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Introduction
The fruit fly pest from the Tephritidae family severely affects fruit crops all around the world. The Tephritids, called true fruit flies, consist of 4,700 species located throughout the temperate, tropical and subtropical regions of the world (Norrbom et al., 2012). There are specifically about 250 species in the Americas which are spread all the way from the south of the United States to the north of Argentina, including the Caribbean islands (Hernández-Ortiz et al., 2010).
The genus Anastrepha Schiner is endemic to the Americas and four species are of economic and quarantine importance due to their high preferred range for cultivated and wild hosts; e.g. A. ludens, A. obliqua, A. serpentina y A. striata (Hernández-Ortiz et al., 2010; SENASICA, 2021). Their preferred hosts include up to 330 species belonging to 48 families, including Anacardiaceae, Cucurbitaceae, Myrtaceae, Rosaceae, Rutaceae, and Sapotaceae (Hernández-Ortiz, 1993; Hernández-Ortiz et al., 2010).
The integrated management of the fruit fly pest caused by Anastrepha has been implemented since 1992 through the National Program and the National Campaign Against Fruit Flies (NCFF), under an international agreement between Mexico, Guatemala, and the United States (Montoya et al., 2010). The program is based on the implementation of phytosanitary measures to control, suppress, and eradicate fruit flies. The monitoring system consists of trapping and detecting larvae in fruits, and the control methods are based on the collection and destruction of infested fruits (mechanical control), spraying of specific baits (synthetic pesticides mixed with hydrolyzed protein; chemical control), and massive releases of sterile flies (sterile insect technique; autocidal control), and natural enemies in priority zones (biological control) (Miyatake, 2011; Montoya et al., 2010; SENASICA, 2021).
The national fruit fly campaign reported that 52% of Mexican territory was a fly free zone (SENASICA, 2021). In the remaining areas the pest persists, and the use of chemical control continues, based on organophosphate insecticides such as Malathion (2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate, diethyl), which has a broad-spectrum toxicity (SENASICA, 2019). This synthetic insecticide is highly neurotoxic, and its chronic exposure induces oxidative stress in mammals (Ali & Ibrahim, 2018; Delgado et al., 2006) and morphological anomalies in early stages of amphibians (Krishnamurthy & Smith, 2011). Also, physiological alterations and protein reduction have been reported in fish (Singh et al., 2004), imbalance in the abundance and composition of species in aquatic ecosystems (Smith et al., 2018), as well as resistance in pest insects (Hsu & Feng, 2006; Jyoti et al., 2014; Magaña et al., 2008).
Alternatives such as botanical pesticides have been suggested as a way of complementing integrated pest management and reducing the harmful effects of synthetic pesticides (Díaz-Fleischer et al., 2017). Botanical pesticides derived from plants have agrochemical potential, because they are selective (i.e., for target insects), biodegradable and harmless to the environment (Amoabeng et al., 2019; Sarkar & Kshirsagar, 2014). In the case of fruit fly pest, various studies have focused on assessing insecticides botanicals. The ethanolic extracts of Annona mucosa had an 80% mortality against Anastrepha fraterculus adults a LC50 728.36 mg L-1 (Stupp et al., 2020). Similarly, the extracts (MeOH-PE) of the fruit of Citrus aurantium had a 76% effectiveness in olive fruit fly adults (Bactrocera oleae) (Siskos et al., 2009).
The Magnoliaceae family is known worldwide for having bioactive compounds, secondary metabolites, with applications in the pharmaceutical, biotechnological, and agri-food industries (Chen et al., 2019; Lee et al., 2011; Poivre & Duez, 2017). Several species of this family have broad-spectrum inhibitory activity against viruses (Fang et al., 2015), bacteria (Jacobo-Salcedo et al., 2011; B. Wu et al., 2018), human pathogenic fungi (Bang et al., 2000), fungi and plant pathogens (Lin et al., 2019; H. Wu et al., 2018), nematodes (Hong et al., 2007), and arthropods (Kelm et al., 1997; Miyazawa et al., 1994; Yang et al., 2015).
It has been shown that several species of Magnolia have bioinsecticidal effects on pest insects. The lignans of the M. fargesii flowers, which inhibit the larval growth of Drosophila melanogaster, stand out among the reported compounds (Miyazawa et al., 1994). Active and isolated compounds (Costunolide, geranial, methyl and isomethyl eugenol, neral, partenolide, and trans-anethole) from different vegetative structures of Magnolia salicifolia, induced 100% larval mortality of the Aedes aegypti (Kelm et al., 1997). In a similar way, crude ethanolic extracts from different vegetative structures of M. dealbata, and M. schiedeana, had insecticidal potential against the Anastrepha ludens fruit fly. In particular, sarcotesta showed the highest insecticidal potential, which reached 96% and 64%, respectively (Flores-Estévez et al., 2013; S. Vásquez-Morales et al., 2015). Therefore, it is essential to assess the insecticidal effect of a greater number of Magnolia species and to expand insecticide bioassays for a greater number of Anastrepha species. The objectives of the present study were: 1) To determine the insecticide-effectiveness of crude ethanolic extracts of sarcotesta of Magnolia perezfarrerae, M. pugana and M. vovidesii against adults of Anastrepha ludens and A. obliqua, 2) To identify the presence of secondary metabolite clusters through qualitative chemical analyses. To this aim, we focused on a system of experimentation of pest feeding assay to based extracts crude of sarcotesta of Magnolia, in addition identify the groups of secondary metabolites using thins layer chromatography.
Materials and methods
Plant material
Four endemic Magnolia species of Mexico were analyzed. Magnolia perezfarrerae is naturally distributed in state of Chiapas, M. pugana in state of Jalisco, M. schiedeana (Schltdl.) and M. vovidesii in state of Veracruz. Magnolias are evergreen trees, except M. vovidesii which is a deciduous tree. The tree height varies between 15 to 25 m, it has a rough cracked greyish bark covered with lichens; glabrous, elliptic, oblong or obovate leaves, with pubescence only on the underside. Flowers white or creamy; in particular, M. vovidesii have pink dots inside them, during the first hours of their opening. The fruit is a dehiscent ellipsoid polyfollicle, containing between 15 to 115 seeds, depending on the species. The physical and climate features of the collection sites are described in Table 1. The M. perezfarrerae was determinate in voucher No. 23948 of herbarium CH - El Colegio de la Frontera Sur. M. pugana and M. vovidesii in process of determination in herbarium XAL - Instituto de Ecología A.C. Further information on taxonomic aspects and collection sites are described in previous research (S. G. Vásquez-Morales et al., 2017; S. G. Vásquez-Morales & Ramírez-Marcial, 2019; Vázquez-García et al., 2002).
Table 1 Characteristics of collection sites in Mexico.
| Species | M. perezfarrerae | M. pugana | M. schiedeana b | M. vovidesii |
|---|---|---|---|---|
| Polyfollicles Collection | March 3rd, 2018 | Match 19th, 2018 and May 17th, 2019 | July 10th, 2018 | August 17th, 2018 |
| Sites (Municipaly) | Ocuilapa de Juárez (Ocozocoautla de Espinosa) | CUCBAa (Zapopan) | La Martinica (Banderilla) | Coyopolan (Ixhuacán de los Reyes) |
| Latitude (N) | 16º50´ 57´´ | 20º 44´51´´ | 19º 34´55´´ | 19º 21´59´´ |
| Longitude (W) | 93º 24´35´´ | 103º 30´46´´ | 96º56´55´´ | 97º04´05´´ |
| Altitude (masl) | 959 | 1 670 | 1 451 | 1 570 |
| Mean annual temperature (ºC) | 22 | 23.5 | 18 | 18 |
| Mean annual rainfall (mm) | 1 000 | 906 | 1 451 | 1 807 |
a Centro Universitario de Ciencias Biológicas y Agropecuarias (CUCBA) of University of Guadalajara.
b Due to the shortage of M. schiedeana seeds, only qualitative chemical tests were assessed.
Laboratory insects
Sterile laboratory flies Anastrepha ludens (Loew) and Anastrepha obliqua (Macquart), from 6 to 15 days of age were used in the bioassays. Flies are mass-produced at Planta MoscaFrut in Metapa de Domínguez, Chiapas, Mexico. These flies are irradiated with Cobalt 60 isotopes at a dose rate of 70 Gy/min (Montoya et al., 2010). Later, they are transferred by air, in pupal stage. The pupae were kept in wooden cages (approximately 25 g on cage) and covered with a cotton mesh of about 900 m3, under laboratory conditions, that is, at a temperature of 25 ºC ± 1 ºC, a relative humidity of 70 ± 30% and a 12-h photoperiod. Ad libitum, they were given purified water (in a container with cotton to prevent them from drowning) and food (table sugar) until they underwent bioassays.
Crude extracts of Magnolia
The seeds of each Magnolia species were extracted from the polyfollicles; then, their sarcotesta (red outer seedcoat) was manually removed. The sarcotesta of each species was placed separately in paper bags and kept for 72 h in a drying oven (Mermmet Incubador IN30; Germany) at 40 ºC for total dehydration. Subsequently, it was grinded in a mortar until pulverization. The preparation of each Magnolia extract consisted of 50 g of pulverized sarcotesta and 250 mL of ethanol 96% (1:5 w.v-1) were added. For each species, six crude Magnolia extracts were prepared and cold-stored (4 ºC) for 72 h. Afterwards, the solvent was removed from each extract and the solvent volume was concentrated in a rotary evaporator (Buchi, Model R-300; Switzeland), set at 40 ºC, with a 1.8 m3/h final vacuum (absolute) and a 5 ± 2 mbar vacuum capacity, until a final extract volume of 10 mL to 22.5 mL was obtained, with an interval of yielder of 2 mg/mL-1 to 4.5 mgmL-1.
Following the same method, six crude extracts of steam, leaves, and flowers of Chrysanthemum grandiflorum Dum. Cours., were obtained. These extracts were used as positive control for their insecticide activity as they contain pyrethroids (Haouas et al., 2012). The C. grandiflorum was purchased at “Mercado Embajadoras, Guanajuato City, Mexico”. Concentrated crude extracts of Magnolia and Chrysanthemum were stored at 4 ºC, in the dark, until evaluation.
Treatments and bioassays
The experimental units were cages (wooden structures covered with cotton meshes of approximately 900 m3) with fifty adult flies (25 females and 25 males) of Anastrepha ludens and A. obliqua. To ensure an adequate intake of the treatments, the flies were deprived of food (table sugar) and kept hydrated with purified water 24 h before each bioassay. For each Magnolia species, reduced crude extracts of sarcotesta were analyzed in three dilutions (0.2, 0.02, 0.002 mg/g) in three different cohorts of each species of Anastrepha. Each bioassay assessed five treatments: 1) 1 g of table sugar mixed with 2 mL of ethanol solution 96% (Negative Control), 2) 1 g of table sugar mixed with 2 mL of reduced crude extract of Chrysanthemum grandiflorum at 0.2 mg/g (Positive Control), 3) 1 g of table sugar mixed with 2 mL of reduced crude extract of sarcotesta of Magnolia at 0.2 mg/g (Dilution 1), 4) 1 g of table sugar mixed with 2 mL of reduced crude extract of sarcotesta of Magnolia at 0.02 mg/g (Dilution 2), and 5) 1 g of table sugar mixed with 2 mL of reduced crude extract of sarcotesta of Magnolia at 0.002 mg/g (Dilution 3). The treatments were applied on 0.07 g of cotton to reduce adherence and facilitate its consumption. For each Magnolia species, three bioassays were performed, on A. ludens and A. obliqua, with five replicates per treatment. For each bioassay, daily mortality was recorded for a period of five consecutive days.
Qualitative chemical profiles determination using TLC
Thin layer chromatography (TLC) experiments were performed using the following polarity systems (v/v); i) hexane (100%), ii) hexane-acetonitrile (75:25%), iii) hexane-acetonitrile (50:50%), iv) ethyl acetate (100%), v) acetonitrile-methanol (50:50%), vi) ethanol (100%), vii) methanol (100%). To this end, 1 mL of each crude extract of sarcotesta (Magnolia perezfarrerae, M. vovidesii, M. pugana and M. schiedeana) was dissolved in 1 mL of each solvent tested, and 10 μL of each sample was applied on silica gel aluminum TLC plates, coated with fluorescent indicator F254 (Merck KEGaA, 64271; Darmstadt Germany). The plates were developed in the different solvents systems for 10 minutes and finally were revealed using p-anisaldehyde (98%). The retention factor (Rf) was estimated for each visible spot, using the equation
For each qualitative test, 1 mL samples of ethanolic extracts of sarcotesta were used. Each test was performed in triplicate according to standard procedure (Domínguez, 1973; Zhang et al., 2019). In these assays, alkaloids, coumarins, flavonoids, phenols, saponins, steroids and terpenes were screened (Table 2).
Table 2 Methodology used to identify secondary metabolites groups by qualitative test.
| Group | Methodology | Positive test | References |
|---|---|---|---|
| Alkaloids | Dragendorff’s reagent of Merck | Turbidity or precipitate formation |
Domínguez, 1973
Mora-Arango et al., 2012 |
| Coumarins | Standard procedure with sodium hydroxide | Green, red, or yellow fluorescence |
Domínguez, 1973
Mora-Arango et al., 2012 |
| Flavonoids | Shinoda´s test | Orange, pink, red, or violet coloration |
Mora-Arango et al., 2012
Zhang et al., 2019 |
| Phenols | Standard procedure with iron chloride | Black, blue, or green coloration |
Domínguez, 1973
Mora-Arango et al., 2012 Zhang et al., 2019 |
| Saponins | Standard procedure with distilled water | Abundant foam was formed and remained stable at least for 5 min | Domínguez, 1973 Mora-Arango et al., 2012 Zhang et al., 2019 |
| Steroids and terpenes | Standard procedure with acetic anhydride and sulfuric acid | Blue, green, red, or violet coloration |
Domínguez, 1973
Mora-Arango et al., 2012 Zhang et al., 2019 |
| Tannins | Gelatin-salt reagent | Turbidity or precipitate formation |
Domínguez, 1973
Mora-Arango et al., 2012 Zhang et al., 2019 |
Statistical Analysis
A completely randomized design was used in all bioassays. The data was analyzed with an analysis of variance (ANOVA, one-way) followed by a LS Means difference Tukey HSD post-hoc test in order to find the effect of the treatments in comparison with the controls in R package Version 3.3.1. (R Core Team, 2013). The natural mortality rate was corrected with the modified formula of Abbott
Results
The ethanolic crude extracts of Magnolia presented a high mortality against Anastrepha ludens and A. obliqua adults. More precisely, the extract of sarcotesta of M. perezfarrerae, in the first dilution (0.2 mg/g), had an insecticide effectiveness of 95% against A. ludens (F= 12.24, df= 3, P<0.05) and of 66% against A. obliqua (F= 4.88, df= 3, P=0.03). These results did not show a significant difference with those shown by the extract prepared with Chrysanthemum grandiflorum (Table 3). Regarding the ethanolic extract of M. pugana, in the first dilution (0.2 mg/g), there was an effectiveness of 93% against A. ludens (F= 1.65, df= 3, P=0.25) and of 91% against A. obliqua with no significant difference between treatments (F= 0.80, df= 3, P=0.52). Regarding the extract from M. vovidesii, in this study it was only tested against A. obliqua and showed an effectiveness of 92% in the first dilution (0.2 mg/g) with no significant difference with the extract of C. grandiflorum (F= 13.75, df= 3, P<0.05; Table 3).
Table 3 Insecticide-effectiveness of crude extracts of sarcotesta of Magnolia against Anastrepha adults in three dilutions, 0.2 mg/g (D1), 0.02 mg/g (D2), and 0.002 mg/g (D3) with negative (NC= Ethanol 96%) and positive control (PC= Chrysanthemum grandiflorum). Mortality percentage and Abbott indices. Active extracts are presented in bold. Mean ± SD. Bars that do not share the same letter are significantly different from controls (P<0.05).
| A. ludens | A. obliqua | |||
|---|---|---|---|---|
| Treatments | % mortality | Abbott index | % mortality | Abbott index |
| M. perezfarrerae | ||||
| NC | 36.26 ± 20.5c | 44.33 ± 10.8b | ||
| PC | 97.86 ± 3a | 97.37 ± 3.4a | 68.8 ± 39.3a | 54.87 ± 48.3ab |
| D1 | 97.6 ± 1a | 95.69 ± 2.5a | 82.26 ± 11.4a | 66.09 ± 24.2a |
| D2 | 67.6 ± 16.4b | 48.92 ± 24.7ab | 74.93 ± 10.3a | 54.07 ± 15.7ab |
| D3 | 40.66 ± 24.4c | 14.32 ± 15.2b | 34 ±13.2b | 0 ± 0b |
| M. pugana | ||||
| NC | 33.86 ± 11.5b | 35.46 ± 10.5c | ||
| PC | 97.6 ± 2.8a | 93.91 ± 8a | 73.6 ± 43.3ab | 65.01 ± 56.3a |
| D1 | 95.6 ± 5.1a | 93.73 ± 7.5a | 95.06 ±5.9a | 91.74 ± 10.8a |
| D2 | 63.6 ± 31.2b | 44.62 ± 49.1a | 78.8 ± 23.9ab | 63.3 ± 43.0a |
| D3 | 66.4 ±25.3b | 44.12 ± 46.7a | 60.8 ± 29.8bc | 36.10 ± 50.0a |
| M. vovidesii | ||||
| NC | 35.86 ± 2.2c | |||
| PC | 98.26 ± 1.8a | 97.31 ± 2.7a | ||
| D1 | 95.06 ± 3.2a | 92.26 ± 5.1a | ||
| D2 | 92.13 ± 7.5a | 87.83 ± 11.3a | ||
| D3 | 55.73 ±10.2b | 30.53 ± 18.7b | ||
The Kaplan-Meier survival analysis demonstrated that crude extracts of Magnolia have a high insecticide-effectiveness from day two to five days of exposure and that there is a significant difference between treatments in A. ludens (M. perezfarrerae P<0.05; M. pugana P<0.05), and A. obliqua (M. perezfarrerae P<0.05; M. pugana P<0.05; M. vovidesii P<0.05) (Fig. S1). It is worth mentioning that the extracts of M. perezfarrerae and M. pugana in the first dilution (D1) showed lower survival of A. obliqua in comparison with C. grandiflorum (CP; Fig. S1A, B).
TLC profiling of Magnolia extract in the different solvent systems indicated the presence of diverse types of phytochemicals as a complex matrix. In general, the number of spots found in some of the mean polarity solvents was more varied than those spots observed in the low polar and polar solvent systems (Fig. 1).
Fig. 1 Thin layer chromatography (TLC), revealed with p-anisaldehyde 98 %, of crude extracts of sarcotesta of Magnolia in seven solvent systems. M. perezfarrerae (1), M. vovidesii (2), M. pugana (3) and M. schiedeana (4). The distance travelled by the compound on the plate (RF).
We observed that diverse phytochemicals on samples traveled different distances up the TLC plate depending on the solvent system chosen. The retention factors (Rf) for each solvent system are detailed in Table S1, and solvent systems are sorted in ascending order. Variations in Rf values of the phytochemicals reflect an idea about their polarity. For example, compounds with high Rf values in less polar solvent have low polarity and with fewer Rf values have high polarity. On the TLC plate using hexane (100%) as the mobile phase was observed a good separation of low polarity compounds only for M. vovidesii (Rf=0.6) and M. pugana (Rf= 0.8) extracts.
In this study, sarcotesta extracts of the four Magnolia species present a greater amount of compounds of medium polarity. A solvent combination such as hexane-acetonitrile (75:25%) and hexane-acetonitrile (50:50%) were good solvent systems that moves different compounds of the mixture off the baseline compared to Ethyl acetate and Acetonitrile-methanol (50:50%).
Using hexane-acetonitrile (75:25%) were identified two compounds in M. perezfarrerae (Rf= 0.38, 0.92), in M. vovidesii were identified four compounds (Rf= 0.60, 0.74, 0.82, 0.94), in M. pugana were identified three compounds (Rf= 0.38, 0.72, 0.92), and were identified two compounds in M. schiedeana (Rf= 0.56, 0.80).
Using hexane-acetonitrile (50:50%) were identified three compounds in M. perezfarrerae (Rf= 0.34, 0.70, 0.94), in M. vovidesii were identified four compounds (Rf=0.30, 0.40, 0.74, 0.82), in M. pugana were identified four compounds (Rf= 0.30, 0.64, 0.74, 0.80), and were identified two compounds in M. schiedeana (Rf= 0.60, 0.82).
On the other hand, in the mobile phases of higher polarity with solvents such as ethanol and methanol, we do not observe a good separation of compounds that migrated upp the TLC plate, and some of them have similar Rf values. This information will drive future experiments in a selection of the appropriate solvent system for further separation, isolation, and identification of compounds from these plant extracts of Magnolia spp.
For the first time, the presence of alkaloids, flavonoids, and phenols in the four species of Magnolia endemic to Mexico is reported on qualitative phytochemical analyses. On the contrary, the tannin test was negative in all species. Extracts of M. perezfarrerae and M. pugana showed a high content of alkaloids, steroids, and terpenes. A medium amount of the three metabolites was detected in M. vovidesii. A low alkaloid content was detected in M. schiedeana; however, the test for steroids and terpenes was negative. A low content of coumarins, which were absent in the other analyzed plant species, was detected in the extracts of M. perezfarrerae and M. pugana. A high flavonoid content was found in M. pugana, a medium concentration in M. vovidesii and M. schiedeana, and low concentration in M. perezfarrerae. High levels of phenolic compounds were found in M. schiedeana, a medium amount in M. perezfarrerae, and a low amount in species of M. pugana and M. vovidesii. The test for saponins was positive only in extracts of M. perezfarrerae, but a low content of these compounds was found (Table 4).
Table 4 Qualitative analysis of secondary metabolites in ethanolic extracts of sarcotesta of Magnolia. Symbols (+), (++), (+++), indicate a low, medium, or high content or the absence (-) of this type of metabolites.
| Species | Alkaloids | Coumarins | Flavonoids | Phenols | Tannins | Saponins | Steroids and terpenes |
|---|---|---|---|---|---|---|---|
| M. perezfarrerae | +++ | + | + | ++ | - | + | +++ |
| M. pugana | +++ | + | +++ | + | - | - | +++ |
| M. vovidesii | ++ | - | ++ | + | - | - | ++ |
| M. schiedeana | + | - | ++ | +++ | - | - | - |
Discussion
Magnolias have a high insecticide potential against Tephritidae. Among botanical pesticides there is a wide range of effectiveness that is determined by the botanical species, its vegetative structures, and the target pest species (Haouas et al., 2012; Hernández-Carlos & Gamboa-Angulo, 2019). In this study, it was observed that extracts of sarcotesta of M. perezfarrerae and M. pugana had more than 93% of insecticide-effectiveness against A. ludens and up to 91% against A. obliqua. This corresponds to the effectiveness shown by other species of Magnolias located in Mexican territory. In a preliminary study the insecticidal potential of M. dealbata (currently M. vovidesii) against A. ludens adults was recorded, with an effectiveness range of 19% to 96%, dry sarcotesta manifested itself as the vegetative structure with the highest effectiveness (Flores-Estévez et al., 2013). Likewise, the vegetative structures of M. schiedeana showed an effectiveness of 0.08 % for the flower and up to 64 % for the sarcotesta of its seeds against A. ludens adults (S. Vásquez-Morales et al., 2015).
Magnolias are known to have secondary metabolites with multiple biological effects (Lee et al., 2011; Sarker et al., 2002). This study confirmed qualitative that the assessed endemic species of Mexico contained alkaloids, flavonoids, phenols, and steroids or terpenes, consistent with chemical profiles reported for the Magnoliaceae family (Sánchez-Velásquez et al., 2016; Sarker et al., 2002). For example, the bark of M. officinalis has been reported to be a rich source of alkaloids (Yan et al., 2013), and the seeds of M. grandiflora contain alkaloids, saponins, and terpenes (Thakur & Sidhu, 2013). It is interesting to mention that M. perezfarrerae, M. pugana and M. vovidesii stood out for their high toxicity against fruit flies, Anastrepha spp; M. perezfarrerae distinguished itself for its high content of alkaloids, steroids, and terpenes, whereas M. pugana and M. vovidesii stood out for their high content of alkaloids, steroids, terpenes, and flavonoids (Table 4).
In nature terpenes are important compounds for plant defense mechanisms against herbivores and it has been suggested that they can be developed as biopesticides (Isman, 2000). Besides their effect on Diptera such as mosquitoes (Maheswaran & Ignacimuthu, 2012) and houseflies (Rossi & Palacios, 2013) has been reported. Several alkaloids have been reported as highly toxic to insects due to their effect on acetylcholinesterase receptors and sodium channels (Albuquerque et al., 2009; Crossthwaite et al., 2017). Our results preliminary suggest that the metabolites groups identified in the assayed Magnolias may contribute to the insecticidal effects reported for the fruit fly; nevertheless, studies of structure elucidation and chemical quantification of the major compounds in the extracts of sarcotesta of Magnolia spp are required.
Likewise, plants produce phenolic and flavonoid compounds as response mechanisms against herbivorous insects and plant pathogens (Ahmed et al., 2019; Bhattacharya et al., 2010). Polyphenolic compounds derived from the phenylpropanoid pathway, such as lignans, honokiol and magnolol, are the main components of Magnolia species; they possess antiviral (Amblard et al., 2006), antibacterial (Jacobo-Salcedo et al., 2011; B. Wu et al., 2018), fungicide (Chen et al., 2019) and insecticide properties (Wang et al., 2019; Yang et al., 2015). Honokiol, magnolol, 5-arylbenzofuran and their derivatives showed insecticidal activity against the black bean aphid (Aphis fabae), the fall webworm (Hyphantria cunea), the moth (Mythimna separata), and the swallowtail butterfly (Papilio palamedes y P. troilus) (Lin et al., 2019; Nitao et al., 1992). Likewise, the active compounds of M. denudata seeds (palmitic acid, linoleic acid and honokiol) showed potent larvicidal effects in Culex pipiens pallens y Aedes aegypti (Wang et al., 2019).
Alonso-Castro et al., (2014) determined the presence of honokiol and magnolol in the seeds of M. dealbata (currently M. vovidesii), so it can be inferred that the ethanolic extracts of sarcotesta of M. vovidesii, used in this study, contain these active insecticidal compounds, because the seeds come from the same location (Table 1). In addition, phenylpropanoid (iso-methyl eugenol) and sesquiterpene lactone (Costunolide) were isolated from hexane extracts of mature fruits of M. salicifolia, both compounds have insecticide activity against Aedes aegypti. Iso-methyl eugenol and costunolide have 0.13 and 0.14 Rf, respectively, in TLC with hexane (Kelm et al., 1997), hence we can extrapolate that both compounds or their derivatives may be present in the sarcotesta of M. vovidesii and M. pugana species (Fig. 1).
Several botanical and synthetic insecticides are inhibitors of acetylcholinesterase, an enzyme that inactivates the neurotransmitter excitation of acetylcholine during synapses, which leads to hyperexcitation in the insect (Hernández-Carlos & Gamboa-Angulo, 2019). It was demonstrated that the magnaldehyde B isolated from the bark of M. officinalis has a potent inhibitory activity against acetylcholinesterase at IC50 values of 12.63 ± 0.51 (Zhang et al., 2019). Likewise, guaiacol and caffeic acid (structurally related phenol compounds of honokiol) inhibit acetylcholinesterase in Aedes aegypti larvae and those treated with honokiol and magnolol showed spots all over their bodies due to damage and rupture of the middle intestine with no nucleus cell organelles and severely damaged mitochondria and plasma organelles with indiscernible appearances (Nitao et al., 1992).
It has been reported that the highest impact of the pest is caused by adult insects, especially females who oviposit their eggs on the fruits (Hernández-Ortiz, 1993; Montoya et al., 2010). However, the control of A. ludens flies at third-instar larval was also studied using the aqueous extract of Annona lutescens stem, obtaining a 95% effectiveness at 72 h of exposure (González-Esquinca et al., 2012).
Conclusions
Our study confirms the insecticide effectiveness of extracts of sarcotesta of M. perezfarrerae, M. pugana and M. vovidesii species against Anastrepha ludens and A. obliqua fruit fly species. The three Magnolia spp investigated showed differences in their phytochemical profile. The insecticide properties of Magnolia can contribute to the integrated management of Tephritids.
