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Article

Entomopathogenic Fungus Treatment Affects Trophic Interactions by Altering Volatile Emissions in Tomato

by
Asim Munawar
1,†,
Haonan Zhang
1,†,
Jinyi Zhang
1,†,
Xiangfen Zhang
1,
Xiao-Xiao Shi
2,
Xuan Chen
1,
Zicheng Li
1,
Xiaoli He
1,3,
Jian Zhong
1,
Zengrong Zhu
1,3,
Yaqiang Zheng
4,* and
Wenwu Zhou
1,*
1
State Key Laboratory of Rice Biology, Ministry of Agricultural and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China
2
Zhejiang Academy of Forestry, Hangzhou 310023, China
3
Hainan Institute, Zhejiang University, Sanya 572000, China
4
Resource and Utilization Research Center of Medicinal Cordyceps, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China
*
Authors to whom correspondence should be addressed.
†
The authors contributed equally to this study.
Agronomy 2025, 15(5), 1161; https://doi.org/10.3390/agronomy15051161
Submission received: 16 April 2025 / Revised: 6 May 2025 / Accepted: 8 May 2025 / Published: 9 May 2025
(This article belongs to the Special Issue Pests, Pesticides, Pollinators and Sustainable Farming)
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Abstract

:
Entomopathogenic fungi (EPFs) can influence plant–insect interactions through complex molecular and chemical mechanisms. This study investigates how EPF treatment of tomato plants modulates volatile organic compound (VOC) emissions and subsequent trophic interactions between tomato plants, the herbivorous pest Phthorimaea absoluta, and the parasitic wasp, Trichogramma chilonis. Our results demonstrate that EPF-treated plants exhibited reduced attractiveness to adult P. absoluta moths, which were actively repelled by EPF-induced VOCs. Conversely, these same plants showed enhanced recruitment of the parasitoid T. chilonis, which demonstrated positive chemotaxis toward the modified VOC profile. Chemical analysis revealed significantly elevated emissions of key VOCs in EPF-treated plants, particularly (E)-β-Caryophyllene, β-phellandrene, and α-Phellandrene. This increase is correlated with enhanced production of defense-related phytohormones, including JA, SA, and JA-Ile, which may regulate VOC biosynthesis pathways. Behavioral response studies using synthetic VOCs and electroantennogram (EAG) measurements confirmed that these EPF-induced VOCs elicited strong olfactory responses in both insect species. To summarize, EPF treatment reshapes multitrophic interactions by strategically modulating plant VOC emissions and activating defense signaling pathways in tomato plants, providing new insights for potential applications in sustainable pest management strategies.

1. Introduction

Entomopathogenic fungi (EPFs) play a significant role in the regulation of insect populations [1]. These EPFs serve as promising biological control agents that can serve as environmentally sustainable alternatives to chemical insecticides [2]. EPFs exhibit insecticidal properties against numerous insect pests, including Myzus persicae, Aleurodicus rugioperculatus, and Leucoptera coffeella [3,4]. In addition to its direct effects on insects, EPF colonization can also lead to indirect effects on herbivores via affecting plant defenses [5] and recently has gained increasing research attention [6].
EPFs have emerged as multi-functional biocontrol agents in agricultural systems, extending beyond their direct pathogenic effects on insect pests [7]. When colonizing plants as endophytes, these EPFs establish complex symbiotic relationships that significantly alter plant physiology, growth patterns, and defense capabilities [8,9]. Endophytic colonization by EPFs induces significant changes in the host plant’s physiology and metabolism, influencing growth, development, and disease resistance by modulating the metabolic pathways that enhance resilience to various biotic and abiotic stresses [10]. These growth-promoting effects are mediated through the fungal production of phytohormones and enzymes that facilitate nutrient solubilization and uptake [11]. During plant development, EPFs enhance the immune system by triggering the synthesis of key secondary metabolites—including phenolics, terpenoids, and alkaloids—that function as natural defenses against herbivore attacks [12,13,14]. These plant-derived metabolites play vital roles in regulating plant–insect interactions by influencing various aspects of plant defense and signaling mechanisms [15]. However, the specific mechanisms by which EPF treatment alters plant defense and how these changes impact trophic interactions remain poorly characterized despite their potential significance for pest management strategies.
VOCs are ubiquitous substances in plants that regulate insect–plant interactions and have been elucidated as a pivotal component of plant defense mechanisms against herbivory [15,16]. When subjected to external stressors, plants activate their direct and indirect defense mechanisms [17]. They synthesize and release intricate blends of VOCs that play a crucial role in mediating indirect resistance against herbivores and their natural enemies [18,19]. Changes in plant physiology and biochemistry induced by EPF colonization can modulate VOC emissions [20,21,22]. EPF colonization has been demonstrated to affect plant VOC emissions [23,24,25], likely due to the shared molecular synthesis pathway between VOCs and jasmonic acid (JA), which serves as a regulatory factor for VOC production [19,26]. Phytohormones, whose levels may be altered by EPF colonization, also influence VOC release. Ultimately, EPF colonization-induced changes in plant VOC emissions can change the behavior of herbivorous insects and their associated natural enemies.
Tomato (Solanum lycopersicum), originating from South America, is the second-largest vegetable crop grown globally. P. absoluta is one of the most devastating pests of Solanaceous plants and is recorded as a major invasive pest worldwide due to its high reproductive capacity, substantial food consumption, and extensive migration behaviors [27,28,29]. Adults preferentially oviposit on stems or leaf blades and the larvae directly consume leaves or fruits upon hatching. Larvae typically mine leaves and consume substantial amounts of mesophyll, leading to reduced photosynthesis. In severe infestations, tomato yields can be reduced by 80–100% [30]. Insecticides, such as pyrethroids and abamectin, are primarily used for pest control in tomatoes, but P. absoluta has developed resistance [31,32]. Concurrently, the injudicious application of pesticides has adverse effects on human health, the agricultural environment, and non-target insects. In these situations, using EPF for the indirect management of herbivore pests provides a better alternative. Trichogramma wasps are widely used as biocontrol agents [33,34,35]. Among these, T. chilonis is a widespread egg parasitoid employed to manage various lepidopteran herbivores [36].
EPF colonization has been shown to induce physiological and biochemical changes in plants, particularly altering their VOC emissions [36,37]. These plant-emitted VOCs serve as important chemical cues for T. chilonis wasps to locate their hosts [36]. Such changes may consequently affect the behavior of both P. absoluta and T. chilonis. In this study, we tested two hypotheses: (I) the EPF colonization of tomato plants may influence their attractiveness to adult herbivorous insects and parasitoids, altering trophic interactions; (II) these altered trophic interactions under EPF colonization may be mediated by changes in VOC emissions. This study may provide insights for understanding the molecular mechanisms of EPF treatment on insect–plant interactions.

2. Materials and Methods

2.1. Plant and Insect Culture

Tomato seeds (S. lycopersicum, cv. ‘Heinz’) were used in the study. The seeds underwent surface sterilization by immersion in 1% NaClO for 5 min, followed by three thorough rinses with sterile water. The sterilized seeds were then placed on sterile moist filter paper to germinate. Once small plantlets developed, they were transferred to 250 mL pots. All the plants were randomly distributed in an environmental chamber maintained at 28 °C, with a 16 h/8 h light/dark cycle and 80% relative humidity.
P. absoluta specimens were obtained from Yuxi City, Yunnan Province, China, and their population was reared for more than 10 generations on tomato plants under controlled conditions: 28 °C, 16 h:8 h light/dark cycle, and 80% relative humidity. The egg parasitoid, T. chilonis, was sourced commercially from Keyun Biocontrol, China, and subsequently reared for more than 4 generations on P. absoluta eggs in laboratory conditions prior to use in experiments. All the experiments were conducted between [06/2020] and [03/2025].

2.2. Fungal Culture and Colonization on Tomato Plants

All the EPF strains used in this study were sourced from the Resource and Utilization Research Center of Medicinal Cordyceps, Guizhou University of Traditional Chinese Medicine, Guiyang, China. The obtained EPFs were activated and cultured on Potato Dextrose Agar (PDA; Haibo Biology, Qingdao, Shandong, China) at 27 °C. To prepare the fungal inoculum, conidia were scraped from the culture using a sterile scraper and suspended in sterile distilled water containing 0.05% TWEEN® 80. The conidia concentration was adjusted to 1 × 108 conidia mL−1 using a hemocytometer. A control solution was prepared using sterile distilled water containing 0.05% TWEEN® 80 without fungal conidia. The suspension was uniformly sprayed on tomato leaves until runoff. To promote conidia germination and colonization, inoculated plants were enclosed in plastic bags for 24 h post-treatment. Among all the strains, Cordyceps fumosorosea (YNKM210801) was selected for an in-depth analysis based on preliminary assessments, as it demonstrated the most pronounced effects on herbivore behavior.
To confirm the endophytic colonization of tomato plants, leaves from both the Cordyceps fumosorosea (YNKM210801)-inoculated and control plants were surface-sterilized in 1% hypochlorite for 5 min. After sterilization, the leaves were cut into 0.5 × 0.5 cm2 pieces and placed on Potato Dextrose Agar (PDA) supplemented with dodine and chloramphenicol following the method described previously [38]. Plates were incubated at 28 ± 1 °C with a 16:8 h light/dark cycle and 85% relative humidity for 15 days. After the incubation period, the presence of C. fumosorosea (YNKM210801) was assessed. The colonization rate was determined by counting the number of leaf pieces showing EPF growth. Final colonization assessment was conducted at 28 days post-inoculation (dpi) following soil drenching treatments. The experiment was repeated three times. The visual evidence of EPF colonization is shown in Figure S1.

2.3. P. absoluta Oviposition and T. chilonis Parasitism Assays

The oviposition preference of P. absoluta and the parasitism choice of T. chilonis were assessed using our previously established bioassay [36] (Figure S2A). In detail, individual cages (65 cm L × 55 cm H × 60 cm W) were set up, each containing one EPF-inoculated plant and one control plant. Pots were covered with aluminum foil to eliminate soil VOC interference. Two newly mated P. absoluta females (24 h post-emergence) were introduced into each cage for a 24 h oviposition period, after which the total number of eggs on each plant was quantified.
For the T. chilonis parasitism assay, standardized egg masses of P. absoluta (N = 150 eggs) were prepared on 30 mm × 30 mm filter paper pieces. These were obtained by allowing gravid P. absoluta females to oviposit on 120 mm × 120 mm filter paper in a 3 L transparent plastic container for 24 h. The oviposited filter papers were then sectioned and eggs counted microscopically, with excess eggs removed to maintain the standard count. These egg masses were affixed to the abaxial surface of apical leaflets on both the inoculated and control tomato plants. Eight pairs of newly emerged T. chilonis were released into each cage. After a 12 h exposure period, the egg-bearing filter papers were transferred to ventilated Petri dishes (BeyoGold, 100 × 20 mm), sealed with Parafilm, and incubated at 28 °C for subsequent parasitism assessment.

2.4. Sampling of Foliar VOC Blends

Foliar VOCs were sampled using Polydimethylsiloxane (PDMS) tubes (Carl Roth, Karlsruhe, Germany) following the methodology detailed in our previous studies [15,36]. Briefly, two clean PDMS tubes and a single leaflet from each plant were enclosed in a sealed plastic chamber for VOC sampling. For constitutive VOC analysis, intact leaves were used. To assess herbivory-induced plant volatiles (HIPVs), the plants were first infested with P. absoluta larvae. A blank treatment consisting of an empty plastic chamber with PDMS tubes was included as a control. The VOC analysis was performed using a thermal desorption-gas chromatography/mass spectrometry (TD-GC-MS) system (TD-100XR, Markes International; Trace 1300 GC, ISQ 7000 single quadrupole MS, Thermo Fisher Scientific, Milan, Italy). The resulting chromatographic peaks were normalized by fresh leaf mass and integrated. Compound identification was conducted using the NIST v and rep libraries integrated within the Chromeleon software v7.2.8 (Thermo Scientific), complemented by comparison with authentic standards.

2.5. Phytohormone Extraction and Quantification

Two fully expanded compound leaves were harvested from the treatment plants and immediately flash-frozen in liquid nitrogen until analysis. Phytohormone extraction and quantification procedures were conducted using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system (LCMS-8040 system, Shimadzu Corporation, Kyoto, Japan) following previously established protocols without modifications [39,40].

2.6. Insect Choice Assay with Y-Tube Olfactometer

The behavioral responses of P. absoluta and T. chilonis to synthetic VOCs were assessed using a glass Y-tube olfactometer system adapted from our previous study [36]. The Y-tube consisted of a 16 cm stem and two 10 cm arms set at a 60° angle, with an inner diameter of 10 mm. Purified air was supplied by an air pump (0.02 MPa) through activated charcoal, distilled water, and silica gel. Airflow rates were set at 0.5 L min−1 for P. absoluta and 0.1 L min−1 for T. chilonis, regulated through Teflon tubing connected to the Y-tube (Figure S2B). Prior to testing, newly emerged females of both species were paired with males for 24 h. Synthetic VOCs were diluted in n-hexane (≥98.0%, Shanghai Aladdin Biochemical Technology Co.Ltd. Xinjinqiao, Pudong, Shanghai) to concentrations of 0.01, 0.05, 0.1, 0.5, and 1.0 μL mL−1. Aliquots were applied to filter paper (4 cm × 2 cm) using a glass micropipette. The treated filter paper was placed in a glass tube (10 cm × 2 cm) connected to one arm of the Y-tube, while a hexane-treated control was placed in the opposite arm. Individual insects were introduced into the stem and allowed to make a choice. Non-responsive individuals were excluded from the analysis. To mitigate positional bias, odor source positions were alternated every five trials and fresh VOC samples were used. The apparatus was cleaned and sterilized after every 10 trials.
For the whole-plant preference tests, a similar setup was employed. The plants were enclosed in transparent plastic sheets, with the upper ends connected to the air source and choice arm via Teflon tubes. The plant pots were covered with aluminum foil to minimize soil VOC interference.

2.7. Electroantennogram (EAG) and Odor Delivery

The EAG responses of P. absoluta and T. chilonis antennae were recorded following the methodology described previously [36]. The apical tip of each antenna was excised with micro-scissors and inserted into a recording electrode containing 0.2 M KCl solution. A similar reference electrode was attached to a micromanipulator and connected to the distal end of the antenna, completing the circuit. The EAG signals were amplified using a high-impedance (>1012 Ω) pre-amplifier coupled to an EAG amplifier (Syntech, AM-02, Kirchzarten, Germany). The amplified signals were then processed and digitized using a signal acquisition interface board (Syntech, IDAC-02).
Odor stimuli were delivered using an air stimulus controller (Syntech, CS-05) with a constant flow (2 L min−1) of humidified air passing over the antenna through a glass tube (20 × 0.5 cm) positioned 20 mm from the preparation. VOCs were introduced via a pipette tip (Corning Life Sciences, Jiangsu, China) inserted 5 cm from the end of the glass tube. During stimulation, 0.4 L min−1 of air was pulsed through the pipette tip into the main airflow for 0.40 s. The test compounds were serially diluted in n-hexane to concentrations of 0.01, 0.05, 0.1, 0.5, and 1.0 μL mL−1 for dose–response studies. Aliquots of 7 μL of each concentration were applied to filter paper strips and inserted into the pipette tip. Odors were presented in order of increasing concentration, with 30 s intervals between applications to allow for antennal recovery. A pipette tip containing 7 μL of n-hexane on filter paper served as a control. The EAG responses were quantified as the amplitude of deflection relative to the control stimulus. Peak amplitudes were marked and expressed as relative amplitudes (mV) compared to the standard response.

2.8. Synthetic VOC Applications and P. absoluta Behavior Assays

To investigate the influence of plant VOCs on P. absoluta behavioral responses, we applied synthetic VOCs to the plant leaf surfaces following the protocols established [15]. (E)-β-Caryophyllene, at a biologically relevant concentration of 1.5 μL mL−1, was precisely dissolved in liquefied lanolin (BBI Life Sciences) to create a standardized treatment solution. The control plants received applications of pure lanolin, with the lanolin aliquot prepared according to methods previously described [41]. Following the (E)-β-Caryophyllene treatment, the plants were immediately subjected to P. absoluta oviposition behavioral assays using the experimental protocols detailed in the preceding sections.

2.9. Statistical Analyses

The total number of eggs oviposited and parasitized was analyzed using a generalized linear model (GLM) with Poisson distribution [42]. Pearson’s chi-square (χ2) tests were used to evaluate differences in the responses of P. absoluta and T. chilonis to odor stimuli. The EAG responses of both species were analyzed using one-way analysis of variance (ANOVA) and means were separated by post hoc comparisons (Tukey’s HSD; p ≤ 0.05). Phytohormone data were analyzed using T-tests. Hierarchical cluster analysis using Euclidean distances and Ward’s method was performed to generate VOC heatmaps; utilizing the ‘pheatmap’ package in R., VOC data were further analyzed using analysis of variance (ANOVA) and principal component analysis (PCA). Statistical analyses were conducted using the R software version 4.0.2.

3. Results

3.1. Effects of EPF Treatment on Herbivore and Parasitoid Behavioral Preferences

We investigated the effects of EPF treatment on plant–insect interactions through oviposition and parasitism choice assays using tomato plants with EPF-colonized roots. Tomato plants treated by Beauveria bassiana (HXBA202002, HX200801, M190901), Metarhizium fkavoviride (HXMF211004), Metarhizium anisopliae (QLSMA201102), Cordyceps cateniobliqua (BMZ2075841), Cordyceps cateinannulata (BNCC150028), and Cordyceps fumosorosea (YNKM210801) received fewer eggs from P. absoluta females compared to control plants (Figure S3A–I). Tomato treated by Metarhizium rileyi (QLSMR211004) received a similar number of eggs as those of untreated plants. Among these EPF strains, C. fumosorosea (YNKM210801) demonstrated the most potent deterrent effect on oviposition (Figure S3G–I). The exogenous application of C. fumosorosea (YNKM210801) to tomato plants also resulted in reduced oviposition by P. absoluta females (GLM: T = 2.22, p = 0.015), confirming the effectiveness of this EPF when applied externally (Figure S4). Additionally, the root application of C. fumosorosea (YNKM210801) significantly altered plant attractiveness to both herbivores and parasitoid wasps. In choice experiments, P. absoluta showed a strong preference for control plants over C. fumosorosea (YNKM210801)-treated plants for oviposition (GLM: T = 2.13, p = 0.019). In contrast, the egg parasitoid T. chilonis significantly preferred C. fumosorosea (YNKM210801)-treated plants for parasitism (GLM: T = 2.39, p = 0.01) (Figure 1A,B). These preference patterns persisted even in plants subjected to the larval feeding of P. absoluta, indicating that herbivore damage did not substantially alter the effects of EPF treatment on plant attractiveness to either herbivores or parasitoids.
To further investigate the olfactory responses of both insect species, we conducted Y-tube olfactometer experiments (Figure 2A). The P. absoluta females exhibited a significant preference for the control plants compared to the C. fumosorosea (YNKM210801)-treated plants (χ2 = 5.58, p = 0.01). Conversely, T. chilonis displayed stronger attraction to the C. fumosorosea (YNKM210801)-treated plants than to the control plants (χ2 = 6.33, p = 0.01). The olfactory responses of both insect species intensified when the plants (both control and C. fumosorosea (YNKM210801)-treated) were subjected to larval feeding (Figure 2B,C). These results suggest that the C. fumosorosea (YNKM210801) treatment modifies the plant VOC profile, thereby influencing plant interactions with herbivores and their natural enemies.

3.2. EPF Treatment Modulates VOC Emissions and Induces Phytohormone Accumulation

To elucidate the mechanisms underlying the altered plant attraction to herbivores and parasitoid wasps following the EPF root treatment, we analyzed the VOC emissions in tomato plants (Figure 3A). The C. fumosorosea (YNKM210801) treatment induced a distinct VOC profile compared to the control plants, characterized by enhanced emissions of several compounds. This effect remained consistent in both the undamaged and herbivory-damaged plants, indicating that herbivore damage did not significantly alter the C. fumosorosea (YNKM210801)-induced VOC emissions. Generally, the VOC levels detected from the undamaged leaves were lower than those from the herbivory-induced leaves. Among the C. fumosorosea (YNKM210801)-enhanced VOCs, (E)-β-caryophyllene and β-phellandrene exhibited higher emissions in the undamaged leaves, with further enhancement observed in both the C. fumosorosea (YNKM210801)-treated and herbivory-damaged leaves. Notably, α-phellandrene was the only VOC significantly enhanced by the combination of the C. fumosorosea (YNKM210801) treatment and herbivory damage, while α-humulene was uniquely enhanced in both the C. fumosorosea (YNKM210801)-treated leaves and C. fumosorosea (YNKM210801) + herbivory damaged leaves (Figure 3A). Additionally, the emissions of several other VOCs including α-pinene, 3-carene, and 2-carene were significantly affected across all the treatments. The principal component analysis (PCA) of the VOC profiles revealed clear treatment-specific signatures, with the first two principal components explaining 94.26% of the total variance (PC1: 84.3%; PC2: 9.96%) (Figure 3B). Furthermore, the C. fumosorosea (YNKM210801)-treated plants exhibited distinct temporal patterns in their VOC emissions. Among the most abundant VOC in tomato, (E)-β-caryophyllene, α-phellandrene, and β-phellandrene demonstrated time-dependent emission profiles (Figure 4A–C). Notably, (E)-β-caryophyllene was the only volatile compound that maintained significant emission levels across all the time points examined (Figure 4A). In contrast, α-phellandrene showed no significant emission at the 6 h time point (Figure 4B), while β-phellandrene emissions were not significant at the 3 h collection period (Figure 4C). These temporal variations in VOC emission patterns suggest that the C. fumosorosea (YNKM210801) treatment induces a complex and dynamic reprogramming of plant secondary metabolism, potentially as part of the plant’s adaptive response to fungal presence.
EPF colonization also significantly influenced phytohormone accumulation in tomato leaves. In the undamaged plants, JA (T8 = 2.70, p = 0.027) and JA-Ile (T8 = 2.84, p = 0.022) levels were significantly higher in the C. fumosorosea (YNKM210801)-treated plants compared to controls (Figure 5A,B). This pattern persisted in the herbivory-damaged plants as well. Interestingly, the SA levels were higher in the undamaged plants than in the damaged plants following the C. fumosorosea (YNKM210801) treatment (T8 = 2.96, p = 0.039) (Figure 5C). These differential responses in phytohormone accumulation suggest that various phytohormones may play distinct roles in mediating plant indirect defense responses to C. fumosorosea (YNKM210801) colonization and herbivory.

3.3. Behavioral and Electrophysiological Responses of Insects to Synthetic VOC Odors

To verify whether the altered VOC profiles are responsible for the observed changes in plant–herbivore–parasitoid interactions, we conducted Y-tube olfactometer and EAG studies using pure VOCs. The Y-tube olfactometer assays revealed distinct behavioral responses of P. absoluta and T. chilonis female adults to the tested VOCs (Figure 6A–F). The olfactory response of T. chilonis demonstrated high sensitivity and attraction to all three tested VOCs with concentration-dependent patterns. For (E)-β-caryophyllene, T. chilonis consistently showed stronger responses at lower concentrations, with a significant decrease in response at 1.0 μL mL−1 (χ2 = 4.79, p = 0.028) (Figure 6A). α-Phellandrene elicited reduced responses at both 0.01 μL mL−1 (χ2 = 4.29, p = 0.038) and 0.5 μL mL−1 (χ2 = 5.23, p = 0.022), with enhanced responses observed at 0.05, 0.1 and 1.0 μL mL−1 concentrations (Figure 6B). For β-phellandrene, T. chilonis exhibited decreased response at 0.1 μL mL−1 (χ2 = 4.93, p = 0.026) while showing stronger responses at the other tested concentrations (Figure 6C). Conversely, compared to the control (n-hexane), all three VOCs exhibited repellent effects on P. absoluta (Figure 6D–F). The olfactory responses to (E)-β-caryophyllene were significant across all the concentrations except 0.05 μL mL−1 (χ2 = 3.59, p = 0.058) (Figure 6D). Responses to α-phellandrene were non-significant at higher concentrations of 0.5 μL mL−1 (χ2 = 3.42, p = 0.064) and 1.0 μL mL−1 (χ2 = 3.47, p = 0.062) while showing the strongest repellent effect at the medium concentration of 0.1 μL mL−1 (χ2 = 6.23, p = 0.01) (Figure 6E). The repellent response of P. absoluta to β-phellandrene was significant across most concentrations, with a decreased effect at 0.5 μL mL−1 (χ2 = 5.16, p = 0.023) and a non-significant response at 0.1 μL mL−1 (χ2 = 3.53, p = 0.060) (Figure 6F). These findings indicate that VOCs differentially modulate the behavior of both the parasitoid and pest species, suggesting their potential application in integrated pest management strategies.
We also investigated whether the direct application of pure VOCs to tomato leaves could alter P. absoluta oviposition behavior (Figure S5A). Notably, the tomato plants treated with (E)-β-caryophyllene exhibited significantly reduced oviposition, with the P. absoluta females laying fewer eggs on these plants compared to the control plants treated with lanolin only (GLM: T = 13.59, p = 0.001; Figure S5B). This finding further supports the role of (E)-β-caryophyllene as an important mediator of plant–insect interactions. It is worth noting that in our previous study, we demonstrated that lanolin application alone does not induce changes in VOC emission patterns in potato plants, confirming that the observed effects were specifically due to the applied VOC.
The EAG studies revealed that both insect species, P. absoluta and T. chilonis, exhibited distinct response patterns to pure VOCs, specifically (E)-β-caryophyllene and β-phellandrene (Figure 7A,B). These findings suggest that these plant-emitted VOCs are detectable and recognized by the olfactory systems of both insects. Notably, T. chilonis displayed a significantly stronger concentration-dependent EAG response to (E)-β-caryophyllene (F3,11 = 61.6, p = 0.001), whereas P. absoluta showed a weaker response at lower concentrations, with a marked increase only at higher concentrations (F3,11 = 110.9, p = 0.001; Figure 7A). Also, both species exhibited EAG responses to β-phellandrene, with T. chilonis demonstrating the highest sensitivity (F3,11 = 101.4, p = 0.001) compared to P. absoluta (F3,11 = 134.7, p = 0.001; Figure 7B). These results indicate that the antennae of both insects are capable of detecting and responding to these specific plant VOCs, albeit with varying sensitivity and intensity.

4. Discussion

EPFs play important roles in crop protection by simultaneously controlling insect pests and altering plant immune responses [43]. These EPFs have the capacity to colonize plant tissues endophytically and can elicit systemic defense responses [44]. EPF colonization triggers a cascade of biochemical and physiological changes within the plant, including the up-regulation of defense-related genes, altered production of secondary metabolites, and priming of the immune system for enhanced resistance against a broad spectrum of pathogens and herbivores [45]. This study investigated the effects of EPF treatment on tomato plant defenses and its consequent impact on plant–insect interactions.
Through choice assay tests and olfactory studies, our findings reveal that EPF treatment significantly alters the plant attractiveness to both herbivores and parasitoid wasps. The EPF treatment of tomato plants significantly reduced the attraction of the female herbivore P. absoluta while enhancing the attraction of the parasitoid wasp T. chilonis. Importantly, these effects persisted and were stronger even when the plants were subjected to herbivory to produce more VOC. These alterations in plant chemotype and subsequent insect behavior suggest that EPF treatment may serve as an effective strategy for enhancing plant indirect defenses against herbivores while promoting the attraction of beneficial insects [1]. Previous studies have explored the effect of EPFs on insect behavior, such as Beauveria bassiana treatment to faba bean seeds which altered the choice preferences and development of the aphid parasitoid, Aphidius colemani [46]. Similarly, EPF applications have been found to affect the behavior and life history of peach aphid, Myzus persicae [47], and the population growth of two-spotted spider mites, Tetranychus urticae [48]. However, the role of plant VOCs mediating insect behavior under such conditions remained largely unexplored. Moreover, it has been discovered that the release of constitutive plant VOC plays an important role in mediating the attraction/repellence of insect species [49,50]. This study found that EPF treatment induces changes in the responses of herbivorous insects and parasitoids for tomato plants, which was likely attained via the alteration of the VOCs by the host, which is involved in the interaction of these organisms. Also, the observed changes in insect preference were consistent in both choice assays and olfactometer experiments, indicating that the effect is primarily mediated through plant VOC emissions rather than visual or tactile cues.
Plant-released VOCs play important roles in regulating plant–insect interactions in agro-ecosystem [16]. EPF-treated tomato plants emitted a distinct VOC blend compared to control plants, with several compounds showing enhanced emissions. Interestingly, the VOC profile alterations persisted even under herbivore attack. Notably, (E)-β-caryophyllene and β-phellandrene exhibited increased emissions in both the undamaged and herbivore-damaged EPF-treated plants. These sesquiterpenes have been previously associated with indirect plant defenses, attracting natural enemies and repelling herbivores [15,36,51]. Our results showed that (E)-β-caryophyllene and β-phellandrene were repellent to P. absoluta while attractive to T. chilonis. Moreover, both insects also responded differently to several other key tomato leaf VOCs, indicating that these VOCs might also be involved in promoting or deterring natural enemies and herbivores [52]. Our olfaction studies provided crucial evidence linking the observed changes in plant VOC profiles to specific insect behavioral and physiological responses. P. absoluta and T. chilonis exhibited distinct preferences for different VOCs, with responses often showing dose dependency [36]. Most tested VOCs were repellent to P. absoluta but attractive to T. chilonis, aligning with the overall pattern of herbivore deterrence and parasitoid attraction observed in whole-plant assays.
The altered plant–insect interactions observed in our study can be largely attributed to the significant changes in VOC profiles induced by EPF treatment. The alterations in VOC profiles and plant–insect interactions were accompanied by significant changes in phytohormone levels following EPF treatment. JA and SA are key regulators of plant responses to biotic stresses [53,54], particularly in mediating defenses against herbivores and necrotrophic pathogens [36,55]. Our results showed that the levels of JA, JA-Ile, and SA were increased in both undamaged and herbivore-damaged EPF-treated plants. This simultaneous activation of both the JA and SA pathways is noteworthy, as these hormones often exhibit antagonistic interactions [56]. However, recent studies have shown that certain beneficial microbes can circumvent this antagonism, leading to a synergistic activation of multiple defense pathways [57]. These changed phytohormone levels likely play a crucial role in modulating the plant VOC emissions, as both JA and SA are known to regulate the biosynthesis of various defense-related VOC [15]. Furthermore, the sustained increase in these defense hormones in the EPF-treated plants, even prior to the herbivore attack, suggests that the fungus may be priming the plant defense responses, potentially enabling a more rapid and robust response to subsequent stresses.
Our findings reveal a significant relationship between (E)-β-Caryophyllene treatment and P. absoluta oviposition behavior. When the tomato plants were treated with (E)-β-Caryophyllene, we observed a marked reduction in oviposition by adult female P. absoluta. This observation aligns with our Y-tube olfactometer assays, which demonstrated that this VOC effectively repels P. absoluta, thus confirming the involvement of (E)-β-Caryophyllene in modulating host preference behaviors. Previous research demonstrated that application of (E)-β-Caryophyllene to potato leaves significantly altered the movement patterns of predatory mites across plant organs [15]. Taken together with our current results, these findings suggest that EPFs mediate an increase in VOC emission in tomato plants, which may serve to prime both the direct and indirect plant defense responses [58]. Unlike traditional pesticides that directly target pests, EPFs appear to enhance the plant’s own defense mechanisms, creating a less favorable environment for herbivores while simultaneously attracting their natural enemies. The use of EPFs as a biocontrol agent could reduce reliance on chemical pesticides, thereby minimizing environmental impacts and slowing the development of pesticide resistance in pest populations [59,60]. This research was conducted under controlled laboratory conditions, which may not fully reflect the complexity of field environments [61]. Future studies should validate these findings in diverse field conditions to assess the robustness and applicability of EPF-induced effects across different environmental contexts. Also, further investigation into the molecular mechanisms underlying the observed changes, particularly the signaling pathways mediating the interaction between EPFs and plant defense responses, could provide deeper insights and potentially identify targets for enhancing these beneficial effects.

5. Conclusions

Our study provides evidence that EPF treatment significantly alters plant–insect interactions in tomato plants through multiple mechanisms. Choice assays and olfactometer experiments consistently demonstrated that the EPF-treated plants repelled the herbivore P. absoluta while attracting the parasitoid T. chilonis, with these effects intensifying under herbivory conditions. A chemical analysis revealed that the EPF treatment induced distinct VOC profile changes, particularly elevating emissions of (E)-β-caryophyllene and β-phellandrene, compounds associated with plant defensive functions. Our olfaction studies directly linked these specific VOCs to the observed behavioral responses, with (E)-β-caryophyllene demonstrably repelling P. absoluta while attracting T. chilonis. Furthermore, oviposition experiments confirmed that the (E)-β-caryophyllene treatment significantly reduced egg-laying by female P. absoluta, providing a mechanistic explanation for the observed herbivore deterrence. These altered plant–insect interactions coincided with significant phytohormonal changes, as the EPF treatment elevated the levels of JA, JA-Ile, and SA in both the undamaged and herbivore-damaged plants. This research highlights the potential of EPFs as a biocontrol strategy that strategically modifies plant defense responses to create less favorable conditions for herbivores while promoting natural enemy attraction (Figure 8).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15051161/s1, Figure S1: Detection of Cordyceps fumosorosea (YNKM210801) colonization on leaves. Figure S2: Schematic drawings of the setup used for the choice experiments. Figure S3: Effect of EPF treatment on the oviposition selection behavior of P. absoluta. Figure S4: Effect of Cordyceps fumosorosea (YNKM210801) spray application on the oviposition selection behavior of P. absoluta for tomato plants. Figure S5: Exogenous application of (E)-β-caryophyllene reduces herbivore oviposition preference in tomato plants.

Author Contributions

Conceptualization, W.Z. and Y.Z.; methodology, A.M., J.Z. (Jinyi Zhang), X.Z., X.-X.S., X.C., X.H., Y.Z., and W.Z.; software, A.M. and J.Z. (Jian Zhong); validation, A.M., J.Z. (Jinyi Zhang), J.Z. (Jian Zhong), X.C., and W.Z.; formal analysis, A.M., Z.L., J.Z. (Jinyi Zhang), X.Z., X.-X.S., X.C., X.H., J.Z. (Jian Zhong), Y.Z., and W.Z.; investigation, A.M., H.Z., and J.Z. (Jinyi Zhang); resources, W.Z. and Z.Z.; data curation, A.M., H.Z., J.Z. (Jinyi Zhang), and Z.L.; writing—original draft preparation, A.M., H.Z., J.Z. (Jinyi Zhang), and W.Z.; writing—review and editing, A.M., H.Z., Z.L., J.Z. (Jinyi Zhang), X.Z., X.-X.S., X.C., X.H., Y.Z., and W.Z.; visualization, A.M. and H.Z.; supervision, W.Z., Z.Z., and Y.Z.; project administration, W.Z. and Z.Z.; funding acquisition, W.Z., A.M., and Z.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Key R&D Program (2023YFC2604500), National Nature Science Foundation of China (Grant Nos. W2433069 and 32272636), and the Yunnan FVF-IPM Joint Lab (no. 202303AP140018).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors are grateful to their colleagues for their help in the experiments and the Yaqiang Zheng for providing EPF strains.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Figure 1. C. fumosorosea (YNKM210801) treatment reduced herbivore moth attraction while increasing parasitoid preference for tomato plants: (A) Oviposition preference of female P. absoluta (n = 24 tests per treatment). (B) Parasitism preference of T. chilonis (n = 25 tests per treatment). Each replicate utilized a new set of plants. The intact and herbivore-induced plants were assessed on separate days. Asterisks (*) denote significant differences between means within the treatments (Tukey HSD following GLM ANOVA, *: p < 0.05; **: p < 0.01). The data are mean ± SE.
Figure 1. C. fumosorosea (YNKM210801) treatment reduced herbivore moth attraction while increasing parasitoid preference for tomato plants: (A) Oviposition preference of female P. absoluta (n = 24 tests per treatment). (B) Parasitism preference of T. chilonis (n = 25 tests per treatment). Each replicate utilized a new set of plants. The intact and herbivore-induced plants were assessed on separate days. Asterisks (*) denote significant differences between means within the treatments (Tukey HSD following GLM ANOVA, *: p < 0.05; **: p < 0.01). The data are mean ± SE.
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Figure 2. C. fumosorosea (YNKM210801) treatment altered olfactory behaviors of herbivores and parasitoids: (A) Schematic illustration of Y-tube olfactometer used to test insect olfactory responses. (B,C) Olfactory responses of P. absoluta females and T. chilonis to control or C. fumosorosea (YNKM210801)-treated plants (n = 100 individuals released per test). Non-responding insects were excluded from analysis. Asterisks (*) indicate significant differences in preference between control and EPF-treated plants (χ2 test; **: p < 0.01).
Figure 2. C. fumosorosea (YNKM210801) treatment altered olfactory behaviors of herbivores and parasitoids: (A) Schematic illustration of Y-tube olfactometer used to test insect olfactory responses. (B,C) Olfactory responses of P. absoluta females and T. chilonis to control or C. fumosorosea (YNKM210801)-treated plants (n = 100 individuals released per test). Non-responding insects were excluded from analysis. Asterisks (*) indicate significant differences in preference between control and EPF-treated plants (χ2 test; **: p < 0.01).
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Figure 3. C. fumosorosea (YNKM210801) treatment enhances VOC emission in tomato plants: (A) Heatmap showing relative abundance of VOCs from control and C. fumosorosea (YNKM210801)-treated tomato plants (n = 6 plants/treatment, 6 h post-treatment). (B) PCA of VOC profiles demonstrating treatment-specific clustering of compound signatures.
Figure 3. C. fumosorosea (YNKM210801) treatment enhances VOC emission in tomato plants: (A) Heatmap showing relative abundance of VOCs from control and C. fumosorosea (YNKM210801)-treated tomato plants (n = 6 plants/treatment, 6 h post-treatment). (B) PCA of VOC profiles demonstrating treatment-specific clustering of compound signatures.
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Figure 4. Emission kinetics of VOCs following C. fumosorosea (YNKM210801) treatment: (A–C) Temporal patterns of highly abundant VOCs emitted from tomato plants treated with C. fumosorosea (YNKM210801) compared to control plants. Asterisks (*) denote significant differences between control and C. fumosorosea (YNKM210801)-treated plants at each time point (t-test, *: p < 0.05 and **: p < 0.01). Data are presented as means ± SE.
Figure 4. Emission kinetics of VOCs following C. fumosorosea (YNKM210801) treatment: (A–C) Temporal patterns of highly abundant VOCs emitted from tomato plants treated with C. fumosorosea (YNKM210801) compared to control plants. Asterisks (*) denote significant differences between control and C. fumosorosea (YNKM210801)-treated plants at each time point (t-test, *: p < 0.05 and **: p < 0.01). Data are presented as means ± SE.
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Figure 5. C. fumosorosea (YNKM210801) treatment induces phytohormone accumulation in tomato plants: (A–C) Phytohormone concentrations in control and C. fumosorosea (YNKM210801)-treated tomato plants (n = 5 plants per treatment). Means between treatment groups were compared using T-tests. Asterisks (*) indicate significant differences between control and treated plants within each treatment (t-test; *: p < 0.05). Bars represent means ± standard error.
Figure 5. C. fumosorosea (YNKM210801) treatment induces phytohormone accumulation in tomato plants: (A–C) Phytohormone concentrations in control and C. fumosorosea (YNKM210801)-treated tomato plants (n = 5 plants per treatment). Means between treatment groups were compared using T-tests. Asterisks (*) indicate significant differences between control and treated plants within each treatment (t-test; *: p < 0.05). Bars represent means ± standard error.
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Figure 6. Herbivore female moths and their egg parasitoids show differential responses to tomato plant VOCs. (A–C) Choice responses of P. absoluta and (D–F) T. chilonis females to varying concentrations of key synthetic tomato VOCs (n = 100 insects tested/concentration). n-Hexane served as control. Non-responding insects were excluded from analysis. Asterisks (*) indicate significant differences in choice response (χ2 test; *: p < 0.05, **: p < 0.01). ns; non-significant.
Figure 6. Herbivore female moths and their egg parasitoids show differential responses to tomato plant VOCs. (A–C) Choice responses of P. absoluta and (D–F) T. chilonis females to varying concentrations of key synthetic tomato VOCs (n = 100 insects tested/concentration). n-Hexane served as control. Non-responding insects were excluded from analysis. Asterisks (*) indicate significant differences in choice response (χ2 test; *: p < 0.05, **: p < 0.01). ns; non-significant.
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Figure 7. EAG responses of herbivore female moth and egg parasitoid antennae to tomato plant VOCs. (A,B) EAG responses of T. chilonis and P. absoluta females to varying concentrations of (E)-β-caryophyllene and β-phellandrene (n = 3 insects tested/concentration). CK indicates antennal response to n-hexane. Different letters denote significant differences between concentrations within each insect species (p ≤ 0.05, Tukey’s HSD following ANOVA). Bars represent means ± standard error.
Figure 7. EAG responses of herbivore female moth and egg parasitoid antennae to tomato plant VOCs. (A,B) EAG responses of T. chilonis and P. absoluta females to varying concentrations of (E)-β-caryophyllene and β-phellandrene (n = 3 insects tested/concentration). CK indicates antennal response to n-hexane. Different letters denote significant differences between concentrations within each insect species (p ≤ 0.05, Tukey’s HSD following ANOVA). Bars represent means ± standard error.
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Figure 8. Graphical abstract. Endophytic fungal (EPF) treatment reduces herbivore preference and enhances the attraction of their egg parasitoid, mediated by the increased emissions of VOCs and phytohormone inductions. The size of the arrows represents the relative intensity of attraction or repellence.
Figure 8. Graphical abstract. Endophytic fungal (EPF) treatment reduces herbivore preference and enhances the attraction of their egg parasitoid, mediated by the increased emissions of VOCs and phytohormone inductions. The size of the arrows represents the relative intensity of attraction or repellence.
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MDPI and ACS Style

Munawar, A.; Zhang, H.; Zhang, J.; Zhang, X.; Shi, X.-X.; Chen, X.; Li, Z.; He, X.; Zhong, J.; Zhu, Z.; et al. Entomopathogenic Fungus Treatment Affects Trophic Interactions by Altering Volatile Emissions in Tomato. Agronomy 2025, 15, 1161. https://doi.org/10.3390/agronomy15051161

AMA Style

Munawar A, Zhang H, Zhang J, Zhang X, Shi X-X, Chen X, Li Z, He X, Zhong J, Zhu Z, et al. Entomopathogenic Fungus Treatment Affects Trophic Interactions by Altering Volatile Emissions in Tomato. Agronomy. 2025; 15(5):1161. https://doi.org/10.3390/agronomy15051161

Chicago/Turabian Style

Munawar, Asim, Haonan Zhang, Jinyi Zhang, Xiangfen Zhang, Xiao-Xiao Shi, Xuan Chen, Zicheng Li, Xiaoli He, Jian Zhong, Zengrong Zhu, and et al. 2025. "Entomopathogenic Fungus Treatment Affects Trophic Interactions by Altering Volatile Emissions in Tomato" Agronomy 15, no. 5: 1161. https://doi.org/10.3390/agronomy15051161

APA Style

Munawar, A., Zhang, H., Zhang, J., Zhang, X., Shi, X.-X., Chen, X., Li, Z., He, X., Zhong, J., Zhu, Z., Zheng, Y., & Zhou, W. (2025). Entomopathogenic Fungus Treatment Affects Trophic Interactions by Altering Volatile Emissions in Tomato. Agronomy, 15(5), 1161. https://doi.org/10.3390/agronomy15051161

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