Toxicity of petiveria alliacea l. On greenhouse whitefly (trialeurodes vaporariorum west.)

Ma. Rosario García-Mateos, Elisa Elizalde Sánchez, Policarpo Espinosa-Robles and Ma. Edna Álvarez-Sánchez

Ma. Rosario García-Mateos. Chemist, Benemérita Universidad Autónoma de Puebla (BUAP), México. M.Sc. in Chemistry, Universidad Nacional Autónoma de México (UNAM). Doctor in Plant Physiology, Colegio de Postgraduados (COLPOS), México. Researcher, Universidad Autónoma Chapingo (UACh). Address: Carretera México-Texcoco km. 38.5, Chapingo, Méx. CP. 56230. México. e-mail: rosgar08@hotmail.com

Elisa Elizalde Sánchez. Agronomist. M.Sc. in Horticulture, UACh, Mexico. Researcher, UACh, Mexico.

Policarpo Espinosa-Robles. Agronomist. UACh. M.Sc. in Horticulture, UACh, Mexico. Researcher, UACh, Mexico.

Ma. Edna Álvarez-Sánchez. Agronomist. M.Sc. and Doctor in Soil Science, COLPOS, Mexico. Researcher, UACh, Mexico.

SUMMARY

Several natural products present in plant extracts have been evaluated for their effectiveness as insecticides. Petiveria alliacea (Phytolaccaceae) is a perennial herb whose distribution includes tropical areas of Mexico and Central America, as well as other regions. Its properties as insecticide and acaricide have been reported. To determine the toxic effects of P. alliacea on the greenhouse whitefly Trialeurodes vaporariorum, mortality rate and LC₅₀ were evaluated in the laboratory and in a greenhouse hydroponic tomato crop. In the laboratory, mortality rates were 98.35% for the aqueous extract and 100% for the methanol and dichloromethane extracts. Under greenhouse conditions, the mortality rates were 58.33, 48.75 and 57.55% for the aqueous, methanol and dichloromethane extracts, respectively. The LC₅₀ for aqueous, methanol and dichloromethane extracts were 4.6, 1.1 and 0.3%, respectively, in the laboratory, and 16.6, 13.3 and 3.5%, respectively, in the greenhouse. The treatments did not interfere with the tomato yield (weight and fruit diameter), nor was there significant difference among them. It is thus inferred that the use of the extracts for T. vaporariorum control is feasible.

Toxicidad de petiveria alliacea l. Sobre la mosca blanca (trialeurodes vaporariorum west.)

RESUMEN

Varios productos naturales presentes en los extractos de plantas han sido evaluados por su efectividad como insecticidas. Petiveria alliacea (Phytolaccaceae) es una planta herbácea perenne distribuida en las zonas tropicales de México y América Central, entre otras regiones, reportada con actividad insecticida y acaricida. Para determinar el efecto tóxico de P. alliacea sobre la mosca blanca Trialeurodes vaporariorum se evaluó el porcentaje de mortalidad y la CL₅₀ en el laboratorio y en un cultivo hidropónico de tomate en invernadero. En el primer caso, la mortalidad encontrada fue 98,35% para el extracto acuoso, 100% para los extractos en metanol y en diclorometano. En condiciones de invernadero, la mortalidad fue 59,33; 48,75 y 57,55% para los extractos acuoso, metanólico y de diclorometano, respectivamente. Las CL₅₀ obtenidas en el laboratorio para los extractos acuoso, metanólico y de diclorometano fueron 4,6; 1,1; 0,3%, respectivamente, y en condiciones de invernadero 16,6; 13,3 y 3,5%, respectivamente. Los tratamientos no interfirieron en el rendimiento de tomate (peso y diámetro por fruto), al no mostrar diferencias significativas entre ellos, infiriendose la viabilidad del uso de los extractos contra T. vaporariorum.

Toxicidade de petiveria alliacea l. Sobre a mosca branca (trialeurodes vaporariorum west.)

RESUMO

Vários produtos naturais presentes nos extratos de plantas têm sido avaliados por sua efetividade como inseticidas. Petiveria alliacea (Phytolaccaceae) é uma planta herbácea perene distribuída nas zonas tropicais do México e América Central, entre outras regiões, reportada com atividade inseticida e acaricida. Para determinar o efeito tóxico de P. alliacea sobre a mosca branca Trialeurodes vaporariorum se avaliou a porcentagem de mortalidade e a CL₅₀ no laboratório e em um cultivo hidropônico de tomate em estufa. No primeiro caso, a mortalidade encontrada foi de 98,35% para o extrato aquoso e de 100% para os extratos em metanol e em diclorometano. Em condições de estufa, a mortalidade foi de 59,33%; 48,75% e 57,55% para os extratos aquoso, metanólico e de diclorometano, respectivamente. As CL₅₀ obtidas no laboratório para os extratos aquoso, metanólico e de diclorometano foram de 4,6%; 1,1%; e 0,3%, respectivamente, e em condições de estufa foram de 16,6%; 13,3% e 3,5%, respectivamente. Os tratamentos não interferiram no rendimento do tomate (peso e diâmetro por fruto), ao não mostrar diferenças significativas entre eles, inferindo-se a viabilidade do uso dos extratos contra T. vaporariorum.

KEYWORDS / Mortality / Petiveria alliacea / Plant extracts / Tomato / Trialeurodes vaporariorum /

Received: 03/08/2006. Modified: 15/12/2006. Accepted: 01/02/2007.

Introduction

Tomatoes, widely demanded for their nutritional characteristics, have been affected by infestation of the pest Trialeurodes vaporariorum, commonly called greenhouse whitefly (Jauset et al., 1998), which markedly reduces production (FAO, 2002; Agoitia, 2003). One alternative for fighting pests such as this one is the use of natural insecticides, active principles isolated mainly from plants (Schmutterer, 1990). The search for natural insecticides is permanent (Lagunes and Rodríguez, 1989), especially in the case of products from plant species that have not been studied extensively.

In Mexico, there are several plants with insecticidal properties; among these, Petiveria alliacea L. (Phytolaccaceae), native to the Caribbean, Central and South America (Cáceres et al., 1998), is commonly known in Mexico as mapurite or hierba del zorrillo, because of the unpleasant odor produced by its leaves, which contain sulfide compounds (De Sousa et al., 1990; Kubec and Musah, 2000; Kubec et al., 2002, 2003). These compounds are believed to have insecticidal activity against the adult insects Cimex lectularius and Musca domestica, it repels termites and acts as an acaricide and nematicide against Meloidogyne spp. (Da-Ponte et al., 1996; Lyndon et al., 1997).

In the literature, no description has been found of the insecticidal activity of the di-, tri-, and polysulfide metabolites of P. alliacea against T. vaporariorum. However, there is empirical evidence suggesting that the greenhouse whitefly is repelled by mapurite in some greenhouse crops. This study was conducted to evaluate the effectiveness of aqueous, methanol, and dichloromethane extracts of leaves of Petiveria alliacea L. in the control of the greenhouse whitefly (Trialeurodes vaporariorum West.) under laboratory and greenhouse conditions in a hydroponic tomato crop.

Materials and Methods

Collection of Petiveria alliacea

Plant material was collected in the community of Rodriguez Clara, Veracruz state, Mexico. This municipality is located at 17º59'00''N and 03º43'08''E, with an average of 95masl. The climate is hot and humid, with mean annual temperature of 24.8ºC, abundant rainfall in summer and early fall, and mean annual rainfall of 1266mm. The species was certified by Stephen D. Kooch, head of the Herbario CHAPA of the Colegio de Postgraduados, Montecillo, Estado de México. An herbarium specimen was donated to the Herbario MEXU of the Universidad Nacional Autónoma de Mexico (register Nº 1042314).

Collection of Trialeurodes vaporariorum

Greenhouse whiteflies were collected in the nymph and adult stages from a tomato field in the experimental station of the Universidad Autónoma Chapingo (UACh), Mexico, and were identified as Trialeurodes vaporariorum Westwood by Juan Fernando Solís Aguilar, Department of Parasitology, UACh.

Preparation of extracts

The plant material was dried in an oven at a constant temperature of 50ºC and later ground mechanically. Different dilutions (10, 15 and 20% w/v) were prepared from the aqueous extract obtained by maceration for 24h at room temperature. Methanol extracts were obtained by macerating leaf material for one week; the crude extract was then concentrated by evaporation reduced pressure, obtaining 23.4g of crude moisture-free extract per 100g dry leaves. In a similar manner a crude extract was prepared with dichloromethane, obtaining 15.7g per 100g dry weight.

Dilutions of different concentrations (1.0, 5.0 and 7.5% v/v) were prepared using 1% Tween-20 as a surfactant. As controls, water and a 0.08% imidacloprid solution.

Laboratory bioassay

A tomato leaf was extended in a 5.5cm Petri dish. A wet cotton ball was placed at the base of each leaf for hydration. The different extracts were sprayed over each leaf and left to dry. Later, 50 adult greenhouse whiteflies, unsexed, were placed in each Petri dish and covered with organza. Each treatment, including the controls, was replicated four times. Live and dead flies were counted every 12h over a period of 48h. Environmental temperature oscillated between 12 and 26ºC, and relative humidity was 60-70%.

Greenhouse bioassay

Tomato (Lycopersicum esculetum, var. Floradade) seeds were sown in polystyrene trays with vermiculite substrate. The germinated seeds were kept for two weeks with sufficient moisture, and application of nutrient solution was initiated at the third week with a concentration of 75%.

Seedlings were transplanted to 9 liter bags in the fourth week, using red volcanic sand substrate. Nine plants (3×3) were grouped with no spaces between plants and the groups were ordered in four columns in 10 rows 30cm apart, for a total of 360 plants in 40 groups. In this period application of the nutrient solution began with a concentration of 100%. The crop was transplanted and in the area of greenhouses of the Departamento de Fitotecnia in the experimental station of UACh, at 10º29'N and 98º53'W, at 2251masl.

Fifty two plants were selected randomly in a complete random design and isolated in entomological cages 150cm high and 40cm diameter, covered with organza. Greenhouse whiteflies were captured with a vacuum cleaner; 100 unsexed greenhouse whiteflies were introduced into each cage. The adults were kept for one week in the cages for adaptation and counted before application of each of the extracts. Extracts were applied only once. Treatments were replicated three times, and water and a 0.08% imidacloprid solution were used as controls.

During the experiment, temperatures of 10-30ºC and relative humidity of 70-75% were recorded. To assess the fly population decrease, readings were taken twice every 12h after the application of extracts. The variables evaluated were number of live and dead greenhouse whiteflies, and median lethal concentration (LC₅₀).

To evaluate possible damage by the extracts and their effect on tomato yield, the plant extracts were applied when the fruits had reached maturity. Yield was expressed in fruit weight (g) and diameter (cm), plant height (cm) and total weight of fruit per plant (g). During crop growth, insecticides were not applied.

Statistical analysis

Analysis of variance (ANOVA) and a test of comparison of means (Tukey, p£0.05) were carried out with the Statistical Analysis System (SAS, version 8.0) software. LC₅₀ was calculated with Probit (Finney, 1972).

Results and Discussion

Percentage of mortality

Percentages of adult greenhouse whitefly mortality observed in the laboratory (Figure 1) were 86.6, 94.1 and 98.3% for the treatments with 10, 15 and 20% aqueous extract, respectively. In the experiments conducted in the greenhouse, mortality was 35.8, 47.5 and 58.3%, respectively, for the same concentrations as those in the laboratory experiment.

In the evaluation of population decrease under greenhouse conditions (Figure 1), significant statistical differences were found for highest concentrations (15 and 20%) of aqueous extract compared with the control and the lowest concentration (10%). The percentage of mortality was similar (47.50, 58.33 and 55.43%, respectively) in the three experiments, with 15 and 20% aqueous extract and 0.08% imidacloprid.

Adult greenhouse whitefly mortality with the methanol extract (Figure 2) was 100% in the laboratory treatments with the highest concentrations (5.0 and 7.5%). These mortalities were found to be statistically different from those caused by each of the other three treatments: 44.1% with the lowest concentration (1.0%), 55.43% with imidacloprid and 2.5% in the control. With the crude dichloromethane extract, the highest mortality (100%) was observed with the highest concentrations (5.0 and 7.5%) in the laboratory.

In the greenhouse, both methanol and dichloromethane extracts had a similar effect. The highest concentrations (5.0 and 7.5%) were significantly different from the lowest concentration (1.0%) and the control (0%). The crude dichloromethane extract caused approximately the same percentages of mortality as the methanol extract, but in a shorter time.

The highest mortality was caused by the pesticide imidacloprid in both laboratory and greenhouse (Figures 2 and 3). The mortality caused by the pesticide was very similar in both cases, in the laboratory (62.5%) and in the greenhouse (55.43%), possibly because of its systemic effectiveness and persistence in the environment (Bernal, 2003). When comparing the performance of the two organic extracts in laboratory and greenhouse, the same trend was found in terms of higher T. vaporariorum mortality (Figures 2 and 3).

The mortality rate of adult flies caused by the three extracts was higher in the treatments in the laboratory experiment, as compared with those conducted in the greenhouse. Lewis and van Emden (1986), however, pointed out that field tests on insects can confirm laboratory results, but laboratory results cannot be extrapolated from those conducted in the field or greenhouse because of diverse factors, such as the alteration of the substances in the environment and the adaptation of the insects to the use of artificial substrates in the laboratory.

Median lethal concentration (LC₅₀)

Figure 4 shows the LC₅₀ for the aqueous, methanol and dichloromethane extracts used in the experiments, under both laboratory and greenhouse conditions. The same trends in toxicity were detected in the organic extracts. The lowest LC₅₀ (3.48%) was found for the dichloromethane extract, while for the aqueous extract the LC₅₀ was 16.62% and for the methanol extract it was 3.26%.

The concentrations required to kill half of the treated population were much lower under laboratory conditions (4.6, 1.1 and 0.3%) than in the greenhouse (16.6, 13.3 and 3.5%). The same trend was found in the assessment of mortality rate. This is because plant extracts are more susceptible to faster degradation under greenhouse conditions than when exposed to the environment (Ortega, 1997); solar radiation, temperature, wind and other environmental factors can act to degrade these substances (Yee and Toscano, 1998; Prabhaker et al., 1999).

The higher mortality observed with the methanol and dichloromethane extracts, compared with the aqueous extract, could be due to their less polar characteristics, which facilitate absorption and translocation by the insect and possible movement through the membranes to the site of action (Ujváry et al., 1992). These results suggest that in the dichloromethane extract, which is more lipophilic than the methanol extract, substances of a non-polar nature can be found, such as those reported by Kubec et al. (2002) in P. alliacea.

Kubec et al. (2002, 2003) reported the identification of non-polar sulfur compounds such as thiosulfinates (allicin) and mono-, di- and trisulfides, derived from cysteine sulfoxides (alliin), which may be responsible for the toxicity observed in the present study. These authors also point out that these compounds become volatile when exposed to the environment. These substances are responsible for the odor of garlic and skunks (allyldisulfides), which is characteristic of P. alliacea (Kubec et al., 2002). The volatility of some of these compounds may also be another of the explanations for the toxicity of the methanol and dichloromethane extracts. Won et al. (2003) demonstrated that many of the essential oils derived from garlic are effective in fighting T. vaporariorum by direct contact. Likewise, Chiam et al. (1999) pointed out that allyldisulfide compounds are the most reactive and odiferous, and are thus attributed with certain insecticidal properties. It can therefore be inferred that the toxicity observed in P. alliacea could be due not only to the lipophilic metabolites present, but also to their volatility, as has been reported in species of Allium sp. There is empirical evidence that suggests that the greenhouse whitefly is repelled by mapurite in some greenhouse crops, and this is attributed to the unpleasant odor produced by its leaves (Alejandro Barrientos Priego, personal communication).

Tomato yield

No significant differences were found in the different variables, fruit weight and diameter, plant height and total fruit weight per plant that express yield in any of the treatments; and it is thus inferred that neither the extracts nor the controls (imidacloprid 0.08% and 0%) affected crop yield under greenhouse conditions in a hydroponic system.

Conclusions

The results showed that under laboratory conditions, the dichloromethane and methanol extracts caused 100% mortality in Trialeurodes vaporariorum adults. Under greenhouse conditions the three extracts showed a similar trend in the percentage of mortality. Both percentage of mortality and the median lethal concentration (LC₅₀) were higher in the laboratory than in greenhouse conditions. All of the extracts evaluated in the laboratory and greenhouse were significantly different from the control treatments. Therefore, the mortality of greenhouse whitefly observed was caused by the P. alliacea extracts.

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