PLANT EXTRACTS

ABSTRACT - Extratos brutos em etanol, clorofórmio e hexano de três plantas de ocorrência freqüente na província de Córdoba, [Aloysia polystachia (Griseb) Moldenke (Verbenaceae), Solanum argentinum Bitter et Lillo (Solanaceae) e Tillandsia recurvata (L.)L. (Bromeliaceae)] foram avaliadas em relação à repelência e deterrência alimentar sobre Sitophilus oryzae (L.). O potencial antialimentar desses extratos foi determinado através do teste de deterrência ou dissuasão nutricional utilizando-se discos constituídos  de trigo. Para cada extrato determinou-se o Coeficiente de Deterrência Total (T). Para a avaliação da repelência foram conduzidos testes utilizando-se papéis de filtro separados em duas partes iguais. Os extratos foram aplicados numa dessas partes em concentração de 0,31 mg.cm^-² e posteriormente determinadas as porcentagens de repelência (PR) de cada extrato. Comparando-se todas as espécies vegetais e solventes avaliados, os extratos em clorofórmio (classe ++++) de A. polystachia seguido pelos extratos em etanol (classe ++++) e em hexano (classe +++) dessa mesma espécie, apresentou o maior efeito deterrente sobre S. oryzae. Observou-se ainda um moderado efeito de repelência dos extratos de S. argentinum e A. polystachia sobre S. oryzae, destacando-se o extrato em hexano de S. argentinum como o mais efetivo repelente (classe 4). Entre os extratos avaliados, o extrato em clorofórmio de A. polystachia apresentou o mayor efeito deterrente e o extrato em hexano de S. argentinum o maior efeito de repelência.

KEYWORDS - Extratos vegetais, repelência, efeito antialimentar. 

RESUMO - Plant extracts in ethanol, chloroform and hexane of three widely distributed plants in the province of Córdoba were evaluated for their repellency and feeding deterrency to Sitophilus oryzae (L.). The plant species studied were, Aloysia polystachia (Griseb) Moldenke (Verbenaceae); Solanum argentinum Bitter et Lillo (Solanaceae) and Tillandsia recurvata (L.) L. (Bromeliaceae).  Antifeedant potential of these extracts was determined by the deterrence or antifeedant test using wheat wafer disks. The Total Coefficient of Deterrence (T) was determined for each extract. Repellency tests were performed using filter-paper circles cut in halves. The extracts were applied on each half at a concentration of 0.31 mg.cm^-2. Percentual Repellency (PR) was determined for each extract. The chloroform extracts of A. polystachia had the strongest antifeedant effect (class ++++) followed by the ethanol extracts (class ++++) and hexane extracts of the same species (class +++). Extracts of A. polystachia had stronger antifeedant effect than S. argentinum and T. recurvata extracts. A moderate repellent effect of S. argentinum and A. polystachia extracts on S. oryzae was observed; the hexane extract of S. argentinum was the one with strongest repellency (class 4). Among the extracts analyzed,  the strongest antifeedant effect occur with the chloroform extract of A. polystachia and the hexane extract of S. argentinum showed the strongest repellency effect. 

PALAVRAS-CHAVE - Crude extracts, repellency, antifeedant effect. 
 

Table 1

Table 2

Table 3

Plants have developed for 400 million years and have acquired effective defense mechanisms that ensure survival under rough environmental conditions and in the presence of natural enemies. Besides a number of morphological protective mechanisms, plants have developed subtile chemical defense mechanisms against insects and other organisms; these defense mechanisms do not generally produce immediate death but do affect common biochemical and physiological functions (Prakash & Rao 1997). Until a few decades ago, plant secondary metabolites were considered substances with no specific function, which only reflected an aspect of biodiversity (Pérez Izquierdo & Ocete 1994). Recent chemical ecology studies have shown that many of these secondary compounds play an important role in plant-insect relations. Some compounds, either separately or synergically, make up a chemical defense barrier in the plant against certain pests and diseases.

Synthetic insecticides have been widely developed and are extensively used because of their effectiveness and easy application and storage. However, their extensive use has brought about severe disadvantages, like environmental disturbances, pest recovery, pest resistance, lethal effects on non-target organisms, and toxicity to users and consumers (Prakash & Rao 1997). Evaluating and using botanical pesticides, either as crude or formulated extracts, is an alternative strategy. Botanical pesticides tend to have broad-spectrum activity, are relatively specific in their mode of action and easy to process and use. Furthermore, they would be safe for higher animals and the environment and could be easily produced by farmers and small-scale industries (Talukder & Howse 1994). According to Prakash & Rao (1997) these pesticides do not contribute to resistance development or pest resurgence, nor do they cause negative effects on non-target organisms; also, they do not affect plant growth, seed viability or food quality of products.

The international literature on the biological properties of crude extracts and isolated secondary substances of plants against different insects and other organisms is abundant. Jilani & Su (1983) and Jilani et al. (1988) conducted insect repellency assays using extracts of different plants on stored-product pests. Talukder & Howse (1994) mentioned the toxic and repellent properties of extracts of Aphanamixis polystachya against S. oryzae.

The effect of a crude extract of Melia azedarach fruits on Tribolium castaneum was studied by Del Tío et al. (1996). Also, Ocete & Pérez (1996) evaluated the effect of extracts of Daphne gnidium and Anagyris foetida on several insect pests. Boeke et al. (2004) evaluated the efficiency of 23 different plant extracts on Callosobruchus maculatus and found repellency of volatile oils.

Prakash & Rao (1997) conducted an extensive revision of plants containing active secondary substances against insects. These authors provide information on 866 plant products with insecticidal activity –repellent, antifeedant, or regulatory of insect growth– used against agricultural pests. Pretheep-Kumar et al. (2004) observed promising results in the protection of rice against S. oryzae using protein-enriched pea flour extract. Rahman & Talukder (2006) studied the bioefficacy of seven plant derivatives on C. maculatus in Bangladesh. They obtained better results with the vegetal oils, mainly of Azadirachta indica. In Brazil, Tavares & Vendramim (2005) studied the bioactivity of Chenopodium ambrosioides on S. zeamais, whereas Oliveira & Vendramim (1999) observed repellent properties of essential oils and powders from three plant species on Zabrotes subfasciatus.

In Argentina this subject has received less attention. Peñaloza (1995) studied the biological effects of crude extracts of six plants on Tribolium castaneum. Novo et al. (1997, 1998) observed the repellent activity of several crude extracts of four native plants against T. castaneum and the antifeedant effect on Anticarsia gemmatalis. Valladares et al. (2003) examined the antifeedant activity of an extract of senescent leaves of Melia azedarach on nine insect species, including S. oryzae. This work explores the presence of secondary substances with biological properties against insects in wild plants of central Argentina with the aim of including them in integrated pest management programs. The aim of this work was to evaluate the repellent and antifeedant properties of crude extracts of common plants from the province of Córdoba, Argentina, against S. oryzae, one of the most important cereal pests of world-wide distribution.

Material and Methods

Preparation of Plant Extracts. Crude extracts of the following plant species were used: Aloysia polystachia (Griseb) Moldenke (Verbenaceae)(leaves); Solanum argentinum Bitter et Lillo (Solanaceae) (leaves), and Tillandsia recurvata (L.) L. (Bromeliaceae) (whole plant). Plant material was air-dried (in shade conditions) and fragmented. Maceration was performed in solvents of different polarity (ethanol, chloroform and hexane) for 72 h with the aim of extracting different plant components. Each extract was then concentrated to dryness using a rotary vacuum evaporator until constant weight was obtained. The extracts obtained were dissolved in the corresponding pure solvent until a 10% (w/v) stock solution was obtained.

Insect. A stored-product pest species, the weevil Sitophilus oryzae (L) (Coleoptera: Curculionidae) was used for the bioassays. Weevils were reared in growth chambers at 27±2°C and relative humidity of 75±5% with alternating light and dark periods of 12 h. Jars covered with a fine piece of cotton cloth were used to allow the passage of air; weevils were fed on wheat grains.

Adults of S. oryzae were placed in the rearing medium for 10-15 days; then they were removed and placed in a new medium to obtain a new progeny and avoid generation overlapping. The medium containing the eggs was placed in the growth chamber until adult emergence. This process was successively repeated with the aim of obtaining homogenous generations. 

Feeding Deterrent Activity Bioassays. The potential of the antifeedant effect of the extracts was determined by the feeding deterrency test described by Talukder & Howse (1994). Wheat wafer disks were used as test food; the disks were saturated by dipping into either solvents only (control C) or into 10mg.mL^-1 solution of each extract (treatment T) and were air-dried for 20 h. Wheat disks were then weighed and 10 weevils were placed on them; the disks were reweighed after 6 days. Food consumption of weevils was recorded under three conditions: 1) on pure food, composed of two untreated disks (CC); 2) on food with possibility of choice between one treated (T) and one untreated (C) disk: choice test, and 3) on food with two treated (TT) disks: non-choice test.

Blank disks treated with the solvent but not offered to weevils were prepared. The experiment was arranged in a randomized complete design with five replications. Disks were dipped for two different time periods: 15 and 30 seconds. Once dried, wheat disks may increase in their weight as a result of water absorption from the surrounding air which was humidified for the normal growth and development of weevils. Hence, a correction procedure was applied. 

Disk weight loss, which was estimated as the amount of food consumed (FC), was calculated by the formula of Serit et al. cited by Talukder & Howse (1994):

where: IW= initial weight of disk after being treated with extract or solvent, FW= final weight of disk, subscript b= blank disk (treated with solvent and not offered to weevils), and subscript s = treated and control disks, on which weevils were released. 

According to the amount of food consumed in the three different tests (CC, TT, and CT) three feeding deterrent activity coefficients were calculated: 

• Absolute coefficient of deterrency A= (CC - TT/CC + TT)*100
• Relative coefficient of deterrency R= (C - T/C + TO)*100
• Total coefficient of deterrency T= A + R

Repellent Activity Bioassays. Repellency tests were conducted following the method proposed by Talukder & Howse (1993, 1994). Filter-paper circles of 9 cm in diameter were cut in half. Extracts were applied on one half at a concentration of 10mg.mL^-1. One mL of solution was uniformly applied with a pipette, in such a way as to have a treated substrate of 0.31 mg.cm^-2 extract. The treated half-circles were air-dried until the solvent was totally evaporated. The treated and the untreated half-circles were placed contiguously on the Petri dishes and 10 adult weevils were released on each dish. Weevils present in each half circle were counted at hourly intervals for 5 h after treatment.  Data were converted to express percentage repulsion (PR) using the following formula: PR (%) = (Nc – 50) x 2, where N_c is the percentage of weevils present in the control half. Positive values (+) indicated repellency and negative values (-) attractancy. Five replications were made of each treatment. Data were analyzed using a two factor completely randomized design using the different plant species and solvents as the two factors. Mean separation of repellent activity of the different extracts and the comparisons analyses between plant species and solvents were made using Fisher’s least significant difference (LSD) test (P0,05) (INFOSTAT, 2001). Mean values were classified according to the following scale: 
 

Results and Discussion

Antifeedant Activity. The extract with the strongest antifeedant effect on S. oryzae was the chloroform extract of A. polystachia, in both treatment times (class ++++). An important antifeedant effect was also observed in the ethanol extracts of A. polystachia (class ++++), and with the hexane extracts of the same species to a lesser degree (class +++) (Table 1). 
 

The analysis of antifeedant effect of each plant, regardless of the solvent or immersion time used, shows that A. polystachia was significantly stronger than S. argentinum and T. recurvata. However, no significant differences were observed between the extracts of T. recurvata and S. argentinum (Table 2). Contrasts between solvents indicate that the hexane extracts were significantly less potent than chloroform and ethanol extracts; however, no significant differences were detected between the two latter extracts (P=0,9429). Contrast between immersion times indicated that they were not significantly different (P=0,6601), suggesting that using either of them renders no difference (Table 2).
 

Similar results of antifeedant effect on S. oryzae and T. castaneum were observed by Talukder & Howse (1993, 1994) with four different extracts of Aphanamixis polystachya, acetone extract being the most significant with a total deterrency coefficient of 159.5. Valladares et al. (2003) also indicated an antifeedant effect of ethanol extract of senescent leaves of Melia azedarach on S. oryzae, with an antifeedant index of 100%. 

Repellency Effects. Among all the combinations of plant species and solvents tested, the hexane extract of S. argentinum was the one with the strongest repellent effect on S. oryzae (class 4) with PR of 63%, followed by the ethanol extract of this species (class 3). Other extracts with significant repellency activity were the chloroform extract of A. polystachia (class 3) and the ethanol extract of T. recurvata (class 3) (Table 3). The percentage of repellency observed during the 5 hours of the test did not show a defined behaviour either between each hour. The factorial analysis indicate significative differences between plant species (P=0,0004), solvents (P=0,0396) and in the interaction plant species vs. solvents (P=0,0006). The analysis of the repellency effect of each plant species, regardless of the solvent used, shows that the extracts of A. polystachia and S. argentinum did not differ significantly but were significantly stronger than T. recurvata (Table 3). Comparisons between solvents used indicate that the repellency effect of ethanol extracts was significantly higher than chloroform extracts but was not significantly different of hexane extracts (Table 3). 
 

In general, the extracts of A. polystachia and S. argentinum had a moderate repellency effect on S. oryzae. These results agree with those obtained by Talukder & Howse (1993, 1994) with extracts of A. polystachya on S. oryzae (1994) and T. castaneum (1993), although the latter species showed a higher repellency effect; the authors also found a higher repellency effect of extracts on S. oryzae, mainly extracts of methanol (67%), acetone (67%), and ethanol (57%). Jilani & Su (1983) reported the repellent effects of extracts of three common plants in Pakistan on three stored-product pests, including Sitophilus granarius. The powdered extract of Curcuma longa had the strongest repellency effect on the three species studied.  Oliveira & Vendramim (1999) observed repellency to vegetal oils and powders on Zabrotes subfasciatus in bean seeds. The strongest repellency was obtained with the oils of Cinnamomum zeylanicum (96.2% repellency) and of Azadirachta indica (89.4% repellency). Pretheep-Kumar et al. (2004) found that an extract of protein-enriched bean flour had a high level of repellency on S. oryzae. They found 76.3% and 91,2% of repellency with a concentration of 0.1% and 1% of the extract, respectively, after 48 h after the assay was initiated. In contrast, Tavares & Vendramim (2005) reported the insecticide activity but a lack of repellent activity of extracts of Chenopodium ambrosioides on S. zeamais; these findings do not agree with the results observed in the present work, in which extracts showed repellent activity but no toxic effects. 

Among the extracts analyzed, the strongest antifeedant effect occur with the chloroform extract of A. polystachia, followed by the ethanol extracts and, to a lesser degree, the hexane extract of the same species. Regardless of the solvent and immersion time used, the extracts of A. polystachia show a stronger antifeedant effect than the extracts of S. argentinum and T. recurvata. The results of the repellency bioassays indicate that the hexane extract of S. argentinum is the one with strongest repellency effect on S. oryzae, with a PR of 63% (class 4). In general, the extracts of S. argentinum and A. polystachia showed a moderate repellency effect on S. oryzae.

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