Growth inhibitory effect of extracts from Reynoutria sp. plants against Spodoptera littoralis larvae
Efecto inhibitorio del crecimiento de los extractos de plantas de Reynoutria sp. contra larvas de Spodoptera littoralis
Roman Pavela1* , Nadezda Vrchotová2 and Bozena Šerá2
1 Crop Research Institute. Drnovská 507. 161 06 Prague 6. Czech Republic *Author for correspondence: (pavela@vurv.cz).
2 Institute of Systems Biology and Ecology AS CR, v.v.i. Branišovská 31. 370 05 Ceské Budejovice. Czech Republic.
]]>Received: July, 2007.
Aproved: May, 2008.
Abstract
Laboratory studies were conducted to determine the effects of methanolic extracts from three Reynoutria species on feeding, development and mortality of larvae S. littoralis. The extracts of all three Reynoutria species did not reveal feeding deterrence in the Spodoptera littoralis larvae. However they significantly decreased the larvae growth and the efficiency of conversion of the ingested and digested food. Conversion of the ingested food was lower, when compared with control larvae. The use of food provoked prolongation of the larvae development and chronic toxicity. Even if extracts of all three plant species were effecting the larvae mortality with the same results (LC50 ranged from 0.5 to 1 mg g–1 in all extracts), the impact on growth inhibition in the larvae varied. The most efficient extract was R.xbohemica interspecific hybrid (LC50 was 1.24 mg g–1), then the extract of R. japonica (LC50 of 6.72 mg g–1), and the least efficient was the R. sachaliensis extract (LC50 of 9.48 mg g-1).
Key words: Reynoutria, Spodoptera littoralis, growth inhibition, botanical insecticides, toxicity test, plant extracts.
Resumen
Se realizaron estudios en laboratorio para determinar los efectos de extractos metanólicos de tres especies de Reynoturia sobre alimentación, el crecimiento y la mortalidad de larvas de S. littoralis. Ninguno de los extractos de las especies de Reynoturia mostró disuasión alimentaria en las larvas de Spodoptera littoralis. Sin embargo, sí provocaron una disminución significativa en el crecimiento de larvas, así como en la eficiencia de conversión del alimento ingerido y digerido. La conversión de alimento ingerido fue menor comparada con las larvas testigo. El uso de alimento ocasionó prolongación del crecimiento de larvas, así como toxicidad crónica. Aunque los extractos de las tres especies de plantas afectaron la mortalidad de las larvas de manera similar (LC50 varió entre 0.5 y 1 mg g-1 en todos los extractos), el impacto en la inhibición del crecimiento de las larvas varió. El extracto más eficiente fue el híbrido inter–específico R. bohemica (LC50 de 6.72 mg g-1), seguido por el extracto R. japonica (LC50 de 6.72 mg g–1); el menos eficiente fue el extracto R. sachaliensis (LC50 de 9.48 mg g–1).
]]> Palabras clave: Reynoutria, Spodoptera littoralis, inhibición del crecimiento, insecticidas botánicos, prueba de toxicidad, extractos de planta.
INTRODUCTION
Due to the increasing problem with resistance, impacts on non–target organisms and residue in food, associated with the use of toxic synthetic insecticides, it is needed to develop ecologically safer botanical insecticides. The search for plants with novel insecticidal constituents is very intensive. Among the plant families studied (Asteraceae, Lamiaceae, Meliaceae, Piperaceae, Rutaceae) crude extracts compounds have shown toxicity (Pavela, 2006), antifeedant activity (Wheeler and Isman, 2001; Sadek, 2003; Pavela, 2004a), presence of insect growth regulators (Akhtar and Isman, 2004; Pavela, 2004b; Pavela, 2005), oviposition deterrence (Dimock and Renwick, 1991; Zhao et al., 1998), suppression of calling behaviour (Khan and Saxana, 1986) and reduction of fecundity and fertility (Pavela et al., 2005). Such a wide variety of effects provides potential alternatives for the use of conventional insecticides.
Some other promising families, e.g. Annonaceae, Apiaceae, Asteraceae, Lamiaceae and Polygonaceae, have documented antifungal, antiviral, and antibacterial properties (Inoue et al., 1992; Konstantinidou–Doltsinis and Schmitt, 1998). Equeous and ethanolic plant extracts from the Giant Knotweed (Reynoutria sachalinensis), protect greenhouse–grown cucumbers, tomatoes and begoniae from powdery mildew fungi (Herger et al., 1988; Neuhaus and Pallut, 1992). These plants contain biologically active compounds, such as catechin, epicatechin, chlorogenic acid, caftaric acid and quercetin derivatives, resveratrol and their derivates (Vrchotová et al., 2004, 2005a and b; Vastano et al., 2000; Yang et al., 2001). Extracts from Reunoutria showed allelopatical, antifungal, antioxidant and antiviral effects (Inoue et al., 1992; Konstantinidou–Doltsinis and Schmitt, 1998; Daayf et al., 2000). Anthraquinones from Reynoutria sp. also act estrogenicaly (Zhang et al., 2006). Nevertheless, the insecticidal effect of extracts from Reynoutria sp. has not been not tested.
The Egyptian cotton leaf worm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), is a major pest on cotton (Gossypium herbaceum), corn (Zea mays L.), peanuts (Arachis hypogea), vegetables, soybean (Glycine max) and other plants cultivated in Egypt, as well as in Mediterranean and Middle East countries (Champion et al., 1977; Ahmad, 1988). The fact that the insect infests more than 87 host plants, from 40 plant families (Brown and Dewhurst, 1975), makes it an example of a serious polyphagous pest. The aim of this work was to present the effects of methanolic extracts from three Reynoutria species on feeding activity, development and mortality of S. littoralis.
MATERIALS AND METHODS
Plant material and its extraction
In Central Europe, the genus Reynoutria is represented by Reynoutria sachalinensis (F. Schmidt) Nakai (Syn.: Polygonum sachalinense F. Schmidt, Pleuropterus sachalinensis (F. Schmidt) H. Gross, Tiniria sachalinensis (F. Schmidt) Janchen), Reynoutria japonica (Houttuyn) Ronse Decraene (Syn.: Pleuropterus cuspidatus H. Gross; Polygonum cuspidatum Siebold & Zuccarini; Reynoutria henryi Nakai; Tiniaria japonica (Houttuyn) Hedberg), and their hybrid Reynoutria X bohemica Chrtek & Chrtková (Syn.: Fallopia X bohemica (Chrtek & Chrtková) J. P. Bailey; Polygonum X bohemicum (Chrtek & Chrtková) Zika & Jacobson; Reynoutria X vivax auct. non J. Schmitz & K. J. Strank).
]]> The leaves of all three Knotweeds were collected in September 2005 in the area of the permanent research plot located in ÄŒeské Budejovice, the Czech Republic. Leaves were dried at laboratory temperature; the material was pulverized and extracted with use of a 100% pure methanol for 45 min (ratio:leaves:methanol; 1:3.5). After centrifugation the sediment was washed three times in methanol (1:1 ratio). Supernatants were collected and filtered trough filter papers. Methanol was eliminated with the flow of nitrogen. The final dry extract yield was 15.8, 15.8 and 11.6% for R. japonica, R. x bohemica and R. sachalinensis.Test insects
These were obtained from a population of S. littoralis, fed with artificial insect diet (Stonefly Industries, Bryan, TX, USA). The colonies were reared at 25±1 °C and 16:8 (L:D) photoperiod.
Bioassays
The test substances were incorporated into the artificial insect diet as follows: 0 (control); 0.23, 0.47; 0.94; 1.88; 3.76 and 7.5 mg g–1. Assay diets (100 g) containing plant extracts were prepared as in the following example: diet containing 7.5 mg g–1 of the extract was prepared from 750 mg of the test substance dissolved in 2 ml of the solvent carrier (MeOH), which was spilled with a pipette into 75 mL of water; after dissolution, 25 g of dry diet was put into the liquid, the solvent was evaporating in a fume hood for 1 h; then, the final diet was refrigerated (4 °C).
Effects of plant extracts on larval development and survival
Newly hatched larvae (n=50) were placed in Petri dishes (24 cm diameter), fed ad libitum the diet containing the concentrations of the extract, and their mortality was recorded. As they grew up, they were divided in several dishes to prevent crowdedness. The length of larval period was calculated and pupae (approximately 24 h old) were weighed.
Effects of crude extract on food consumption and utilization
Larvae reared on a control diet were weighed after the fourth molt (<24 h), and placed individually in glass tubes (30 mm diameter; 100 mm long). They were fed controlled amounts of food containing 0 (control), 0.23, 0.94; 3.76 and 7.5 mg g–1 of the extract (n=30 for each concentration) and were feed 3 d, which is a period a little shorter than instar duration. Fifth instar larvae were chosen for the test because the feeding activity was more or less steady throughout this stadium, whereas sixth instar larvae may stop feeding before the completion of the 3 d experiment.
At the end of the experiment, the larvae were weighed and their faeces and uneaten food were dried and weighed. Fresh weights of faeces and consumed food were determined from dry weight vs. fresh weight curves. The nutritional indices: relative consumption rate (RCR), relative grown rate (RGR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD) and approximate digestibility (AD) were calculated as follows:
]]> RCR = I/BaT; RGR = B/BaT; ECI = (B/I) X 100; ECD = [B/ I–F)]X 100; and AD = [(I–F)/I] X 100, where I = weight of food consumed; Ba = arithmetic mean of insect weight during the experiment; T = feeding period (d); B = change in body weight; F = weight of faeces during the feeding period (Waldbauer, 1968; Farrar et al., 1989).The test of extracts with antifeedant properties
The antifeedant activity of crude extracts against the larvae (3rd instar) was evaluated through no–choice and choice tests using an artificial diet disc. The test was performed by placing one control and one treated diet disc (for choice test) or two treated diet disc (for no–choice test), containing 0.23, 0.94; 3.76 and 7.5 mg extracts per g fresh diet. The diet discs (2.5 cm diameter; 8 mm thick) were placed in a plastic Petri disc area (9 cm diameter); the distance between discs was about 2 cm.
Twenty larvae were placed in the middle of each dish and there were 4 replicates for each treatment. After 72 h, the remnants of diet discs were collected and dried separately at 70 °C to a constant weight. The amount of consumed food was calculated from the initial fresh weight and the dry weight of each disc, using a standard curve of the relation between fresh weight and dry weight of different sized dish pieces.
The feeding deterrence index (FDI) was calculated as:
FDI = [(C–T)/(C + T)]X100
where, C and T are the control and treated diet disc consumed by the insect.
Experimental conditions and statistical analysis
The experiments were carried out at 25±1 °C, 65–72% RH and 16:8 (L:D) photoperiod. All of them were repeated four times using larvae from different generations. The results were evaluated by ANOVA and Turkey's honest significant difference (HSD) test (p<0.05). The mortality of larvae and pupae, and growth inhibitory effect (RGR indexes) in each replicate was determined for S. littoralis. The lethal doses (LD50) were calculated using the Spearman–Karber method with Abbot correction (Hamilton et al., 1977).
]]> RESULTS
Effects of plant extracts on larval development and survival
When the neonate larvae were fed diet containing different concentrations of the extracts from all tested plant species, the larval period increased due to the extract concentration. The shortest larval period (160 d) was observed for the control group, while the longest period (34.33 and 32.23 d) was that of larvae fed on diet containing 3.76 mg g–1 of the extract of R.xbohemica and R. sachaliensis, and 26.40 d for larvae fed 1.88 mg g–1 of the extract of R. japonica (Tables 1–3) (Tables 1, 2, 3). The larval period for the larvae fed 7.5 mg g–1 (for all tested extracts) and 3.75 mg g–1 of extract R. japonica was not obtained, as no larvae survived to pupation. Significant increase (p<0.05) of larval period was caused by diets containing 0.23 mg g–1 or more (in addition to extract of R. japonica). Survival, calculated at the end of larval period, depended on the concentration of the extracts in the food: 97% of larvae fed on control diet; total survival until the hatching of adults was over 92%.
The mortality of larvae fed on the diet containing 0.23, 0.47; 0.94; 1.88; 3.76 and 7.5 mg g–1 of R. japonica was 29, 37, 64, 73, 100 and 100% (Table 1); for extract of R. x bohemica was 15, 24, 33, 68, 85 and 100% (Table 2); and for extract of R. sachalinensis was 27, 37, 45, 65, 76 and 100% (Table 3). Significant increase (p<0.05) of pupal mortality was caused by diets containing 3.76 mg g–1 (for extract of R. x bohemica and R. sachalinensis). The lethal concentrations (LC50) value for R. japonica, R. sachaliensis and R. x bohemica was 0.58, 0.80 and 1.22 mg g–1 (Table 8). The body weight of pupae was significantly reduced (p<0.05) after larval feeding on diets containing 0.23 mg g–1 extract of R. x bohemica (Table 2) and R. sachalinensis (Table 3) and 0.47 mg g–1 extract of R. japonica (Table 1) or higher doses.
Effects of crude extracts on food consumption and utilization
The extracts of Reynoutria sp. reduced the nutritional indices RGR, ECI ECD and AD of the fifth instar larvae (Tables 4, 5, 6). There was a significant reduction (p<0.05) at the doses 0.23 mg g–1 or higher (in addition to AD index to extract of R. x bohemica) of all extracts. The relative consumption rate (RCR) was significantly increased (p<0.05) by doses 0.23 mg g–1 or higher of the extract of R. sachalinensis and R. japonica, and by doses 3.76 mg g–1 or higher of extract of R. x bohemica (Table 4, 5, 6). The effective doses reduced larval growth by 50 % (LC50) in relation to the control: 1.24, 6.72 and 9.48 mg g–1 for R. x bohemica, R. japonica and R. sachaliensis (Table 8).
The antifeeding activity of extracts
Strong deterrent effects of extracts against larvae S. littoralis were not observed (Table 7). There was a direct relationship between doses of tested extracts and feeding deterrence. In the choice test the larvae preferred the control food. Even if differences between single concentrations were not significant, it seems that the concentration of extracts stimulated intake of the food. The highest food attractiveness (24.12%) was for the highest test dose (7.5 mg g–1) in the R. japonica extract. In the no–choice test there was a deterrent effect.
]]>DISCUSSION
Even if extracts of the three Reynoutria species did not affect the feeding deterrence of the Spodoptera littoralis larvae, they significantly decreased the larval growth and the efficiency of conversion of the ingested and digested food. Conversion of the ingested food was mostly minor, compared to the control group. The low utilization of food caused prolongation of the larval development and chronic toxicity. Even if extracts of the three plant species were influencing the larval mortality with the same effects (LC50 was 0.5 to 1 mg g–1 in all extracts), the impact on growth inhibition of larvae varied. The most efficient extract was the R. x bohemica intespecific hybrid (LC50 1.24 mg g–1), then the extract of R. japonica (LC5050, 6.72 mg g–1), and the least efficient was the extract from R. sachaliensis (LC50 9.48 mg g–1). Generally, larval growth inhibition is caused by the antifeedant effect which decreased food ingestion or even prevented feeding.
Both the ECI and ECD decrease was proportional to the extract concentration increase; however, the feeding deterrence index was not determined. ECI is an overall measure of an insect's ability to utilize the ingested food for growth: a drop in ECI indicates that more food is being metabolized for energy and less is being converted to body substance (i.e., growth). ECD also decreases as the proportion of digested food metabolized for energy increases. Decreasing ECI and ECD values indicate that ingested Reynoutria sp. extract also elicit some chronic toxicity.
Similar results were also seen with hirtin and T. connaroides extract when tested against Peridroma saucia (hirtin and extract) and S. litura (extract) (Xie et al., 1994; Wheeler and Isman, 2001). Extracts of methylalcohol may contain an array of substances which probably could make the artificial diets for polyfagous moths larvae (vitamins, chlorophyll). Furthermore, they also contain extracts of substances which may reduce growth, even they are not considered as antifeedants (Caroll et al., 2006).
Ghumare and Mukherjee (2005) found that S. litura larvae exhibited preference for food contained (–)–α–pinene, (–)–β–pinene, alphamethrin, D–limonene, and cineole, even though they showed negative effects on the growth of S. litura larvae. Taste aversion has been described as an adaptive specialization of learning, which possibly should provide polyphagous species with an adaptive advantage (Ratcliffe et al., 2003). Besides, contrary to the hypothesis that generalists should develop aversion to toxic substances on palatable plants, Spodoptera littoralis as well as S. litura (Ghumare and Mukherjee, 2005) do not exhibit the food aversion shown by certain slugs, caterpillars or grasshoppers (Gelperin, 1975; Dethier, 1980; Bernays and Lee, 1988; Behmer et al., 1999).
The leaves of Knotweeds species contain many biologically active compounds: catechin, epicatechin, chlorogenic acid, caftaric acid, derivatives of caffeic acid and many derivatives of quercetin (Vrchotová et al. 2004, 2005 a, b). The contents of phenolic compounds from ground parts of these species are very different; some phenolic compounds (Vrchotová et al., 2004) are summarized in Table 9. The fresh aerial parts of R. sachalinensis contain quinones, for example emodin (72 mg kg–1 fresh weight), physcion (22 mg kg–1) (Inoue et al., 1992).
Even if trials of the studied extracts were not performed, it is probable, based on previously performed analysis, other possible impacts of some substances on the Spodoptera larvae as reported by other authors. For example, Lindroth and Peterson (1988) showed that rutin and quercetin cause some mortality and rutin reduced growth rates consumption and digestion efficiency of larvae Spodoptera eridania. For isoflavonoids tested in combinations with chlorogenic acid, the combinations containing judaicin and maackiain were most active, and chlorogenic acid enhanced the antifeedant activity of isoflavonoids against Heliocoverpa armigera (Simmonds and Stevenson, 2001). In our extracts there were several phenolic compounds and their numerous glycosides. Besides the so far known substances, they contain leaves and their extracts, and a large number of unknown substances. We have to focus on determining the substances which are part of the shoot parts of plant because the main interest is focused on the under–ground plant organs for the content of stilbens (resveratrol and transresveratrols), which is not present in the shoot of plants. It is probable that some of the phenolic substances, or their compounds, may significantly decrease phytophagous of the plants.
CONCLUSION
]]> The Reynoutria extracts directly affected the growth and, in higher doses, caused chronic toxicity of Spodoptera littoralis larvae. Besides this direct impact, the extracts from these plants may indirectly increase its insecticidal effectiveness if they applied onto the plants directly. These extracts may elicitate several phenolic substances of a defensive characters with the plants, which may increase the total effectiveness of the agents. Nevertheless, it is probable that the Reynoutria sp. extracts, used in plant protection due to their fungicidal effects, may significantly reduce the number of pests.
ACKNOWLEDGEMENTS
Funding for this research was sponsored by the MSMT funding no. 1P05ME764, MSMT OC D28.001 and from the Research Intention Program of ISBE AS CR AV0Z60870520.
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