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Introduction
Maize Zea mays L. is a crop of great importance for food in Mesoamerican cultures; its availability requires that the crop be stored for extended periods of time. It is under these storage conditions that several insects feed on the grain and cause economic losses (Safitri et al. 2019). The corn weevil, Sitophilus zeamais (Motschulsky) and the large grain borer Prostephanus truncatus (Horn), are the most important pests of stored corn (Birkinshaw and Smith, 2000; Ribeiro et al. 2007; Demissie et al. 2015) causing losses of up to 30 % (Martinez et al. 2013). To reduce damage to stored grains, physical control methods (extreme temperatures, controlled atmospheres) and chemical methods (phosphine) are used, the latter can cause adverse effects on humans such as blisters, headaches, dizziness, bronchitis, and motor problems, among others (Reyna et al. 2012). An alternative to this problem could be plant extracts for the management of warehouse pests, but it is necessary to generate information to understand their action in toxicological aspects, as well as their extraction and application methods (Athanassiou et al. 2014b).
It is recognized beforehand how plants and other biological products have benefited humanity (Akkoc et al. 2019). In the last 35 years, several plant species with insecticidal properties and low environmental impact, such as those associated with traditional agroecosystems, have been studied in Latin America (Pérez et al. 2017). In specific, plant extracts for pest management of stored products offer a traditional and economically viable alternative use. A parallel aspect is to update regulations to recognize these alternatives and continue promoting the development and application of this kind of product (Stevenson et al. 2014).
For stored grain pests, the use of powders as a preventive action has been evaluated (Baek et al. 2013) since it is reported that once the insect is introduced into the grain, the extracts substantially diminish impact (Athanassiou and Kavallieratos, 2014a). In detail, for S. zeamais, the ethanolic extract of Pimienta pseudocaryophyllus (Myrtaceae) reduced the damage and, therefore, could be used as a bioinsecticide (Ribeiro et al. 2015). Although infusions may represent a more ecological (solvent-free) alternative, Lannacone et al. (2005) report that none of the aqueous extracts of Coriandrum sativum L. (Apiaceae), Caesalpinia spinosa (Mol.) Kuntze (Fabaceae), Bidens pilosa L. (Asteraceae) and Sambucus peruviana HBK (Caprifoliaceae) on adults of S. zeamais obtained more than 40 % mortality.
The effectiveness of plant extracts is associated with secondary metabolites such as terpenes, steroids, coumarins, flavonoids, phenolic acids, lignins, xanthones, and anthraquinones (Ogunleye et al. 2007). These metabolites have been related to plant families that can function as biopesticides. Thus, in grasses there are cyanohydrins in the leaves and phenols in the roots; for the Brassicaceae family are mainly glucosinates such as isothiocyanates; in the Meliaceae (Neem), terpenoids, limonoids, and flavonoids such as azadirachtin (Kokalis-Burelle and Rodríguez-Kábana, 2006). Likewise, herbivores can develop their own survival strategies. Pérez et al. (2008) found metabolites in larvae of the butterfly Battus philenor (L.) from its host Aristolochia californica (Torrey), produced with the purpose of frightening predation. Currently, many of the new products of plant origin with the ability to control pests are phenolics and limonoids from plants of the Meliaceae (azadirachtin) and Leguminosae (rotenone) families (Cespedes et al. 2016); being more and more reports of plant extracts as a low environmental impact alternative for the management of insect pests (González and Horianski, 2018). Considering the food and cultural importance of maize cultivation in Mexico, the need to promote the use of local, accessible biotic resources, as well as to have sustainable alternatives for pests in stored grains; this study aimed to evaluate the effects of plant extracts associated with the maize agroecosystem for the control of two of the pests of stored maize, S. zeamais, and P. truncatus.
Material and Methods
Plant collection and identification.
Whole plants were collected in three sites cultivated with maize in three municipalities of the State of Aguascalientes: Pabellón de Arteaga (N21º59.836" W102º15.888"), with irrigation system and high technology; Jesús María (N22º10.72" W102º17.648"), with intermediate technology and rainfed cultivation; and El Llano (N21º42.41" W102º09.55") with low technology and rainfed system. All the plants present at the edges of the plots and between the crop rows were collected: before sowing, during its development, and after harvest. For each of the collection points, the complete plants (aerial part and roots) were placed in polyethylene bags and transported to the Instituto Tecnológico El Llano Aguascalientes (ITEL) for subsequent identification, which was corroborated with the support of specialists from the Herbarium of the Instituto Tecnológico de Tlajomulco de Zúñiga, Jalisco.
Insect rearing.
Adult insects of S. zeamais and P. truncatus were obtained from the previous rearing and were reproduced in the ITEL laboratory by placing 10 pairs of each species in 0.5 L jars with 150 g of corn kernels at 22.5 2.5 ºC, 25-30 % R.H. and a 12:12 (light-dark) period (Hincapié et al. 2008), until the necessary number of insects was obtained for laboratory studies.
Extract preparation.
Whole plants were dried in the shade at room temperature (20-25 ºC) and constant weight. They were crushed with a commercial Hamilton stainless steel mixer until 500 g of plant material was obtained. From this material, two extraction methods were used: infusion and ethanol extraction. For extraction by infusion, 1 L of distilled water was heated at 80 °C for 10 min with constant stirring and 500 g of each plant was added (Benítez-Benítez et al. 2019). Subsequently, it was filtered and made up to 1 L with distilled water in sterile dark glass bottles, and then stored at low temperature (2 ºC) until use. For ethanol extraction, 500 g of the ground plant was taken and kept for five days in 1 L of ethanol (95 %) at cold (12 °C) and with shaking every 24 h (Tenorio-Rodríguez et al. 2017). The extracts were filtered, volumized to 1 L with ethanol (95 %), and stored at room temperature, in amber containers.
Bioassays with the olfactometer.
For this bioassay, only adult specimens of S. zeamais were used, employing a two-way "Y" shaped glass olfactometer with an inner diameter of 15 mm and a 90° angle between the arms. The insects were introduced individually in the base of the olfactometer, connected to a vacuum pump (Barnant Co. Model 400-3901) with a flow rate of 25 ml min-1 and where at the end of each arm was placed a kitazate flask (125 ml) in one of them were placed the extracts (2 ml) as olfactory stimuli, impregnated in pieces of filter paper (2 x 6 cm) and in the other was placed ethanol or distilled water as control. The conditions for the bioassays were: 230 lux illumination, 22 °C +/- 2 °C temperature, and 29 +/- 5 % RH.
The maximum time for each bioassay was 5 min, recording the time and the distance traveled by the insect through the arm of the olfactometer. The olfactometer was rotated 180° on its axis after each bioassay to rule out orientation bias. Twelve replicates were performed and with each treatment change, the device was cleaned with phosphate-free detergent and water, rinsed with acetone, and oven-dried for 30 min (Vuts et al. 2018). An analysis of variance and the mean comparison test (Tukey α=0.05) were performed. With the exception of Melilotus albus Medik, which was not included in the olfactometry test of ethanolic and infused extracts, those that presented the best results as attractants and repellents were chosen to be considered in the evaluation of effectiveness against pests in stored corn.
Effect of extracts against corn borer weevils under laboratory conditions.
A completely randomized design with 10 treatments and three replications was used, the experimental unit was a 500 ml wide-top plastic jar with 20 adult insects of variable age and 250 g of corn kernels previously examined to ensure their sanitation. The extracts were applied topically at a rate of 5 ml of the base solution per bottle (Ribeiro et al. 2014). For this purpose, the corn kernels were spread on a surface and with a sprinkler the experimental solution was sprayed homogeneously, allowing the kernels to dry before emptying them into the jar and adding the insects. Mortality was recorded at 72 h and the number of dead insects was counted and verified with the help of a dissecting needle (Adarkwah et al. 2017). The jars were covered with a muslin cloth (Cardoso-Almeida et al. 2014) and kept at room temperature (22+/-2.5 °C). The recorded data were subjected to analysis of variance, and Tukey's test (α=0.05).
Results and Discussion
Plant collection and identification.
A total of 16 plants were identified, located in 11 families, in the three municipalities of the state, associated with maize cultivation (Table 1). Samples of these plants are catalogued and stored in the herbarium of the Instituto Tecnológico de Tlajomulco.
Table 1 Plant species asociated to maize agroecosystem on three Aguascalientes municipalities: Pabellón de Arteaga (Pab), Jesús María (JM) and El Llano (Ll).
| Common name | Scientific name | Family (Location) |
|---|---|---|
| Rodadora | Atriplex suberecta | Chenopodiaceae (Pab) |
| Cebadilla criolla | Bromus catharticus | Poaceae (Pab) |
| Trompetilla | Bouvardia ternifolia | Rubiaceae (Pab) |
| Hierba del perro | Brickellia veronicifolia | Asteraceae (Pab) |
| Alfombrilla | Verbena (= Glandularia) bipinnatifida | Verbenaceae (Pab) |
| Malva | Malva parviflora | Malvaceae (JM) |
| Alfalfilla amarilla | Melilotus indicus | Fabaceae (JM) |
| Escoba amarga | Parthenium hysterophorus | Asteraceae (JM) |
| Kikuyo | Pennisetum clandestinum | Poaceae (JM, Ll) |
| Tabaquillo | Nicotiana glauca | Solanaceae (JM) |
| Gualda | Reseda luteola | Resedaceae (JM, Ll) |
| Lengua de vaca | Rumex crispus | Polygonaceae (Ll) |
| Salvia | Salvia ballotiflora | Lamiaceae (Ll) |
| Hierba mora | Solanum nigrescens | Solanaceae (Ll) |
| Borraja | Sonchus oleraceus | Asteraceae (Ll) |
| Alfalfilla blanca | Melilotus albus | Fabaceae (Ll) |
Olfactometry results.
Figure 1 shows the results of attraction and repellency of pest insects to the infusion (A) and ethanolic (B) extracts. For the infusions, repellency of up to 80 % was obtained with the B. catharticus extract, followed by V. bipinnatifida with 40 % repellency. While the highest attraction (80 %) was obtained with A. suberecta and R. luteola (Figure 1A), followed by B. ternifolia, M. parviflora, P. clandestinum, with 75 % attraction. Particularly, the compounds involved with this repellency of the Alfombrilla (V. bipinnatifida) are flavonoids and phenolic acids (Umber, 1980). Attraction to plant stimuli that are not their natural host, in some insects such as weevils, presents a wide range of responses as they become attracted to fruits such as apples and peach (Nornberg et al. 2018). Repellency, on the other hand, is likely to be caused by the presence of volatile alkaloids (Patiño-Bayona et al. 2021). For ethanolic extracts, the highest repellency occurred with P. hysterophorus, (80 %) followed by P. clandestinum (75 %) and N. glauca (70 %) (Figure 1B). B. catharticus, obtained the highest percentage of attraction (83 %). Extracts of P. hysterophorus have also been recommended for the management of the diamondback moth, Plutella xylostella (L.) by showing promising toxicity on larvae after 96 h (Reddy et al. 2017).
Figure 1 Attraction and repelence percent on S. zeamais adult olfactometry tests to plant extracts in infusion (A) and in ethanol (B)
Toxicity evaluation of the extracts in the laboratory.
The results showed that the infusion extract of B. catharticus obtained the highest percentage of mortality in the two insect pests, 57.5 % for S. zeamais and 70 % for P. truncatus. For S. zeamais, alcohol extracts of N. glauca, P. clandestinum, and R. luteola also presented worthy performance, causing more than 42 % mortality in this insect. This effect of Gualda (R. luteola) is related to its secondary metabolites containing sulfur and nitrogen (Burger et al. 2017). In P. truncatus, two of the extracts in alcohol presented outstanding results by causing mortality of 62.5 % (R. luteola) and 50 % (S. ballotiflora); as well as the alcoholic extracts of N. glauca and P. clandestinum, which caused more than 40 % mortality (Table 2). The use of infused plant extracts and ethyl alcohol is an alternative to the use of synthetic pesticides, due to their effectiveness against stored grain pests, according to Román-Farje et al. (2017), who evaluated infused V. bipinnatifida and ethanol extract of M. indicus against two maize weevils and reported that for S. zeamais the highest damage to maize grains was 3 % with the infused extract, while for P. truncatus there was higher grain damage with the alcohol extract (17 %). De Souza et al. (2014) mention that plants of the Fabaceae family, such as Dimorphandra mollis Benth. have good insecticidal properties against the corn weevil, S. zeamais. Other studies have reported good results using plant extracts for the management of the corn weevil S. zeamais and other insect pests (Lannacone and Quispe 2004). Souza et al. (2018) mention that the difference in damage of S. zeamais is due to the hardness characteristics of maize kernels of different varieties. Several species of the genus Melilotus, such as Melilotus indicus L. and Melilotus albus, have been reported to contain several bioactive compounds such as terpenes, sterols, and polyphenols (Romo-Asunción et al. 2016). De Souza et al. (2009)mention that the presence of several metabolites in an extract causes a summative effect, which increases insecticidal activity and decreases the traditional resistance problems of synthetic products. Therefore, the toxicity of an extract will be greater than that of an isolated metabolite and it should not be ruled out that in future studies the effects reported here may differ if other active principles are extracted according to the polarity of the solvent used (Lizarazo et al. 2008).
Table 2 S. zeamais and P. truncatus mortality after the ethanolic and infusion extracts application.
| Treatment | Extract | (%) Mortality | |
|---|---|---|---|
| S. zeamais | P.truncatus | ||
| B. catharticus | Infusión | 57.5(5.0 a | 70.0(5.0 a |
| N. glauca | Alcohol | 47.5(5.0 a | 42.5(9.6 ab |
| P. clandestinum | Alcohol | 45.0(5.0 a | 42.0(2.2 ab |
| R. luteola | Alcohol | 42.5(5.0 a | 62.5(17.3 a |
| V. bipinnatifida | Infusión | 25.0(5.0 b | 22.5(5.0 bc |
| S. ballotiflora | Alcohol | 17.5(5.7 bc | 50.0(0.9 ab |
| M. indicus | Alcohol | 12.5(5.0 bcd | 35.0(9.5 ab |
| P. hysterophorus | Alcohol | 7.5(5.7 cd | 35.0(5.7 ab |
| M. albus | Infusión | 2.5(0.5 cd | 35.0(5.7 ab |
| Control | water | 0(0 d | 0(0 c |
Means followed by the same letter do not differ significantly (P≤0.05, Tukey test).
As mentioned by Athanassiou et al. (2014b), the commercial use of plant extracts still has to address several aspects, such as patents and intellectual rights, differences in extraction methods, and formulation of active ingredients. Although there are advances, standardization of extraction techniques and homogeneity in formulations is essential. The main properties of pesticides of vegetable origin are that they are biodegradable and exert low toxicity to vertebrates, which makes them highly friendly to the environment of stored grains. Obtained results in this work can be integrated as a low environmental impact strategy to implement an agroecological pest management program and promote optimal corn storage under a sustainable agricultural management scheme.
Conclusions
In the maize agroecosystem studied in Aguascalientes, 16 associated plant species distributed in 11 families were identified. Experimentally, nine of these species produced greater repellency in laboratory olfactometry bioassays; from which the effect on the mortality of the two stored corn pests, S. zeamais, and P. truncatus, was evaluated. The infusion extract of Bromus catharticus (Poaceae) showed the greatest effect in reducing the two stored corn pests by decreasing infestation by 57-70 %. Additionally, Reseda luteola (Resedaceae) showed good results for P. truncatus, with a mortality of 62.5 %. Alcoholic extracts of three other plants, N. glauca, P. clandestinum, and S. ballotiflora, caused mortality of the two insect pest species between 42-50 %. In general, five of the nine plants evaluated against the two stored corn pests showed promising results, so that, according to how effectively these plants perform under storage conditions, could be used in stored corn pest management programs to replace chemically synthesized pesticides.
