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Anales del Instituto de la Patagonia

versión On-line ISSN 0718-686X

Anales Instituto Patagonia (Chile) vol.47 no.1 Punta Arenas  2019

http://dx.doi.org/10.4067/S0718-686X2019000100017 

ARTÍCULOS CIENTÍFICOS

Antifeedant activity of alkaloid extracts from calafate ( Berberís microphylla , G. Forst, 1789) against diamondback moth larvae ( Plutella xylostella , Linnaeus, 1758)

Actividad antialimentaria de los extractos de alcaloides de calafate (Berberís microphylla, G. Forst, 1789) sobre larvas de la polilla de la col (Plutella xylostella, Linnaeus, 1758)

Loreto Manosalva1 

Ana Mutis2 

Rubén Palma3  4 

Víctor Fajardo5 

Andrés Quiroz2 

1 Facultad de Ciencias, Universidad de Magallanes, Avenida Bulnes 01850, Punta Arenas, Chile. E-mail: loreto.manosalva@umag.cl

2 Laboratorio de Química Ecológica, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile.

3 Instituto de Ciencias Biológicas, Universidad de Talca, Talca, Chile.

4 Millennium Nucleus Centre in Molecular Ecology and Evolutionary Applications in the Agroecosystems, Universidad de Talca, Talca, Chile.

5 Laboratorio de Productos Naturales, Universidad de Magallanes, Punta Arenas, Chile.

Abstract:

In laboratory by dual choice test, stem and root alkaloid extracts of Berberis microphylla showed antifeedant activity against third-instar Plutella xylostella larvae. Stem extracts showed significantly lower leaf consumptions at concentrations of 500 and 1000 mg/L, as illustrated by antifeedant index of 72 and 97% respectively, whereas, root extracts were significantly active at 100, 500 and 1000 mg/L with antifeedant index of 68, 85 and 99% respectively. In addition, the pure berberine identified as the major alkaloid in the plant extracts showed significant lower food consumption by larvae only at concentration of 1000 mg/L, but the pure palmatine not affect feeding at any concentration. The results suggest that alkaloids extract of B. microphylla have promising antifeedant activity and could by incorporated in an integrated pest management (IPM) programs for P. xylostella.

Keywords: antifeedant activity; alkaloid extracts

Resumen:

En laboratorio, mediante prueba de doble elección los extractos de alcaloides obtenidos de tallos y raíces de Berberis microphylla mostraron actividad antialimentaria frente a larvas de tercer estadio de Plutella xylostella. Los extractos de tallos mostraron consumos foliares significativamente más bajos en concentraciones de 500 y 1000 mg/L con un índice antialimentario de 72 y 97% respectivamente, mientras que los extractos de raíces fueron significativamente más activos a 100, 500 y 1000 mg/L con un índice antialimentario de 68, 85 y 99% respectivamente. Además, berberina identificada como el principal alcaloide de los extractos tuvo significativamente un menor consumo de alimento por parte de las larvas mostrando su efecto solo a la concentración de 1000 mg/L. Por otra parte, palmatina no tuvo efecto en la conducta de alimentación de las larvas a ninguna de las concentración evaluadas. Los resultados sugieren que los extractos de alcaloides obtenidos de B. microphylla tienen una prometedora actividad antialimentaria y podrían ser incorporados en un programa de manejo integrado de plagas (MIP) para P. xylostella.

Palabras clave: actividad antialimentaria; extractos de alcaloides

Introduction

Insect pests are one of the biotic factors that cause significant crop losses worldwide, where the use of synthetic pesticides (eg. organophosphates and pyrethroids) is a control strategy used traditionally for to pest control (Birch et al. 2011; Zhang et al. 2011; Chareonviriyaphap et al. 2013). However, the chemical control has become less effective in the last decades because of its rapid developing resistance (Shelton et al. 1993). In this sense, the indiscriminate use of synthetic pesticide chemicals has given rise to many ecological problems, among which, toxic residues and harm to mammals are the most crucial (Carriger et al. 2006; Aktar et al. 2009; Damalas & Eleftherohorinos, 2011). For this reason, the use of naturally derived plant products emerges as environmentally friendly alternatives to synthetic pesticide, with applications as botanical insecticides, antifeedants and insect growth regulators (Isman, 1994, 2006; Sarkar & Kshirsagar, 2014; Kedia et al. 2015).

In the case of natural antifeedant, has as an advantage that it never kills target insects directly allowing them to be available to their natural enemies and thus help in the maintenance of the natural balance (Jeyasankar et al. 2012). Several investigators reported that alkaloids are strong natural insect antifeedants (González-Coloma et al. 1998; Brem et al. 2002; Mao & Henderson, 2007; Cornelious et al. 2009; Sani et al. 2014). In this sense, Berberís genus are characterized to produced alkaloids with an interesting spectrum of insecticide and antifeedant properties (Tewary et al. 2005; Quevedo et al. 2007; Rehman et al. 2018). Berberís microphylla (known also B. buxífolía), is a native plant growing abundantly in southern regions of Chile and Argentina (Patagonia) with medicinal values attributable to the presence of alkaloids (Pitta-Álvarez et al. 2008; Domínguez et al. 2012; Manosalva et al. 2016). However, studies on the biological activity of B. microphylla against insects have not been reported.

For this reason, the aim of this study was to evaluate the antifeedant activity of stem and root alkaloid extracts from B. mícrophylla against larvae of Plutella xylostella. In addition, two synthetic compounds, berberine and palmatine previously identified in B. mícrophylla (Manosalva et al. 2014) were evaluated.

Materials and methods

Plant materíal

Stems and roots samples of B. microphylla were collected during the flowering season (December of 2011) near the shores of Deseado Lake, Province of Tierra del Fuego (54°22’12.4”S; 68°45’45.0”W). A voucher specimen was deposited in the herbarium at the Universidad de Concepción (Voucher N° CONC 178057). The plant material was vacuum-packed and stored at -20°C for further study.

Alkaloids extraction

In this procedure, oven dried and powdered leaves (100 g), stems (300 g) and roots (300 g) of B. microphylla were sequentially extracted (24, 48 and 72 h) with methanol at room temperature. Methanolic extracts were evaporated in vacuo at 40°C, and the residue reconstituted with 200 mL 10% HCl for 1 h under agitation (orbital shaker, MS- NOR, Taiwan), and allowed to stand for 12 h at 10°C prior to filtering. The filtrate was washed with CHCl3 (5 x 100 mL). The aqueous phase was adjusted to pH 10 with NH4OH and extracted with CHCl3 (5 x 100 mL). Finally, the solvent was evaporated under reduce pressure for obtaining dried extract containing alkaloids. The solvent was evaporated for obtaining crude extract containing alkaloids.

Alkaloid standards

Berberine chloride (purity, >98%), palmatine chloride (purity, >97%) and all analytical grade solvents used for extractions, were purchased from Sigma Aldrich (St. Louis, USA).

Insects

The larvae of P. xylostella were obtained from the laboratory of the Biological Chemistry and Crop Protection Department, Rothamsted Research Station (Harpenden, UK). Culture of larvae were maintained on cabbage plants at 25 ± 2°C, under a 16:8 light: dark cycle. One-day- old third instar larvae were selected for antifeedant assay.

Antifeedant assay

The antifeedant activity of stem and root alkaloid extracts of B. microphylla and pure compounds (berberine and palmatine) identified in the plant extracts were assessed via dual-choice bioassays using third-instar larvae of P. xylostella. In the dual-choice test, each insect was given a choice between a treated and an untreated cabbage leaf dics. If the substance acted as an antifeedant, insects would then eat a greater amount of the untreated leaves. Concentrations of 100, 500 and 1000 mg/L of each plant extracts and 10, 100 and 1000 mg/L of pure compounds were prepared with ethanol to avoid damaging the leaves, and this solvent used as control. Each treatment was separately applied on half of a fully opened leaf by dipping both sides of the leaf blade up to the midrib in the appropriate solution. The leaf disc (5 cm diameter) was then placed on a filter paper into a tray and kept in the fume cupboard for ca. 20 min to dry. Thereafter, the leaf was placed in a Petri dish padded with a moist filter paper marked on one side to distinguish the treated half of the leaf. Five third-instar P xylostella larvae previously starved for 24 h were then introduced and the dishes kept in the laboratory for 12 h. Each treatment and control were replicated ten times. The eaten leaf area of the treated leaf portion and that of control were recorded separately, and expressed in square centimeters (cm2) using coordinate paper (Ling et al. 2008).

Furthemore, the percentage antifeedant (AFI) of compounds was calculated using an antifeedant index (AFI) (Krishnakumari et al. 2001):

AFI (%) = (1- T / C) x 100

where C is the leaf area consumed in control and T is the leaf area consumed in treatments.

Statistical analysis

Insecticide and antifeedant activities of alkaloid extracts from B. microphylla and pure compounds were statistically analyzed by ANOVA (P <0.05) with Sigma Plot test.

Results

The results of antifeedant activity of alkaloid extracts from B. microphylla and pure compounds are presented in (Table 1). Stem and root alkaloid extracts at concentration of 500 and 1000 mg/L significantly reduced leaf consumptions by P xylostella larvae. Thus, at 500 mg/L, stems and root extracts elicited a decrease the insect feeding behavior with antifeedant index of 72% and 85% respectively. At de concentration of 1000 mg/L, stem and root extracts deterrent feeding by larvae of P xylostella with antifeedant index of 97 and 99% respectively (Figure 1). The antifeeding activity depending on the concentration of the plant extracts and all the activities are dose dependent.

Table 1 Antifeedant activity of alkaloid extracts from B. microphylla and pure compounds against the third instar larvae of P. xylostella. 

Fig. 1 Photograph of antifeedant activity of root alkaloid extract of B. microphylla at 1000 mg/L after 12 h treatment by via dual-choice bioassays with third-instar larvae of P. xylostella. (a) control, (b) treated leaf. 

Furthemore, the P xylostella larvae were fed with leaves treated with berberine and palmatine (Figura 2). Berberine exhibited significant antifeedant activity only at the highest concentration of 1000 mg/L, whereas palmatine did not show such activity.

Fig. 2 Structure of berberine (a) and palmatine (b) identified in stem and root extracts of B. microphylla. 

Discussion

Our results showed that B. microphylla stem and root alkaloid extracts have antifeedant activity against P xylostella larvae because reducing leaf consumptions. Some investigators have reported feeding deterrent activity of plant alkaloids against P xylostella.Banaag et al. (1997) shown that alkaloid fractions from Dioscorea hispida not only inhibited the feeding of Plutella xylostella larvae, but had toxic activity and disrupted of adult emergence. In the same way, Guo et al. (2014) found that two alkaloids (7-demethoxytylophorine and 6-hydroxyl- 2,3-dimethoxy phenanthroindolizidine), isolated from Cynanchum komarovii were active against the third-instar larvae of P xylostella as insecticides, antifeedants and growth inhibitors. Other study by Paulraj et al. (2014) reported that leaf fraction from Adhatoda vasica contain alkaloids called vasicine acetate and 2-Acetyl benzylamine that present antifeedant activity against P xylostella larve. Furthermore, in relation to biological activity of Berberis species has been report that alkaloids identified in stem and leaf extracts of B. glauca were showed antifeedant effects on larvae 3rd y 4th stage of the moth Spodoptera sunia, as well as toxic effects (Moreno-Murillo et al. 1995).

In previous studies, we have reported that stem and root extracts of B. microphylla showed a complex mixture of isoquinoline alkaloids. The roots contain of the following alkaloids: allocryptopine, berberine, calafatine, jatrorrhizine, palmatine, protopine, reticuline and thalifendine. Stems also contain of the following alkaloids: allocryptopine, berberine, isocorydine, jatrorrhizine, protopine, scoulerine and thalifendine (Manosalva et al. 2014). In this sense, two synthetic alkaloids, berberine and palmatine identified in stem and root extracts were tested, where only berberine reduced larval feeding of P xylostella.Shields et al. (2008), evaluated feeding deterrents of nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine and strychnine) against Lymantria dispar larvae. Results suggested that berberine and aristolochic acid were the two most potent feeding deterrents. The results obtained for berberine are interesting because this compounds is a major constituent in stems and roots of Berberis plants (Ivanovska et al. 1996). Park et al. (2000) indicating that berberine and palmatine identified in roots of Coptis japónica had antifeeding activity against Hyphantria cunea larvae when treated by leaf-dipping assay. They suggested that the antifeeding activity was much more pronounced in applications of mixtures of berberine and palmatine, indicating a synergistic effect.

Considering that the alkaloid extracts from stem and root of B. microphylla are a complexely mixture of alkaloids (Manosalva et al. 2014) the antifeedant activity against P. xylostella it could be related to the synergistic effect among alkaloids present in the extracts. Future studies will be focused in identifying other bioactive components present in plant extracts of B. microphylla against P xylostella larvae to allow the development of botanical insecticides with greater effectiveness than the crude plant extracts evaluated in this study.

Conclusion

Alkaloid extracts from stems and roots of B. microphylla showed antifeedant activity against larvae of P. xylostella suggesting extracts have compounds that could be contributing towards feeding suppression against the P xylostella larvae.

Acknowledgments:

The authors would like to acknowledge CONICYT scholarship N° 21100561 awarded to LM, Grant N° AT24121407, Doctoral Internship N° 75120095 and Internal proyect N° 027202, Universidad de Magallanes. We are grateful to Ph D. Michael Birkett from Department of Biological Chemistry, Rothamsted Research, Harpenden, England, for provide us with the biological material and for support in designed the experiment.

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Recibido: 10 de Octubre de 2018; Aprobado: 13 de Noviembre de 2019

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