Botanical extracts can be an alternative to synthetic insecticides for pest management, owing to their efficiency and environmental safety. The lethal times (LT50 and LT90), loss of grain weight, and repellence of Sitophilus zeamais (Coleoptera: Curculionidae) were evaluated when extracts from 5 plant species from the Brazilian Cerrado biome [Adenocalymma nodosum (Bignoniaceae) leaves, astilbin from Dimorphandra mollis (Fabaceae) flowers, Psychotria prunifolia (Rubiaceae) leaves, Senna obtusifolia (Fabaceae) leaves, and Tithonia diversifolia (Asteraceae) flowers] were applied to a surface and to food. The weevils exposed to a surface treated with extracts from T. diversifolia flowers (106.43 h) and P. prunifolia leaves (119.68 h) at 2% (m.v-1) presented the lowest LT50 and LT90 values, respectively. Corn grains treated with the extract from P. prunifolia leaves at 2% (w.w-1) exhibited fastest mortality of S. zeamais individuals with lower values of LT50 and LT90 than the other treatments. All the botanical extracts repelled S. zeamais after 30 min of application; however, the extracts from S. obtusifolia leaves, P. prunifolia leaves, and T. diversifolia flowers at 2% (m.v-1) repelled this insect after 6 min. Moreover, at 90 and 120 min after the beginning of the experiment, the extracts from P. prunifolia leaves and T. diversifolia flowers repelled the adult weevils. The extracts from A. nodosum leaves, P. prunifolia leaves, and T. diversifolia flowers applied to corn grains showed class III repellency according to the Preference Index for S. zeamais and less grain weight loss, when compared with astilbin from D. mollis flowers and S. obtusifolia leaves. Therefore, it was concluded that the extracts from T. diversifolia flowers and P. prunifolia leaves were the most promising in controlling S. zeamais, with the potential for application in programs for the integrated management of this pest.
Synthetic insecticides are used to protect agricultural and forest crops as well as stored grains and byproducts (Tavares et al. 2010a, 2010b). However, the repetitive use of the same active component may lead to resistant insect populations and toxic residues adhering to food (Pimentel et al. 2010; Tavares et al. 2012). The use of botanical extracts and essential oils to control insects is increasing because these natural products present smaller effects on native fauna and flora, greater availability, and faster degradation, when compared with synthetic compounds (Tavares et al. 2011; Fouad et al. 2014).
Maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is an important insect pest, especially of corn grain, Zea mays L. (Poaceae), both in the field and in storage (Tavares et al. 2013a). The extracts from several plant parts (bark, flowers, leaves, roots, seeds, and stems) of the families Euphorbiaceae, Fabaceae, Lamiaceae, Monimiaceae, Verbenaceae, and Zingiberaceae have shown efficacy against the adults and larvae of S. zeamais (Fouad et al. 2012; Tavares et al. 2013a). However, only a few bioprospection studies of compounds useful as natural insecticides have been carried out (Tavares et al. 2009, 2013b).
Cerrado, a type of savannah and an important Brazilian biome, is rich in endemic flora. Plants from Cerrado are used in agriculture, folk medicine, and pharmaceutical industry, thus motivating the search for insecticides from these plants. Adenocalymma nodosum (Silva Manso) L.G. Lohmann [= Memora nodosa (Silva Manso) Miers] (Bignoniaceae) and Psychotria prunifolia (H.B.K) Steyerm. (Rubiaceae) are plants used in folk medicine to treat several human diseases (Soares et al. 2010; Tavares et al. 2013b). Dimorphandra mollis Benth. (Fabaceae) contains the flavonoid astilbin and its seeds are toxic to cattle, Bos primigenius Bojanus (Artiodactyla: Bovidae) (Cintra et al. 2002, 2005). Senna obtusifolia (L.) Irwin & Barneby (Fabaceae) has anti- fungal and anti-bacterial properties (Doughari et al. 2008), and Tithonia diversifolia (Hemsl.) A. Gray (Asteraceae) has antidiabetic, anti-inflammatory, and anti-malarial properties (Ambrósio et al. 2008).
Botanical extracts of the families Asteraceae, Bignoniaceae, Fabaceae, and Rubiaceae affect the biology, behavior, and reproduction of coleopteran and lepidopteran pests (Asawalam et al. 2008; Tavares et al. 2012), and reduce the growth of their populations (Fazolin et al. 2010). The aim of the present study was to identify the compound astilbin, extracted and purified from D. mollis flowers, and determine its lethal time to kill 50% (LT50) and 90% (LT90) of the maize weevil, S. zeamais, and its repellency potential, besides other ethanolic extracts from 4 Cerrado plant species at 2% (m.v-1 or w.w-1).
Material and Methods
Experimental Site
The experiment was carried out in the Laboratory of Integrated Pest Management-Grains (LIPMGrains), Department of Agricultural Engineering (DEA), the Federal University of Viçosa (UFV), Viçosa, Minas Gerais, Brazil, in an environmentally controlled room at 25 ± 1 °C, 70 ± 10% RH and a 12:12 h L:D photoperiod.
Sitophilus zeamais and Corn Grains
The weevils S. zeamais were obtained from stock cultures of the Laboratory of Integrated Pest Management- Grains (LIPM-Grains), DEA-UFV, where this species was reared with corn grains (13% humidity). Individual weevils were isolated for 24 h without food before the experiment.
The corn was grown and harvested at the UFV Experiment Farm where no insecticide was used, and stored in an environmentally controlled chamber at 5 °C until the beginning of the experiment.
Plants Collection and Deposit
Adenocalymma nodosum leaves, D. mollis flowers, P. prunifolia leaves, S. obtusifolia leaves, and T. diversifolia flowers were removed with a pocketknife from the flowering plants in February 2013 in a part of Cerrado (a protected area) at S 18° 9′-W 47° 56′, and an altitude of 835 m in Catalão, Goiás, Brazil. These materials were placed in twenty plastic bags inside polystyrene (C8H8)n boxes lined with ice cubes and taken to the laboratory, where they were stored at 2 °C in a refrigerator. The plants were identified after examination of their reproductive organs, according to keys and descriptions (Watson & Dallwitz 1992 onwards).
Samples of these plants (a voucher specimen and 2 duplicates) were deposited in the herbarium of the Institute of Biological Sciences (ICB) at the Federal University of Goiás (UFG), Samambaia campus in Goiânia, Goiás, Brazil (Table 1).
Preparation of Crude Extracts
A total of 600 g of fresh material of each plant species were left under direct sunlight in 1-L ethanol (C2H6O) for 7 days to extract its compounds, and the yield of the plant extracts was verified on a dry weight basis after concentration in vacuum (Table 1). The crude extract obtained after the extraction was stored in 50-mL plastic tubes covered with black paper and kept at 2 °C in a refrigerator.
Extraction of Astilbin from D. mollis Flowers
Astilbin (Fig. 1) was isolated from D. mollis flowers. This compound was extracted thrice with dichloromethane (DCM, CH2Cl2) and twice with ethanol, before being concentrated in vacuum. The ethanolic extract of astilbin was separated in a recycling High Performance Liquid Chromatography (HPLC) [Asahipak GS-310 P polymetric column, mobile phase of methanol (CH3OH) with a flow of 7 mL.min-1, and UV detection at 290 nm] and recrystallized with methanol to yield yellow crystals. The structural characterization of astilbin was based on comparisons with the physical and spectral data of its crystals (De Brito et al. 1995; Han et al. 1998).
Analysis of Nuclear Magnetic Resonance (NMR) 1H (200 MHz) and of NMR 13C (50 MHz) (Figs. 2A and 2B) was carried out in a Brüker spectrometer (ARX-200) with tetramethylsilane (TMS, C4H12Si) as an internal standard. The mass spectra were obtained in positive mode Atmospheric Pressure Chemical Ionization (APCIMS) in a mass spectrometer (Micromass Quattro LC). The melting point of the astilbin crystals was determined as 190–191 °C, [α]25 D= -12.8° (c 1.0, methanol), m/z= 451 [M + H]+ (APCI-MS, positive mode), and the NMR of 1H and 13C NMR were compatible with the published data (De Brito et al. 1995; Han et al. 1998). The yield of the plant extracts was calculated by dividing the mass obtained from that of the initial material after concentration in a vacuum (Tavares et al. 2013c).
Table 1.
Species, botanic family, plant part, yield of the extract residue (%, w.w-1), and voucher code for each species collected from the Brazilian cerrado biome and deposited in the herbarium of the Institute of Biological Sciences at the Federal University of Goiás, Samambaia Campus, Goiânia, Goiás, Brazil.
Preparation of Solutions from Crude Extracts
Solutions were prepared after dilution of the dry botanical extracts with absolute ethanol (Merck KGaA) until 2% (m.v-1 or w.w-1) concentration. These solutions were shaken with a Branson 2510 sonifier for 220 min in SET DEGAS and homogenized with a Fisher Vortex Genie 2™ shaker at “speed eight” for 10 min (Tavares et al. 2009).
Toxicity on a Treated Surface
A total of 50 µL of the botanical extracts (A. nodosum leaves, astilbin from D. mollis flowers, P. prunifolia leaves, S. obtusifolia leaves, and T. diversifolia flowers) at 2% (m.v-1) (Table 1) or absolute ethanol (control) were applied to disks of filter paper (5 cm diam) with a precision micro-syringe (Hamilton 1 mL), and left for 10 min at 25 ± 1 °C to evaporate the solvent. Subsequently, the treated papers were placed in Petri dishes (5 cm diam) with twenty 90-day-old unsexed S. zeamais adults. These dishes were placed in an environmentally controlled chamber (25 ± 1 °C, 70 ± 10% RH, and 12:12 h L:D photoperiod). The mortalities of the weevils were evaluated after 1, 3, 6, 9, 12, 24, 48, 72, 96, and 120 h (Tapondjou et al. 2005). A total of 10 replications was carried out.
Toxicity on Treated Corn Grains and Weight Loss of the Corn
The toxicities of botanical extracts (A. nodosum leaves, astilbin from D. mollis flowers, P. prunifolia leaves, S. obtusifolia leaves, and T. diversifolia flowers) mixed with food (corn grains) were evaluated by applying 50 µL of the extract at 2% (w.w-1) (Table 1) or absolute ethanol (control) in 10 g of corn grains. Plastic jars (100 mL) with the respective treatments were shaken for 15 s to mix the extracts or ethanol with the corn grains. The treated grains were left for 10 min at 25 ± 1 °C to evaporate the solvent. Subsequently, twenty 90-day-old unsexed S. zeamais adults were left to feed on the corn grains, treated or untreated, in an environmentally controlled chamber (25 ± 1 °C, 70 ± 10% RH, and 12:12 h L:D photoperiod). The mortalities of the weevils were recorded after 1, 6, 9, 12, 24, 48, 72, 96, and 120 h (Betancur et al. 2010). The weight loss of the corn grains offered to the insects was recorded after 30 days (Tavares et al. 2013b). A total of 10 replications was carried out.
Insect Repellency
Repellency tests were conducted in Petri dishes (14 cm diam) containing filter paper (Whatman no 1) of the same size as the dishes. To half of the papers, 1 mL of each of the botanical extracts (A. nodosum leaves, astilbin from D. mollis flowers, P. prunifolia leaves, S. obtusifolia leaves, and T. diversifolia flowers) at 2% (m.v-1) was uniformly applied (Table 1) and absolute ethanol (control) was applied to the rest of the filter papers. The dishes were left for 10 min at 25 ± 1 °C to evaporate the solvent. Subsequently, twenty 90-day-old unsexed S. zeamais adults were placed in the center of each dish. The numbers of weevils in the control (NC) and treatment (NT) dishes were recorded after 30, 60, 90, and 120 min (Islam et al. 2009). A repellency assay was carried out in an environmentally controlled chamber (25 ± 1 °C, 70 ± 10% RH, and 12:12 h L:D photoperiod). A total of 10 replications was carried out.
Statistical Analyses
The data on the toxicity of the botanical extracts on contaminated surfaces and grains, determined with six treatments (five plant extracts and one control) and 10 replications, were analyzed by using regression equations, and the 95% confidence limits were calculated by PROBIT analysis (Finney 1971). The LT50 and LT90 of the insects were obtained by PROC PROBIT (Supplier: UFV). The total insects in the NC and NT plates were evaluated 30, 60, 90, and 120 min after the beginning of the repellency experiment, and the results were submitted to Analysis of Variance (one-way ANOVA) and the means were compared with the “t” test at 5% probability (SAS Institute 1997).
The percentage of repellency (PR) was obtained by using the equation: PR = [(NC – NT) ÷ (NC + NT)] × 100, and classified into class 0 (PR ≤ 0.1%), class I (PR = 0.1–20%), class II (PR = 20.1–40%), class III (PR = 40.1–60%), class IV (PR = 60.1–80%), and class V (PR = 80.1–100%) (Benzi et al. 2009).
Results
Extracts
The yield of astilbin from D. mollis flowers (7.60%) was higher than that from P. prunifolia leaves (1.64%), A. nodosum leaves (3.91%), S. obtusifolia leaves (4.90%), and T. diversifolia flowers (7.56%) (Table 1). The signals in the spectra with their multiplicities and coupling constant (number that determines the strength of the force exerted in an interaction) found for astilbin (Fig. 1) were characteristic of flavonoids (Figs. 2A and 2B).
Toxicity of the Treated Surface
The extracts from all 5 plant species investigated caused mortality of S. zemais adults. The LT50 for S. zeamais on the surface treated with the extracts from T. diversifolia flowers was approximately 1.12-, 1.29-, 1.34-, and 1.52-fold lower (106.43 h) than that for S. zeamais on the surface treated with the extracts from P. prunifolia leaves, A. nodosum leaves, astilbin from D. mollis flowers, and S. obtusifolia leaves, respectively (Table 2). On the other hand, the LT90 for S. zeamais adults on grains treated with extracts from P. prunifolia leaves (167.56 h) was approximately 1.45-, 1.52-, 1.72-, and 2.16-fold lower than that for S. zeamais adults on grains treated with the extracts from A. nodosum leaves, T. diversifolia flowers, astilbin from D. mollis flowers, and S. obtusifolia leaves, respectively (Table 2).
Toxicity of the treated grains
The extract from P. prunifolia leaves mixed with corn grains was more toxic (LT50= 89.73 h and LT90= 171.19 h) to the adult maize weevils than those from T. diversifolia flowers, S. obtusifolia leaves, A. nodosum leaves, and astilbin from D. mollis flowers (the toxicity was 1.25-, 1.25-, 1.36-, and 1.54-fold higher with respect to LT50 and 1.04-, 1.14-, 1.31-, and 1.47-fold higher with regard to LT90, respectively) (Table 2).
Repellency
The botanical extracts from Cerrado repelled S. zeamais adults after 30 min of exposure (Fig. 3A) and those of S. obtusifolia leaves (P = 0.03), P. prunifolia leaves (P = 0.0008), and T. diversifolia flowers (P = 0.005) repelled this weevil after 60 min of exposure (Fig. 3B). Furthermore, the extracts from P. prunifolia leaves [(P = 0.004) and (P = 0.02)] and T. diversifolia flowers [(P = 0.02) and (P = 0.04)] repelled the maize weevil adults after 90 and 120 min, respectively (Figs. 3C and 3D).
Fig. 2.
A. Nuclear Magnetic Resonance (NMR) 1H (200 MHz) spectrum of astilbin, and of NMR 13C (50 MHz) spectrum of astilbin.
The extracts from A. nodosum leaves exhibited class III repellency after 30 min, while those from P. prunifolia leaves and T. diversifolia flowers exhibited similar class of repellency after 60 and 90 min of treatment against S. zeamais adults, respectively (Table 3).
Grain Weight Loss
The extracts from P. prunifolia leaves (3.14 ± 0.48%), T. diversifolia flowers (3.28 ± 0.45%), and A. nodosum leaves (4.78 ± 0.23%) exhibited higher grain weight loss caused by maize weevil feeding after 30 days (P = 0.0002) (Table 4).
Discussion
The yield of astilbin extract from D. mollis flowers collected from Catalão was found to be similar to that from the same plant collected from Rio Claro and Corumbataí, São Paulo, Brazil (Cintra et al. 2002, 2005), demonstrating that D. mollis contains the same amount of astilbin, irrespective of its geographical location. The yield of the extract from A. nodosum leaves collected on Feb 2012 from Catalão was similar to that extracted from this plant collected on Feb 2011 in the same municipality (Menezes et al. 2014), which indicates that content of the extract in this species is similar over time and over a wide geographic ranges. These results show that plants collected from Cerrado may produce high yields of extracts.
Table 2.
Plant extracts, LT50 (H) and LT90 (H) (Variation Interval), Mean ± Standard Error (Se) of Mortality, Χ;2, And P of the effects of the extracts from plants collected from the Brazilian Cerrado Biome on a treated surface or corn grains on Sitophilus zeamais adults.
Fig. 3.
Selection by Sitophilus zeamais adults of treated and control corn grains after 30 (A), 60 (B), 90 (C), and 120 (D) min of exposure. *Significant values; n.s. non-significant values at 5% probability by the “t” test. Treatments consisted of ethanol extracts of T. diversifolia flowers, S. obtusifolia leaves, P. prunifolia leaves, astilbin isolated from the extract of D. mollis flowers, and A. nodosum leaves, all from the Brazilian Cerrado biome. In the control, the corn grains had been treated with absolute ethanol.
The higher susceptibility of S. zeamais adults to the extract of T. diversifolia flowers on a treated surface with a lower LT50 value (106.43 h) agrees with the previous finding indicating 100% mortality of Callosobruchus maculatus F. (Coleoptera: Bruchidae) adults with extracts of this plant at 3, 4, and 5% (m.v-1) (Adedire & Akinneye 2004). Furthermore, the LT50 values for S. zeamais adults exposed to a surface treated with essential oils of Lantana camara L. (Verbenaceae) and Tephrosia vogelii Hook (Fabaceae) were noted in 6 days (with rates of 7.5–10% w.w-1) and between 7 and 8 days (with rates of 2.5–5% w.w-1), respectively (Ogendo et al. 2005). Sesquiterpene lactones [containing sesquiterpenoids (made up of 3 isoprene units) and a lactone ring], such as eudesmanolides and germacranolides, were isolated from Tithonia rotundifolia (Mill.) S. F. Blake (Asteraceae), which might be responsible for the insecticidal activity of the extracts of this plant (Bohlmann et al. 1981). In addition, chromone (a derivative of benzopyran with a substituted keto group on the pyran ring), dinorxanthane sesquiterpene, and other substances isolated from T. diversifolia roots have been noted to exhibit insecticidal activities (Kou & Lin 1999). It has been reported that the extracts from T. diversifolia have low toxicity to mammals when compared with synthetic insecticides (Bohlmann et al. 1981).
In the present study, the extract from P. prunifolia leaves mixed with corn grains presented toxicity to S. zeamais adults in a shorter time (LT50 and LT90), similar to the hexanic CH3(CH2)4CH3 extract from T. vogelii mixed with food, which reduced the population of S. zeamais by 50% after 5 days of application (Koona et al. 2007). Medicinal herbs have shown anti-feeding and insecticidal activity against stored grain pests (Liu et al. 2007). In a previous study, the extract from Peumus boldus Molina (Monimiaceae) leaves at 15 and 20% (m.v-1) caused 100% mortality of the maize weevil after 15 days of contact with treated food (Bustos-Figueroa et al. 2009). It has been reported that monoterpene indole alkaloids and strictosamide (alkaloid glycoside), besides being important as chemotaxonomic markers for the taxonomy of the species of this genus (Simões-Pires et al. 2006; Petacci et al. 2012), are the main compounds of the extracts from the leaves of the genus Psychotria (Kerber et al. 2008). These compounds also present biological activities against microorganisms that cause diseases in humans and act as a stimulant of the central nervous system (Figueiredo et al. 2010). Furthermore, the extract from Cinchona ledgeriana Moens. (Rubiaceae) leaves was noted to contain abundant monoterpene indole alkaloids that reduced the growth as well as development and increased the mortality of Spodoptera exigua Hübner (Lepidoptera: Noctuidae) larvae that were fed on a treated artificial diet (Aerts et al. 1992).
Table 3.
Plant extracts, number of Sitophilus zeamais (Coleoptera: Curculionidae) adults repelled by treated filt er paper surface after 30, 60, 90, and 120 min, and class of repellency of the extracts from plants collected from the Brazilian Cerrado Biome.
In the present study, the greatest effect of the extract from P. prunifolia leaves on corn grains suggested a higher efficiency of ingestion owing to the protective cuticle that prevents contact of the insecticide with its action site (Lopez et al. 2009). Products with a greater residual effect or sequential application, such as extracts from T. diversifolia, are recommended because they have better efficiency when applied against weevils (Thornham et al. 2007), and the use of insecticides acting on ingestion can increase the mortality and repellency of Coleoptera pests in stored grains (Bozsik 2006).
The greater repellency effect of the extract from P. prunifolia leaves and T. diversifolia flowers on S. zeamais adults, especially after 1 h of treatment, suggested a wide spectrum of action from the extracts of these plants. In a previous study, the crude extract of T. diversifolia also repelled immature Chlosyne lacinia Geyer (Lepidoptera: Nymphalidae) on a treated surface (Ambrósio et al. 2008).
The highest class of repellency (class III) achieved with extracts from P. prunifolia leaves and T. diversifolia flowers against the maize weevil was found to be similar to the repellency effect exhibited by 40.1–60% concentration of the extracts from Ocimum kenyense Ayob. ex A. J. Paton, Ocimum grattisimum L. (Lamiaceae), and Ricinodendron heudelotii (Baill) Pierre ex Pax (Euphorbiaceae) leaves on this insect (Bekele et al. 1997; Asawalam et al. 2008).
The least weight loss in corn grains treated with the extracts from P. prunifolia leaves and T. diversifolia flowers after 30 days of feeding confirmed that secondary compounds in the extracts of these plants produced repellent or anti-feeding activity in S. zeamais adults. The presence of rotenones (substance classified by the World Health Organization as moderately hazardous) in the R. heudelotii extracts repelled S. zeamais and C. maculatus, and decreased the weight loss in corn and common bean Phaseolus vulgaris L. (Fabaceae) grains treated with the extracts of this plant (Udo & Epidi 2009), suggesting that the anti-feeding activity of the botanical extracts can be efficient in protecting stored grains (Nerio et al. 2010). In conclusion, the extracts from T. diversifolia flowers and P. prunifolia leaves caused higher mortality and repellency on S. zeamais adults in corn grains, and thus have the potential for use in the integrated management of this pest in storage.
Table 4.
Mean Weight Loss ± Standard Error (%) and variation interval of corn grains treated with extracts from plants collected from the Brazilian Cerrado Biome after 30 days of feeding by Sitophilus zeamais adults and control.
Acknowledgments
To Dr. Aristônio Magalhães Teles (Department of Botany, Federal University of Goiás, Goiânia, Goiás, Brazil) for plant identification. To “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)”, “The Academy of Sciences for the Developing World (TWAS)”, “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)”, and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for financial support. To Global Edico Services of India for revising and editing the manuscript.
