Assessment of toxicity on the basis of total phenolic content in oleander leaves ( Nerium oleander L . ) against Myzus persicae ( Sulzer , 1776 ) ( Hemiptera : Aphididae )

Nerium oleander is an evergreen flowering shrub or small tree distributed widely in the Mediterranean region. It is also a source of polyphenols and cardenolides ?? with insecticidal effect which could be a safe alternative of chemical control of insect pests. In the present work, five concentrations (0 %, 1 %, 2.5 %, 5 %, and 10 %) of ethanolic extract from Nerium oleander leaves were evaluated for its insecticidal effect against 3 to 4 days old Myzus persicae individuals under laboratory conditions. Obtained results showed a significant insecticidal effect with 70 % of mortality at the highest concentration (10 %). Total phenolic content of leaf ethanolic extract of this plant was 1721.36 mg gallic acid equivalent 100 g dry matter. The results obtained suggest that we could make bioinsecticides based on leaves ethanolic extracts from N. oleander which rich in polyphenols for use eventually in integrated pest management.


INTRODUCTION
Chemical pesticides used in plant protection have hazardous effects on human health and environment.Phytochemicals and plant extracts have long been a subject of research in an effort to develop alternatives to conventional insecticides but with reduced health and environmental impact (Dancewicz et al., 2011).Most plant species which are used for plant protection contain ingredients which inhibit the development of insects, hinder their feeding (antifeedants) or act as repellents and confusants (Rojht et al., 2009, Laznik et al., 2010, Rojht et al., 2012.).Polyphenols are a reputed and large phytochemical family with many interesting properties such plant resistance to insect pests (Bennett, 1996, Ding et al., 2000), antioxidant activity (Emmons and Peterson, 2001), and organoleptic properties (Es-Safi et al., 2003).
The green peach aphid, Myzus persicae (Sulzer, 1776) is found throughout the world.In addition to attacking plants in the field, the aphid readily infests vegetables and ornamental plants grown in greenhouses (Capinera, 2011).It is also an insect model for many studies (Ji et al., 2016).Its management is generally based upon the use of synthetic insecticides (Ciarla et al., 2005).
Consequently, this study aims to assess the toxicity of Nerium oleander L. ethanolic leaf extract against M. persicae and determine the content of phenolic compounds of this extract due to its richness in these molecules which implicated in pest control in order to valorize the role of this plant in this field.

MATERIALS AND METHODS
N. oleander leaves were collected in April 2014 at the flowering stage from Batna in the East of Algeria.It is located at 35° 61´ N, 6° 24´ E with an elevation of 641 meters above sea level.Leaves were dried at 50 °C and ground to a fine powder in a mortar grinder (Retsch RM 200).The air-dried and finely ground leaves (80 g) were extracted by successive extraction three times.Indeed, the powder was stirred with 400 ml of ethanol at room temperature for 30 min.The mixture was filtered three times to obtain three filtrates.These three filtrates were mixed and brought together in a sand bath (N'guessan et al., 2009).These series of operations resulted in a concentrated solution.The extract was kept at +4 C° until toxicity test on the aphid and determination of total phenolic content.

Aphid collecting and rearing
Last stage larvae of green peach aphid have been collected collected from Biskra in the South East of Algeria at 34° 52´ N, 5° 45´ E in April 2014 where they were living on Malva sp.Mass rearing of the green peach aphid was started on broad beans (Vicia faba L.) in a greenhouse.Each plant was inoculated with an apterous adult when emerging in the morning.Aphids were collected after 10 days by brushing them carefully from the leaves.

Preparation of dilutions
Five dilutions have been made: 0, 1, 2.5, 5 and 10 %.These were prepared by adding the methanol according to the protocol of Singh et al. (2012).

Bioassay test
To assess the insecticidal effect of N. oleander leaf extract, 15 M. persicae larvae (3 to 4 days old) per treatment were placed in a Petri dish (90 mm) containing three leaves of Vicia faba soaked in these different concentrations with three replications.The experiment was carried out under laboratory conditions (25 ± 1 °C, 50 ± 5 %).The mortality was determined after 24 h from the beginning of exposure.When no leg or antennal movements were observed, insects were considered dead (Salari et al., 2010).

Total phenolic content
The total phenolic content of the ethanolic extract of N. oleander L. was measured by the method described by Juntachote (2007).0.5 ml of the extract was added to 5 ml of distilled water and vortexed for 1 min using a vortex mixer (Janke & Kunkel IKA, Model: VF2, Germany). 1 ml of Folin and Ciocalteu's Phenolic reagent was added and mixed well.After 5 min, 1 ml of saturated sodium carbonate solution was added and the mixture was vortexed again.The sample was allowed to develop a blue colour for 1 h.The absorbance was measured at 640 nm using a spectrophotometer (UV-120-01; Shimadzu Co., Kyoto, Japan).A standard curve was prepared at the same time with gallic acid (Sigma-Adrich GmbH, Sternheim, Germany) at concentrations ranging from 0 to 0.2 mg ml -1 .The quantity of total phenolic content in the sample was calculated as gallic acid equivalent by using the standard curve.

Statistical analysis
Logistic regression analysis was employed to predict the probability that the augmentation of ethanolic extract concentrations would increase mortality of M. persicae individuals.The predictor variable was ethanolic extract concentrations (1 %, 2.5 %, 5 %, and 10 %).The Chisquare value will determine whether there is a difference between the current model and the interceptonly model.We used the statistical program Statistica 8 (StatSoft, Inc., Tulsa, OK) for all analyses.
Statistical analysis indicated the presence of dependence between the mortality and the different concentrations (p < 0.01).The Chi-square value for the difference between the current model and the intercept-only model is highly significant.Thus, we can conclude that mortality is related to ethanolic extract concentrations.
In fact, Table 1 shows the logistic regression coefficient, Wald test and odds ratio.Using a 0.05 criterion of statistical significance showed that all concentrations had significant effect (Table 1).A concentration of 10 % caused 73 % of mortality on green peach aphid (Figure 1) with a LC 50 of 7.2 % while LC 90 was 9.85 %.  2011).The extract hydro-alcoholic of its leaves administred to larvae of the tribe Rhizotrogini (Coleoptera: Scarabaeidae) had an effect on the protein content of the haemolymph and acetylcholine esterase activity (Madaci et al., 2008).
Based on the absorbance values of phenolic extract reacting with the Folin-Ciocalteu, and compared to the standard gallic acid solution, the result of the quantitative analysis of total phenolic compounds was 1721.36 mg gallic acid equivalent 100 g -1 of DR (dry matter) (Figure 2).High toxicity against green peach aphid individuals was due probably to the high amounts of phenolic compounds present in the extract.Indeed, many authors have highlighted the importance of polyphenols for aphid control which support our findings.Indeed, Laznik (2010) tested cinnamic acid against Aphis pomi (De Geer, 1773) and found that this molecule has showed aphicidal properties.Also, two flavanoles and one flavanone were found to be active as aphicid against the woolly apple aphid, Eriosoma lanigerum (Hausmann, 1802) by Ateyyat et al. (2012).In fact, larval mortality was higher than that obtained against apterous adults.The increase in the concentration of polyphenols has resulted in a remarkable augmentation in the larval mortality rate.However, works on aphicidal activity of N. oleander are very scarce.Goławska (2012Goławska ( , 2008) ) reported the effectiveness of polyphenols against Acyrthosiphon pisum (Harris, 1776).El-Akhal et al. (2015) tested the ethanolic extract of N. oleander on culicid mosquitoes and noticed toxic effects on their larvae.Indeed, the lowest concentration necessary to achieve 100 % mortality of Culex pipiens (Linné, 1758) larvae was evaluated at 160 mg ml -1 .

CONCLUSIONS
Leaf ethanolic extract of N. oleander was effective against green peach aphid and this efficacy is probably due to the high content of this extract on polyphenols.Thus, the results obviously show that will be possible to develop new biopesticides based on high content of these molecules in integrated pest management programs to reduce the use of conventional insecticides.Nevertheless, further research is needed on the phytotoxicity of these molecules and toxicity against non-target species.

Figure 1 :
Figure 1: Average mortality (% ± standard error) of Myzus persicae larvae with leaf extracts from Nerium oleander with several concentrations after 24 hours

Figure 2 :
Figure 2: Total phenolic content in leaves of Nerium oleander

Table 1 :
Logistic regression predicting mortality from concentrations and lethal concentration (LC 50 and LC 90 %)