Printer Friendly

Phytochemical characterization and bioactivity of ethanolic extracts on eggs of citrus blackfly/Caracterizacao fitoquimica e bioatividade de extratos etanolicos em ovos de mosca-negra-dos-citros.


Citrus blackfly (Aleurocanthus woglumi Ashby, 1915), an exotic pest widely disseminated in Brazil, is capable of infecting more than 300 species including fruit trees and ornamental plants. Citrus species, considered its primary hosts, are highly susceptible to infestation by this pest (EPPO, 2008; RAGA et al., 2016; ALVIM et al., 2016). Citrus blackfly eggs are creamy white until the second day after laying, resemble curved rods, with spiral-shaped, concentrating on the abaxial surface of leaves, adhered by a short peduncle. Usually on the seventh day after laying, eggs are orange, while on the eighth day, longitudinal hatching lines are observed. Average incubation period lasts 15 days and mean viability is 88% at 27.4 [+ or -] 1.1[degrees]C and 79.4 [+ or -] 4.6% RH (PENA et al., 2009). Bioprospecting is essential for discovering alternative biotechnological solutions aiming to produce botanical insecticides (SACCARO JUNIOR, 2011). The objectives of this study were to determine the content of secondary metabolites (carotenoids, flavonoids, phenolic compounds, and tannins) of Argemone mexicana L., Ipomoea carnea Jacq. subsp. Fistulosa (Martius ex Choisy), Amorimia rigida (A.Juss.) W R. Anderson, Ricinus communis L. and Syzygium aromaticum (L.) Merr. & L. M. Perry, and evaluate the bioactivity of the ethanolic extracts on citrus blackfly eggs.


The exsiccates of the studied plants were deposited and registered in the Universidade Estadual de Santa Cruz (UESC) Herbarium: 19126 (Mexican poppy), 19128 (pink morning glory), 19127 (amorimia), 18835 (castor bean) and 18821 (clove tree).

Phytochemical bioprospecting of the plant extracts was carried out at the Laboratory of Natural Products and Organic Synthesis (LPPNS), at the UESC, Ilheus, Bahia. Total phenolic compounds, total flavonoids, condensed tannins, and carotenoids were quantified using UV-VIS (Nova[R] 1600UV) spectrometry of the leaves of Mexican poppy, pink morning glory, amorimia, and castor bean, and the peduncle of the clove flower buds. All analyses were performed in triplicates. Plants were dried in the shade and shredded in a blender. We used 0.2g of plant material for extraction with 50mL of acetone (KIMURA & RODRIGUES-AMAYA, 2003) for the determination of carotenoids. After clarification with ZnS[O.sub.4] and Ba [(OH).sub.2], the compound was subjected to extraction with petroleum ether, and the carotenoid content was determined spectrophotometrically at 450nm. For flavonoids, we extracted 0.25g of plant material with methanol (PEIXOTO et al., 2008). Subsequently, we added glacial acetic acid, 2% pyridine methanolic solution, and Al[Cl.sub.3]. After 30 min, the spectrophotometric analysis was performed at 420nm. A calibration curve with 10, 30, 50, 70 and 100gg [mL.sup.-1] methanolic solutions of rutin was prepared from the standard stock solution (5000gg [mL.sup.-1]). This curve was represented by the following equation f (y) = 0.0075 x + 01, [r.sup.2] = 0.9349; the limit of detection was 0.480[micro]g [mL.sup.-1] and the limit of quantification was 1.60[micro]g [mL.sup.-1]. For determining the phenolic content, we extracted 0.3g of the plant material with ethanol (FURLONG et al., 2003). After clarification with ZnS[O.sub.4] and Ba [(OH).sub.2], the extract was reacted with Folin-Ciocalteou and [Na.sub.2]C[O.sub.3].

After 30 minutes of reaction, the spectrophotometric reading was performed at 773nm. A calibration curve was prepared using 5, 10, 72.5, 145.0, and 217.5[micro]g [mL.sup.-1] ethanolic solution of gallic acid, which were was prepared from the standard stock solution (5000[micro]g [mL.sup.-1]). This curve was represented by the equation f (y) = 0.0034 x--0.0194; [r.sup.2] = 0.9995; the limit of detection was 1.97[micro]g [mL.sup.-1] and the limit of quantification was 5.0[micro]g [mL.sup.-1]. For the determination of condensed tannins, we extracted 0.125g of plant material with 80% aqueous acetone solution, using a protocol adapted from PEREZ et al. (1999). The extract reacted with vanillin and concentrated HCl. After 20min, the spectrophotometry was performed at 500nm. A calibration curve with 5, 35, 65, 95 and 125[micro]g [mL.sup.-1] standard catechin solution was prepared from the stock solution of 140[micro]g [mL.sup.-1]. This curve was represented by the equation f (y) = 0.0024 x + 0.2173, [r.sup.2] = 0.9990; the limit of detection was 0.23[micro]g [mL.sup.-1] and the limit of quantification was 5.0[micro]g [mL.sup.-1].

To evaluate the bioactivity of treatments, Pera sweet orange [Citrus sinensis L. Osbeck] leaves with eggs were kept in Petri dishes with moistened filter paper and sealed with plastic film. Each sample unit consisted of 30 eggs of citrus blackfly. Leaf areas containing the eggs were demarcated (VIEIRA et al., 2013). Eggs used in these experiments were laid no longer than 24h earlier, and had a creamy white appearance. The plant extracts were prepared from shredded dry material, 10g of each species were removed and placed in an Erlenmeyer flask containing 100mL of absolute alcohol for 8d. Thereafter, they were strained on filter paper and the pure ethanolic extracts (100%) were kept in amber glass. In order to prepare the treatments, pure ethanolic extracts were diluted with distilled water to 0.5, 1.0, 5.0, and 10% concentrations. Additionally, we used the commercial product Bioneem[c] diluted at the same concentrations. Distilled water was used as a control. Three applications of each treatment (n=7) were made by leaf immersion (30s per leaf). The application interval was 7s. Subsequently, the Petri dishes containing the leaves with eggs were closed, sealed with plastic film, identified, and allocated in BOD incubators (25 [+ or -] 1[degrees]C; RU 60 [+ or -] 5% and 12h of photophase). Egg mortality was evaluated weekly under a stereoscopic microscope (56x) after the first application of the treatments. Eggs that did not hatch until the 21st day were considered non-viable (JESUS et al., 2013, LIMA et al., 2013; VIEIRA et al., 2013).


Phytochemical bioprospecting

Data on carotenoid, flavonoid, and phenolic compounds of I. carnea, R. communis, A. mexicana, A. rigida, and S. aromaticum are presented in figure 1. S. aromaticum showed high phenolic content, possibly due to the presence of eugenol, a volatile phenylpropanoid. It is the main compound repored in the steam of this species, as are [beta]-caryophyllene (3.61%) and eugenyl acetate (3.76%), whereas a-humolene (0.60%) is a minor component (AFONSO et al., 2012). Further, the tannin content of this species was high (Figure 1). In the other species, negative values were obtained for tannin content, probably because the extracts were based on leaves (Figure 1). Phenolic compound level of I. carnea was 228.7mg [g.sup.-1] (Figure 1). The phenolic constituents were not identified, but some studies have reported that the catechol content is between 45 and 75mg [g.sup.-1] and flavonoid levels are high (SAHAYARAJ & RAVI, 2008; KHATIWORA et al., 2010; JAIN et al., 2016). Among the species evaluated in this study, R. communis had the highest flavonoid content (Figure 1). Moreover, presence of glycosides, quercetin, empferol, and kaempferol, which are phenolic compounds, have been reported in other studies (CHEN et al., 2008; PACHECO-SANCHEZ et al., 2012). The high content of tannins and phenolic compounds in S. aromaticum, and the high content of flavonoids in R. communis may be associated to the insecticidal potential of these species.


Except for the extract of A. mexicana, all the treatments had significant differences in citrus blackfly egg survival at 10% concentration (Table 1). Bioneem[c] caused more than 90% egg mortality (Table 1), and a control efficiency of more than 80% at all concentrations (Figure 2), probably due to the action of azadirachtin and other compounds present in leaves and fruit of the neem tree (MARTINEZ, 2003). After leaf immersion in Bioneem[c], 100% of citrus blackfly eggs darkened. Moreover, the commercial product Rot-Nim[R] (0.5%) has been reported to cause citrus blackfly egg mortality of 80.2% (SILVA et al., 2012), which indicated that neembased products can efficiently control citrus blackfly. Extract of S. aromaticum (5 and 10% only) caused egg mortality higher than 80% (Table 1) and control efficiency of greater than 80% at 10% concentration (Figure 2), possibly due to the presence of eugenol (phenolic compound) and flavonoids (AFONSO et al., 2012). The highest control efficiency (81.6%) for the extract of R. communis was observed at 10% concentration (Figure 2), causing eggs mortality above 90% (Table 1). These results are supported by VIEIRA et al. (2013), who reported a decrease in the viability of citrus blackfly eggs as the commercial castor oil concentration increased. The extract of A. rigida caused citrus blackfly egg mortality higher than 70.0% at 5% and 10% concentrations only (Table 1). However, this species had low control efficiency (Figure 2). Furthermore, A. mexicana caused citrus blackfly egg mortality lower than 80.0% at all concentrations, which is similar to the results obtained for A. rigida species (Table 1, Figure 2). MARTINEZ-TOMAS et al. (2015) reported that the aqueous extract of A. mexicana applied to eggs of the whitefly (Hemiptera: Aleyrodidae) caused a 97.64% reduction of the nymph population. Citrus blackfly egg viability is approximately 65-95% (PENA et al., 2009), which justifies 40% mortality in the control (Table 1).


The species A. rigida, R. communis, and S. aromaticum presented higher levels of carotenoids, flavonoids, and phenolic compounds, respectively. The commercial oil Bioneem[c] is effective in controlling citrus blackfly eggs. Ethanolic extracts of S. aromaticum and R. communis have insecticidal potential on citrus blackfly eggs, acting as alternative control agents for this species, besides subsidizing and contributing to its integrated management.

Received 02.08.17 Approved 08.21.17 Returned by the author 09.16.17


The authors are grateful for the support of the Embrapa Cassava and Tropical Fruits and the Programa de Posgraduacao em Producao Vegetal (PPGPV).


Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES).


ALVIM, R.G. et al. Dissemination of Aleurocanthus woglumi in citrus plants, its natural enemies and new host plants in the state of Rio de Janeiro, Brazil. Ciencia Rural, v.46, n.11, p.1891-1897, 2016. Available from: <http://dx.doi.oig/10.1590/0103-8478cr2015U01>. Accessed: Jan. 13, 2017. doi: 10.1590/0103-8478cr20151101.

CHEN, Z. et al. Simultaneous determination of flavones and phenolic acids in the leaves of Ricinus communis Linn. by capillary electrophoresis with amperometric detection. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, v.863, n.1, p.101-106, 2008. Available from: <>. Accessed: Jan. 19, 2017. doi: 10.1016/jjchromb.2008.01.002.

EPPO (EUROPEAN PLANT PROTECTION ORGANIZATION). EPPO quarantine pest. Aleurocanthus woglumi. Available from: < woglumi/ALECWO_dspdf>. Accessed: Feb. 08, 2017.

FURLONG, E.B. et al. Evaluation of the potential of phenolic compounds in plant Tissues. Vetor, v.13, n.1, p. 105-114, 2003. Available from: <>. Accessed: Jan. 19, 2017.

JAIN, A. et al. Evaluation of In-vitro cytotoxic and antioxidant activity of methanolic extracts ofIpomoea carnea and Alternanthera sessilis. International Journal of Bioassays, v.5, n.8, p.4763-4766, 2016. Available from: <>. Accessed: Jan. 19, 2017. doi: 10.21746/ijbio.2016.08.008.

JESUS, S.C.P. de et al. Insecticide and activity ofvegetal extracts on the silverleaf whitefly (Bemisia tabaci). Revista em Agronegocios e Meio Ambiente, v.6, n.1, p.117-134, 2013. Available from: <>. Accessed: Jan. 19, 2017.

KHATIWORA, E. et al. Spectroscopic determination of total phenol and flavonoid contents of Ipomoea carnea. International Journal of ChemTech Research, v.2, n.3, p.1698-1701, 2010. Available from: <

chemtech/chemtechvol2.3july-sept210/CT=50%20(1698-1701). pdf>. Accessed: Jan. 19, 2017.

KIMURA, M. et al. Carotenoid composition of hydroponic leafy vegetables. Journal of Agriculture and Food Chemistry, v.51, n.1, p.2603-2607, 2003. Available from: <http://hdl.handle. net/11449/67266>. Accessed: Jan. 19, 2017. doi: 10.1021/jf020539b.

LIMA, B.M.F.V. et al. Evaluation of plant extracts in the control of whitefly Bemisia tabaci biotype B in squash. Revista Ciencia Agronomica, v.44, n.3, p.622-627, 2013. Available from: <http://>. Accessed: Jan. 19, 2017. doi: 10.1590/S1806-66902013000300026.

MARTINEZ, S.S. The use of Nim in coffee and other cultures. Revista Agroecologia Hoje, v.4, n.21, p. 13-14, 2003. Available from: <>. Accessed: Jan. 16, 2017.

MARTINEZ-TOMAS, S.T. et al. Evaluation of three plant extracts in the whitefly population in organic tomato growing in greenhouses. Entomologia Mexicana, v.2, n.1, p.371-375, 2015. Available from: < EA/PAG%20%20371-375.pdf>. Accessed: Jan. 19, 2017.

NAKANO, O. et al. Entomologia economica. Sao Paulo, Piracicaba: Livroceres, 1981. 314p.

PACHECO-SANCHEZ, C. et al. Effect of Ricinus communis extracts on weight and mortality of Scyphophorus acupunctatus (Coleoptera: Curculionidae). International Journal of Applied Science and Technology, v.2, n.1, p.83-94, 2012. Available from: < pdf>. Accessed: Jan. 06, 2017.

PEIXOTO, TJ. da S.P.S. Validation of spectrophotometric methodology for quantify flavonoid content in Bauhinia cheilantha (Bongard) Steudel. Revista Brasileira de Ciencias Farmaceuticas, v.44, n.4, p.683-689, 2008. Available from: <http://>. Accessed: Jan. 08, 2017. doi: 10.1590/S1516-93322008000400015.

PENA, M.R. et al. Biology of the citrus blackfloy, Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae), in three host plants. Neotropical Entomology, v.38, p.254-261, 2009. Available from: <>. Accessed: Apr. 29, 2017. doi: 10.1590/S1519-566X2009000200014.

PEREZ, D.M. et al. Determination of the tannin content of four sorghum varieties with quantitative methods. Revista Brasileira de Zootecnia, v.28, n.3, p.453-458, 1999. Available from: <http://>. Accessed: Jan. 12, 2017. doi: 10.1590/S1516-35981999000300003.

RAGA, A. et al. Population dynamic of citrus blackfly, Aleurocanthus woglumi (Hemiptera: Aleyrodidae), in Tahiti Lime in the eastern of the State of Sao Paulo, Brazil. Annual Research & Review in Biology, v.11, n.1, p.1-7, 2016. Available from: <http://www.journalrepository. org/media/journals/ARRB_32/2016/Sep/Raga1112016ARRB28668. pdf>. Accessed: Feb. 08, 2017. doi: 10.9734/ARRB/2016/28668.

SACCARO JUNIOR, N.L. The rules of access to genetic resources and benefit sharing: disputes inside and outside Brazil. Ambiente e Sociedade, v.14, n.1, p.1-16, 2011. Available from: <http://dx.doi. org/10.1590/S1414-753X2011000100013>. Accessed: Jan. 19, 2017. doi: 10.1590/S1414-753X2011000100013.

SAHAYARAJ, K.; RAVI, C. Preliminary phytochemistry of Ipomea carnea jacq. and Vitex negundo Linn. Leaves. International Journal of Chemical Sciences, v.6, n.1, p.1-6, 2008. Available from: <http:// pdf>. Accessed: Jan. 14, 2017.

SILVA, L.D. et al. Use of vegetable oils in the control of the citrus black fly, Aleurocanthus woglumi (Hemiptera: Aleyrodidae). Revista Colombiana Entomologia, v.38, n.2, p. 182-186, 2012. Available from: <>. Accessed: Jan. 19, 2017.

VIEIRA, D.L. et al. Application of commercial oils for ovicidal control of Aleurocanthus woglumi Asbhy. Bioscience Journal, v.29, n.5, p.1126-1129, 2013. Available from: <http://www.seer.ufu. br/index.php/biosciencejournal/article/viewFile/21923/13006>. Accessed: Jan. 19, 2017.

Bruno Marcus Freire Vieira Lima (1) *, Rosilene Aparecida de Oliveira (2), Emerson Alves dos Santos (3), Maria Aparecida Leao Bittencourt (4), Olivia Oliveira dos Santos (5)

(1) Universidade Estadual de Santa Cruz (UESC), Campus Soane Nazare de Andrade, Rodovia Jorge Amado, Km 16, Salobrinho, 45662-900, Ilheus, BA, Brasil. E-mail: * Corresponding author.

(2) Departamento de Ciencias Exatas, Universidade Estadual de Santa Cruz (UESC), Ilheus, BA, Brasil.

(3) Ciencias Biologicas, Universidade Estadual de Santa Cruz (UESC), Ilheus, BA, Brasil.

(4) Departamento de Ciencias Agrarias e Ambientais Rod. Jorge Amado, Universidade Estadual de Santa Cruz (UESC), Ilheus, Bahia, Brasil.

(5) Doutorado em Agronomia, Universidade Estadual do Sudoeste da Bahia (UESB), Vitoria da Conquista, BA, Brasil.
Table 1--Aleurocanthus woglumi egg mortality (%) under different
treatments, after 21 days of application in laboratory (25 [+ or -]
1[degrees]C; RH 60 [+ or -] 5% and 12h of photophase).

Treatments      0.5%                      1.0%

I. carnea       65.5% [+ or -] 4.7 bB      64.5% [+ or -] 8.1 bcB
R. communis     60.2% [+ or -] 5.14 bC     78.4% [+ or -] 13.79 bB
A. mexicana     68.1% [+ or -] 9.82 bA     74.1% [+ or -] 8.29 bA
A. rigida       66.9% [+ or -] 13.54 bA    65.5% [+ or -] 15.94 bA
S. aromaticum   78.2% [+ or -] 10.76 bAB   67% [+ or -] 3.24 bB
Bioneem [c]     94.1% [+ or -] 19.13 aA    94.3% [+ or -] 28.12 aA
Control         44.3% [+ or -] 3.6 cA      39.5% [+ or -] 3.39 cA

Treatments      5.0%                      10.0%

I. carnea       77.6% [+ or -] 6.9 Ba      79.1% [+ or -] 6.81 bA
R. communis     76.4% [+ or -] 14.24 bB    93.8% [+ or -] 16.6 bA
A. mexicana     71.9% [+ or -] 14.07 bcA   71.9% [+ or -] 6.14 bcA
A. rigida       74.99% [+ or -] 19.78 bA   77.2% [+ or -] 24.37 bA
S. aromaticum   83.9% [+ or -] 10,2 bA     88% [+ or -] 29.33 bA
Bioneem [c]     96.6% [+ or -] 14.20 aA    97.2% [+ or -] 13.88 aA
Control         49.9% [+ or -] 6.8 cA      56% [+ or -] 3.56 cA

Values followed by the same uppercase letters on the rows and
lowercase letters in a column do not differ statistically from each
other, according to Tukey's honestly significant difference (HSD) test
at the 5% probability level.

Figure 1--Carotenoid, flavonoid (rutin), and phenolic compound
content (mg [g.sup.-1]) of the different plant species.

Carotenoids(mg [g.sup.-1])

I. carnea       0,2
R. communis     0,4
A. Mexicana     0,35
A. rigida       0,55
S. aromaticum   0,1

Flavanoids (Rutina)(mg [g.sup.-1])

I. carnea       141
R. communis     477
A. Mexicana     343
A. rigida       201
S. aromaticum   95

Phenolic compounds (mg [g.sup.-1])

I. carnea       229
R. communis     91
A. Mexicana     176
A. rigida       169
S. aromaticum   1282

Note: Table made from bar graph.

Figure 2--Efficiency (%) of different treatments based on plant
species on egg mortality of Aleurocanthus woglumi after three
applications by leaf immersion (NAKANO; SILVEIRA NETO; ZUCCHI, 1981).

                10%    5%     1%     0,50%

I. carnea       64,0   53,5   31,0   13,3
R. communis     81,6   57,0   54,3   18,6
A. Mexicana     46,5   43,0   41,4   25,7
A. rigida       55,3   43,9   35,3   30,1
S. aromaticum   80,7   69,3   48,3   47,8
Bioneem[c]      94,7   93,9   86,2   84,1

Note: Table made from bar graph.
COPYRIGHT 2017 Universidade Federal de Santa Maria
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Lima, Bruno Marcus Freire Vieira; de Oliveira, Rosilene Aparecida; dos Santos, Emerson Alves; Bitten
Publication:Ciencia Rural
Date:Nov 1, 2017
Previous Article:LESS ovariohysterectomy in cats using a new homemade multiport/Ovario-histerectomia por LESS em gatas com um novo multiportal artesanal.
Next Article:Antibodies against rabies virus in dogs with and without history of vaccination in Santa Maria--RS-Brazil/Anticorpos contra o virus da raiva em caes...

Terms of use | Privacy policy | Copyright © 2022 Farlex, Inc. | Feedback | For webmasters |