Plant material

Rhizomes of H. flavum (2.01 kg) were collected from Kaili City (107°55′ E, 26°35′ N), Guizhou Province, China, in March 2019. The identification of the collections was verified by Dr. Q.R. Liu (College of Life Science, Beijing Normal University). Voucher specimens (BNU-dushushan-2019-03-03) were deposited in the herbarium of the College of Resources, Science, and Technology (Faculty of Geographical Science, Beijing Normal University).

Insects

Tribolium castaneum and L. serricorne were reared in 250-ml glass containers filled with flour and yeast (10:1, w/w). Liposcelis bostrychophila was reared in a conical flask with a mixture of flour, milk powder, and active yeast (10:1:1, w/w/w). Insects were reared in total darkness in incubators maintained at 30 ± 1°C and 70% ± 10% relative humidity (RH). All bioassays were performed on 2-wk-old adults removed from the colonies.

Essential oil extraction

Rhizomes of H. flavum were crushed and subjected to hydrodistillation for 6 h in a Clevenger-type apparatus to obtain the essential oil. The volume was determined by dehydration with anhydrous sodium sulfate after extraction. The essential oil was stored in an airtight glass vial at 4°C until used.

Gas chromatography and mass spectrometry

Gas chromatography–mass spectrometry (GC-MS) analysis was performed on a Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector (GC-FID) and a capillary column of DB-5MS (30 m × 0.25 mm × 0.25 μm). The injector temperature was maintained at 250°C. The injected volume was 1 μL of 1% solution (diluted in n-hexane). The carrier gas was helium, and the flow rate was 1.0 mL/min. The oven temperature was held at 50°C for 3 min then increased to 290°C at 10°C/min. The vaporizer temperature was 250°C, and the spectral scanning range was 45–650 m/z in full scan mode. GC-MS and GC-FID were performed under the same operating conditions, and the retention indices were calculated with a homologous series of n-alkanes (C₅–C₃₆). The relative percentages of individual compounds were determined by the GC-FID peak area percentage. The mass spectrum and retention index were compared with the mass spectra in NIST 05 (Standard Reference Data, Gaithersburg, MD).

Fumigant bioassay

The fumigant toxicity bioassay with T. castaneum and L. serricorne were conducted following the methods of Liu and Ho (1999), and the methods of Zhao et al. (2012) were used for L. bostrychophila. Preliminary testing established the range of concentrations used against each of the pest insects: 1.93, 2.89, 4.33, 6.5, and 10% for T. castaneum and 1.28, 1.93, 2.89, 4.33, and 6.5% for L. serricorne. Each concentration experiment was replicated five times with n-hexane as the solvent and negative control.

For the bioassays with T. castaneum and L. serricorne, 10 μL of the solution was dropped on a filter paper disk (Whatman, 2 cm in diameter) and allowed to evaporate for 20 s. The treated filter paper was attached to the inside of the screw cap of a 25-ml glass vial (diameter 2.5 cm, height 5.5 cm). Ten adult insects were placed in each glass vial. The screw cap was attached to the vial and sealed with Parafilm® (Amcor, Zurich, Switzerland). The vials were maintained in darkness at 30 ± 1°C and 70 ± 10% RH, and mortality of the target insect was recorded 24 h later.

For L. bostrychophila, 10 adults were placed in a glass vial (8 mL), and the vial was placed in a glass jar (250 mL). The filter paper strip (3.5 cm × 1.5 cm) was attached to the cap of the jar, and 10 μL of the test solution was applied to it. The lid of the jar was tightly screwed down and sealed with Parafilm. Vials and jars were kept at room temperature, and mortality was recorded after 24 h.

Contact toxicity bioassay

The bioassay methods used with T. castaneum and L. serricorne were similar to those of Liu and Ho (1999). Based on preliminary testing, a range of sequential dilutions of bioassay concentrations was established for T. castaneum (1.28, 1.93, 2.98, 4.33, and 6.5%) and L. serricorne (0.89, 1.33, 2.0, 3.0, and 4.5%) using n-hexane as the diluent. For each concentration, a 0.5-μL droplet of solution was placed on the dorsum of the thorax of each of the 10 insects treated with each concentration. Treated insects were transferred individually to glass vials.

The contact toxicity bioassay with L. bostrychophila was conducted as described by Zhao et al. (2012). Preliminary testing established the range of concentrations appropriate for the assay: 0.8, 0.9, 1.56, 2.33, and 3.5%. For each concentration, 300 μL of the solution was applied to a filter paper disk (5.5 cm diameter) and allowed to evaporate for 20 s at room temperature. The filter paper disk was then glued to the bottom of a 5.5-cm petri dish, and 10 L. bostrychophila adults from the laboratory colony were placed inside the dish. To prevent L. bostrychophila from escaping, the inner wall of the dish was coated with Fluon® (AGC Chemicals Co., Tokyo, Japan), and the dish was covered with a glass lid.

For each bioassay, n-hexane was used as the solvent and negative control. Treated insects were maintained in darkness at 30 ± 1°C and 70 ± 10% RH, and mortality was recorded after 24 h of exposure. Each bioassay was conducted five times.

Repellent bioassay

The area preference method of Liu and Ho (1999) was used for the repellent bioassays. For T. castaneum and L. serricorne, the test concentrations of 0.13, 0.63, 3.15, 15.73, and 78.63 nL/cm² were prepared by serial dilutions in n-hexane. Filter paper disks (9 cm in diameter) were cut into two equal halves. One half was treated with 500 μL of the test solution, and the other half was treated with 500 μL of n-hexane as a control. After air drying for 30 s, the treated and control filter paper halves were placed side by side on the bottom of a 9-cm-diameter petri dish. Twenty adult insects were placed in the middle of the dish, which was then covered with its top. The number of insects on each half of the filter paper in each dish was counted at 2 and 4 h after initial exposure.

The same method was used for bioassays with L. bostrychophila; however, some adjustments to the protocol were made. A smaller filter paper disk (5.5 cm in diameter) was used, and the concentrations of the test solutions were 0.10, 0.51, 2.53, 12.63, and 63.17 nL/cm². The filter paper disks were halved, treated with 150 μL of the test solution or n-hexane (control), allowed to air dry for 30 s, and placed in the bottom of petri dishes as previously described. Twenty adult insects were added to the center of the dish, which was then covered. The number of insects on each half of the filter paper was counted after 2 and 4 h. Treatments were conducted five times.

Statistical analysis

In the fumigation toxicity and contact toxicity bioassays, probit analysis was used with the mortality data to calculate median lethal concentration (LC₅₀) (fumigation and contact), median lethal dose (LD₅₀) (contact), the 95% confidence intervals (CIs) for each, and other related parameters. In the repellency bioassays, percent repellency (PR) was calculated with the numbers of adult insects on either the treated or control filter paper using the formula PR = [(Nc − Nt)/(Nc + Nt)] × 100, where Nc is the number of insects in the control group and Nt is the number of insects in the treatment group. PR values were normalized by converting to arcsine and square root values before being subjected to a one-way analysis of variance. Treatment means were subjected to Tukey’s honestly significant difference test, with statistical significance set at P < 0.05. All data were processed by SPSS 20.0 (IBM, Armonk, NY).

Chemical composition of essential oil

The yield of essential oil obtained from H. flavum rhizomes by hydrodistillation was 0.17% (v/w) each. The oil was pale yellow with a density of 0.89 g/mL. Twenty-four compounds were identified by GC-MS, accounting for 90.52% of the essential oil extracted. These compounds were grouped into five classes: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and others (Fig. 1). The major components were β-pinene (33.52%), linalool (15.56%), and 1,8-cineole (11.20%) (Table 1). Terpenes were the most abundant compounds, among which monoterpenes (43.41%) and sesquiterpenes (37.25%) accounted for a large proportion and had a strong and unique aroma.

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Table 1. Chemical composition of the essential oil from H. flavum rhizome.

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These results are similar to those of Fan et al. (2017) in their analysis of the chemical composition of four Hedychium species collected from Guangdong Province, China. Their analysis revealed that the major components of the essential oil from H. flavum flower were linalool, 1,8-cineole, (E)-ocimenone, and α-farnesene, in agreement with our results. Fan et al. (2017) further speculated that based on the compounds in the essential oils and the intensity of their odor, the aromatic intensity of Hedychium species may be closely tied to volatile terpene compounds. The aroma intensity of the essential oil of H. flavum rhizomes may be associated with linalool and 1,8-cineole.

In contrast to those findings, Tian et al. (2020) identified 77 components collected from H. flavum in Guangxi Province, China. Of those compounds, the dominant ones were coronation E (20.3%), β-pinene (16.8%), (E)-nerolidol (11.8%), and linalool (8.5%); coronation E was identified for the first time. Tian et al. (2023) found 55 volatile compounds in an essential oil extracted from H. flavum flowers collected from Nayong, Bijie, Guizhou Province, China. The predominant constituents were β-pinene (20.2%), α-pinene (9.3%), and α-phellandrene (8.3%). Hedychium flavum collected from Vietnam yielded 49 volatile compounds, which accounted for 99.3% of the total constituents of the essential oil. The predominant constituent was α-pinene (21.8%) (Thanh et al. 2014).

The number of compounds we identified from H. flavum rhizomes was lower than the numbers reported by Tian et al. (2020, 2023) and Thanh et al. (2014), and the primary compounds differed among these studies. These differences may be linked to climate and environmental conditions at the collection sites (Figueiredo et al. 2008). Rawat et al. (2020) also found that the percentages of β-pinene and 1,8-cineole were negatively correlated with elevation and, thus, another possible contributing factor in the observed differences. The percentage of 1,8-cineole during the rainy season and the summer was higher than that in winter (Bhardwaj et al. 2019). Prinsloo and Nogemane (2018) also noted that chemical composition and physical and chemical properties of the essential oil may differ among the seasons. Given that the chemical composition of the essential oils changes with environmental conditions, geographic location, habitats, plant growth stage, growing season, and extraction methods, resulting changes could impact bioactivity (Figueiredo et al. 2008). The composition of H. flavum essential oils is especially variable, and choice of optimal planting time, geographic location, and harvest time can significantly improve the yield of bioactive essential oils, thus enhancing the potential for the development of related industries.

Fumigant toxicity

The fumigant toxicity of the essential oil from H. flavum rhizomes and its three primary chemical constituents against T. castaneum, L. serricorne, and L. bostrychophila was determined by concentration–mortality response bioassays. By using nonoverlapping 95% CIs as indicators of statistical significance, we determined that the fumigant activity of 1,8-cineole was significantly superior to that of the other three solutions against T. castaneum (Table 2). Linalool was superior to β-pinene and the H. flavum essential oil, and these were statistically equal in their fumigant toxicity. Liu and Ho (1999) reported an LC₅₀ of 1.8 mg/L air for methyl bromide against T. castaneum.

Table 2. Fumigant toxicity of essential oil from H. flavum (EOHFR) and its major constituents against T. castaneum (TC), L. serricorne (LS), and L. bostrychophila (LB) at 24 h.

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For L. serricorne, the fumigant toxicity of the four test solutions was 1,8-cineole > linalool = H. flavum essential oil > β-pinene (Table 2). Phosphine had an LC₅₀ of 9.2 × 10⁻³ mg/L air for L. serricorne (Yang et al. 2014). We also determined that 1,8-cineole was superior to linalool as a fumigant against L. bostrychophila; however, we were unable to calculate the LC₅₀ values of β-pinene or the H. flavum essential oil because 50% concentrations of each did not cause 100% mortality of L. bostrychophila (Table 2). Liu et al. (2013) obtained an LC₅₀ of 1.4 × 10⁻³ mg/L air for dichlorvos against L. bostrychophila.

Although we did not find significant differences among the three insect species in their responses to the chemicals, 1,8-cineole, the most abundant compound in H. flavum essential oil, had effective fumigant activity against the three insect pests (Table 2). Our finding support those of similar studies; 1,8-cineole, as a major component of a variety of plants such as Stachys riederi Chamisso, Eucalyptus globulus Labill, and Zanthoxylum armatum de Candolle, exhibited excellent fumigant toxicity against L. bostrychophila, Oryzaephilus surinamensis L., and T. castaneum. The toxic effect of 1,8-cineole also was comparable to that of synthetic insecticides (Lee et al. 2000, Quan et al. 2018, Wang et al. 2015). Therefore, this compound might play a critical role in the fumigant bioactivity of the essential oil.

Mortality responses to the other three compounds varied among the three pest species. In other studies, fumigant toxicity responses to essential oils also were highly variable among coleopteran stored product pests (Feng et al. 2023, Li et al. 2023). Our results are similar to those of Cao et al. (2018), who found that linalool and β-pinene were toxic for insects, but the same concentration of essential oil did not have insecticidal activity. One postulation is that antagonistic interactions occur among the components of essential oils, thus neutralizing at least a portion of the fumigant toxicity (Ukeh and Urnoetok 2011). Monoterpenes are volatile compounds with the potential to be used against insects in fumigants products, so the total monoterpene concentrations of essential oils can affect their insecticidal activities (Lee et al. 2003).

We also observed that dead insects fumigated with the essential oil often appeared black and had their wings spread. This response may be due to the action of small monoterpene molecules, indicating that the essential oil and its components may have neurotoxic effects on target insects. Subsequent studies could include assessment of the bioactivity of combinations of components against target insects to explore optimal usage.

Contact toxicity

The contact toxicity of the four test substances was determined by the dose–mortality responses of T. castaneum and L. serricorne and the concentration–mortality responses of L. bostrychophila. The significance of differences among the LD₅₀ or LC₅₀ values of the four substances for each of the three species was determined by nonoverlap of the 95% CIs. The relative contact toxicity of the substances against T. castaneum was 1,8-cineole = β-pinene = H. flavum essential oil > linalool (Table 3). No significant differences were detected among the LD₅₀ values of the four substances against L. serricorne.Yang et al. (2014) determined the contact toxicity of pyrethrins against T. castaneum and L. serricorne adults, with LD₅₀ values of 0.3 and 0.2 µg/insect, respectively.

Table 3. Contact toxicity of essential oil from H. flavum (EOHFR) and its major constituents against T. castaneum (TC), L. serricorne (LS), and L. bostrychophila (LB) at 24 h.

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The LC₅₀ values of the four substances against L. bostrychophila indicated that the H. flavum essential oil was more effective than the three main constituents of the essential oil alone, with relative responses of H. flavum essential oil > linalool = 1,8-cineole > β-pinene (Table 3). Yang et al. (2014) also reported an LC₅₀ of 18.7 µg/cm² for pyrethrins against L. bostrychophila adults.

Although the toxicity of the essential oil extracted from the rhizomes of H. flavum was lower than that of the positive controls, this essential oil appears to have greater contact toxicity against T. castaneum and L. serricorne than do essential oils extracted from other plants in the family Zingiberaceae. Chen et al. (2018) reported that the essential oil from Alpinia katsumadai Hayata seed had contact toxicity against T. castaneum and L. serricorne with LD₅₀ values of 52.6 and 17.4 μg/adult, respectively. Zingiber zerumbet Smith essential oil had contact toxicity against L. serricorne with an LD₅₀ value of 48.3 μg/adult (Wu et al. 2017). Our results clearly indicate that the toxicity response of L. bostrychophila to the combination of the components exceeded that of the components used individually. The synergistic or additive toxicity of monoterpene mixtures to insects was manifested in increased epidermal permeability, and essential oils may have a more significant neurosuppressive effect than do synthetic insecticides. Therefore, the essential oil of H. flavum may be useful as a plant insecticide.

López and Pascual-Villalobos (2010) also noted that monoterpenes, especially linalool that we identified in the H. flavum essential oil, have antiacetylcholinesterase properties, accounting for the high mortality in treated stored product insects. In our contact toxicity assay, we also observed insect behaviors, such as tremors, agitation, and the spreading of wings, that are indicative of exposure to neurotoxins. These behaviors were also reported by Kostromytska et al. (2011). We speculate that the mode of action of certain essential oils against insects may be neurotoxic. The synergistic or additive effects of multiple plant secondary metabolites are undoubtedly one of the most effective defense systems against stored product insects.

Repellent activity

Various concentration-dependent and time-dependent responses were observed in the repellency of the four test substances against the three stored product insect pests (Fig. 2). As concentration decreased, the repellent effect also decreased and reached the lowest activity at the lower concentrations tested. β-Pinene, linalool, and 1,8-cineole, as the major components of H. flavum essential oil, had some repellent effects on the three stored product insects, similar to previous reports (Cao et al. 2018, You et al. 2015, Zhang et al. 2015). Of the substances tested, H. flavum essential oil was most consistent in repelling the three insect species. This effect may be related to the synergistic or additive effect of the compounds in the oil. For example, Chen et al. (2021) confirmed the synergistic repellent effect of four individual components: α-thujone, (+)-camphor, 1,8-cineole, and α-caryophyllene. This synergism can enhance the bioactivity of mixtures of chemicals, such as seen with essential oils. Synergism has also been reported for the repellency of mixtures of essential oils. Liu et al. (2006) reported synergism in the repellency of the mixture of essential oils from Artemisia princeps Pampanini and Cinnamomum camphora (L.) against Sitophillus oryzae L. and Bruchus rugimanus Boheman, which was significantly higher than the repellency of the individual oils.

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Essential oils and their volatile components commonly act in a vapor phase, which imparts a repellent action and drives pests away from the odorous source. However, this repellent barrier diminishes relatively quickly, thus limiting the effectiveness of these repellents. The repellent activity of essential oils, especially its duration, is highly dependent on the chemical content of the essential oil (Zhu et al. 2001). More research is needed to understand the mechanism underlying the interactions among the components of an essential oil and how they enhance its repellent properties. Although the positive control (N,N-diethyl-meta-toluamide) in this assay was more repellent against these three species of stored product insects, concerns about environmental contamination and human safety regarding commercial pesticides may serve as impetus for continued development of essential oils for the management of insect pests.

Concluding remarks

The essential oil extracted from the rhizomes of H. flavum and the three major components of this oil (β-pinene, linalool, and 1,8-cineole) had significant fumigant, contact toxicity, and repellent activity against T. castaneum, L. serricorne, and L. bostrychophila. The lethal toxicity of this essential oil and its three main components suggests that they have potential for development in the management of stored product insect pests. Although the essential oil and its terpenoids individually had relatively effective fumigant, contact toxicity, and repellent activity against the three insects, the modes of action should be studied to determine the best mechanisms to enhance the effectiveness of the essential oil, including exploring possible synergistic mixtures.