Endophytic Beauveria bassiana Blastospores Enhance Susceptibility of Ostrinia furnacalis to Chlorantraniliprole via Modulating Immune-Related Pathways

TL;DR

A fungus called Beauveria bassiana makes corn borer larvae more vulnerable to insecticides by weakening their defenses.

Contribution

Blastospores of B. bassiana enhance insecticide effectiveness by modulating immune pathways in Ostrinia furnacalis.

Findings

Larvae feeding on fungus-colonized maize showed reduced food intake, slower growth, and higher mortality.

Blastospores suppressed protective enzymes and detoxification genes while activating stress-response genes.

Transcriptomic analysis linked increased insecticide susceptibility to downregulated detox genes and upregulated HSP70.

Abstract

The Asian corn borer (Ostrinia furnacalis) is a major pest that causes significant damage to maize crops worldwide. Current control methods rely heavily on chemical insecticides, which can lead to environmental concerns and pest resistance. In this study, we explored an eco-friendly alternative using the beneficial fungus Beauveria bassiana. We found that when corn borer larvae fed on maize plants colonized by the fungal blastospores, they ate less, grew more slowly, and had higher mortality. Importantly, these larvae became more sensitive to the common insecticide chlorantraniliprole, meaning lower doses could be effective. Our findings indicate that the fungus weakens the larval defense system by suppressing protective enzyme activities and detoxification gene expression, while simultaneously activating stress-responsive genes. This research demonstrates that combining endophytic fungi with reduced amounts of chemical insecticides could provide a more sustainable and environmentally friendly approach for managing corn borer pests in agriculture.

Endophytic colonization by entomopathogenic fungi represents a promising sustainable pest management strategy. This study investigated the physiological and molecular effects of maize endophytically colonized by Beauveria bassiana (strain BbOFDH1-5) conidia and blastospores on the Asian corn borer, Ostrinia furnacalis. We evaluated larval development, protective enzyme activities, and subsequent susceptibility to the insecticide chlorantraniliprole, utilizing gut transcriptomic analysis to elucidate the underlying mechanism. Results demonstrated that larvae fed on maize colonized by either spore type, particularly blastospores, exhibited significantly reduced food consumption, body weight, and survival rates. Additionally, the activities of protective enzymes (SOD, POD, CAT) were markedly suppressed. Feeding on blastospore-colonized maize significantly increased larval susceptibility to chlorantraniliprole. Transcriptomic analysis revealed that this enhanced sensitivity is linked to the downregulation of detoxification-related genes, ABCC4, and the upregulation of stress-response genes such as HSP70. These findings confirm that B. bassiana blastospores can synergistically enhance the efficacy of chemical insecticides by impairing larval physiology and suppressing gut-associated resistance mechanisms, providing a novel basis for integrated pest management.

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1. Introduction

Lepidopteran pests (Lepidoptera) pose a serious threat to agriculture, forestry, and stored products worldwide [1,2,3]. Their larval stages cause substantial economic losses and ecological damage primarily through feeding on plant tissues, boring into fruits, and facilitating pathogen transmission [4,5,6]. Prominent examples include Spodoptera frugiperda, Plutella xylostella, and Ostrinia furnacalis [7,8]. Current management strategies for lepidopteran pests rely on chemical control, host-plant resistance breeding, optimized agricultural practices, and biological control. Among chemical approaches, novel insecticides such as diamides have demonstrated high efficacy. Chlorantraniliprole, in particular, has shown excellent control performance against multiple lepidopteran pests, including O. furnacalis, P. xylostella, and Chilo suppressalis [9,10]. However, prolonged dependence on single or multiple classes of chemical insecticides has resulted in serious challenges, most notably the rapid evolution of pest resistance [11,12]. These issues not only undermine the long-term sustainability of pest management programs but also pose significant risks to the environment, non-target organisms, and pest population dynamics [13]. Therefore, biological control has emerged as an increasingly important component of sustainable lepidopteran pest management strategies [14,15,16,17].

Entomopathogenic fungi, particularly Beauveria bassiana, are widely applied as biological control agents against insect pests. The classical infection process involves conidia adhering to the insect cuticle, followed by germination and differentiation into germ tubes, infection pegs, hyphae, or appressoria that penetrate the host integument. After invasion, fungal hyphae proliferate within the hemocoel and secrete toxic metabolites, ultimately leading to host death [18]. Conidia are the primary reproductive structures formed on solid substrates or host surfaces and are highly hydrophobic, facilitating dispersal via air currents or physical contact. However, their production typically depends on solid-state fermentation, which is relatively time-consuming and labor-intensive [19,20]. In contrast, blastospores are produced through liquid fermentation and exhibit more rapid germination [21]. Despite this advantage, direct infection via conidial attachment to the cuticle remains the most widely recognized infection route for B. bassiana [22]. Traditionally, blastospores have been considered poorly suited for external infection due to their low hydrophobicity and limited cuticle adhesion capacity [23], and are therefore rarely used in conventional biological control applications. In practice, the deployment of B. bassiana and other entomopathogenic fungi is further constrained by poor environmental stability, including sensitivity to high temperatures, desiccation, and ultraviolet radiation, which collectively reduce spore survival, infection efficiency, and field persistence. Moreover, suboptimal application technologies limit control consistency, as only a small fraction of sprayed spores successfully contact and infect target insects, while many remain on plant surfaces, increasing the effective cost of pest suppression [24,25,26,27]. Recent studies have demonstrated that both conidia and blastospores of B. bassiana can endophytically colonize plants, forming symbiotic associations that modify plant volatile profiles and influence the oviposition behavior of herbivorous insects [28]. Nevertheless, the consequences of this symbiotic interaction for pest growth and development, particularly its potential to alter susceptibility to chemical insecticides, remain largely unexplored.

Based on this background, we hypothesized that ingestion of B. bassiana could suppress pest growth and development while increasing susceptibility to chemical insecticides, thereby offering a potential strategy to reduce field application rates of synthetic pesticides. To test this hypothesis, larvae of the Asian corn borer, O. furnacalis, were used as the model species. Endophytic B. bassiana–maize systems were established by root drenching with either conidia or blastospores, and larvae were fed leaves from colonized plants. Larval performance was evaluated by measuring food consumption, body weight changes, and survival rates. The combined toxicity of fungal exposure and chemical control was subsequently assessed by treating larvae with the insecticide chlorantraniliprole. In addition, activities of key protective enzymes, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were quantified, and gut transcriptomic analyses were conducted to elucidate the underlying physiological and molecular mechanisms. Therefore, this study aimed to investigate the plant-mediated effects of endophytic B. bassiana (using both aerial conidia and blastospores) on the Asian corn borer (O. furnacalis), and to elucidate the underlying mechanisms that could enhance integrated pest management. The specific objectives were to: (1) evaluate the effects of feeding on B. bassiana-colonized maize on larval growth, development, and protective enzyme activities; (2) assess the combined effect of B. bassiana blastospores and the insecticide chlorantraniliprole on larval survival; and (3) analyze the gut transcriptomic response of larvae to blastospore exposure to identify key molecular mechanisms involved in altered insecticide susceptibility.

2. Materials and Methods

2.1. Materials

The B. bassiana strain used in this study was the wild-type strain BbOFDH1-5 (GenBank accession No. ANFO01). This strain was originally isolated in 2008 from a cadaver of the Asian corn borer (Ostrinia furnacalis) collected in Dehui City, Jilin Province, China. The strain is preserved at the China General Microbiological Culture Collection Center (CGMCC; Beijing, China) under accession number CGMCC No. 15673. Maize (Zea mays) plants of the cultivar ‘Ken Nian No. 1’ (KN1) were used in all experiments. Seeds were purchased from Jilin Continental Seed Industry Co., Ltd. (Jilin, China). A laboratory colony of the Asian corn borer, O. furnacalis, was used in this study. This standard susceptible strain was provided by the Institute of Plant Protection, Jilin Academy of Agricultural Sciences (Jilin, China). The technical-grade insecticide chlorantraniliprole (CAP; 98% purity) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

2.2. Preparation of B. bassiana Spore Suspensions

For conidial suspension preparation, B. bassiana strain BbOFDH1-5 was cultured on Potato Dextrose Agar (PDA) plates and incubated at 25 °C under 80% relative humidity in darkness for three weeks. Conidia were harvested from the culture surface and suspended in sterile deionized water containing 0.05% (v/v) Tween-80. The suspension was filtered through a sterile funnel packed with sterile glass wool to remove mycelial debris, followed by two washes with sterile ddH_2_O via centrifugation at 4000 r/min. The final conidial concentration was adjusted to 1 × 10^8^ spores/mL using a hemocytometer. All experiments were conducted using freshly prepared conidial suspensions.

For blastospore suspension preparation, conidia scraped from PDA cultures were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of Sabouraud Dextrose Yeast (SDY) liquid medium. Cultures were incubated on a rotary shaker at 25 °C and 200 r/min for 84 h. The resulting culture broth was filtered through four layers of sterile gauze to remove mycelia and then centrifuged at 12,000 r/min for 15 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in sterile aqueous solution containing 0.05% (v/v) Tween-80, followed by vortexing for 1 min to ensure complete dispersion. Blastospore concentration was adjusted to 1 × 10^8^ spores/mL using a hemocytometer. All experiments employed freshly prepared blastospore suspensions.

2.3. Endophytic Colonization of Maize by B. bassiana and Its Detection

Three treatment groups were established: a non-inoculated control group (Control), a group inoculated by root drenching with a B. bassiana conidial suspension (AC), and a group inoculated by root drenching with a B. bassiana blastospore suspension (BS), following the method described by Sui et al. [29]. Plants in the AC and BS groups were inoculated by applying 20 mL of the corresponding spore suspension (1 × 10^8^ spores/mL) via root drenching once daily for three consecutive days, whereas control plants received an equal volume of 0.05% (v/v) Tween-80 solution. Twenty-four hours after the final drenching, maize leaves were collected to assess endophytic colonization.

Leaf surfaces were sterilized using a five-step surface-sterilization protocol, after which the leaf margins were excised. Nine segments (1 cm^2^ each) were cut from the remaining leaf tissue. After excess surface moisture was removed with sterile filter paper, the segments were placed on culture medium and incubated for 7 days. The emergence and growth of B. bassiana from the leaf tissues were monitored and recorded daily [29]. Each treatment consisted of ten plants with three independent replicates, resulting in a total of 90 plants. The colonization rate was calculated using the following formula:

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2.4. Effects of Endophytic Colonization by Different B. bassiana Spore Types on the Growth and Development of O. furnacalis Larvae

To evaluate the plant-mediated effects of B. bassiana strain BbOFDH1-5 conidia and blastospores on O. furnacalis larvae, three feeding treatments were established: larvae fed leaves from non-inoculated maize plants (Control), maize plants endophytically colonized by B. bassiana conidia (AC), and maize plants colonized by B. bassiana blastospores (BS). Maize seeds were surface-sterilized with 1% (v/v) NaClO and 75% (v/v) ethanol, rinsed thoroughly with sterile water, and germinated at 26 °C in darkness. After germination, seedlings were transplanted into autoclaved potting substrate and maintained in a climate-controlled chamber until reaching the third to fifth leaf stage [29].

For the feeding bioassay, 100 newly hatched larvae were used per treatment group (300 larvae in total). After starvation for 24 h, each larva was individually placed in a centrifuge tube containing a fresh leaf disc from the corresponding treatment group. Leaf discs were replaced every two days, and the remaining leaf material was weighed to determine food consumption. Larval body weight was recorded every five days, and survival and development were monitored until all individuals died. Larvae that died as a result of mechanical injury during handling were excluded from subsequent analyses. Biological effects were assessed by comparing cumulative survival rates, body weight changes, and leaf consumption among the treatment groups.

2.5. Effects of Different B. bassiana Spore Forms on the Activities of Protective Enzymes in O. furnacalis

To assess the effects of different B. bassiana spore forms on protective enzyme activities in O. furnacalis, third-instar larvae were starved for 24 h and subsequently fed maize leaves from the corresponding treatment groups, as described in Section 2.4, in plastic rearing boxes. Three biological replicates were established for each treatment, with 30 larvae per replicate. After 24 h of feeding, larvae were collected, surface-sterilized with 75% (v/v) ethanol, immediately frozen in liquid nitrogen, and stored at −80 °C until analysis. The activities of SOD, POD, and CAT were determined using commercial assay kits (Total Superoxide Dismutase Assay Kit, Cat. No. A001-1-1; Peroxidase Assay Kit, Cat. No. A084-3-1; Catalase Assay Kit, Cat. No. A007-1-1; Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China) according to the manufacturer’s instructions.

2.6. Combined Effects of B. bassiana Blastospores and Chlorantraniliprole on the Survival of O. furnacalis

To evaluate the combined effects of B. bassiana blastospores and chlorantraniliprole at LC_10_, LC_50_, and LC_100_ concentrations on the survival of O. furnacalis larvae on potted maize, a bioassay was conducted following the method of Sun et al. [30]. Eight treatments were established: sterile water control (Control), B. bassiana blastospore-colonized maize alone (BS), chlorantraniliprole at LC_10_ alone (LC_10_), BS combined with LC_10_ chlorantraniliprole (BS + LC_10_), chlorantraniliprole at LC_50_ alone (LC_50_), BS combined with LC_50_ chlorantraniliprole (BS + LC_50_), chlorantraniliprole at LC_100_ alone (LC_100_), and BS combined with LC_100_ chlorantraniliprole (BS + LC_100_).

Experiments were conducted in a controlled greenhouse maintained at 25 ± 1 °C, 70 ± 5% relative humidity, and a 14L:10D photoperiod. Each experimental unit consisted of ten maize seedlings at the five- to seven-leaf stage, with three biological replicates per treatment, yielding a total of 24 experimental units. Healthy third-instar larvae were starved for 24 h and then introduced at a density of five larvae per plant (50 larvae per unit). Following infestation, plants were sprayed with 10 mL of chlorantraniliprole solution at the designated concentrations (LC_10_ = 0.038 mg/L, LC_50_ = 0.123 mg/L, LC_100_ = 0.30 mg/L), while control plants were treated with sterile water [31]. Cumulative larval survival rates were calculated as described in Section 2.4.

2.7. Transcriptomic Analysis of the Gut Response of O. furnacalis to B. bassiana Blastospores

To examine the effects of B. bassiana blastospores on the gut of O. furnacalis, two treatments were established: larvae fed non-inoculated maize leaves (Control) and larvae fed maize leaves endophytically colonized by B. bassiana blastospores (BS). For each treatment, 30 fourth- to fifth-instar larvae were randomly selected, starved for 24 h, and dissected under aseptic conditions on a clean bench to collect gut tissues [32]. Collected samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction.

Total RNA was extracted from gut tissues, and RNA integrity was assessed prior to library construction. RNA sequencing libraries were prepared and sequenced on an Illumina HiSeq platform by Novogene Co., Ltd. (Beijing, China). Three biological replicates were included for each treatment. After quality control, library concentrations were measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and samples were diluted to 1.5 ng/μL. Insert sizes were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and effective library concentrations were accurately quantified by qRT-PCR. Libraries were pooled according to effective concentration and target sequencing depth and subsequently subjected to Illumina sequencing.

Raw sequencing reads were assembled to generate transcript sequences, which were hierarchically clustered using the Corset program to produce a unified reference transcript set for downstream analyses. Differential gene expression analysis was performed using DESeq2 (version 1.20.0). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the clusterProfiler R package (version 3.8.1) based on the identified differentially expressed genes. The sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE316879. To validate the transcriptomic results, the Ostrinia furnacalis actin gene (Actin, muscle) was used as an internal reference, and the relative expression levels of the HSP70 and ABCC4 genes were measured via qRT-PCR following the method described by Sui et al. (2024) [33]. The primer sequences used are listed in the Appendix A.

2.8. Data Analysis

All experimental data were processed and analyzed using R software version 4.4.3 with the stats package. Differences in leaf consumption and larval body weight among treatment groups were evaluated using non-parametric statistical tests. Survival data were analyzed using the Kaplan–Meier method, and differences among survival curves were assessed with the log-rank test. Prior to statistical analyses, underlying assumptions were examined, including normality using the Shapiro–Wilk test and homogeneity of variances using Levene’s test.

3. Results

3.1. Effects of Endophytic Colonization by B. bassiana on the Growth and Development of O. furnacalis Larvae

Both conidia and blastospores of B. bassiana successfully colonized maize plants endophytically following root-drench inoculation. The distinct morphologies of the aerial conidia and blastospores used in this study are shown in Supplementary Figure S1. Re-isolation of the fungus from surface-sterilized leaf tissues cultured on PDA at 24 h post-inoculation confirmed the presence of B. bassiana within plant tissues. Colonization rates reached 36.67% in the AC treatment and 46.67% in the BS treatment (Figure 1A).

Significant differences in cumulative survival were observed among O. furnacalis larvae fed maize leaves from the different treatments (Figure 1B). Survival rates were comparable among groups during the initial observation period but declined significantly in both the AC and BS groups compared with the Control group over time (p < 0.05). The AC group exhibited a lower final cumulative survival rate than the BS group. Consistently, LT_50_ values in the AC and BS groups were reduced by 15.78% and 3.45%, respectively, relative to the Control group.

Feeding on B. bassiana-colonized maize significantly affected larval leaf consumption (Figure 1C). On days 1, 3, and 5, leaf consumption in both AC and BS groups was lower than that in the Control group, with a more pronounced reduction observed in the BS group. Specifically, consumption in the BS group decreased by 31.1%, 40.7%, and 69.75% on the respective days compared with the Control (p < 0.05). By day 7, leaf consumption in the BS group was significantly lower than that in the AC group, and consumption in the AC group also differed significantly from the Control (p < 0.05).

Larval weight gain was markedly suppressed by exposure to B. bassiana blastospores (Figure 1D). Larvae in the BS group exhibited significantly lower body weights than those in the AC group across the first, second, and third instars (p < 0.05). No significant difference in body weight was detected between the Control and AC groups. The strongest inhibitory effect occurred during the second instar, during which larvae in the BS group were 39.86% and 51.79% lighter than those in the Control and AC groups, respectively.

3.2. Effects of Different B. bassiana Spore Forms on the Activities of Protective Enzymes in O. furnacalis

Feeding on maize leaves endophytically colonized by B. bassiana significantly suppressed the activities of key protective enzymes (SOD, CAT, and POD) in O. furnacalis larvae (Figure 2). After 24 h of feeding, SOD activity in third-instar larvae was significantly lower in both the AC and BS groups than in the Control group (p < 0.05). Specifically, SOD activity was reduced by 5.59% in the AC group and by 12.81% in the BS group relative to the Control (Figure 2A). CAT activity also differed significantly among treatments (Figure 2B). Compared with the Control group, CAT activity decreased by 28.47% in the BS group and by 9.33% in the AC group. Moreover, CAT activity in the BS group was 21.11% lower than that in the AC group (p < 0.05). Similarly, POD activity was significantly reduced in both fungal treatment groups relative to the Control (Figure 2C). The magnitude of reduction was 27.43% in the BS group and 22.48% in the AC group; however, no significant difference in POD activity was detected between the AC and BS groups.

3.3. Combined Toxicity of B. bassiana and Chlorantraniliprole

Toxicity assays showed a pronounced enhancement of insecticidal efficacy when B. bassiana blastospores were combined with chlorantraniliprole, particularly at the LC_50_ concentration (Figure 3). On day 2 after treatment, survival rates of O. furnacalis larvae across treatments followed the order: Control > BS > LC_10_ > LC_10_ + BS > LC_50_ > LC_50_ + BS > LC_100_ = LC_100_ + BS (p < 0.0001). The survival rate in the LC_50_ + BS group was 42.65% lower than that observed in the LC_50_-alone group. Consistent with these findings, all larvae in the LC_50_ + BS group died by day 6, whereas mortality in the LC_50_-alone group was complete only by day 8, indicating a marked acceleration of lethal effects in the combined treatment. Accordingly, the LT_50_ value for the LC_50_ + BS group (2.53 ± 0.07 days) was substantially shorter than that for the LC_50_-alone group (3.27 ± 0.13 days). These results indicate that B. bassiana blastospores significantly accelerate chlorantraniliprole-induced mortality at the LC_50_ concentration in third-instar O. furnacalis larvae.

3.4. Transcriptomic Analysis of the Gut Response of O. furnacalis to B. bassiana

De novo transcriptome sequencing of the O. furnacalis gut generated 39.38 Gb of high-quality data, with Q20 and Q30 values exceeding 99% and 97%, respectively, indicating high sequencing reliability. The overall distribution of FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values under different experimental conditions is shown in Figure 4A. GO enrichment analysis was performed for functional classification of all unigenes. Among the 180,934 unigenes obtained, 51,088 were selected and categorized into three major GO domains: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Within the BP category, genes were predominantly enriched in cellular process, metabolic process, biological regulation, regulation of biological process, localization, response to stimulus, multicellular organismal process, and viral process. CC annotations were mainly associated with cellular anatomical entity and protein-containing complex. MF annotations were primarily enriched in binding, catalytic activity, transporter activity, ATP-dependent activity, molecular function regulator activity, structural molecule activity, transcription regulator activity, molecular transducer activity, and molecular adaptor activity (Figure 4B). In parallel, unigene sequences were mapped to the KEGG database, resulting in the assignment of 14,762 unigenes to 245 KEGG pathways. Pathways significantly enriched with DEGs relative to the background were identified (Figure 4C). Differential expression analysis using DESeq2, with thresholds of padj ≤ 0.05 and |log_2_FoldChange| ≥ 1.0, identified 240 DEGs in the BS versus Control comparison, including 176 upregulated and 64 downregulated genes (Figure 4D).

GO enrichment analysis of these DEGs revealed distinct functional patterns between upregulated and downregulated genes. Upregulated DEGs were mainly associated with transporter activity, transmembrane transport, thylakoid, sulfur compound metabolic process, oxidoreductase activity, molecular function regulator activity, and lipid metabolic process (Figure 5A). In contrast, downregulated DEGs were predominantly enriched in transporter activity, transmembrane transport, toxin activity, sulfur compound metabolic process, reproductive process, oxidoreductase activity, hydrolase activity, and carbohydrate metabolic process (Figure 5B).

KEGG pathway enrichment analysis further showed that upregulated DEGs were significantly enriched in pathways including Antigen processing and presentation, IL-17 signaling pathway, Th17 cell differentiation, Phagosome, Viral myocarditis, Pantothenate and CoA biosynthesis, and Folate biosynthesis (Figure 5C). Downregulated DEGs were enriched across a broader range of pathways, including Peroxisome, Vitamin digestion and absorption, Glycosaminoglycan degradation, Antifolate resistance, Galactose metabolism, Starch and sucrose metabolism, Retinol metabolism, Bile secretion, Porphyrin and chlorophyll metabolism, Pentose and glucuronate interconversions, ABC transporters, Amino sugar and nucleotide sugar metabolism, Longevity regulating pathway—multiple species, Drug metabolism—other enzymes, cAMP signaling pathway, Lysosome, and Protein processing in endoplasmic reticulum (Figure 5D).

Validation of the transcriptomic data by qRT-PCR showed that the expression level of HSP70 was significantly upregulated (Figure 5E), while that of ABCC4 was significantly downregulated (Figure 5F) in the BS treatment group compared to the control group (p < 0.001). This agreement between the qRT-PCR and transcriptomic results confirms the reliability and accuracy of the sequencing data.

4. Discussion

Within ecosystems, plants, microorganisms, and insects form complex and tightly interconnected interaction networks. Previous studies have shown that microorganisms can indirectly influence the behavior and fitness of herbivorous insects by modulating plant defense responses and nutritional composition [34]. Consistent with this framework, the present study demonstrates that feeding on maize leaves colonized by different spore types of B. bassiana significantly reduced food consumption, body weight gain, and cumulative survival of O. furnacalis larvae. In parallel, the activities of key protective enzymes (SOD, POD, and CAT) were markedly suppressed, with the strongest effects observed in the blastospore treatment. Moreover, both laboratory bioassays and pot experiments confirmed that larvae feeding on the blastospore–maize symbiotic system exhibited significantly increased susceptibility to chlorantraniliprole, as reflected by reduced tolerance at both LC_10_ and LC_50_ concentrations. Gut transcriptomic analyses further revealed significant downregulation of genes involved in insecticide resistance and detoxification, including ABCC4 and HSP70. These findings indicate that B. bassiana blastospores can enhance the sensitivity of O. furnacalis larvae to chemical insecticides by inhibiting growth and development and suppressing the expression of key resistance-related genes in the gut.

The entomopathogenic fungus B. bassiana produces both conidia and blastospores, each of which has potential applications in biological pest control [32]. Previous research has established that conidia are the primary infectious propagules responsible for insect mortality through cuticular penetration, nutrient competition, toxin production, and host–pathogen interactions [35]. However, this infection route is often constrained under field conditions by environmental factors such as ultraviolet radiation and extreme temperature or humidity, which markedly reduce spore survival and germination [24,25]. In contrast, blastospores are readily produced via liquid fermentation and exhibit greater water solubility, and emerging evidence suggests that they may be better adapted to the insect gut environment, potentially facilitating more efficient infection [36]. Supporting this notion, Lai et al. demonstrated that a B. bassiana variety could infect lepidopteran larvae through both cuticular and intestinal routes [37]. Ingestion of B. bassiana-colonized maize tissue by corn borer larvae may enable fungal infection via the gut, thereby partially circumventing the environmental limitations associated with cuticle-mediated infection [37]. The results of the present study are consistent with this interpretation. Intestinal infection may allow the fungus to avoid direct exposure to external stressors such as temperature fluctuations and ultraviolet radiation [38,39]. The contribution of this gut-mediated infection pathway warrants further experimental verification.

Recent studies have demonstrated that B. bassiana can endophytically colonize plants and form symbiotic associations, thereby exerting biological functions beyond direct pest infection. For example, although O. furnacalis adults preferentially oviposit on maize plants treated with B. bassiana conidia, the survival of their offspring on these hosts is significantly reduced [28]. In addition, maize leaves colonized by either conidia or blastospores have been reported to repel larval feeding and alter olfactory responses in O. furnacalis [40]. Mantzoukas and Grammatikopoulos reported that three species of entomopathogenic fungi enhanced sorghum resistance, leading to increased larval mortality and reduced relative growth rates [41]. Consistent with these observations, our results show that maize leaves colonized by different spore forms of B. bassiana exerted pronounced adverse effects on O. furnacalis larvae, including reduced food intake, inhibited weight gain, and decreased cumulative survival. Compared with conidia, blastospores exhibited a higher colonization rate in maize and produced more pronounced inhibitory effects on larval development. Sui et al. further demonstrated that B. bassiana blastospores can proliferate within Arabidopsis thaliana following colonization, enabling sustained regulation of host plants [42]. This property may explain the stronger effects of the maize–blastospore symbiotic system observed in this study and highlights its potential for developing more stable and efficient fungal-based insect control strategies. The specific mechanisms underlying blastospore colonization and potential penetration within the gut of O. furnacalis warrant further investigation.

In response to environmental stress, insects can activate altered immune responses and initiate a series of defensive reactions [43]. SOD, POD, and CAT play central roles in protecting insects from oxidative damage by maintaining reactive oxygen species at low levels [44]. In this study, feeding on B. bassiana-colonized maize leaves significantly reduced the activities of these protective enzymes in O. furnacalis larvae. Specifically, 24 h after feeding, larvae in both AC and BS groups exhibited markedly lower SOD, CAT, and POD activities compared with the Control group. Such reductions likely impair the ability of larvae to scavenge reactive oxygen species, increasing susceptibility to oxidative damage [45]. Environmental stressors can disrupt oxygen homeostasis in insects and inhibit the synthesis of protective enzymes, thereby weakening physiological adaptability and constraining growth [46,47]. Disruption of the oxidative defense system may further interfere with normal metabolic processes, manifesting as reduced feeding, delayed growth and development, and increased mortality [48]. Endophytic colonization by B. bassiana, potentially through the release of secondary metabolites or direct interactions with gut tissues, may also impair midgut enzyme function and immune signaling pathways, reducing the insect’s capacity to cope with environmental stress [49]. The observed decline in protective enzyme activities reflects not only physiological deterioration at the individual level but also a potential threat to population sustainability, supporting the application of endophytic entomopathogenic fungi as effective biological control agents.

In this context, integrating entomopathogenic fungi with chemical insecticides represents a promising strategy to enhance control efficacy while mitigating environmental impacts [50]. Chemical insecticides such as chlorantraniliprole, an anthranilic diamide, remain highly effective against lepidopteran larvae but increasingly face challenges associated with resistance development and environmental concerns arising from excessive use [51,52,53]. In the present study, combined application of B. bassiana and chlorantraniliprole produced synergistic or additive toxic effects. The fungal exposure significantly shortened the LT_50_ of chlorantraniliprole at the LC_50_ concentration, indicating that co-application can accelerate insecticidal action and potentially reduce the amount of chemical input required. This synergistic interaction is consistent with previous reports, including the enhanced lethality observed with B. bassiana combined with alpha-cypermethrin against Tenebrio molitor [54] and the high mortality achieved in whitefly nymphs using a B. bassiana–neem combination [55]. Beyond conventional external spraying approaches, the strategy employed here exploits the endophytic colonization capacity of B. bassiana, thereby harnessing its multifunctional roles in promoting plant growth, deterring herbivores, and enhancing disease resistance [56,57,58]. Combining endophytic entomopathogenic fungi with chemical insecticides offers a potentially sustainable pest management approach that can improve control efficacy while reducing reliance on chemical pesticides.

To elucidate the mechanisms by which B. bassiana influences the gut of O. furnacalis, a de novo transcriptomic analysis of larval gut tissues was conducted. The results revealed significant downregulation of ABCC4 within the ABC transporter pathway, suggesting that B. bassiana may impair larval survival and pupation by suppressing the expression of resistance-related genes. Increasing evidence indicates that members of the ABCC subfamily play critical roles in mediating insect resistance to toxic compounds [59], and proteins belonging to the ABCB, ABCC, and ABCG families have been widely implicated in insecticide resistance across multiple insect species [60,61]. Silencing of SeABCB1, SeABCB4, and SeABCB9 has been shown to increase insect mortality following insecticide exposure [62]. These findings support our observation that B. bassiana not only reduces the survival of O. furnacalis but may also compromise its chemical resistance capacity. Moreover, ABCC4 has been identified as a key resistance mechanism capable of directly effluxing insecticides such as ivermectin, thereby lowering intracellular concentrations and reducing toxicity [63]. Therefore, downregulation of ABCC4 observed in this study may increase gut sensitivity to insecticides by limiting efflux capacity and enhancing pesticide accumulation.

In addition, suppression of genes associated with the “Drug metabolism—other enzymes” pathway may further contribute to the reduced survival of O. furnacalis following combined exposure to B. bassiana and chemical insecticides. The insect gut represents the primary interface for contact and absorption of dietary xenobiotics and is therefore enriched in diverse biotransformation enzymes that facilitate detoxification [64]. Beyond digestion, gut epithelial cells play important roles in immune recognition and stress responses, and gut tissues are known to express high levels of immune-related genes, including serine proteases, their homologs, and peptidoglycan recognition proteins [65,66]. Infection of the gut by B. bassiana may disrupt these detoxification and immune processes, thereby weakening the insect’s capacity to cope with chemical stressors. Furthermore, previous studies have shown that exposure to the diamide insecticide flubendiamide significantly upregulates HSP70 expression in O. furnacalis, which has been associated with altered insecticide sensitivity [11]. In line with these findings, our study detected significant upregulation of HSP70 in the gut following B. bassiana infection, suggesting that fungal infection may enhance susceptibility to chlorantraniliprole through stress-response pathways. This mechanism may partly explain the shortened LT_50_ observed under combined treatment conditions. These results indicate that HSP70 expression may be linked to insect tolerance to insecticides and could serve as a potential molecular indicator for monitoring resistance development. Notably, the differential expression of both ABCC4 and HSP70 was independently validated by qRT-PCR, with results consistent with the transcriptomic trends, thereby reinforcing the reliability of our sequencing data and the proposed molecular mechanisms.

5. Conclusions

In conclusion, feeding on the B. bassiana–maize symbiotic system significantly inhibited the growth and development of O. furnacalis larvae. This inhibitory effect was associated with suppression of protective enzyme activities (SOD, POD, and CAT) and downregulation of resistance-related genes such as ABCC4, collectively weakening larval defensive and detoxification capacities. Blastospores exerted stronger effects than conidia in suppressing larval feeding and growth, likely attributable to their more rapid germination and higher infection efficiency. Gut transcriptomic analyses further indicated that B. bassiana may enhance larval sensitivity to chemical insecticides through a dual regulatory mechanism involving downregulation of detoxification-associated genes (e.g., ABCC4) and upregulation of stress-response genes (e.g., HSP70). Together, these findings elucidate the physiological and molecular basis underlying the synergistic interaction between B. bassiana and chlorantraniliprole and provide a theoretical framework for the development of integrated microbial–chemical strategies aimed at reducing pesticide inputs in sustainable pest management systems.