Introduction
Insect pests are a major threat to global agriculture, causing significant crop losses and impacting food security. As the global population continues to grow, the demand for agricultural products intensifies, making it essential to adopt effective pest control strategies (Dhaliwal et al., Reference Dhaliwal, Jindal and Mohindru2015; Ali et al., Reference Ali, Abdellah and Eletmany2023c). Insect pests affect a wide range of crops, including vegetables, cereals, and fruits, often resulting in reduced yield, lower quality, and higher production costs (Dhaliwal et al., Reference Dhaliwal, Jindal and Mohindru2015). Among the numerous pest species, aphids, whiteflies, and caterpillars are particularly notorious for their ability to cause direct damage by feeding on plant tissues and indirectly by transmitting plant viruses (van Emden and Harrington, Reference van Emden, Harrington, van Emden and Harrington2007; Yadav and Rathee, Reference Yadav, Rathee and Omkar2020; Puzanova et al., Reference Puzanova, Novikov, Bilyk, Sharmagiy, Plugatar and Oberemok2023). The reliance on chemical insecticides to control these pests has led to the emergence of pesticide resistance, environmental contamination, and negative impacts on non-target organisms, underscoring the need for alternative, sustainable pest management strategies (Bass et al., Reference Bass, Puinean, Zimmer, Denholm, Field, Foster, Gutbrod, Nauen, Slater and Williamson2014; Bras et al., Reference Bras, Roy, Heckel, Anderson and Karlsson Green2022).
Brassica crops are some of the most economically significant crops worldwide (Ahuja et al., Reference Ahuja, Rohloff and Bones2010; Rai et al., Reference Rai, Yadav, Kumar, Sharma, Kumar, Rai, Chittaranjan and Trilochan2022). These crops are essential for human nutrition, as they provide valuable sources of vitamins, minerals, and dietary fibre (Rai et al., Reference Rai, Yadav, Kumar, Sharma, Kumar, Rai, Chittaranjan and Trilochan2022). Additionally, Brassica crops, such as canola and rapeseed, are crucial for oil production, contributing to global food and industrial markets (Rakow Reference Rakow, Eng-Chong and Carl2004). Despite their economic importance, these crops are highly susceptible to a range of pests, including aphids, caterpillars, and flea beetles, which can lead to substantial yield losses and reduced quality (Imran, Reference Imran2018; Ali et al., Reference Ali, Tonğa, Islam, Mir, Mukarram, Konôpková and Chen2024b). Among these pests, aphids, particularly Myzus persicae, have been identified as one of the most damaging due to their capacity to transmit plant viruses and their tendency to infest the tender tissues of Brassica plants, impacting both crop health and overall productivity (Yang et al., Reference Yang, Ali, Ahmad, Yang, Huang, Alam, Khan, Ghramh, Rahman, Tonğa and Zhao2024).
M. persicae Sulzer (Hemiptera: Aphididae), commonly known as the peach-potato aphid, is a highly polyphagous pest that affects a wide range of plants, including many economically important crops such as Brassica species (Ali, Reference Ali2023; Van Emden et al., Reference Van Emden, Eastop, Hughes and Way1969). M. persicae feeds on plant sap, weakening plants, stunting growth, and causing the yellowing of leaves, known as chlorosis. This aphid species is particularly problematic in Brassica crops because of its ability to transmit over 100 plant viruses, including the Cucumber mosaic virus and Turnip yellows virus, further exacerbating crop losses (Singh and Joshi, Reference Singh and Joshi2020; Ali et al., Reference Ali, Bayram, Mukarram, Zhou, Karim, Hafez, Mahamood, Yusuf, King and Adil2023a). Due to its reproductive capacity and rapid population growth, M. persicae can quickly increase in number, making control efforts more challenging (Jeon et al., Reference Jeon, Kang, Kim, Yang and Kim2008; Drizou et al., Reference Drizou, Bruce, Ray and Graham2018). The use of synthetic chemical insecticides has been the traditional approach to manage M. persicae, but the development of pesticide resistance and concerns over environmental health have prompted the search for more sustainable control options (Avé et al., Reference Avé, Gregory and Tingey1987; Bass et al., Reference Bass, Puinean, Zimmer, Denholm, Field, Foster, Gutbrod, Nauen, Slater and Williamson2014; Drizou et al., Reference Drizou, Bruce, Ray and Graham2018).
Induced plant defence mechanisms, wherein plants activate their inherent defence systems in response to herbivory or other stressors, have emerged as a promising strategy for enhancing pest resistance (Dicke and Hilker, Reference Dicke and Hilker2003). These defences can involve physical changes such as the production of toxic compounds or alterations in leaf structure, as well as chemical changes, including the release of volatile organic compounds (VOCs) that can attract natural enemies or repel herbivores (Agrawal, Reference Agrawal1999; Ballhorn et al., Reference Ballhorn, Kautz and Schädler2013; Barton, Reference Barton2016; Meena et al., Reference Meena, Yadav, Sonigra, Nagda, Mehta, Swapnil and Marwal2022; Traw and Bergelson, Reference Traw and Bergelson2003). To date, several important plant-derived compounds have been identified, with previous studies showing that exogenous application of plant secondary metabolites, such as jasmonates (e.g. jasmonic acid, cis-Jasmone [CJ], prohydrojasmon, methyl jasmonate) and salicylates (e.g. methyl salicylate [MeSA], salicylic acid), and even plant extracellular DNA (Oberemok et al., Reference Oberemok, Useinov, Skorokhod, N V, Novikov, Makalish, Yatskova, Sharmagiy, Golovkin and Gninenko2022) can affect the viability of insects, modulate insect behaviour and performance (Holopainen et al., Reference Holopainen, Heijari, Nerg, Vuorinen and Kainulainen2009), including deterring feeding and oviposition (Boughton et al., Reference Boughton, Hoover and Felton2006; Quadrel et al., Reference Quadrel, Urbaneja-Bernat, Holdcraft and Rodriguez-Saona2024), representing direct plant defence (Conboy et al., Reference Conboy, McDaniel, George, Ormerod, Edwards, Donohoe, Gatehouse and Tosh2020; Rani and Murali-Baskaran, Reference Rani and Murali-Baskaran2025). Additionally, these compounds induce indirect plant defence by promoting the release of an induced volatile blend, which enhances the recruitment of biological control agents (Ali et al., 2024). This study aims to explore whether methyl chavicol (MC) can enhance the natural defence mechanisms of Brassica plants against M. persicae, thus providing an eco-friendly and sustainable alternative to chemical pesticides.
MC, a phenylpropanoid found in several plant species, has been shown to exhibit insecticidal properties (Tian and Dang, Reference Tian and Dang2021). While its insecticidal properties have been demonstrated against various pests, including Spodoptera frugiperda (De Menezes et al., Reference De Menezes, Carvalho, Alves, De Carvalho, Aazza, de Oliveira Ramos, Jebp and Bertolucci2020), Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae (Chang et al., Reference Ling Chang, Kyu Cho and Li2009), its role in inducing plant defence remains unexplored. Specifically, the potential of exogenous MC application in Brassica crops to enhance resistance against M. persicae and its natural enemy, the parasitoid wasp A. gifuensis, has not yet been investigated. Therefore, the main objective of this study is to investigate the effects of exogenous MC application on Brassica plants on the performance (survival and fecundity) and behaviour of M. persicae, and the behavioural responses of its natural enemy A. gifuensis. We hypothesise that MC application will: (1) suppress M. persicae performance and alter its behaviour, while (2) enhance the behavioural responses and parasitism capacity of A. gifuensis, making it a promising tool for integrated pest management (IPM) strategies. This study is both timely and essential for advancing sustainable pest management in Brassica crops, where balancing aphid control with the conservation of natural enemies is critical. By examining the effects of the exogenous application of MC on plants and assessing the performance and behaviour of M. persicae and A. gifuensis on treated and untreated plants, this study will provide valuable insights into how plant-mediated defence inducers influence pest resistance and biological control interactions. The findings could contribute to the development of more sustainable pest management strategies, reducing reliance on chemical pesticides while enhancing plant resilience and overall crop health.
Materials and method
Insect
An initial colony of aphids, Myzus persicae Sulzer (Hemiptera: Aphididae), was obtained from the company Qu Shou Natural (Nanyang, China). The colony was subsequently maintained in the laboratory on potted B. rapa plants within insect-rearing cages (60 × 60 × 60 cm) under controlled environmental conditions (22 ± 5°C, 45 ± 5% RH, photoperiod 16L:8D). To ensure a healthy and vigorous aphid population, fresh, B. rapa plants were provided regularly, and heavily infested or senescent plants were promptly replaced. The colony was maintained for several generations before experiments were initiated, and care was taken to avoid overcrowding and ensure consistent food quality throughout the rearing period. The same rearing protocol was used in previous studies (Ji et al., Reference Ji, Ali, Feng, Zhao, Liu, Khan, Ghramh, Tonğa, Li and Chen2025; Yang et al., Reference Yang, Ali, Ahmad, Yang, Huang, Alam, Khan, Ghramh, Rahman, Tonğa and Zhao2024). An initial colony of the parasitoid A. gifuensis Ashmead (Hymenoptera: Braconidae) was sourced from aphid mummies purchased from Better Life Co. Ltd., Yunnan, China. The parasitoids were reared on B. rapa plants infested with M. persicae under controlled environmental conditions (20°C, 45 ± 5% RH, photoperiod 16L:8D). After emergence, parasitoids were provided with a 1:1 honey–water solution as a food source. To maintain colony health and performance, new M. persicae-infested plants were introduced weekly, and old plants were removed to ensure optimal host quality and availability. Female parasitoids (three- to five-day-old, mated and naïve) were used in all behavioural experiments. The same rearing protocol was used in previous studies (Ahmad et al., Reference Ahmad, Ali, Alam, Abbas, Huang, Zhao, Hamza, Ali, Khan and Ghramh2025; Ji et al., Reference Ji, Ali, Feng, Zhao, Liu, Khan, Ghramh, Tonğa, Li and Chen2025).
Plant
Brassica rapa plants were grown from seeds purchased from Hebei Dayu Seeds Company, Cangzhou, China. Plants were grown in a controlled environment room (20 ± 5°C, 45 ± 5% RH, photoperiod 16L:8D). Plants were individually cultivated in 14 cm pots filled with soil (Jiangsu Siweibo Biotechnology Co., Ltd. China). Experiments were conducted using four-week-old plants (BBCH 14 growth stage).
Treatment
Plants were sprayed with an aqueous emulsion of MC (Xinhua Biological Products, Beijing, China). A stock solution of MC was prepared at 100 mg/L, which was selected based on preliminary testing that showed no significant effects at lower concentrations (25, 50, and 75 mg/L). Tween-80, a surfactant, was added to the solution at 0.05% (v/v) to help dissolve MC and promote even distribution and adherence on the leaf surface. Control plants were treated with deionised water containing the same concentration of Tween-80 (Xinhua Biological Products, Beijing, China). The effects of MC were tested by spraying the plants using an Oshide spray bottle (100 mL; Zhengzhou Xinrui Tongda Metal and Material Co., Ltd., Zhengzhou, China). For each treatment, three trigger pulls of the spray formulation (∼250 μL) were applied to each plant from a distance of 30 cm (fig. 1.1) (Bruce et al., Reference Bruce, Martin, Pickett, Pye, Smart and Wadhams2003; Ali et al., Reference Ali, Covaci, Roberts, Sobhy, Kirk and Bruce2021, Reference Ali, Wei, Mahamood, Zhou, King, Zhou and Shamsi2023b). To ensure even coverage, plants were rotated during spraying. After spraying, plants were left for 24 h before being used in the experiments. To prevent plant–plant interactions, control and MC-treated plants were placed in separate compartments. All experiments were conducted under controlled environmental conditions (25°C, 50 ± 10 % RH, 16 L: 8 D).
Experimental set-up for assessing the effects of methyl chavicol (MC) on aphid behaviour and plant-insect interactions. (1) Exogenous application of MC to Brassica plants using a hand sprayer. (2) Clip cage bioassay for evaluating aphid survival and fecundity. (3) Settlement bioassay where treated (MC) and control (C) plants were placed in a vial with 50 winged aphids positioned centrally in the cage. (4) Y-tube olfactometer set-up for testing olfactory responses of aphids and parasitoids to plant volatiles. (5) Parasitoid foraging bioassay in a non-choice arena to record time spent by parasitoids on MC-treated and control plants. (6) Parasitism bioassay to assess parasitoid oviposition behaviour. (7) Plant traits assessment including measurements of plant height, leaf length and width, root length, and fresh biomass in both MC-treated and control Brassica rapa plants.
Aphid performance bioassay
The performance of M. persicae was evaluated on control and MC-treated Brassica plants using clip-cages (25 mm diameter) (as shown in fig. 1.2). The experiment was conducted 24 h post-treatment to assess aphid survival and fecundity. In each replicate, two clip cages were attached to the abaxial side of the second and third oldest leaves of a plant. Each clip cage contained ten adult wingless aphids. The plants were maintained in a controlled environment room (25 ± 5°C, 45 ± 10% RH, 16 L: 8 D) for 48 h. After this period, the leaves with clip cages were cut, and the cages were carefully removed to record the number of live adults and nymphs produced. A total of twenty replicates were conducted for each treatment (MC and control plants).
Aphid settlement bioassay
The aphid settlement procedure was adopted with slight modifications from Ali et al. (Reference Ali, Covaci, Roberts, Sobhy, Kirk and Bruce2021). In this choice test bioassay, MC-treated and control plants were placed in an insect cage (60 × 60 × 60 cm Kaipiao, Taobao) and were kept in a controlled environment room (20 ± 5°C, 50 ± 10% RH, 16 L : 8D). Each cage contained four plants (two MC-treated and two control) positioned alternately (as shown in fig. 1.3). A plastic vial (2 cm in diameter × 5 cm in height) containing 50 adult alate aphids was placed in the centre of the cage, opened and then the cage was left undisturbed for 24 h. The number of settled aphids on each plant was recorded after 24 h for assessment. A total of fifteen replicates were conducted.
Olfactometer bioassay
To assess the behavioural responses of M. persicae and A. gifuensis towards MC-treated and control plants, a Y-tube olfactometer was used. The olfactometer consisted of a Y-shaped glass tube with a 10 cm stem and 15 cm arms, each with a 2 cm internal diameter, allowing insects to move toward odours from the upwind end. Air was pumped through an activated charcoal filter before being split between the two arms of the olfactometer. A flow meter regulated the airflow in each arm at 200 mL/min (Yang et al., 2024). Two glass jars (30 × 20 cm) with two openings were used: one for releasing air from the charcoal filter into the jar and another for connecting to the Y-tube arms (as shown in fig. 1.4). Glass jars containing either MC-treated or control plants were connected to the Y-tube with PTFE tubing to release plant volatiles into the olfactometer, allowing insect responses to be recorded. Insects were collected 2 h before the start of the experiment. Each insect was given ten minutes to make a choice. If an insect moved at least 1/3 of the distance down an arm, it was considered to have made a choice and was included in the analysis. If an insect did not cover that distance, it was deemed non-responsive and excluded from the analysis. The Y-tube olfactometer and the glass jars were cleaned thoroughly with 70% ethanol and allowed to air dry between each combination. To eliminate directional bias, plant treatment positions were alternated after every five releases, and fresh plants were used for each subsequent set of releases. Additionally, the set-up’s bias was tested by comparing blank air (i.e. no odour) against blank air, ensuring that any observed preference was not due to equipment bias. All bioassays were conducted under controlled laboratory conditions (20 ± 5°C, 50 ± 10% RH, 16 L : 8D) between 10:00 and 16:00 h. Thirty replicates were conducted for each treatment.
Parasitoid foraging bioassay
To assess the behavioural response of the parasitoid A. gifuensis towards MC-treated and control plants, a foraging behavioural bioassay was conducted. A single female parasitoid was released from a vial onto a leaf of a plant placed in an open-fronted cage (40 cm × 40 cm), as shown in fig. 1.5. The total time spent by the parasitoid on treated and untreated plants was recorded. The observation was terminated when the parasitoid flew away from the plant or after 60 min. Fifteen replicates were performed for each treatment. To avoid any bias, treated and control replicates were observed alternately. All experiments were conducted between 10:00 a.m. and 02:00 p.m.
Parasitoid performance
The effect of MC treatment of B. rapa on the parasitism rate of A. gifuensis was evaluated using an experimental set-up (as shown in fig. 1.6). Two separate plastic containers (20 cm in height × 15 cm in width), each containing a plant, were connected by a glass tube (5 cm internal diameter, 20 cm long) with a middle opening for parasitoid release, which was sealed with cotton after the release of the parasitoids. Each plant was infested with 50 adult M. persicae, introduced 2 h prior to parasitoid release. A total of 4 naïve female A. gifuensis (2–3 days old) were released at the midpoint of the connecting glass tube, allowing them to choose between the treated and control plants (Ali et al., Reference Ali, Bayram, Mukarram, Zhou, Karim, Hafez, Mahamood, Yusuf, King and Adil2023a). After 24 h, the parasitoids were removed, and the plants were enclosed in ventilated bags to allow for the development of aphid mummies. The experiment was conducted under controlled environmental conditions (20 ± 5°C, 45% RH, 16L:8D). The number of mummified aphids on treated and control plants was recorded after 12 days. In total, twenty replicates were conducted.
Plant traits
Plant growth parameters were evaluated using 4-week-old B. rapa plants grown under controlled environmental conditions (25°C, 50 ± 10% RH, 16L:8D photoperiod). Plants were sprayed either with MC or with deionised water containing 0.05% Tween-80 as a control. Observations were recorded before spraying (initial) and 7 days after spraying (final), wherever applicable. Each treatment included ten replicates.
Plant height
Plant height was measured as the vertical distance in centimetres from the soil surface to the tip of the longest leaf using a calibrated ruler (fig. 1.7A). Measurements were taken before treatment (initial height) and 7 days after treatment (final height). Ten replicates were recorded for both control and MC-treated plants.
Leaf dimensions
Leaf length was measured from the base of the petiole to the leaf tip, and leaf width was recorded as the maximum lateral span perpendicular to the midrib (fig. 1.7B). Both parameters were measured in centimetres using a ruler. Measurements were taken before treatment (initial) and 7 days after treatment (final). Leaf length and width were analysed separately. Ten replicates were recorded for both control and MC-treated plants.
Root length
After harvest, roots were gently washed to remove soil residues, blotted dry, and measured linearly from the root-shoot junction to the tip of the longest intact root using a ruler (fig. 1.7C). Measurements were taken 7 days after treatment. Ten replicates were performed for both control and MC-treated plants.
Fresh biomass
Whole plants (excluding roots) were carefully removed and immediately weighed using a precision balance (fig. 1.7D). Shoots were gently dried to remove excess surface moisture before weighing. Fresh biomass was recorded in milligrams. Measurements were taken at the final time point, 7 days after treatment. Ten replicates were conducted for both control and MC-treated plants.
Statistical analyses
The count and continuous data were modelled using a generalised linear model (GLM) with Poisson and Gaussian distribution, respectively, to avoid non-normal distributions (Dunn and Smyth, Reference Dunn and Smyth2018), while the data regarding choice tests in olfactometer equipment were analysed using Chi-square test. The statistics of GLMs were derived using Anova function of car package. The foraging duration of the parasitoid was modelled using Kaplan–Meier estimators and subjected to a Log-rank test for treatment effect since survival analysis is the best choice to model the datasets containing censored data points. The survival analysis was performed employing survival and survminer packages (Therneau, Reference Therneau2021; Kassambara et al., Reference Kassambara, Kosinski, Biecek and Fabian2022). Figures were generated using the ggplot2 package with support from dplyr, ggsignif, and ggpubr packages (Wickham, Reference Wickham2016). RStudio (v 2024.09.01) was used as the integrated development environment of R (v4.4.2) (R Development Core Team, 2024).
Results
Aphid performance and behaviour
The number of M. persicae survived on plants treated with MC did not differ from control plants (ꭓ 21 = 0.30, P = 0.584, fig. 2A). In contrast, the fecundity of aphids on the host plants treated with MC was significantly higher than those maintained on control plants (ꭓ 21 = 74.03, P < 0.001, fig. 2B). Interestingly, the number of aphids settled on both control and MC (ꭓ 21 = 0.59, P = 0.44) and those made choice between olfactometer arms loaded with control and MC treated host plant volatiles did not differ (ꭓ 2 = 0.06, P = 0.796, fig. 2C and D).
The number of surviving adults (A), fecundity (B), settlement (C) on host plants treated with methyl chavicol compared to control plants along with choices (D) over olfactometer arms loaded with volatiles from host plant treated with methyl chavicol and control plants. Ns represents non-significant results while triple asterisks indicate a significant treatment effect (P < 0.001).
Parasitoid foraging and parasitism capacity
The foraging duration of the A. gifuensis on host plants treated as control or with MC did not differ in our survival analysis that took the time to leave the host into accounts as the event (ꭓ 21 = 1.50, P = 0.22, fig. 3A). However, the parasitism performance of the parasitoid on host plants treated with MC significantly differed from those maintained on control plants (ꭓ 21 = 29.08, P < 0.001, fig. 3B). Similarly, the parasitoid exhibited a clear preference toward olfactometer arm loaded with volatiles of host plants treated with MC over control arms (ꭓ 2 = 6.53, P = 0.011, fig. 3C).
The survival probability of time to leave the host plant for parasitoid when tested on host plants treated with methyl chavicol compared to control plants (A) along with choices (D) over olfactometer arms loaded with volatiles from host plant treated with methyl chavicol and control plants. The significance value of long rank test is provided as P = 0.22 on panel A while double and triple asterisks on panel C and B, respectively demonstrate the level of significance of generalised linear model and chi-square test, respective (*** = P < 0.001, ** = P < 0.01).
Plant traits
Plant height and leaf dimension
Neither plant height (χ2df=1 = 0.05, P = 0.825) nor leaf length (χ2df=1 = 1.40, P = 0.236) and width (χ2df=1 = 0.07, P = 0.787) were significantly different before treatments (fig. 4A, B, C, respectively), while MC significantly reduced all traits (χ2df=1 = 4.75, P = 0.029; χ2df=1 = 31.66, P < 0.001; χ2df=1 = 9.15, P = 0.002, respectively) (fig. 4A′, B′, C′).
Mean (±SEM) plant height (A and A′) along with leaf length (B and B′) and width (C and C′) of Brassica rapa after methyl chavicol treatments. The asterisks on each panel indicate the significance level as *** = P < 0.001, ** = P < 0.01, and * = P < 0.05 while ns means there was not a significant treatment effect.
Plant root length
Post treatment root length of B. rapa plant was significantly reduced by MC (χ2df=1 = 6.95, P = 0.008, fig. 5).
Mean (±SEM) root length of Brassica rapa plant after methyl chavicol treatments. The asterisks on the panel indicate the significance level as ** = P < 0.01.
Plant fresh biomass
Fresh biomass of B. rapa plant was significantly reduced by MC treatment (χ2df=1 = 5.81, P = 0.016, fig. 6).
Mean (±SEM) fresh biomass of Brassica rapa plant after methyl chavicol treatments. The asterisks on the panel indicate the significance level as * = P < 0.05.
Discussion
The results of this study provide partial support for our hypothesis, which proposed that MC would reduce M. persicae performance and behaviour while enhancing the behavioural responses of the parasitoid A. gifuensis. While MC did not affect aphid survival, it significantly increased their fecundity. On the other hand, MC enhanced the parasitism capacity of A. gifuensis and attracted the parasitoid to MC-treated plants, supporting the idea that MC can influence both aphid and parasitoid behaviour, albeit in different ways.
In aphid performance bioassay, number of aphids surviving on MC-treated plants did not differ from control plants, suggesting that MC does not directly affect aphid mortality. This finding contrasts with the insecticidal effects often seen with other plant-derived compounds, such as those found in some jasmonates and salicylates (Erbilgin et al., Reference Erbilgin, Krokene, Christiansen, Zeneli and Gershenzon2006; Holopainen et al., Reference Holopainen, Heijari, Nerg, Vuorinen and Kainulainen2009; Ali et al., Reference Ali, Mukarram, Ojo, Dawam, Riyazuddin, Ghramh, Khan, Chen, Kurjak and Bayram2024a). However, the increased fecundity observed on MC-treated plants is an interesting result, which contradicts with the previous role of MC as insecticidal properties (Chang et al., 2009; De Menezes et al., Reference De Menezes, Carvalho, Alves, De Carvalho, Aazza, de Oliveira Ramos, Jebp and Bertolucci2020; Chan et al., Reference Chan, Ho and Sit2022). For instance, direct exposure to MC caused 100% mortality in S. frugiperda within 24 h (De Menezes et al., Reference De Menezes, Carvalho, Alves, De Carvalho, Aazza, de Oliveira Ramos, Jebp and Bertolucci2020). Similarly, López et al., (Reference López, Jordán and Pascual-Villalobos2008) demonstrated that the toxicity of essential oils depended on their chemical composition, with MC-rich fractions exhibiting insecticidal activity against stored grain pests. Furthermore, Chang et al., (2009) reported that MC exhibited rapid insecticidal activity against C. capitata, B. dorsalis, and B. cucurbitae, demonstrating a steep dose–response relationship. One potential explanation for this enhanced aphid reproductive performance is that, in the current study, MC was applied to plants 24 h before aphid exposure, potentially leading to an indirect effect rather than direct toxicity. Instead of acting as a direct insecticidal agent, MC may have induced physiological changes in the plant that altered its defensive responses. This could have resulted in a reduction of plant defensive compounds or an increase in nutritional quality, ultimately favouring aphid reproduction. Such changes align with the concept that exogenous influences can alter plant secondary metabolite levels, thereby affecting plant–herbivore interactions (Pavarini et al., Reference Pavarini, Pavarini, Niehues and Lopes2012). The absence of significant effects on aphid settlement and response to volatiles suggests that MCs influence on aphid performance was not mediated through changes in host plant volatile cues. Instead, it is more likely that MC modified the plant’s metabolic state in a way that benefited aphid development and fecundity.
Our hypothesis that MC would influence parasitoid behaviour was more strongly supported, as parasitism capacity was significantly greater on MC-treated plants, suggesting that MC treatment makes the plant more favourable for parasitoids. Additionally, the parasitoid’s preference for the olfactometer arm connected with glass jar providing volatiles from MC-treated plants further supports the idea that MC might induce plant volatiles that attract parasitoid. Given that plants were treated with MC 24 h before exposure to insects and there was no direct contact between the parasitoid and the applied MC solution, the observed behavioural responses (preference and enhanced parasitism) are unlikely to be due to the parasitoid directly perceiving residual MC itself. MC is volatile in nature (Tian and Dang, Reference Tian and Dang2021), and this 24 h period likely allowed for significant dissipation of any unabsorbed or unmetabolised compound from the leaf surface. Therefore, the enhanced attraction and parasitism performance of A. gifuensis are more plausibly attributed to secondary plant metabolites or VOCs induced or modulated within the plant by the MC treatment. These findings align with previous studies demonstrating that plant volatiles play a crucial role in mediating tritrophic interactions, and that induced plant defences can enhance the recruitment of pests natural enemies (Heil, Reference Heil2008; Turlings and Erb, Reference Turlings and Erb2018). For instance, the exogenous application of plant-derived elicitors, such as methyl jasmonate on cotton, enhances plant defence by increasing volatile emissions and extrafloral nectar production, which attract natural enemies and influence tritrophic interactions (Williams and Rodriguez-Saona, Reference Williams, Rodriguez-Saona and Castle del Conte2017; Munawar et al., Reference Munawar, Xu, Abou El-Ela, Zhang, Zhong, Mao, Chen, Guo, Zhang and Sun2023). Similarly, wheat treatment with the plant-derived compound MeSA enhances the recruitment of A. gifuensis by inducing volatile emissions (Xiao et al., Reference Xiao, Liu, Liu, Wang, Zhan and Liu2022). Furthermore, the exogenous application of CJ has been shown to enhance plant defence by attracting and improving the performance of natural enemies across various crops, including Diaeretiella rapae in Brassica (Ali et al., Reference Ali, Covaci, Roberts, Sobhy, Kirk and Bruce2021), scelionid parasitoids in soybean (Vieira et al., Reference Vieira, Moraes, Borges, Sujii and Laumann2013), and A. gifuensis in potato (Khurshid et al., Reference Khurshid, Inayat, Basit, Mobarak and Liu2024).
The results suggest that MC has contrasting effects on M. persicae and A. gifuensis. While MC increases aphid fecundity, it simultaneously enhances the parasitism behaviour of parasitoid. One possible explanation for the increase in aphid fecundity is that MC may modulate or alter plant defences, thereby creating a more favourable environment for aphid reproduction. This aligns with the concept of modifying plant defence responses upon the application of plant natural compounds (Pavarini et al., Reference Pavarini, Pavarini, Niehues and Lopes2012; Nishad et al., Reference Nishad, Ahmed, Rahman and Kareem2020). This could be beneficial for the aphid population, but the enhanced parasitism observed suggests that the reduced plant defences may also make aphids more susceptible to natural enemies. The preference of A. gifuensis for MC-treated plants further supports the idea that MC triggers a plant-mediated response that improves parasitoid recruitment. The increased parasitism, despite no effect on foraging duration, suggests that MC may improve the plant’s ability to attract natural enemies without altering parasitoid search efficiency, as observed with other plant-derived compounds (Ozawa et al., Reference Ozawa, Shiojiri, Sabelis, Arimura, Nishioka and Takabayashi2004; Bruinsma et al., Reference Bruinsma, Posthumus, Mumm, Mueller, Van Loon and Dicke2009). This highlights the potential of MC in IPM strategies to improve biological control through the enhanced recruitment of parasitoids.
However, the increased fecundity of M. persicae on MC-treated plants presents a significant agronomic risk. Should natural enemy populations be insufficient to exert adequate top-down control, this reproductive boost could accelerate aphid outbreaks and potentially exacerbate crop damage. This implies that MC applications may only be effective in IPM systems with robust natural enemy communities. In fields with established conservation biological control practices, such as floral resource provisioning, hedgerow maintenance, or reduced pesticide use, MC could synergise with existing natural enemies to offset the initial reproductive advantage gained by aphids. Conversely, in simplified agricultural landscapes with low parasitoid pressure, standalone MC applications risk being counterproductive. Future field studies should therefore evaluate MC efficacy across gradients of natural enemy abundance and landscape complexity to delineate appropriate deployment scenarios.
The observed reductions in plant height, leaf dimensions, root length, and fresh biomass of B. rapa treated with MC provide physiological context for the enhanced aphid fecundity. This suppression of growth metrics suggests a resource reallocation within the plant, potentially reducing investment in structural development. Such a shift may inadvertently concentrate nutrients or alter phloem sap composition, improving host quality for M. persicae and facilitating the observed increase in reproductive output. This aligns with the broader pattern where exogenous chemical applications can induce physiological trade-offs influencing plant–insect interactions (Huot et al., Reference Huot, Yao, Montgomery and He2014; Qian et al., Reference Qian, Peng, Han, Ren, Zhan and Zhu2014; Hernández et al., Reference Hernández, Gomez, Cerutti, Arana and Silva2015). For instance, Qian et al., (Reference Qian, Peng, Han, Ren, Zhan and Zhu2014) demonstrated that high-dose ascorbate application in Arabidopsis suppressed growth and antioxidant capacity while increasing oxidative stress markers, reconfiguring resource allocation away from development. Similarly, Hernández et al., (Reference Hernández, Gomez, Cerutti, Arana and Silva2015) reported growth inhibition in melatonin-treated Arabidopsis at elevated concentrations despite enhanced stress tolerance, illustrating a trade-off between growth and defence investment. Notably, despite these morphological changes indicative of altered plant physiology, the MC treatment did not compromise the plant’s ability to attract A. gifuensis or support its parasitism efficacy. The concurrent increase in aphid fecundity and parasitoid performance on MC-treated plants underscores the compound’s role in modulating plant-mediated interactions within this tritrophic system, enhancing both pest and natural enemy metrics despite a measurable cost to plant growth.
While MC shows promise as a plant defence modulator, its effects differ notably from other established plant defence elicitors like CJ and MeSA. CJ typically suppresses aphid performance directly, reducing survival, fecundity, and settlement in Brassica (Ali et al., Reference Ali, Covaci, Roberts, Sobhy, Kirk and Bruce2021) and other crops, while simultaneously enhancing parasitoid attraction. Similarly, MeSA applications at effective concentrations (e.g. 100 mg/L) reduce aphid survival and settlement in Brassica and induce volatile emissions that repel herbivores (Ali et al., Reference Ali, Wei, Mahamood, Zhou, King, Zhou and Shamsi2023b). In contrast, MC enhanced M. persicae fecundity in this study, suggesting it may temporarily improve host quality for aphids. However, like CJ and MeSA, MC strongly recruited natural enemies (A. gifuensis), demonstrating a conserved advantage across elicitors. Advantages of MC may include its broader compatibility with natural enemies under field conditions, given its structural similarity to plant volatiles that mediate tritrophic interactions. Unlike MeSA, which showed variable effects on parasitoids (D. rapae) in Brassica, MC consistently enhanced parasitoid foraging and parasitism. Disadvantages include its potential to boost aphid reproduction if natural enemies are absent, whereas CJ/MeSA offer direct suppression. Economically, MC is commercially available as a food/flavour additive, potentially lowering registration barriers compared to newer synthetics. Future studies should compare field efficacy, residual activity, and cost-benefit ratios across elicitors, especially since CJ and MeSA have established field success in crops like wheat and cotton (Bruce et al., Reference Bruce, Martin, Pickett, Pye, Smart and Wadhams2003; Tonğa et al., Reference Tonğa, Çakmak, Şeker, Temiz and Bayram2020).
While this study provides valuable insights, it has several limitations. For instance, it focuses on only one Brassica species, preventing conclusions about whether the observed effects are species- or cultivar-dependent. The study used a single concentration of MC (100 mg/L) and assessed effects only at 24 h post-treatment, which limits dose-response interpretation and fails to capture the temporal dynamics of MC effects (e.g. persistence at 72 h or 7 days). Additionally, the study does not evaluate the long-term effects of MC on aphid populations or assess plant physiological responses such as growth parameters, visual damage symptoms, or morphophysiological changes that might explain the increased aphid fecundity. Future research should investigate whether these results vary across different plant species or cultivars, and whether the increased fecundity observed in this study leads to higher aphid densities over time, which could impact the effectiveness of MC in pest management strategies. Moreover, understanding the chemical and genetic mechanisms behind these changes is crucial. Our inference that MC modulates plant defence lacks direct biochemical validation through measurements of defense biomarkers (e.g. antioxidant enzymes, defence gene expression, or secondary metabolite accumulation). Investigating the specific volatile compounds released by MC-treated plants alongside these physiological markers would offer deeper insights into these mechanisms. Evaluating MC effects under field conditions, where environmental variability may influence outcomes, is also necessary to determine its practical applicability in IPM.
Conclusion
This study demonstrates that exogenous MC modulates B. rapa defences with dual effects: enhancing M. persicae fecundity while simultaneously increasing A. gifuensis parasitoid attraction and parasitism rates. These outcomes, coupled with MC-induced suppression of plant growth traits (height, leaf dimensions, root length, biomass), suggest resource reallocation improves host suitability for aphids yet bolsters tritrophic recruitment of natural enemies. While MC shows promise for IPM in systems with robust parasitoid populations, where enhanced biological control may offset aphid reproductive gains, its efficacy depends critically on natural enemy abundance. Future research must validate field performance, characterise underlying volatile-mediated mechanisms, and assess long-term ecological trade-offs to optimise MC deployment in sustainable Brassica crop protection.
Acknowledgements
The authors would like to express their gratitude to University Higher Education Fund under research support Program for Central labs at King Khalid University through the project number CL/PRI/A/4.
Author contributions
Xi Xian Chen: Methodology, Experiments; Jamin Ali: Conceptualisation, Writing-Original Draft, Review and Editing, Visualisation; Khalid Ali Khan: Review and editing; Hamed A. Ghramh: Review and editing; Vol Oberemok: Writing-Original Draft; Review and editing; Adil Tonğa: Conceptualisation, Data Analysis, Visualisation and Data Curation, Writing – Original Draft Review and editing.
Conflict of interests
The authors declare no conflict of interest.
Availability of data and materials
Available upon reasonable request.
Ethical approval
Not applicable.
