Acquisition and Preparation of Tagetes erecta Plant Material

Tagetes erecta was gathered in the flowering season (Oct and Nov 2009) in the Tepoztlán Municipality, Morelos State, Mexico, and a sample was deposited at the Autonomous University of the State of Morelos Herbarium (HUMO) with registry number 27192. Later, the collected plant material was dried at room temperature in a dark room, and the leaves and flowers were separated in order for these to be triturated with the help of a pulvex plastic hand mill Model 95 (Molinos Pulvex, S.A. de C.V, México) to obtain pulverized samples of each of the plant's parts.

Insects - Spodoptera frugiperda

The fall armyworm larvae were provided by the Entomology Department, Biotic Products Development Center of the National Polytechnic Institute (CEPROBI-IPN) laboratory nursery. The larvae were maintained in a Precision model 818 incubation chamber at a temperature of 27 ± 1 °C, with 60–70% RH at 12:12 h L:D, and reared on a meridic diet (Burton & Perkins 1987). Second-generation (F₂) larvae were used for all experiments, and 100 neonate larvae were used for each treatment.

Meridic Diet for Rearing Spodoptera frugiperda

We prepared the fall armyworm meridic diet (Burton & Perkins 1987) for mass rearing and for conducting the bioevaluations with the following ingredients: Peruvian bean (Phaseolus vulgaris L.) (11.27%), wheat germ (5.16%), beer leavening (3.28%), ascorbic acid (0.32%), sorbic acid (0.10%), methyl parahydroxybenzoate (0.20%), 0.94% of formaldehyde at 10%, agar (1.40%), water for the beans (43.53%), and water for the agar (33.80%).

Crude Extract Preparation of Tagetes Flowers and Leaves

For preparation of the crude extracts, the flower and leaf material (500 g of each), dry pulverized separately, were placed in a 500 mL round, flat-bottomed flask, into which one of the following solvents was added until the material was covered completely: n-hexane (Fermont), or acetone (Mallinckrodt), or ethanol (J. T. Baker). Maceration was performed for 3 d.

Once maceration had been carried out, the solid material was separated from the liquid, and the solvent was eliminated by reduced-pressure distillation (21 torr) in a Buchi 205 rota-evaporator with the purpose of obtaining the 2 plant crude extracts (flowers and leaves). The solvents were removed by evaporation in the process of obtaining extracts. The extracts stored in amber glass bottles to avoid photolysis and stored at -20 °C in a freezer, until they were used in the bioassays.

Bioassays

Each of the various crude extracts of T. erecta flowers and leaves (n-hexane, acetone and ethanol) was incorporated into the above meridic diet at final concentrations of 500 ppm and the effects of these preparations on the development and survival of S. frugiperda larvae were bioassayed. Each of the control diets was prepared with 1mL either of n-hexane, acetone or ethanol. Diet ingredients and the concentrate extracts were mixed following the protocol of Franco et al (2006), and the prepared mixture was dispensed into cylindrical plastic containers (3 cm high × 3.5 cm diam) with 15 mL per container. Once the diet had cooled and solidified, 1 neonate larva was placed in each container with the aid of a fine camel hair brush. This process was replicated 2 times with 50 neonate larvae per replication. The containers were randomly arranged in a climactic chamber under the same conditions as used for rearing the laboratory colony of S. frugiperda.

The experimental design was completely randomized with 7 treatments and 2 replications (n = 100 larvae). Response variables were as follows: larval weights at 7 and 14 d, larval development, larval mortality, pupal viability and adult emergence. Mortality was calculated by means of Abbot's formula (Abbot 1925). The statistical analysis carried out was the analysis of variance (ANOVA) and mean comparison, i.e., mean ± standard deviation (MSD) (P = 0.5).

Effect of Tagetes Leaf Extracts on Weights of S. frugiperda Larvae

In Table 1 are shown the mean weights of larvae treated with the various T. erecta flowers and leaf extracts at 7 d. Statistically significant differences occurred between the mean weights of larvae in the untreated control (0.0186 g) and those of larvae treated with leaf extracts with n-hexane, acetone and ethanol, which had mean weights of 0.0033, 0.0019, and 0.0014 g, respectively. Larvae treated with leaf extracts had significantly lower weights than those fed the corresponding floral extracts.

At 14 d, the mean weight of control larvae was 0.3140 g, while the mean weights of larvae treated with the leaf extracts of n-hexane, acetone and ethanol were merely 0.1669, 0.1844, and 0.0746 g, respectively. Thus the profound effect of T. erecta leaf extracts on retarding gains in larval weight first demonstrated at 7d was corroborated. These data suggest that the leaf extracts may inhibit feeding, and hence they may be antifeedants. At 14 d the ethanol leaf extract clearly had the most drastic effect. Probably the toxic components in the leaf extracts include phagodeterrents.

Argueta & Vazquez (1994) reported that the leaves of T. erecta contain essential oils, which have been identified as geraniol, limonene, linalool and its acetate, menthol, ocimene, beta-phellandrene, dipentene, alpha and beta-pinene and tagetona. According Coll & Esquivel (2009), these compounds have insect antifeedant activities.

TABLE 1.

EFFECT OF EXTRACTS OF TAGETES ERECTA FLOWERS AND LEAVES INCORPORATED INTO MERIDIC DIET ON THE WEIGHTS OF SPODOPTERA FRUGIPERDA LARVAE AT 7 AND 14 D. ALL EXTRACTS HAD BEEN INCORPORATED INTO THE MERIDIC DIET AT 500 PPM.

These results are in agreement with those obtained by Picman (1986), who performed a review on the biological activity of the sesquiterpenes, and concluded that some sesquiterpenes may possess feeding inhibitory activity. There are other studies on this effect carried out on lepidopterans. Blaney et al. (1990) concluded that feeding inhibition is caused by azadirachtin and limonoids. They conducted a study on the effects of azirdiractin and related compounds on feeding by 4 noctuid species (Spodoptera littoralis Boisduval, S. frugiperda, Heliothis virescens F., and H. armigera Hübner), and reported severe antifeedant activity against S. littoralis and, to a lesser degree, against S. frugiperda, H. virescens, and H. armigera.

Caballero (2004) evaluated the antifeedant activity caused by 3 groups of terpenoids (neo-clerodane diterpenes, neo-clerodane sesquiterpenes, and limonoids) against Spodoptera exigua Hübner and the coleopteran, Leptinotarsa decemlineata Say, with results that suggested to us that we would expect to identify an antifeedant effect caused by terpenes against S. frugiperda, because it is congeneric with S. exigua. The latter is possible because both species share morphological, phylogenetic, and ecological characters, and they cope with similar secondary metabolites.

Effects of Tagetes Flower and Leaf Extracts on Larval mortality

In Table 2 are shown the observed percentages of mortality of S. frugiperda larvae treated with the T. erecta flower and leaf extracts made with n-hexane, acetone and ethanol. In the control (no extract) T. erecta, mortality was 0%, the flower extracts cause low mortality, while the leaf extracts caused substantial mortality (Table 2). According to the criterion proposed by Silva et al. (2003), plants and/or extracts with promise as bioinsecticides are those that cause a mortality > 40%. According to this criterion, T. erecta leaf extracts with hexane, acetone, or ethanol all were bioinsecticidal, because they produced mortalities of 48, 60, and 72%, respectively. Therefore, T. erecta has the toxic potential to be considered for as a source of bioinsecticides to control the fall armyworm.

TABLE 2.

EFFECT OF EXTRACTS OF TAGETES ERECTA FLOWERS AND LEAVES INCORPORATED INTO MERIDIC DIET ON THE MORTALITY OF SPODOPTERA FRUGIPERDA LARVAE. ALL EXTRACTS HAD BEEN INCORPORATED INTO THE MERIDIC DIET AT 500 PPM.

Effects of Tagetes Extracts on Duration of Larval Development

Table 3 shows the effects of the 6 phyto-extracts on the duration of S. frugiperda larval development. The durations of the larval stadia in the acetone flower and acetone leaf extract treatments did not differ significantly from that of the control (18.2 d ± 0.169e). Duration of the larval stadia was prolonged the most by the n-hexane flower extract treatment (41 d ± 2.407a) followed by the ethanol leaf extract (33.9 d ± 9.815b) treatments. The apparent slight prolongations of duration of the larval stadia in the ethanol flower, acetone flower and acetone leaf extract treatments did not differ significantly from each other. These results suggest that the n-hexane flower and ethanol leaf extracts possess metabolites with a phytoecdysteroid effect (Alonso 1998).

TABLE 3.

EFFECT OF EXTRACTS OF TAGETES ERECTA FLOWERS AND LEAVES INCORPORATED INTO MERIDIC DIET ON THE DURATION OF DEVELOPMENT OF SPODOPTERA FRUGIPERDA LARVAE. ALL EXTRACTS HAD BEEN INCORPORATED INTO THE MERIDIC DIET AT 500 PPM.

Effects of Tagetes Flower and Leaf Extracts on % Mortality of Pupae

Data in Table 4 demonstrate that the T. erecta flower extracts were not highly toxic to S. frugiperda pupae with the ethanol flower extract causing no mortality and the n-hexane flower and acetone flower extracts causing only 12% and 11% mortality, respectively. On the other hand, the various T. erecta leaf extracts caused pupal mortalities in the range of 57 to 80%; indeed the acetone leaf extract caused the greatest mortality. In each treatment the various T. erecta leaf extracts caused combined mortalities of larvae and pupae in the range of 80 to 97% (Table 4).