Toxicity of Andira paniculata (Fabaceae) Extracts to Helicoverpa armigera (Lepidoptera: Noctuidae)

Helicoverpa armigera is one of the most important pests of soybean crop in Brazil. The purpose of this work was to evaluate the effect of organic Andira paniculata extracts on its biology, feeding and the attractiveness of soybean plants to H. armigera. Hexane, dichloromethane, ethyl acetate and hydroalcoholic fractions at concentrations of 0.01, 0.1, 0.5 and 1% were evaluated. For the biological parameters the period and viability of larval stage, caterpillar weight at ten days, period, viability and pupal weight at 24 hours, total longevity, LC50 and deterrence were evaluated. The non-preference for feeding and attractiveness, the number of caterpillars and the dry matter consumed in each fraction of the extracts were evaluated. The extract of A. paniculata in hexane (0.01%) resulted up to 85% mortality of H. armigera. The A. paniculata extract did not affect the larval period, weight, pupal period and mortality or the consumption of H. armigera. The hydroalcoholic extracts obtained the better results for deterrence. The A. paniculata extract in dichloromethane fraction had the lowest LC50. The A. paniculata extracts in the hexane fractions (0.1%), ethyl acetate (0.01 and 0.5%) and hydroalcoholic (0.01 and 0.5%) were fagodeterrents for H. armigera. Thus, A. paniculata extract in hexane fraction is the most promising for use in the control of H. armigera in soybean.

The insecticide deterrent effect of several plant extracts has been studied in the control of agricultural pests. Very few studies of this nature have been carried out for the pest H. armigera. One of them by Baskar et al. (2009) reported deformities in the body of H. armigera treated with hexane extract of Atalantia monophylla. The insecticide effect in the population of Spodoptera frugiperda by extracts of Andira paniculata has already been studied by Pereira (2012).
The purpose of this work was to evaluate the toxicity (deterrence, larval and pupal mortality, and inhibition of development) of organic extracts of A. paniculata leaves, on H. armigera.

Material and Methods
The research was carried out in the Laboratory of Agricultural Entomology of the Goiano Federal Institute, Campus Urutai, in Urutaí City, Goiás State, Brazil. The assays were performed in a laboratory at a temperature of 25±2 °C, relative humidity of 70±10%, and photoperiod of 12 hours.

Rearing and Maintenance of the Insect
H. armigera larvae were fed on an artificial diet. The insects used in the experiment were obtained from a laboratory of a Brazilian company called Bug Biological Agents. After the eclosion of eggs, the larvae continued to feed on the artificial diet until they reached the third instar. Between zero and six hours after ecdysis to the third instar, parts of the soya leaves treated with different extracts of A. paniculata were offered to the larvae as food. The soya used was the conventional ND 7337, grown in a greenhouse, in 5.0 L pots, containing substrate subsoil base, sand and manure, at a ratio of 2:1:1. No pest control with insecticides was carried out during the development of the soya crop in the greenhouse.

Plant and Extract Preparation
A. paniculata leaves were obtained from trees found in the cerrado region in the municipality of Anápolis City, Goiás, Brazil. After collection, the plant matter was dried in a forced air oven at 50 °C for five days. After this, it was ground and submitted to an exhaustive extraction with ethanol. The filtrate from this extraction was collected and the organic solvent evaporated in a vacuum at a temperature of around 40 °C, producing the crude ethanolic extract.
To prepare the extracts, 1.0 g of the following fractions were used: hexane, ethyl acetate and hydro alcoholic, in addition to a 0.46 g fraction of dichloromethane. The fractions APFE-H, APFE-D, and APFE-A were dissolved in acetone and water (1:1), and the fraction APFE-W was dissolved in ethanol and water (1:1), all in a 1% concentration. Test tubes with the solutions were placed in ultra-sound to provide complete dissolution.
Soya leaf discs (2.5 cm of diameter) were immersed in concentrations of the extracts and control for 30 seconds, after which, they were placed outside for 1.5 hours, under dry paper towel to ensure complete evaporation of the solvents used.

Bioassay-Antibiosis Effect
Newly hatched H. armigera larvae were individualized in 9.0 cm diameter Petri dishes with moist filter paper. The larvae were fed with soya leaves treated with plant extracts at different concentrations. These dishes were kept under controlled conditions (temperature 25±2 °C, UR 70±10%, and photoperiod of 12 hours), where the leaves were replaced daily.
Larval mortality was recorded daily until pupal phase. The surviving larvae were weighed 10 days after the beginning of the experiment and the pupae were removed from the dishes 24 hours after development, then weighed and replaced in the dish for the emergence of adults. The moths were not given food during the adult phase.
The biological variables evaluated were: a) larval phase: period and viability of larval phase, and larval weight 10 days after feeding; b) pupal phase: period and pupal viability, and weight at 24 hours of age; c) total cycle. A completely randomized design with 17 treatments and 20 repetitions was used.

Bioassay-Antixenosis Effect
In free choice attractivity trials with third instar larvae, soya leaf discs (2.5 cm in diameter) treated with plant extract, were distributed equidistant in trays (arena with 350 mm of diameter) and lined with moist filter paper, where the larvae had access to food.
For the dry matter consumed, two leaf discs (2.5 cm 2 diameters) were removed equidistantly from the soybean leaves. One was offered to the insects and the other, known as the aliquot, was oven dried at 60 °C for 48 hours. The dry matter consumed by H. armigera larvae was determined by the difference between this rate and the remaining portion of the disc consumed.
The deterrence percentage of the discs of soya leaves treated with different extracts for the H. armigera larvae were determined according to Singh and Pant (1980): Preference index for H. armigera on the soya leaf discs treated with plant extracts was determined according to Kogan and Goeden (1970): PI = 2A/(M+A); being, PI = preference index, A = leaf area treated with extract and consumed by the larvae, M = leaf area not treated with plant extracts consumed by larvae.

Statistical Analysis
The data were submitted to ANOVA test and the means compared by the use of Scott-Knott to 5% probability, using R software (R Core Team, 2016).

Results
The larval weight of H. armigera fed with soya treated with different A. paniculata extracts was significantly affected (P < 0.0001) by the other variables analyzed (Table 1). The duration of the larval period (P = 0.8498), and pupal period (P = 0.3916), pupal weight (P = 0.1798), and total life cycle (P = 0.8991) were not influenced by the plant extract fractions. Larval weight was lower in larvae fed with the following fractions: Hexane 0.01 and 0.10%, Dichloromethane 0.10%, Acetate Ethyl 0.5 and 1%, and Hydro alcoholic 0.10 and 1%, which differed statistically from the other concentrations, presenting higher means, varying between 0.09 and 0.11 g. Note. Mean±Standard deviation error followed by the same letter, do not differ statistically between themselves by the use of Scott-Knott test at 5% probability. * Significant at 1 and 5% probability, respectively. 1 Insufficient number of repetitions for a reliable statistical analysis due to the mortality in pupal phase or emergence of adults with visible anomalies. ns Non significant.
The A. paniculata extracts did not cause significant changes in either the duration of the period, nor pupal weight, nor in the duration of the H. armigera life cycle (Table 1). However, they influenced H. armigera larval mortality (P < 0.0109), whereas pupal mortality did not differ statistically from each other (P = 1501) ( Table 2). Regarding larval mortality, the highest means were found in the 0.01, 0.10 and 0.5% Hexane fractions, which reached 85, 70 and 60%, respectively. In 0.1% Dichloromethane fraction with 65% mortality, and in 0.5% Acetate Ethyl fraction, which reached 70% mortality. Larval mortality varied between 25 and 50% in the other fractions, which did not differ statistically from the control treatment. jas.ccsenet.