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New insecticide delivery method for the control of Sitophilus zeamais in stored maize
Journal of Stored Products Research 83 (2019) 185e190
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
New insecticide delivery method for the control of Sitophilus zeamais in stored maize n a, b, M.P. Zunino a, b, V.L. Usseglio a, b, M.L. Peschiutta a, b, V.D. Brito a, b, *, F. Achimo J.A. Zygadlo a, b tedra de Química Orga nica, Av. V rdoba, Facultad de Ciencias Exactas, Físicas y Naturales, Departamento de Química, Ca Universidad Nacional de Co elez rdoba, Argentina Sarsfield 1611, X5016GCA, Co b rdoba, Av. V rdoba, Instituto Multidisciplinario de Biología Vegetal (IMBIV), CONICET-Universidad Nacional de Co elez Sarsfield 1611, X5016GCA, Co Argentina a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2019 Received in revised form 19 June 2019 Accepted 22 June 2019
Maize kernel deterioration caused by the action of insects has led to an urgent need to develop new control methods against the maize weevil, Sitophilus zeamais, one of the major pests found in silo bags during the storage of maize. Here, we evaluated the insecticidal efficiency of plasticized and unplasticized cotton matrices (deliveries), loaded with R-(þ)-pulegone, ()-carvone, 2-decanone and trans-2hexenol against S. zeamais. R-(þ)- pulegone was the only compound that produced weevil mortality. Plasticized delivery loaded with R-(þ)-pulegone achieved a 90% mortality on the 12th day, with mortality values recorded of above 96% over the course of 30 days. R-(þ)-pulegone from plasticized delivery was released more slowly compared to unplasticized delivery. Moreover, delivery loaded with R-(þ)-pulegone did not show phytotoxicity in maize kernels. Hence, due to its effectiveness against the weevil and the lack of phytotoxic activity against maize kernels, plasticized delivery loaded with R-(þ)-pulegone represents a promising material for S. zeamais control. However, large-scale studies are needed in order to evaluate its potential use in grain storage systems. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Maize kernels Cotton matrix Delivery Plasticizer Insect control
1. Introduction Maize is one of the most cultivated crops, with there being currently 183.73 million hectares cultivated with maize worldwide and an annual production of 46 million metric tons, according to the Foreign Agricultural Service USDA (2019). This high yield requires simple and efficient storage systems such as silo bags. This system provides adequate storage conditions, thus enabling conservation and good grain quality. One of the major pests found in silo bags is the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) (Lamboni and Hell, 2009), and both the larvae and adults deteriorate the corn grains through their feeding and reproductive habits (Tefera et al., 2011), with this damage favoring the occurrence of several phytopathogenic fungi that synthetize different harmful
rdoba, Facultad de Ciencias * Corresponding author. Universidad Nacional de Co Exactas, Físicas y Naturales, Departamento de Química, C atedra de Química lez Sarsfield 1611, X5016GCA, Co rdoba, Argentina. Org anica, Av. Ve E-mail address: [email protected] (V.D. Brito). https://doi.org/10.1016/j.jspr.2019.06.013 0022-474X/© 2019 Elsevier Ltd. All rights reserved.
mycotoxins (Chulze, 2010; Nesci et al., 2011). Fumigation is a traditional method widely used to control post-harvest insects. An effective fumigant pesticide should not leave any residues in grains or affect the nutritional value, with its removal by aeration carried out after use (Plimrner, 1982; Lee et al., 2001). However, this traditional method of application results in a great deal of waste. According to Akelah (1996), the 90% of the applied agrochemicals never reach their objective causing serious contamination problems. Therefore, any controlled-release mechanism that can minimize the required doses of insecticides is useful (Langer, 1980). In recent years, there has been an increased focus on searching for new organic insecticides and novel methods of application to control S. zeamais in silo bags. Related to this, the use of polymers to provide a controlled long-term delivery of insecticides is a rapidly emerging field. Moreover, insecticides incorporated into cotton matrices and protected by a polymer multilayered structure have been used as repellents or attractants against stored product insects (Akelah, 2013). These cotton matrices are potentially useful as packaging containers or as insect resistant barriers for stored food products (Akelah, 1996). In addition, some adhesives combined
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with insecticides have already been used as protectors against termites and other insects (Jobic, 2009). Previous studies found that R-(þ)-pulegone and ()-carvone (Herrera et al., 2015), 2-decanone (Zunino et al., 2015) and trans-2hexenol (Calvimonte, 2016) have a high fumigant insecticide effect against the maize weevil. However, it remains to be elucidated the best compound and method to apply in a maize kernel storage system. Thus, the aim of this study was to determine the fumigant insecticidal activity of these compounds against the maize weevil by using unplasticized and plasticized insecticide delivery methods.
compound treatment) and a positive control (delivery loaded with dichlorvos, 0.06 mL/L air, Peschiutta et al. (2016)) were also performed. This compound was used as the positive control due to its well-known insecticidal activity and high vapor pressure. Insect mortality was quantified at 6, 12, 16, 20, 27 and 30 days after the application of treatments (exposure time) and mortality percentages were calculated. To avoid the loss of the treatment compounds, independent bags were analyzed at each exposure time. Five replicates were performed for each compound and exposure time.
2. Materials and methods
2.4. R- (þ)-Pulegone release from the deliveries
2.1. Insects
To determine the capacity of the plasticized and unplasticized deliveries to retain R-(þ)-pulegone, we evaluated the diffusion of this compound (proven to be effective against weevils) out of the storage system. Polypropylene bags have a nominal pore size ranging from 80 to 150 mm, which allows gaseous exchange (POREX, 2018). After 24 h, we randomly selected bags of plasticized and unplasticized deliveries loaded with R-(þ)-pulegone, and controls. Each bag was then introduced into a vial (50 mL) closed with a septum and placed in a bath at 25 C for 20 min. Compounds released to the headspace of the vial were captured by solid-phase microextraction (polydimethylsiloxane, SPME; Supelco, Bellefonte, PA, USA) and injected into a GC-MS for identification and quantification. This was performed using a GC-MS Perkin Elmer 600, equipped with a mass selective detector in the electron impact mode (70 eV) and a DB-5 column (30 m 0.25 mm x 0.25 mm; Elite 5 MS Perkin Elmer). The injection port was operated at 250 C in a splitless mode, and the temperature program used was 60 C for 5 min, ramped up to 170 C at 4 C/minute, and then 170e240 C at 20 C/min. Helium was used as a carrier gas with a constant flow of 1 mL/min. The temperature of GC/MS interface was 200 C, and the scan range was 40e350 amu. Identification of the volatile compounds was performed by comparing their mass spectra with the NIST-08 Mass Spectral Library (US National Institute of Standards and Technology) and by comparing their retention index (RI) with authentic standards. The RI of R-(þ)-pulegone was obtained after analysis of the C9eC20 nalkanes series (1 mL; Sigma Aldrich Co. Buenos Aires, Argentina). The amount of R-(þ)-pulegone released out of the bags was estimated using a standard calibration curve.
For all the experiments, unsexed Sitophilus zeamais adults were used. The insects were reared over several generations in sealed containers (10 L) with free-insecticide maize kernels, under laboratory controlled conditions (28 ± 2 C and 70 ± 5% relative humidity). All bioassays were carried out under the same laboratory conditions and in complete darkness (FAO, 1974). 2.2. Description of deliveries and chemical products Two different systems to deliver R-(þ)-pulegone, ()-carvone, 2-decanone and trans-2-hexenol were tested. A 1 cm2 cotton matrix (fabric) of 1 mm thick (from now on referred to as unplasticized) and a cotton matrix impregnated with a plasticizer (from now on called plasticized) were used. The plasticizer used was vinyl adhesive, an aqueous emulsion based on polyvinyl acetate. Compounds were incorporated into the delivery system and then dipped into the plasticizer (21.5 mg) and left to dry for 5 min. Scanning electron microscope (SEM) images of three plasticized and unplasticized cotton matrices were obtained using a FE-SEM Sigma microscope at an accelerating voltage of 10.00 kV, and processed using the ImageJ 1.47 k Software. The physical characteristics of the plasticized and unplasticized deliveries were determined from 10 fibers forming cotton yarns. The compounds R-(þ)- pulegone, ()-carvone, 2-decanone, trans-2-hexenol and polyvinyl acetate were purchased from Sigma Aldrich (Buenos Aires, Argentina), and the compound 2,2dichlorovinyl dimethyl phosphate (dichlorvos; >98%, Chemotecnica S.A, Buenos Aires, Argentina) was used as the reference insecticide, which were selected because of their high fumigant activity against S. zeamais (Herrera et al., 2015; Zunino et al., 2015; Calvimonte, 2016). The chemical properties of the selected compounds, such as lipophilicity (Log P: Logarithm of the octanol/water partition coefficient) and vapor pressure (VP), were obtained from the ChemSpider database (ChemSpider, 2018). The chemical structures, VP and LogP of these compounds are shown in Fig. 1. 2.3. Bioassays Assays were carried out using polypropylene bags (4 25 cm), as they are versatile transparent non-toxic materials. In addition, polypropylene prevents moisture transfer and has good organoleptic, chemical and resistance properties (POREX, 2018). For the bioassays, 20 g of maize kernels and 10 adult weevils were placed in each bag. Then, each delivery (plasticized and unplasticized) was loaded with the treatment compound (R(þ)-pulegone, ()-carvone, 2-decanone or trans-2-hexenol) at a dose of 127.2 mL/L air (9 times the LD95 of R-(þ)-pulegone against S. zeamais at 24 h, according to Herrera et al. (2015)). A negative control (plasticized and unplasticized deliveries without
2.5. Phytotoxicity assay After 7 and 28 days of the experiment, maize grains from each treatment were extracted and randomly selected to evaluate the phytotoxicity of each delivery, using a standard germination test (Agrawal, 1980). Seven grains per Petri dish (three Petri dishes per treatment) were placed on filter paper moistened with 1 mL of distilled water. The seeds were maintained at 24 C and the germination of the kernels was observed after 9 days. 2.6. Statistical analysis All data were analyzed to assess normality using the ShapiroWilks test, and homogeneity of the variances was determined using the Levene test before performing an ANOVA. The Student's ttest was used to compare the physical characteristics of the deliveries and the mortality percentage of S. zeamais among treatments (plasticized and unplasticized deliveries for each exposure time). A two-way ANOVA and Tukey HSD posteriori test (P < 0.01, Di Rienzo et al. (2017)) were used for the phytotoxicity assays.
M.L. Peschiutta et al. / Journal of Stored Products Research 83 (2019) 185e190
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Fig. 1. Molecular structures of the compounds and 2,2-dichlorovinyl dimethyl phosphate. The values shown of Log P (Logarithm of the octanol/water partition coefficient) and VP (Vapor pressure, at 25 C) were obtained using ChemSpider (2018).
3. Results The physical characteristics of the deliveries did not show significant differences (P ¼ 0.92) between the thickness of fibers forming cotton yarn of plasticized and unplasticized deliveries (15.11 ± 0.54 and 15.23 ± 1.04 mm, respectively). In addition, no significant differences were found in yarn thickness between both matrices (273.20 ± 6.26 and 272.57 ± 6.51 mm, respectively; P ¼ 0.95) or in the spaces between the networks of yarns for plasticized and unplasticized deliveries (0.05 ± 0.01 and 0.03 ± 0.01 mm2, respectively; P ¼ 0.10). These features are shown in the scanning microscopy images (Fig. 2). The plasticized and unplasticized deliveries loaded with R(þ)-pulegone were the only treatments that caused insect mortality over time (Fig. 3). Plasticized delivery loaded with R(þ)-pulegone had mortality values of 96%, similar to those produced by the positive control at the end of the experiment (30 days). Moreover, insecticide bioassays with deliveries loaded with R-(þ)-pulegone showed that the toxic activity increased with
exposure time (Fig. 3). Plasticized delivery loaded with R(þ)-pulegone achieved a 90% mortality at day 12, and remained above 96% over the course of 30 days. In contrast, unplasticized delivery loaded with R-(þ)-pulegone showed mortality values of 30% recorded on day 12, and attained a 68% mortality by the end of the experiment (Fig. 3). R-(þ)-pulegone from plasticized delivery was released more slowly (0.04 mL, Fig. 4d) than the unplasticized delivery (0.07 mL, Fig. 4c), with the former allowing a high percentage of mortality from day 12. Plasticized and unplasticized deliveries loaded with 2-decanone (P ¼ 0.92), ()-carvone (P ¼ 1) or trans-2-hexenol (P ¼ 0.86) exhibited mortality values below 6% throughout the experiment. The plasticized and unplasticized deliveries loaded with dichlorvos reached mortality values of 100% on days 6 and 12, respectively. The plasticizer itself showed no insecticidal effect (negative control, P ¼ 0.11) and had mortality values below 2% (data not shown). An interaction between the compound assessed and exposure time (P < 0.001, Table 1) was found in the phytotoxicity tests (Table 1). The plasticized and unplasticized deliveries loaded with
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Fig. 2. Scanning electron microscopy (SEM) images obtained using an FE-SEM Sigma on: unplasticized (a, c) and plasticized (b, d, e) cotton matrix section (fabric) (a, b) fibers form yarn, (c, d) detail of the fibers, (e) binding of the fibers to each other by the action of the plasticizer. The scale bar represents 200 mm (a, b), 20 mm (c, d) and 2 mm (e).
trans-2-hexenol were the most phytotoxic, with low percentages (less than 60%) or a complete lack of germination. Deliveries loaded with R-(þ)-pulegone did not show phytotoxicity in maize kernels (Table 1). 4. Discussion In the present study, we found that only R-(þ)- pulegone incorporated in the cotton matrices produced weevil mortality. It is widely known that R-(þ)-pulegone presents high toxicity and is lethal to different species of insects even in extremely small amounts (Franzios et al., 1997; Rossi et al., 2011; Herrera et al., 2015; Peschiutta et al., 2017). Also, R-(þ)-pulegone is not as volatile as others organic compounds but is highly lipophilic, tending to be more toxic (Philippou et al., 2016; Peschiutta et al., 2017). Furthermore, this kind of compounds are less selective in binding to proteins, in some cases being chemically reactive and are extensively metabolized (Winiwarter et al., 2007; Eckert and Trinh, 2016).
The effectiveness of cotton fibers to retain volatile compounds is an important feature for determining the insecticidal effect over time. This depends mainly on the total compound retained in the deliveries after drying and on the release rates (Langer, 1980). We found that R-(þ)-pulegone treatment with plasticizer improved its insecticidal activity, suggesting that plasticized delivery could be a suitable material for controlling the release of the bioactive compounds. It has been previously shown that the incorporation of organic compounds in solid polymers permits a controlled release of volatile compounds over an extended period of time (Herrera et al., 2017). Abdel-Mohdy et al. (2008) reported that cyclodextrin and an insecticide binding to each other to form an inclusion complex, where the insecticide acts as an agent molecule in the center of the hydrophobic interior of the cyclodextrin. This inclusion complex was incorporated into the delivery and allowed the insecticide to remain attached, even after repeated washing, thereby prolonging the insecticidal efficacy of the matrix. On the other hand, deliveries loaded with R-(þ)-pulegone did not show phytotoxicity in maize kernels, which is a very important
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Table 1 Phytotoxicity (% of maize germination) of plasticized and unplasticized delivery loaded with R-(þ)-pulegone, ()-carvone, 2-decanone and trans-2-hexenol after 7 and 28 days of delivery exposure (100% indicates complete germination). Cotton matrix without compound treatment was used as negative control and delivery with dichlorvos (0.06 mL/L air) was used as positive control.
R-(þ)-Pulegone 2-decanone ()-carvone Trans-2-hexenol Negative control Positive control
Phytotoxicity (germination, %)
7 days
28 days
Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized
92.8 ± 4.1 92.8 ± 4.1 92.8 ± 4.1 100.0 ± 0.0 92.8 ± 4.1 85.7 ± 0.0 0.0 ± 0.0 * 57.1 ± 4.1 * 100.0 ± 0.0 100.0 ± 0.0 85.71 ± 8.25 92.86 ± 4.13
100.0 ± 0.0 100.0 ± 0.0 92.8 ± 4.1 100.0 ± 0.0 100.0 ± 0.0 92.8 ± 4.1 0.0 ± 0.0 * 14.2 ± 8.2 * 100.0 ± 0.0 100.0 ± 0.0 85.71 ± 0.00 100.00 ± 0.00
Values are expressed as means ± SE. * Indicate the highest phytotoxicity (< 60% germinated) according to Tukey HSD test, (P < 0.05). Fig. 3. Mortality percentages of Sitophilus zeamais for plasticized and unplasticized deliveries loaded with R-(þ)-pulegone at a dose of 127.2 mL/L. Vertical bars represent the mean mortality value ± SE (n ¼ 5) of plasticized (black bars) and unplasticized (white bars) deliveries over time. Significant differences between treatment within each time are indicated as: ***P < 0.001and *P < 0.05 (t-test).
feature for potential use of this compound against the maize weevil. In agreement with this result, previous studies showed that this compound presents a low phytotoxicity in plants (Herrera et al., 2015; Chaimovitsh et al., 2017). Hence, given the effectiveness against the weevil and the lack of phytotoxic activity against
maize kernels, plasticized delivery loaded with R-(þ)-pulegone could serve as a potential tool for controlling S. zeamais in a grain storage system, such as in silo bags. The search for novel insecticides and new methods of delivery to control S. zeamais will continue as this insect is still one of the most economically important pests in stored grains. Certain synthetic insecticides have been replaced because of increasing public demand for safer and more effective products, among other causes (Buczkowski and Schal, 2001; Agrovoz, 2018). New active organic ingredients and innovative delivery tools have consequently emerged as providing an effective means of managing pests
Fig. 4. Gas chromatography/mass spectrometry (GC/MS) chromatograms indicating the release of R-(þ)-pulegone. Peaks: 1: R-(þ)-pulegone: (a) unplasticized delivery without treatment compound (control), (b) plasticized delivery without treatment compound (control), (c) unplasticized delivery loaded with R-(þ)-pulegone (Release rate: 3.08*103 mL/h), (d) plasticized delivery loaded with R-(þ)-pulegone (Release rate: 1.64*103 mL/h).
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(Peschiutta et al., 2016; Herrera et al., 2017). From the results of the present study, we propose the use of cotton fabric (delivery) impregnated with R-(þ)-pulegone and a polyvinyl adhesive plasticizer as an effective alternative for weevil control in a grain storage system. However, further research is still necessary in order to evaluate the efficacy of this delivery method on a larger scale and under field conditions. Acknowledgements This work complies with Argentinean laws. The authors also declare that there is no conflict of interest. Financial support for this work came from the following sources: FonCyT (PICT 2012-2146), CONICET (PIP 11220120100661CO) and Universidad Nacional de rdoba. We thank Dr. Paul Hobson, native speaker, for revising the Co manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jspr.2019.06.013. References Abdel-Mohdy, F., Fouda, M.M., Rehan, M., Aly, A., 2008. Repellency of controlledrelease treated cotton fabrics based on cypermethrin and prallethrin. Carbohydr. Polym. 73, 92e97. Agrawal, R.L., 1980. Seed Technology. Seed Technology. Agrovoz, 2018. Prohíben el uso de un insecticida que se USA en el almacenamiento de granos. http://agrovoz.lavoz.com.ar/agricultura/prohiben-el-uso-de-uninsecticida-que-se-usa-en-el-almacenamiento-de-granos. Akelah, A., 1996. Novel utilizations of conventional agrochemicals by controlled release formulations. Mater. Sci. Eng. C 4, 83e98. Akelah, A., 2013. Functionalized Polymeric Materials in Agriculture and the Food Industry. Springer. Buczkowski, G., Schal, C., 2001. Method of insecticide delivery affects horizontal transfer of fipronil in the German cockroach (Dictyoptera: blattellidae). J. Econ. Entomol. 94, 680e685. Calvimonte, H., 2016. Microorganismos y Plantas como Fuentes Productoras de Bioalcoholes para el Control de Sitophilus zeamais, Facultad de Ciencias Exactas, rdoba, Co rdoba, Argentina, p. 39. Físicas y Naturales. Universidad Nacional de Co Chaimovitsh, D., Shachter, A., Abu-Abied, M., Rubin, B., Sadot, E., Dudai, N., 2017. Herbicidal activity of monoterpenes is associated with disruption of microtubule functionality and membrane integrity. Weed Sci. 65, 19e30. ChemSpider, 2018. Search and share chemistry. http://www.chemspider.com/. Chulze, S., 2010. Strategies to reduce mycotoxin levels in maize during storage: a review. Food Addit. Contam. 27, 651e657. Di Rienzo, J.A., Casanoves, F., Balzarini, M.G., Gonzalez, L., Tablada, M., C.W.,R, 2017. n 2017. Universidad Nacional de Co rdoba, Argentina. InfoStat Versio Eckert, C.A., Trinh, C.T., 2016. Biotechnology for Biofuel Production and Optimization. Elsevier.
FAO, 1974. Metodo provisional para gorgojos adultos importantes en cereals almacenados con malation o lindano. Boletín Fitosanitario de la FAO, 22, 127e137. Metodo N 15 la FAO. Franzios, G., Mirotsou, M., Hatziapostolou, E., Kral, J., Scouras, Z.G., MavraganiTsipidou, P., 1997. Insecticidal and genotoxic activities of mint essential oils. J. Agric. Food Chem. 45, 2690e2694. ~ i, M.L., Gan ~ an, N.A., Zygadlo, J.A., 2017. An insecticide formulation Herrera, J.M., Gon of terpene ketones against Sitophilus zeamais and its incorporation into low density polyethylene films. Crop Protect. 98, 33e39. ~ an, N.A., Lucini, E.I., Herrera, J.M., Zunino, M.P., Dambolena, J.S., Pizzolitto, R.P., Gan Zygadlo, J.A., 2015. Terpene ketones as natural insecticides against Sitophilus zeamais. Ind. Crops Prod. 70, 435e442. Jobic, S., 2009. Wood adhesive comprising an insecticide. BASF agro B.V., arnhem (NL), wadenswil (CH). Patent No. US 7,604,813 B2. https://patentimages.storage. googleapis.com/b3/36/18/211c5934d9ab78/US7604813.pdf. Lamboni, Y., Hell, K., 2009. Propagation of mycotoxigenic fungi in maize stores by post-harvest insects. Int. J. Trop. Insect Sci. 29, 31e39. Langer, R., 1980. Invited review polymeric delivery systems for controlled drug release. Chem. Eng. Commun. 6, 1e48. Lee, S.-E., Lee, B.-H., Choi, W.-S., Park, B.-S., Kim, J.-G., Campbell, B.C., 2001. Fumigant toxicity of volatile natural products from Korean spices and medicinal plants towards the rice weevil, Sitophilus oryzae (L). Pest Manag. Sci. 57, 548e553. Nesci, A., Montemarani, A., Passone, M.A., Etcheverry, M., 2011. Insecticidal activity of synthetic antioxidants, natural phytochemicals, and essential oils against an Aspergillus section Flavi vector (Oryzaephilus surinamensis L.) in microcosm. J. Pest. Sci. 84, 107e115. Peschiutta, M., Arena, J., Ramirez Sanchez, A., Gomez Torres, E., Pizzolitto, R., Merlo, C., Zunino, M., Omarini, A., Dambolena, J., Zygadlo, J., 2016. Effectiveness of Mexican oregano essential oil from the Dominican Republic (Lippia graveolens) against maize pests (Sitophilus zeamais and Fusarium verticillioides). AgriScientia 33, 89e97. Peschiutta, M., Pizzolitto, R., Ordano, M., Zaio, Y., Zygadlo, J., 2017. Laboratory evaluation of insecticidal activity of plant essential oils against the vine mealybug, Planococcus ficus. Vitis 56, 79e83. Philippou, D., Borzatta, V., Capparella, E., Moroni, L., Field, L., Moores, G., 2016. The use of substituted alkynyl phenoxy derivatives of piperonyl butoxide to control insecticide-resistant pests. Pest Manag. Sci. 72, 1946e1950. Plimrner, J., 1982. Pesticides for stored products. In: Mastumura, F., Krishma Murti, C. (Eds.), Biodegradation of Pesticides. Plenum Press, New York, pp. 239e255. POREX, 2018. © Porex Corporation. http://www.porex.com/technologies/materials/ porous-plastics/polypropylene/. Rossi, Y., Canavoso, L., Palacios, S., 2011. Molecular response of Musca domestica L. to Mintostachys verticillata essential oil, (4R)(þ)-pulegone and menthone. Fitoterapia 83, 336e342. Tefera, T., Mugo, S., Likhayo, P., Beyene, Y., 2011. Resistance of three-way cross experimental maize hybrids to post-harvest insect pests, the larger grain borer (Prostephanus truncatus) and maize weevil (Sitophilus zeamais). Int. J. Trop. Insect Sci. 31, 3e12. USDA, U.S.D.o.A., 2019. World Agricultural Production. Office of Global Analysis, International Production Assessment Division (IPAD), Washington, DC. €m, M., Ungell, A.-L., Andersson, T., Zamora, I., 2007. Use of Winiwarter, S., Ridderstro Molecular Descriptors for Absorption, Distribution, Metabolism, and Excretion Predictions. Zunino, M.P., Herrera, J.M., Pizzolitto, R.P., Rubinstein, H.c.R., Zygadlo, J.A., Dambolena, J.S., 2015. Effect of selected volatiles on two stored pests: the fungus Fusarium verticillioides and the maize weevil Sithophilus zeamais. J. Agric. Food Chem. 63, 7743e7749.
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
New insecticide delivery method for the control of Sitophilus zeamais in stored maize n a, b, M.P. Zunino a, b, V.L. Usseglio a, b, M.L. Peschiutta a, b, V.D. Brito a, b, *, F. Achimo J.A. Zygadlo a, b tedra de Química Orga nica, Av. V rdoba, Facultad de Ciencias Exactas, Físicas y Naturales, Departamento de Química, Ca Universidad Nacional de Co elez rdoba, Argentina Sarsfield 1611, X5016GCA, Co b rdoba, Av. V rdoba, Instituto Multidisciplinario de Biología Vegetal (IMBIV), CONICET-Universidad Nacional de Co elez Sarsfield 1611, X5016GCA, Co Argentina a
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2019 Received in revised form 19 June 2019 Accepted 22 June 2019
Maize kernel deterioration caused by the action of insects has led to an urgent need to develop new control methods against the maize weevil, Sitophilus zeamais, one of the major pests found in silo bags during the storage of maize. Here, we evaluated the insecticidal efficiency of plasticized and unplasticized cotton matrices (deliveries), loaded with R-(þ)-pulegone, ()-carvone, 2-decanone and trans-2hexenol against S. zeamais. R-(þ)- pulegone was the only compound that produced weevil mortality. Plasticized delivery loaded with R-(þ)-pulegone achieved a 90% mortality on the 12th day, with mortality values recorded of above 96% over the course of 30 days. R-(þ)-pulegone from plasticized delivery was released more slowly compared to unplasticized delivery. Moreover, delivery loaded with R-(þ)-pulegone did not show phytotoxicity in maize kernels. Hence, due to its effectiveness against the weevil and the lack of phytotoxic activity against maize kernels, plasticized delivery loaded with R-(þ)-pulegone represents a promising material for S. zeamais control. However, large-scale studies are needed in order to evaluate its potential use in grain storage systems. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Maize kernels Cotton matrix Delivery Plasticizer Insect control
1. Introduction Maize is one of the most cultivated crops, with there being currently 183.73 million hectares cultivated with maize worldwide and an annual production of 46 million metric tons, according to the Foreign Agricultural Service USDA (2019). This high yield requires simple and efficient storage systems such as silo bags. This system provides adequate storage conditions, thus enabling conservation and good grain quality. One of the major pests found in silo bags is the maize weevil, Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) (Lamboni and Hell, 2009), and both the larvae and adults deteriorate the corn grains through their feeding and reproductive habits (Tefera et al., 2011), with this damage favoring the occurrence of several phytopathogenic fungi that synthetize different harmful
rdoba, Facultad de Ciencias * Corresponding author. Universidad Nacional de Co Exactas, Físicas y Naturales, Departamento de Química, C atedra de Química lez Sarsfield 1611, X5016GCA, Co rdoba, Argentina. Org anica, Av. Ve E-mail address: [email protected] (V.D. Brito). https://doi.org/10.1016/j.jspr.2019.06.013 0022-474X/© 2019 Elsevier Ltd. All rights reserved.
mycotoxins (Chulze, 2010; Nesci et al., 2011). Fumigation is a traditional method widely used to control post-harvest insects. An effective fumigant pesticide should not leave any residues in grains or affect the nutritional value, with its removal by aeration carried out after use (Plimrner, 1982; Lee et al., 2001). However, this traditional method of application results in a great deal of waste. According to Akelah (1996), the 90% of the applied agrochemicals never reach their objective causing serious contamination problems. Therefore, any controlled-release mechanism that can minimize the required doses of insecticides is useful (Langer, 1980). In recent years, there has been an increased focus on searching for new organic insecticides and novel methods of application to control S. zeamais in silo bags. Related to this, the use of polymers to provide a controlled long-term delivery of insecticides is a rapidly emerging field. Moreover, insecticides incorporated into cotton matrices and protected by a polymer multilayered structure have been used as repellents or attractants against stored product insects (Akelah, 2013). These cotton matrices are potentially useful as packaging containers or as insect resistant barriers for stored food products (Akelah, 1996). In addition, some adhesives combined
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with insecticides have already been used as protectors against termites and other insects (Jobic, 2009). Previous studies found that R-(þ)-pulegone and ()-carvone (Herrera et al., 2015), 2-decanone (Zunino et al., 2015) and trans-2hexenol (Calvimonte, 2016) have a high fumigant insecticide effect against the maize weevil. However, it remains to be elucidated the best compound and method to apply in a maize kernel storage system. Thus, the aim of this study was to determine the fumigant insecticidal activity of these compounds against the maize weevil by using unplasticized and plasticized insecticide delivery methods.
compound treatment) and a positive control (delivery loaded with dichlorvos, 0.06 mL/L air, Peschiutta et al. (2016)) were also performed. This compound was used as the positive control due to its well-known insecticidal activity and high vapor pressure. Insect mortality was quantified at 6, 12, 16, 20, 27 and 30 days after the application of treatments (exposure time) and mortality percentages were calculated. To avoid the loss of the treatment compounds, independent bags were analyzed at each exposure time. Five replicates were performed for each compound and exposure time.
2. Materials and methods
2.4. R- (þ)-Pulegone release from the deliveries
2.1. Insects
To determine the capacity of the plasticized and unplasticized deliveries to retain R-(þ)-pulegone, we evaluated the diffusion of this compound (proven to be effective against weevils) out of the storage system. Polypropylene bags have a nominal pore size ranging from 80 to 150 mm, which allows gaseous exchange (POREX, 2018). After 24 h, we randomly selected bags of plasticized and unplasticized deliveries loaded with R-(þ)-pulegone, and controls. Each bag was then introduced into a vial (50 mL) closed with a septum and placed in a bath at 25 C for 20 min. Compounds released to the headspace of the vial were captured by solid-phase microextraction (polydimethylsiloxane, SPME; Supelco, Bellefonte, PA, USA) and injected into a GC-MS for identification and quantification. This was performed using a GC-MS Perkin Elmer 600, equipped with a mass selective detector in the electron impact mode (70 eV) and a DB-5 column (30 m 0.25 mm x 0.25 mm; Elite 5 MS Perkin Elmer). The injection port was operated at 250 C in a splitless mode, and the temperature program used was 60 C for 5 min, ramped up to 170 C at 4 C/minute, and then 170e240 C at 20 C/min. Helium was used as a carrier gas with a constant flow of 1 mL/min. The temperature of GC/MS interface was 200 C, and the scan range was 40e350 amu. Identification of the volatile compounds was performed by comparing their mass spectra with the NIST-08 Mass Spectral Library (US National Institute of Standards and Technology) and by comparing their retention index (RI) with authentic standards. The RI of R-(þ)-pulegone was obtained after analysis of the C9eC20 nalkanes series (1 mL; Sigma Aldrich Co. Buenos Aires, Argentina). The amount of R-(þ)-pulegone released out of the bags was estimated using a standard calibration curve.
For all the experiments, unsexed Sitophilus zeamais adults were used. The insects were reared over several generations in sealed containers (10 L) with free-insecticide maize kernels, under laboratory controlled conditions (28 ± 2 C and 70 ± 5% relative humidity). All bioassays were carried out under the same laboratory conditions and in complete darkness (FAO, 1974). 2.2. Description of deliveries and chemical products Two different systems to deliver R-(þ)-pulegone, ()-carvone, 2-decanone and trans-2-hexenol were tested. A 1 cm2 cotton matrix (fabric) of 1 mm thick (from now on referred to as unplasticized) and a cotton matrix impregnated with a plasticizer (from now on called plasticized) were used. The plasticizer used was vinyl adhesive, an aqueous emulsion based on polyvinyl acetate. Compounds were incorporated into the delivery system and then dipped into the plasticizer (21.5 mg) and left to dry for 5 min. Scanning electron microscope (SEM) images of three plasticized and unplasticized cotton matrices were obtained using a FE-SEM Sigma microscope at an accelerating voltage of 10.00 kV, and processed using the ImageJ 1.47 k Software. The physical characteristics of the plasticized and unplasticized deliveries were determined from 10 fibers forming cotton yarns. The compounds R-(þ)- pulegone, ()-carvone, 2-decanone, trans-2-hexenol and polyvinyl acetate were purchased from Sigma Aldrich (Buenos Aires, Argentina), and the compound 2,2dichlorovinyl dimethyl phosphate (dichlorvos; >98%, Chemotecnica S.A, Buenos Aires, Argentina) was used as the reference insecticide, which were selected because of their high fumigant activity against S. zeamais (Herrera et al., 2015; Zunino et al., 2015; Calvimonte, 2016). The chemical properties of the selected compounds, such as lipophilicity (Log P: Logarithm of the octanol/water partition coefficient) and vapor pressure (VP), were obtained from the ChemSpider database (ChemSpider, 2018). The chemical structures, VP and LogP of these compounds are shown in Fig. 1. 2.3. Bioassays Assays were carried out using polypropylene bags (4 25 cm), as they are versatile transparent non-toxic materials. In addition, polypropylene prevents moisture transfer and has good organoleptic, chemical and resistance properties (POREX, 2018). For the bioassays, 20 g of maize kernels and 10 adult weevils were placed in each bag. Then, each delivery (plasticized and unplasticized) was loaded with the treatment compound (R(þ)-pulegone, ()-carvone, 2-decanone or trans-2-hexenol) at a dose of 127.2 mL/L air (9 times the LD95 of R-(þ)-pulegone against S. zeamais at 24 h, according to Herrera et al. (2015)). A negative control (plasticized and unplasticized deliveries without
2.5. Phytotoxicity assay After 7 and 28 days of the experiment, maize grains from each treatment were extracted and randomly selected to evaluate the phytotoxicity of each delivery, using a standard germination test (Agrawal, 1980). Seven grains per Petri dish (three Petri dishes per treatment) were placed on filter paper moistened with 1 mL of distilled water. The seeds were maintained at 24 C and the germination of the kernels was observed after 9 days. 2.6. Statistical analysis All data were analyzed to assess normality using the ShapiroWilks test, and homogeneity of the variances was determined using the Levene test before performing an ANOVA. The Student's ttest was used to compare the physical characteristics of the deliveries and the mortality percentage of S. zeamais among treatments (plasticized and unplasticized deliveries for each exposure time). A two-way ANOVA and Tukey HSD posteriori test (P < 0.01, Di Rienzo et al. (2017)) were used for the phytotoxicity assays.
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Fig. 1. Molecular structures of the compounds and 2,2-dichlorovinyl dimethyl phosphate. The values shown of Log P (Logarithm of the octanol/water partition coefficient) and VP (Vapor pressure, at 25 C) were obtained using ChemSpider (2018).
3. Results The physical characteristics of the deliveries did not show significant differences (P ¼ 0.92) between the thickness of fibers forming cotton yarn of plasticized and unplasticized deliveries (15.11 ± 0.54 and 15.23 ± 1.04 mm, respectively). In addition, no significant differences were found in yarn thickness between both matrices (273.20 ± 6.26 and 272.57 ± 6.51 mm, respectively; P ¼ 0.95) or in the spaces between the networks of yarns for plasticized and unplasticized deliveries (0.05 ± 0.01 and 0.03 ± 0.01 mm2, respectively; P ¼ 0.10). These features are shown in the scanning microscopy images (Fig. 2). The plasticized and unplasticized deliveries loaded with R(þ)-pulegone were the only treatments that caused insect mortality over time (Fig. 3). Plasticized delivery loaded with R(þ)-pulegone had mortality values of 96%, similar to those produced by the positive control at the end of the experiment (30 days). Moreover, insecticide bioassays with deliveries loaded with R-(þ)-pulegone showed that the toxic activity increased with
exposure time (Fig. 3). Plasticized delivery loaded with R(þ)-pulegone achieved a 90% mortality at day 12, and remained above 96% over the course of 30 days. In contrast, unplasticized delivery loaded with R-(þ)-pulegone showed mortality values of 30% recorded on day 12, and attained a 68% mortality by the end of the experiment (Fig. 3). R-(þ)-pulegone from plasticized delivery was released more slowly (0.04 mL, Fig. 4d) than the unplasticized delivery (0.07 mL, Fig. 4c), with the former allowing a high percentage of mortality from day 12. Plasticized and unplasticized deliveries loaded with 2-decanone (P ¼ 0.92), ()-carvone (P ¼ 1) or trans-2-hexenol (P ¼ 0.86) exhibited mortality values below 6% throughout the experiment. The plasticized and unplasticized deliveries loaded with dichlorvos reached mortality values of 100% on days 6 and 12, respectively. The plasticizer itself showed no insecticidal effect (negative control, P ¼ 0.11) and had mortality values below 2% (data not shown). An interaction between the compound assessed and exposure time (P < 0.001, Table 1) was found in the phytotoxicity tests (Table 1). The plasticized and unplasticized deliveries loaded with
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Fig. 2. Scanning electron microscopy (SEM) images obtained using an FE-SEM Sigma on: unplasticized (a, c) and plasticized (b, d, e) cotton matrix section (fabric) (a, b) fibers form yarn, (c, d) detail of the fibers, (e) binding of the fibers to each other by the action of the plasticizer. The scale bar represents 200 mm (a, b), 20 mm (c, d) and 2 mm (e).
trans-2-hexenol were the most phytotoxic, with low percentages (less than 60%) or a complete lack of germination. Deliveries loaded with R-(þ)-pulegone did not show phytotoxicity in maize kernels (Table 1). 4. Discussion In the present study, we found that only R-(þ)- pulegone incorporated in the cotton matrices produced weevil mortality. It is widely known that R-(þ)-pulegone presents high toxicity and is lethal to different species of insects even in extremely small amounts (Franzios et al., 1997; Rossi et al., 2011; Herrera et al., 2015; Peschiutta et al., 2017). Also, R-(þ)-pulegone is not as volatile as others organic compounds but is highly lipophilic, tending to be more toxic (Philippou et al., 2016; Peschiutta et al., 2017). Furthermore, this kind of compounds are less selective in binding to proteins, in some cases being chemically reactive and are extensively metabolized (Winiwarter et al., 2007; Eckert and Trinh, 2016).
The effectiveness of cotton fibers to retain volatile compounds is an important feature for determining the insecticidal effect over time. This depends mainly on the total compound retained in the deliveries after drying and on the release rates (Langer, 1980). We found that R-(þ)-pulegone treatment with plasticizer improved its insecticidal activity, suggesting that plasticized delivery could be a suitable material for controlling the release of the bioactive compounds. It has been previously shown that the incorporation of organic compounds in solid polymers permits a controlled release of volatile compounds over an extended period of time (Herrera et al., 2017). Abdel-Mohdy et al. (2008) reported that cyclodextrin and an insecticide binding to each other to form an inclusion complex, where the insecticide acts as an agent molecule in the center of the hydrophobic interior of the cyclodextrin. This inclusion complex was incorporated into the delivery and allowed the insecticide to remain attached, even after repeated washing, thereby prolonging the insecticidal efficacy of the matrix. On the other hand, deliveries loaded with R-(þ)-pulegone did not show phytotoxicity in maize kernels, which is a very important
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Table 1 Phytotoxicity (% of maize germination) of plasticized and unplasticized delivery loaded with R-(þ)-pulegone, ()-carvone, 2-decanone and trans-2-hexenol after 7 and 28 days of delivery exposure (100% indicates complete germination). Cotton matrix without compound treatment was used as negative control and delivery with dichlorvos (0.06 mL/L air) was used as positive control.
R-(þ)-Pulegone 2-decanone ()-carvone Trans-2-hexenol Negative control Positive control
Phytotoxicity (germination, %)
7 days
28 days
Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized Plasticized Unplasticized
92.8 ± 4.1 92.8 ± 4.1 92.8 ± 4.1 100.0 ± 0.0 92.8 ± 4.1 85.7 ± 0.0 0.0 ± 0.0 * 57.1 ± 4.1 * 100.0 ± 0.0 100.0 ± 0.0 85.71 ± 8.25 92.86 ± 4.13
100.0 ± 0.0 100.0 ± 0.0 92.8 ± 4.1 100.0 ± 0.0 100.0 ± 0.0 92.8 ± 4.1 0.0 ± 0.0 * 14.2 ± 8.2 * 100.0 ± 0.0 100.0 ± 0.0 85.71 ± 0.00 100.00 ± 0.00
Values are expressed as means ± SE. * Indicate the highest phytotoxicity (< 60% germinated) according to Tukey HSD test, (P < 0.05). Fig. 3. Mortality percentages of Sitophilus zeamais for plasticized and unplasticized deliveries loaded with R-(þ)-pulegone at a dose of 127.2 mL/L. Vertical bars represent the mean mortality value ± SE (n ¼ 5) of plasticized (black bars) and unplasticized (white bars) deliveries over time. Significant differences between treatment within each time are indicated as: ***P < 0.001and *P < 0.05 (t-test).
feature for potential use of this compound against the maize weevil. In agreement with this result, previous studies showed that this compound presents a low phytotoxicity in plants (Herrera et al., 2015; Chaimovitsh et al., 2017). Hence, given the effectiveness against the weevil and the lack of phytotoxic activity against
maize kernels, plasticized delivery loaded with R-(þ)-pulegone could serve as a potential tool for controlling S. zeamais in a grain storage system, such as in silo bags. The search for novel insecticides and new methods of delivery to control S. zeamais will continue as this insect is still one of the most economically important pests in stored grains. Certain synthetic insecticides have been replaced because of increasing public demand for safer and more effective products, among other causes (Buczkowski and Schal, 2001; Agrovoz, 2018). New active organic ingredients and innovative delivery tools have consequently emerged as providing an effective means of managing pests
Fig. 4. Gas chromatography/mass spectrometry (GC/MS) chromatograms indicating the release of R-(þ)-pulegone. Peaks: 1: R-(þ)-pulegone: (a) unplasticized delivery without treatment compound (control), (b) plasticized delivery without treatment compound (control), (c) unplasticized delivery loaded with R-(þ)-pulegone (Release rate: 3.08*103 mL/h), (d) plasticized delivery loaded with R-(þ)-pulegone (Release rate: 1.64*103 mL/h).
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(Peschiutta et al., 2016; Herrera et al., 2017). From the results of the present study, we propose the use of cotton fabric (delivery) impregnated with R-(þ)-pulegone and a polyvinyl adhesive plasticizer as an effective alternative for weevil control in a grain storage system. However, further research is still necessary in order to evaluate the efficacy of this delivery method on a larger scale and under field conditions. Acknowledgements This work complies with Argentinean laws. The authors also declare that there is no conflict of interest. Financial support for this work came from the following sources: FonCyT (PICT 2012-2146), CONICET (PIP 11220120100661CO) and Universidad Nacional de rdoba. We thank Dr. Paul Hobson, native speaker, for revising the Co manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jspr.2019.06.013. References Abdel-Mohdy, F., Fouda, M.M., Rehan, M., Aly, A., 2008. Repellency of controlledrelease treated cotton fabrics based on cypermethrin and prallethrin. Carbohydr. Polym. 73, 92e97. Agrawal, R.L., 1980. Seed Technology. Seed Technology. Agrovoz, 2018. Prohíben el uso de un insecticida que se USA en el almacenamiento de granos. http://agrovoz.lavoz.com.ar/agricultura/prohiben-el-uso-de-uninsecticida-que-se-usa-en-el-almacenamiento-de-granos. Akelah, A., 1996. Novel utilizations of conventional agrochemicals by controlled release formulations. Mater. Sci. Eng. C 4, 83e98. Akelah, A., 2013. Functionalized Polymeric Materials in Agriculture and the Food Industry. Springer. Buczkowski, G., Schal, C., 2001. Method of insecticide delivery affects horizontal transfer of fipronil in the German cockroach (Dictyoptera: blattellidae). J. Econ. Entomol. 94, 680e685. Calvimonte, H., 2016. Microorganismos y Plantas como Fuentes Productoras de Bioalcoholes para el Control de Sitophilus zeamais, Facultad de Ciencias Exactas, rdoba, Co rdoba, Argentina, p. 39. Físicas y Naturales. Universidad Nacional de Co Chaimovitsh, D., Shachter, A., Abu-Abied, M., Rubin, B., Sadot, E., Dudai, N., 2017. Herbicidal activity of monoterpenes is associated with disruption of microtubule functionality and membrane integrity. Weed Sci. 65, 19e30. ChemSpider, 2018. Search and share chemistry. http://www.chemspider.com/. Chulze, S., 2010. Strategies to reduce mycotoxin levels in maize during storage: a review. Food Addit. Contam. 27, 651e657. Di Rienzo, J.A., Casanoves, F., Balzarini, M.G., Gonzalez, L., Tablada, M., C.W.,R, 2017. n 2017. Universidad Nacional de Co rdoba, Argentina. InfoStat Versio Eckert, C.A., Trinh, C.T., 2016. Biotechnology for Biofuel Production and Optimization. Elsevier.
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