UHPLC-OrbiTrap MS Characterization of Phenolic Profiles in French Marigold Extracts and Analysis of Their Antifeedant Activity against Colorado Potato Beetle

Abstract

French marigold is an aromatic plant rich in polyphenolic secondary metabolites, which pesticidal potential was examined in this study. Ultra-high-performance liquid chromatography (UHPLC) connected with OrbiTrap mass spectrometer (MS) identified 113 phenolics and revealed the most detailed phytochemistry of French marigold published so far. Depending on plant material (flowers or leaves) and solvents used for extraction (water, methanol, dichloromethane), the phenolic composition varied. Methanol extract of flowers, with 89 identified phenolics and high antioxidant activity statistically comparable with positive control Trolox, was chosen for testing of antifeedant potential against the 3rd and 4th instars of Colorado potato beetle (CPB). A significant reduction in final body mass of 4th larval stage fed with potato leaves coated with methanol extract of flowers in the concentration of 10 mg/mL was observed (157.67 mg vs. 182.26 mg of controls fed with non-treated leaves). This caused delayed molting since treated larvae reached the maximal mass a day after controls and this delay persisted during the entire larval development. Continuous feeding caused a 25% decline in digestive proteolytic activity of the 4th instar in comparison to controls. The results suggest that French marigold methanol extract of flowers could be proposed as a promising antifeedant for CPB management, with an impact on the reduction in the environmental footprint associated with synthetic pesticide application.

Keywords: Leptinotarsa decemlineata Say; Tagetes patula L.; biological insecticides; insect protease activity; phytochemical profile.

Figure 1

Number of identified phenolic acids, phenolic acid glycosides, flavonoids, and flavonoid glycosides in water, methanol (MeOH), and dichloromethane (DCM) extracts of flowers and leaves of French marigold. Pies represent relative distribution of different groups of phenolics in each extract (number of compounds per extract = 100%).

Figure 2

Distribution of different phenolics in extracts of French marigold flowers and leaves obtained in water, methanol (MeOH), and dichloromethane (DCM). Heatmaps represent values for scaled areas obtained from full scan MS for each compound (1 in the color legend corresponds to the maximal peak area detected for each compound).

Figure 3

Effect of French marigold methanol extract of flowers on CPB larval growth and digestive activity. Larvae of 2nd instar hatched from eggs (A) were fed with potato leaves coated with the extract in concentrations 1 mg/mL (T1) or 10 mg/mL (T10) or with control leaves. Larval mass gain (B) was recorded daily during the entire larval development, from the 2nd through 4th instar to the prepupal stage (given stage borders are for controls). Digestive protease activity (C) in midguts of 3rd and 4th instars fed with T1 and T10 coated potato leaves was analyzed and presented relative to activity measured in control larvae fed with non-coated leaves. All results are presented as means ± standard errors (n = 15 for (B) and n = 3 for (C)), and values with an asterisk (*) were different than corresponding controls according to Student’s t-test (p ≤ 0.1) for each day of measurement (for (B)) or larval stage (for (C)) separately. Bars represent 1 cm.

Figure 4

Effect of French marigold methanol extract of flowers on CPB larval development. The potato leaves coated with extract in concentrations of 1 mg/mL (T1) and 10 mg/mL (T10), were used as food for CPB larvae during development from 2nd, through 3rd, and 4th instars. The overall developmental pattern was compared with larvae fed on control leaves. Results are presented as a percentage of larvae in the particular developmental stage for each day during the entire development. Bars represent 1 cm.