Aphis fabae. Some of these compounds possess insecticidal activity or inhibit the growth of insects larvae such as the tobacco moth, and the mosquito Caulax pipiens pallens 4). Other monoterpenes have cytostatic action and antibacterial activity against Bacillus sphaericus, Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa and Proteus sp.5). Plocamene and costatol are terpenes that inhibit the oxidative phosphorylation 6).

The tomato moth Tuta absoluta is a microlepidopteran that affects tomato crops. The female oviposites a large number of eggs (10 - 50), on the leaf, shoots or fruits approximately 10 hours after copula. The larvae feed on parenchyma tissue, transform in pupae and become adults after 12 or 15 days. Shizaphis graminum (Rondani) is a worldwide pest aphid of a number of graminaceous crops.

The purpose of this work was to test the insecticidal activity of mertensene, violacene and derivatives against the tomato moth and the greenbug of cereals.

EXPERIMENTAL

General Methods. IR spectra were obtained with a Perkin-Elmer 1650/ FTIR spectrometer in CHCl3 solutions. EIMS spectra were taken on a Micromass Autospect spectrometer. 1H NMR and 13C NMR spectra were measured employing a Bruker AMX 300 instrument operating at 300 MHz for 1H NMR and at 75.13 MHz for 13C NMR, using TMS as internal standard. HPLC separations were performed with a Merck-Hitachi L-6200 Intelligent pump and a L-4000 UV detector using a Hibar column. The gel filtration column (Sephadex LH-20) used hexane-MeOH-CHCl3 (2:1:1) as eluent solvent. Merck silica gel 7734 and 7729 were used for column chromatography. The spray reagent for TLC was H2SO4: MeOH (1:9).

Extraction and Isolation. Plocamium cartilagineum (Linnaeus) Dixon was collected by a scuba diver off the coast of Algarrobo (Chile) at a depth of 18 m. A sample of the specimen has been deposited at the Museo de Historia Natural, Santiago de Chile. The dried alga (1100 g) was extracted exhaustively with acetone at room temperature to give a dark residue (40 g). This extract was chromatographed by flash chromatography on silica gel. The fraction eluted with hexane: EtOAc (95: 5) was further separated by filtration chromatography and Si-gel chromatography to yield mertensene 1 (131 mg). The fraction eluted with hexane: EtOAc (4:1) (2.57 g) was further separated by filtration chromatography to give violacene 2 (235 mg).

Compound 3. Mertensene (50 mg) was treated with bromine in CCl4 at room temperature for 12 hr. The reaction mixture was washed with 5% NaOH. The CCl4 layer was dried over Na2SO4 and evaporated to a residue. The residue was crystallised in n-hexane to obtain 32 mg of 3 ( 64% ). White crystals, mp. 106 º (hexane) ;IR (CHCl3) n max 3000, 2950, 1470, 1105, 1017, 823, 752, 705, 682, 600 cm-1; 1H NMR (300 MHz, CDCl3); d 1.37 (3H, s), 1.74 (3H, s), 2.38 (1H, ddd, J=3.6; 4.0; 14.0 Hz), 2.57 (1H, m), 2.62 (1H, d, J= 14.6 Hz), 2.87 (1H, d, J = 14.6 Hz). 4.15 (1H, dd, J= 4.0; 13.0 Hz), 4.49 (1H, dd, J=3.6; 3.9 Hz), 4.82 (1H, d, J=1.1 Hz), 6.23 (1H, d, J=1.1 Hz). 13C NMR: 20.9 (CH3), 26.1 (CH3), 40.4 (CH2), 46.5 (CH2), 47.5 (CH2) 55.8 (CH), 57.0 (CH), 66.6 (CH), 70.7 (C), 72.6 (CH). EIMS m/z (rel.int.) :488 (2), 486 (1), 484 (3), 482 (12), 480 (20), 478 (14), 476 (2), 407 (10), 405 (2), 403 (1), 401 (2), 261 (4), 259 (15), 257 (65), 197 (30) 195 (14), 193 (60), 187 (5), 185 (3) .

Compounds 4 and 5. Mertensene (60 mg) dissolved in 50 mL of CH2Cl2 was treated overnight with H2 using Pt/C as a catalyst. After usual work up, and purification on silica gel column, compounds 4,19 mg ( 32 %) and 5, 24 mg ( 40 %) were isolated. Compound 4. White crystals; mp.480 (hexane); IR (CHCl3) n max 2950, 1475, 1330, 1140, 1050, 825, 680 cm-1.1H NMR (300 MHz, CDCl3) d : 1.17 (3H, s), 1.74 (3H, s), 2.12 (1H, d, J=14.1 Hz), 2.18 (1H, m), 2.40 (1H, m), 2.45 (1H, m), 2.48 (1H, d, J=14.1 Hz), 2.60 (1H, ddd, J=4.1; 4.4; 13.9 Hz), 3.55 (2H, t, J=.8.3 Hz), 3.92 (1H, dd, J=4.1; 12.6 Hz), 4.14 (1H, dd, J=4.4; 12.6. Hz) .13C NMR: 21.1 (CH3), 26.1 (CH3), 39.6 (C), 40.9 (CH2), 47.1 (CH2), 52.2 (CH2), 57.3 (CH), 67.4 (CH2), 67.5 (CH), 71.2 (C). EIMS m/z (rel.int.) :260 (1), 258 (1), 256 (1), 219 (4), 217 (1), 215 (1), 209 (1), 207 (1), 205 (3), 187 (5), 185 (5), 183 (1), 173 (36), 171 (100), 169 (2), 137 (2), 135 (72), 133 (4), 107 (39), 105 (24), 103 (10), 93 (18), 91 (35), 89 (12) . Compound 5. White crystals; mp. 780 (hexane); IR (CHCl3) n max. 2960, 1480, 1330, 1140, 1060, 820, 680 cm-1.1H NMR (300 MHz, CDCl3) d:0. 86 (3H, dd, J=7.5; 7.5 Hz), 1.08 (3H, s), 1.27 (1H, m), 1.62 (1H, m), 1.72 (3H, s), 2.03 (1H, d, J=14.4 Hz), 2.38 (2H, m), 2.60 (1H, m), 3.92 (1H, dd, J=4.4; 8.2 Hz), 4.14 (1H, dd, J=4.4; 8.2 Hz). 13C NMR: 7.8 (CH3), 21.3 (CH3), 26.2 (CH3), 36.8 (CH2), 40.6 (C), 41.4 (CH2), 51.5 (CH2), 57.9 (CH), 68.0 (CH), 72.1 (C). EIMS m/z (rel.int.) 292 (1), 290 (4), 288 (22), 286 (12), 251 (8), 249 (3), 183 (5), 181 (42), 180 (23), 179 (4), 178 (16), 136 (100), 134 (60), 102 (65), 101 (18), 100 (12), 99 (4).

Plant material. Tomato plants (cultivar Rome) were grown in greenhouse under 15 - 20 ° and a 12/12h light/dark photoperiod. When plants had 3 or 4 leaves they were transplanted to pots, situated into entomological cages and infected with larvae of the tomato moth. Barley plants (cultivar Aramir) were grown in pots under control conditions of temperature (25°) of 12/12 h light/dark photoperiod and irrigated with Hoagland solution.

Aphids. Schizaphis graminum were collected from colonies maintained on Hordeum distichum cv Aramir grown in chamber under 12/12h light/dark photoperiod and 25 ± 4°C.

Tomato larvae of T. absolute were collected from colonies maintained on tomato plants cv Rome in a grown in chambers under 12/12 h, light/dark a photoperiod and 15-20 °.

Feeding assays. Aphid feeding assays were done using holidic diets placed between two layers of parafilm M7). The diet consisted of solutions containing 35% sucrose, aminoacids and mineral salts, adjusted to pH 6 dissolved in H2O-Me2CO (2:3). Compounds were added to the diet at different concentrations and the mortality of aphids was determined at different times. Six or eight samples containing 10 aphids each were used for each treatment.

Larvae of T. absolute were fed with leaves taken from tomato variety Rome sprayed with different concentration of compounds dissolved in acetone. The leaves were placed in a Petri disc that contain a moistened filter paper. The solution (1mL) was sprayed on the leaves faces. After 15 minutes when, the acetone was evaporated, one larva was placed on each leaf. Each treatment was repeated 10 times. The quantity of leaf eaten by the larva and the mortality of insect were measured after 24 h.

The quantity of leaf eaten was determined as follow: each leaf was weighed. The contour of the leaf was drawn on a milimetric paper. After the assay the damage area of the leaf was measured.

  Damaged area x Weigh of the leaf
Quantity of leaf eaten =
------------------------------------------------------------
  Total area of the leaf

Statistical analysis of data: We performed ANOVAS and Tukey test to evaluate the existence of differences among treatments.

RESULTS AND DISCUSSION

Chemical analysis. Two halogenated monoterpenes, mertensene 1 and violacene 2, (Fig 1) were isolated and identified espectroscopically by comparison with authentic samples from acetone extracts of P. cartilagineum. Compounds were purified by chromatography (Sephadex LH-20 and silica gel columns).


Fig. 1

Derivatitation of compounds. Compound 1 was treated with bromine in carbon tetrachloride at room temperature to give the dibromo derivative 3. Its structure was confirmed by spectroscopic methods (see experimental). The addition of hydrogen to 1 was carried out at room temperature in presence of Pd/C catalyst. Two products were obtained and separated by column chromatography on silicagel (4 and 5). The spectroscopic data confirm the addition of hydrogen with chlorine loss at carbon 8 in compound 5 (Fig 1).

Effect of compounds on aphid mortality. The insecticide activity of the compounds on Schizaphis graminum was measured by offering the insect an artificial diet containing the compounds. Violacene was the most toxic compound (Table I). Mortality measured after 12, 24 and 48 h of assay was similar to the insecticide (Demeton S-metil) and significantly higher than the mortality obtained in the control and with the other compounds (p< 0.05). Violacene caused over a 90% of mortality after 48 hours. Mertensene had only a slight effect on aphids. Dibromomertensene 3 and dihydromertensene 4 did not affect aphid mortality. When 1 was present in diets at concentrations of 250 ppm it caused minor mortality on aphids than 100 ppm at 12 and 24 hours. However after 48 hours the mortality was the same in both 100 and 250 ppm. These results suggest that mertensene at 250 ppm could be feeding deterrent and the aphids dead after 48h due to starvation.

Effect of compounds on aphid reproduction. Compounds 3 and 4 affected significantly the reproduction index of greenbug after 72h decreasing this index around 55% with respect to control. Violacene caused only a light reduction with respect to control. Mertensene did not affect aphid reproduction (see table II).

Table II. Effect of Monoterpenes of P. cartilagineum on aphid.

Effect of compounds on tomato moth. -The results in Table III show that the amount of leaf eaten by larvae in those sprayed with mertensene or dibromomertensene at 100, 250, 500 ppm or with violacene at 250 and 500 ppm was significantly lower than controls. The percentage of damage in the leaves sprayed with the compounds at the indicated concentrations was significantly lower than in control leaves (the total leaf area was considered as 100%) Larvae mortality was significantly higher in assay with leaves containing mertensene at 250 and 500 ppm, or dibromomertensene at 100, 250 and 500 ppm or violacene at 500 ppm, than in control leaves. No damage was observed in the leaves sprayed with the compounds at 500 ppm. This fact suggests that these compounds may act as antifeedant at 500 ppm.

ACKNOWLEDMENTS

This work was supported by FONDECYT (Grant Nº 1990935).

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