AaCyt b Point Mutation and Overexpression of the Alternative Oxidase (AOX) Gene Conferred Moderate to High Level Resistance to Azoxystrobin in Alternaria alternata, the Causal Agent of Ginseng Leaf and Stem Blight Disease

Abstract

Ginseng Alternaria leaf and stem blight (GALSB), caused by Alternaria alternata, poses a severe threat to ginseng cultivation. Although azoxystrobin is a cornerstone fungicide for GALSB management, the emergence of widespread adaptive resistance has severely curtailed its field efficacy. This study integrated molecular, transcriptomic, and genetic approaches to unravel the underlying resistance mechanisms. Targeted gene sequencing and molecular docking revealed that resistant strains harbor a conserved G143A point mutation in the AaCyt b protein. This mutation weakens the azoxystrobin–AaCyt b protein binding affinity by elevating the binding energy from −8.31 to −7.08 kcal/mol. Additionally, comparative transcriptomics and RT-qPCR demonstrated pronounced upregulation of the alternative oxidase gene (AaAOX) and core energy metabolism pathways in resistant strain TYC8-2, with AaAOX expression increasing 4.45–6.91-fold. Fungicidal inhibition of AOX via salicylhydroxamic acid (SHAM) restored fungal sensitivity, increasing azoxystrobin sensitivity by 11.66-fold. Crucially, genetic knockout of AaAOX enhanced sensitivity by approximately 2.7 × 10⁴-fold. Phenotypic assays further established AaAOX as a multifunctional regulator; the AaAOX mutant exhibited attenuated virulence on ginseng leaves and increased sensitivity to oxidative and osmotic stresses (NaCl, H₂O₂, NaAc). The G143A mutation in AaCyt b and the transcriptional overexpression of AaAOX contribute independently to drive azoxystrobin resistance in A. alternata. These findings provide comprehensive mechanistic insights to guide resistance surveillance, rational fungicide application, and precision prevention of GALSB in ginseng cultivation. We conclude that the G143A mutation in AaCyt b and the transcriptional overexpression of AaAOX act independently to drive azoxystrobin resistance in A. alternata. These findings provide comprehensive mechanistic insights to guide resistance monitoring, optimize fungicide applications, and develop precision strategies for GALSB management.

1. Introduction

Panax ginseng C. A. Meyer is a perennial medicinal herb belonging to the Araliaceae family. Valued for abundant bioactive constituents, particularly ginsenosides, ginseng has long been recognized as an economically crucial cash crop that underpins the agricultural industry in Northeast China. Nevertheless, large-scale commercial cultivation of ginseng is severely constrained by a wide range of above- and belowground phytopathogenic diseases. Among these biotic stressors, ginseng Alternaria leaf and stem blight (GALSB), a destructive foliar disease primarily caused by Alternaria alternata, A. panax, and A. tenuissima [1], represents the most dominant aboveground disease affecting ginseng plants worldwide. GALSB has consistently caused substantial economic losses to the ginseng industry: this disease normally triggers a 20–30% annual yield reduction [2,3,4], and severe epidemic outbreaks can result in up to 70% yield loss, irreversible plant death, and deteriorated commercial quality of ginseng products [2,5].

Currently, chemical fungicides remain the primary strategy for controlling GALSB, with quinone outside inhibitor (QoI) fungicides, such as azoxystrobin, kresoxim-methyl, and pyraclostrobin, being the most extensively used. QoI fungicides exert specific antifungal effects by targeting the quinone oxidation (Qo) pocket of cytochrome b (Cyt b) within mitochondrial complex III. The binding interaction interferes with the electron transfer process from ubiquinol to cytochrome c1, thereby disrupting core cellular respiratory functions and ultimately inhibiting fungal growth [6,7]. Due to their single-site mode of action against pathogens, QoI fungicides impose a moderate-to-high risk of resistance emergence. Fungal resistance to QoI fungicides is predominantly governed by two well-documented molecular mechanisms: point mutations in the Cyt b gene and transcriptional overexpression of the AOX gene, which encodes a terminal oxidase responsible for initiating the alternative respiratory pathway [8]. Within the Alternaria genus, the amino acid substitution of glycine to alanine at the 143rd residue (G143A) of the Cyt b protein is a typical mutation tightly correlated with high-level QoI resistance. This G143A mutation also serves as a conserved resistance marker across diverse phytopathogens, including Blumeria graminis, Sphaerotheca fuliginea, Plasmopara viticola, and Pseudoperonospora cubensis [7,9,10,11]. Beyond G143A, multiple other Cyt b mutations have been verified to confer tolerance to azoxystrobin in phytopathogenic fungi. These resistance-related mutations cover single-point variants (F129L, G137R, G137S, L299F, L275F) and combined mutation haplotypes (N256S + L299F and L275F + L299F), which have been reported in Pyrenophora tritici-repentis and A. solani [12,13,14,15,16].

As a cyanide-resistant terminal oxidase embedded in the inner mitochondrial membrane, AOX acts as a core component of the alternative respiratory pathway. It stabilizes mitochondrial function and maintains intracellular metabolic homeostasis under respiratory inhibition by rerouting redundant electrons from the blocked mainstream respiratory chain [17,18,19]. The stress-alleviating and resistance-regulating functions of AOX have been validated in multiple fungal species. For instance, enhanced AOX expression rescues impaired electron transport, sustains mitochondrial membrane potential, and stabilizes intracellular ATP levels in Candida albicans confronted with respiratory stress [20]. Similarly, the upregulation of AOX contributes to pyraclostrobin resistance in Lasiodiplodia theobromae [21], and genetic deletion of AOX significantly increases the sensitivity of F. graminearum to pyraclostrobin [11].

Although prior studies have evaluated the biological sensitivity of field-isolated A. panax strains to azoxystrobin in ginseng-growing regions [22], the resistant level and molecular regulatory mechanisms responsible for azoxystrobin resistance in ginseng-derived A. alternata remain poorly defined. Specifically, functional evidence linking Cyt b target-site mutations and AOX-dependent alternative respiration to QoI resistance is still absent in this specialized ginseng–Alternaria pathosystem.

Additionally, the global metabolic adaptive responses that enable A. alternata to survive under QoI fungicide stress have yet to be comprehensively elucidated. Given the over-reliance on QoI fungicides in ginseng production and the high inherent risk of resistance development in pathogenic fungi, uncovering the resistance mechanisms is indispensable for achieving long-term and sustainable GALSB management. Accordingly, the present study focuses on characterizing the roles of AaCyt b target-site variations and AOX-mediated bypass respiration in azoxystrobin resistance, as well as exploring the transcriptomic and metabolic signatures associated with fungal resistance phenotypes.

The findings of this study will systematically decipher the dual regulatory mechanisms of target-site mutation and AOX-mediated non-target resistance in A. alternata, providing a solid theoretical basis for resistance surveillance, optimized field disease management, and the development of targeted and sustainable control strategies against GALSB.

2. Materials and Methods

2.1. Fungicides and Strains

Three QoI fungicides, azoxystrobin (98% technical concentrate, TC), kresoxim-methyl (97% TC), and pyraclostrobin (96% TC) produced by Hebei Ruiyao Biotechnology (Hebei, China); picoxystrobin (96% TC) produced by Jiangsu Fubiya Chemical (Jiangsu, China), were dissolved in acetone to prepare 1 × 10⁴ μg/mL stock solutions. Salicylhydroxamic acid (SHAM, 99% TC, Shanghai Jieshikai Biotechnology, Shanghai, China) was dissolved in 70% ethanol to make the same concentration stock solutions. Above fungicides and SHAM had 0.1% (v/v) Tween-80 added prior to use and were stored at 4 °C for subsequent assays.

Between 2018 and 2022, 140 Alternaria alternata (Aa) strains were isolated from ginseng-producing regions in Jilin Province (Tonghua, Baishan, Jilin, Changchun, and Dunhua Cities). The frequency distribution of Alternaria alternata EC₅₀ values of 140 A. alternata strains to azoxystrobin did not follow a normal distribution (Shapiro–Wilk test, W = 0.946, p ˂ 0.001) (Figure S1). Based on the sensitivity baseline of A. alternata to azoxystrobin (25.83 μg/mL) established by Huang Yanfei (2016) [23], resistance levels of sensitive, moderately resistant, and highly resistant for Alternaria alternata strains were classified according to resistance factor (RF) values [24]. The frequency of resistance levels among the 140 strains is shown in Table S1. In 2024, seven azoxystrobin-resistant mutant strains, including a highly resistant strain (TYC8-1) and moderately resistant strains (TYC8-2, TYC4-1, TYC4-2, TYC4-3, TJN12-1, TJN5-1), and two sensitive strains (TJH1-16-1, TND1-8.2-1) were generated from six wild-type strains (YC8, YC4, JN12, JN5, JH1-16, ND1-8.2) via azoxystrobin acclimatization and UV mutagenesis (Table S2). All strains were preserved at −20 °C using the filter paper block method in the Laboratory of Green Prevention and Control for Chinese Herbal Medicine Diseases, Jilin Agricultural University.

2.2. Detection of Point Mutations in AaCyt b Gene of A. alternata Strains

The AaCyt b gene sequences (ID: CC77DRAFT_1010553) were retrieved from the genomes of 15 A. alternata strains (7 resistant mutants, 2 sensitive strains, and 6 wild-type strains). PCR primers (Table S2) were designed using Primer 5.0 and synthesized by Sangon Biotech (Shanghai, China). Genomic DNA from A. alternata was used as the template for PCR amplification of AaCyt b using primers ACytb-F/ACytb-R. The 25 μL PCR system included 12.5 μL 2× Phanta Max Master Mix, 1 μL each primer (10 μM), 1 μL template DNA (100 ng), and 9.5 μL ddH₂O. Thermal cycling conditions were 95 °C for 3 min; 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; final extension at 72 °C for 5 min. Amplicons of the expected size were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Sequences were assembled using Chromas Pro 2.1.3 (weak terminal signals trimmed) and aligned with DNAMAN 9.0 to identify mutations.

2.3. Molecular Docking Experiment of Azoxystrobin with AaCyt b Protein

The amino acid sequence of AaCyt b in A. alternata was obtained from UniProt. Homology modeling of the 3D structure was performed using SWISS-MODEL [25], and the PDB file was downloaded. The initial receptor model was refined via PyMOL 3.7.3. A site-directed mutagenesis was introduced to replace glycine (G) at position 143 with alanine (A) to generate the G143A mutant. Energy minimization and structural optimization were conducted to obtain wild-type and mutant (G143A) AaCyt b receptor models. For molecular docking, the azoxystrobin ligand was prepared using ChemDraw 19 and Chem3D, and the receptor was processed with AutoDock Tools 1.5.7. Flexible docking was performed using AutoDock 4.2.6 (100 runs) with Quick Vina-W to define the binding site [26]. Binding affinity was evaluated via binding energy, and the optimal conformation was selected.

2.4. Azoxystrobin-Resistant A. alternata RNA-Seq Analysis

To elucidate the resistance mechanism, RNA-Seq was performed on two resistant, genetically stable strains (TYC8-1, TYC8-2) and their parental wild-type strain (YC8). Strains were inoculated into 100 mL potato dextrose broth (PDB) and treated with azoxystrobin (100 μg/mL) in triplicate. Cultures were incubated at 25 °C in the dark with shaking (160 rpm) for 48 h and 60 h. Mycelia were harvested, washed with ddH₂O, vacuum-dried, and sent to Novogene (Beijing, China) for RNA extraction, library construction, and sequencing.

Differential gene expression (DGE) analysis was conducted using DESeq2 (alignment to the reference genome, expression quantification, and statistical testing). GO functional enrichment was performed with GOseq, and KEGG pathway annotation was achieved via KEGGscape. Protein–protein interactions (PPIs) of DEGs were predicted using the STRING database, and core modules/hub proteins were identified via Cytoscape 3.10.3 with the MCODE 2.0.3 plugin [27]. Pathview 1.42.0 (Bioconductor 3.19, 2024) and KEGGscape 0.9.2 were used to identify resistance-associated genes.

2.5. Detecting AaAOX and AaCyt b Expression in A. alternata Resistant Mutants and Parental Strains

RT-qPCR was performed to quantify the transcript levels of AaAOX in A. alternata resistant mutants (TYC8-1, TYC8-2, TYC4-1, TYC4-2, TYC4-3, TJN12-1, TJN5-1, TJH1-16-1, TND1-8.2-1) and their corresponding parental strains (YC8, YC4, JN12, JN5, JH1-16, ND1-8.2). Fungal mycelia were first cultured on potato dextrose agar (PDA) at 25 °C for 5 days. Subsequently, 8 mm mycelial plugs were transferred into 100 mL potato dextrose broth (PDB) supplemented with 100 μg/mL azoxystrobin or an equal volume of solvent (blank control), with three biological replicates for each treatment. After incubation for 60 h at 25 °C in the dark with shaking at 160 rpm, total RNA was isolated using the RNAprep Pure Plant Kit (DP441, Tiangen Biotech, Beijing, China). The quantitative RNA was reverse-transcribed into first-strand cDNA using a reverse transcription kit (Tolo Biotech, Shanghai, China).

The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH, ID: CC77DRAFT_1011328) was used as the internal reference gene. Gene-specific primers for AaAOX, AaCyt b and AaGAPDH were designed and synthesized (Table S3). The RT-qPCR reaction was carried out in a 20 μL reaction system: 10 μL 2× SYBR Green qPCR Master Mix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 1 μL cDNA template, and 8.0 μL RNase-free ddH₂O.

The qPCR amplification conditions were set as follows: initial denaturation at 95 °C for 30 s; followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. A melting curve analysis was performed from 65 °C (1 min) to 95 °C (15 s) to verify the specificity of amplified products. Each sample was assayed with three technical replicates. The relative gene expression levels of AaAOX and AaCyt b were calculated using the 2^−ΔΔCt method.

2.6. Effect of AaAOX Inhibitor SHAM on the Sensitivity of A. alternata to Azoxystrobin

To validate the role of AaAOX in resistance, the mycelial growth rate method [28] was used to assess the sensitivity of 140 A. alternata strains to azoxystrobin (0, 1, 10, 50, 100, 500, and 1000 μg/mL) alone or combined with SHAM (150 μg/mL). Plates were incubated at 25 °C for 7 days, and colony diameters were measured. Inhibition rates were calculated as Formula (1), and EC₅₀ values were further calculated via SPSS (version 27.0).

Relative inhibition rate (%) = 100 − ((colony diameter of treatment group − disk diameter)/(colony diameter of control group − disk diameter)) × 100

(1)

Changes in sensitivity across strains were compared to clarify the correlation between AaAOX upregulation and azoxystrobin resistance.

2.7. Functional Verification of AaAOX in A. alternata

2.7.1. Construction of AaAOX Knockout and Complementation Vectors

The AaAOX gene sequence was extracted from the published reference genome, and the corresponding flanking sequences were mapped using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 15 March 2024) alignment. Specific primers for amplifying the upstream and downstream homologous arms, hygromycin resistance cassette, and gene complementation fragments were designed with Primer Premier 5.0 software (Table S3). Genomic DNA isolated from the wild-type (WT) strain was adopted as the template for polymerase chain reaction (PCR) amplification. All amplified PCR products were subsequently purified using a commercial DNA gel extraction kit according to the manufacturer’s instructions.

The standard 50 μL PCR system was configured as follows: 25 μL of 2× TransStart FastPfu Fly PCR SuperMix, 1 μL of each primer (10 μM), 1 μL of template DNA (100 ng), and nuclease-free water to a final volume of 50 μL. The cycling protocol consisted of an initial denaturation at 98 °C for 1 min, followed by 35 cycles of 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 30 s. A final extension was conducted at 72 °C for 1 min, and the reaction was maintained at 4 °C for temporary preservation.

Homologous recombination vectors were constructed via a yeast-mediated cloning approach. For genetic transformation, conidial suspension of the WT strain at a concentration of 1 × 10⁶ conidia/mL was co-cultivated with Agrobacterium tumefaciens (GV3101) solution (OD₆₀₀ = 0.8) at 22 °C under dark conditions for 36 h. Positive knockout transformants were screened on PDA medium supplemented with 50 μg/mL hygromycin, while complementary transformants were selected on PDA medium containing 5 μg/mL azoxystrobin. All putative transformants were further validated by PCR amplification targeting the AaAOX gene and hygromycin resistance cassette, and the transcriptional expression level of the AaAOX gene was quantified via RT-qPCR.

2.7.2. Phenotypic Analysis of Transformants

Fungicide sensitivity: Transformants and WT were inoculated on PDA amended with picoxystrobin (1000, 10 μg/mL), azoxystrobin (10, 0.1 μg/mL), kresoxim-methyl (1 μg/mL), and pyraclostrobin (0.1 μg/mL). Colony diameters were measured after 7 days (25 °C), and sensitivity was compared.

Pathogenicity: Healthy 4-year-old Panax ginseng (cv. Damaya) leaves were disinfected and inoculated with transformants/WT. Lesion development was observed after 7 days of moist incubation (25 °C).

Stress tolerance: Mycelial plugs (7-day-old) were inoculated on PDA amended with 1 M NaCl, 2.4 mM H₂O₂, 0.05% Congo red (CR), 1.5 M sorbitol (SO), 0.05% SDS, 1.2 M KCl, or 1 M NaAc. Colony morphology and growth rates were assessed after 7 days (25 °C) to evaluate stress responses.

2.8. Statistical Analysis

Microsoft Excel 2021, PilotEdit Pro 2013, and TBtools 2022 were used for data organization and analysis. Graphs were generated with Origin 2025. Statistical analyses were performed in SPSS (version 27.0). Data are presented as mean ± SD. One-way analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) test were used to analyze significant differences. All treatments were performed in three biological replicates.

3. Results

3.1. AaCyt b Gene Mutations in Partial Azoxystrobin-Resistant Mutants

Sequence alignment of the AaCyt b gene in A. alternata between azoxystrobin-resistant mutants and their parental strains revealed that four resistant mutants harbored identical target-site amino acid substitutions (Figure 1A–C). Specifically, the highly resistant (HR) strain TYC8-1 and moderately resistant (MR) strains TYC4-1, TYC4-2, and TJN12-1 carried a nucleotide substitution at codon 143 (GGT→GCT), resulting in a glycine-to-alanine (G143A) change in the encoded AaCyt b protein. A silent mutation was identified in strain TND1-8.2-1 at codon 145 (ACA→ACT), which did not alter the amino acid sequence. No target-site mutations were detected in the remaining resistant mutants.

3.2. Decreased Binding Affinity Between Azoxystrobin and AaCyt b Protein Due to G143A Mutation

In the wild-type strain YC8, the AaCyt b protein forms a tight complex with azoxystrobin through specific amino acid residues within a hydrophobic pocket. This interaction stabilizes six hydrogen bonds with the pyrimidine ring, a critical structural moiety of azoxystrobin, resulting in a predicted binding energy of −8.31 kcal/mol. In the resistant strain TYC8-1, the G143A substitution replaces glycine with alanine; the methyl group of alanine interferes with the native binding mode, reducing hydrogen bonding interactions within the pocket and disrupting the para-positional π-π conjugation. Consequently, the predicted binding energy decreases from −8.31 to −7.08 kcal/mol (Figure 1D,E). Collectively, these structural and energetic changes diminished hydrogen bonding, impaired π-π conjugation, and had a less favorable (higher) binding energy, significantly weakening azoxystrobin affinity for the AaCyt b protein in TYC8-1, thereby conferring azoxystrobin resistance in A. alternata.

3.3. Upregulation of AaAOX Gene and Key Energy Metabolism-Related Genes in Resistant Strains

Transcriptome profiles at 48 h and 60 h exhibited consistent trends; thus, sequencing data from both time points were integrated for combined analysis. Differential expression analysis was performed using DESeq2 across three pairwise comparisons: YC8 vs. TYC8-2, YC8 vs. TYC8-1, and TYC8-2 vs. TYC8-1. Genes with |log₂FoldChange| > 2 and False Discovery Rate (FDR) adjusted p-value < 0.05 were designated as differentially expressed genes (DEGs; Table S4; Figures S2 and S3; NCBI BioProject database under accession number PRJNA1477664). Among these comparisons, 1089 DEGs (450 upregulated, 639 downregulated) were identified in YC8 vs. TYC8-2; 863 DEGs (389 upregulated, 474 downregulated) in YC8 vs. TYC8-1; and 926 DEGs (374 upregulated, 552 downregulated) in TYC8-2 vs. TYC8-1. Volcano plots revealed substantial transcriptional divergence between the two resistant strains (TYC8-2 and TYC8-1; Figure 2A–C), while heatmap analysis demonstrated greater similarity between the two resistant strains than between either and the wild-type strain YC8 (Figure 2D). Collectively, these results suggest that distinct resistant mutants may harbor shared resistance-associated genes.

GO enrichment analysis of differentially expressed genes (DEGs) revealed distinct functional enrichment patterns between the two comparison groups. In the YC8 vs. TYC8-1 group, DEGs were significantly enriched in: (1) molecular functions, including cofactor binding and iron ion binding; (2) cellular components, such as the extracellular region and plasma membrane; and (3) biological processes, particularly organic acid metabolism and α-amino acid metabolism. In contrast, the YC8 vs. TYC8-2 group showed DEGs enriched in molecular functions (e.g., transmembrane transporter activity, carbon-carbon lyase activity, coenzyme binding), cellular components (e.g., actin cytoskeleton, mitochondrial membrane), and associated biological processes (Figure 3A,B).

KEGG pathway enrichment analysis revealed distinct metabolic pathway involvements between the two comparisons. In the YC8 vs. TYC8-1 group, DEGs were significantly enriched in pathways including secondary metabolite biosynthesis, glycolysis (EMP)/gluconeogenesis, pyruvate metabolism, and fatty acid degradation. For the YC8 vs. TYC8-2 group, DEGs were enriched in secondary metabolite biosynthesis, carbon metabolism, glycolysis (EMP)/gluconeogenesis, and amino acid metabolism. Notably, both comparisons shared prominent enrichments in secondary metabolism, carbon metabolism, and glycolysis (EMP)/gluconeogenesis, key pathways linked to azoxystrobin resistance (Figure 3C,D).

Protein–protein interaction (PPI) network analysis revealed distinct alterations in metabolic processes. Compared with the wild-type YC8, TYC8-2 exhibited significant changes in glycolysis (EMP)/gluconeogenesis, secondary metabolite biosynthesis, carbon metabolism, and pyruvate metabolism. Relative to TYC8-1, TYC8-2 displayed marked differences in biological oxidation, glutathione metabolism, and EMP-associated metabolic pathways (Figure 3E,F).

In the STRING-derived network (confidence score ≥ 0.90), proteins linked to the EMP pathway accounted for 40.3% (31/77) of interaction nodes in the TYC8-2 vs. YC8 comparison. Notably, key upregulated enzymes included phosphoenolpyruvate carboxykinase (CC77DRAFT_1021809, 5.5-fold), hexokinase (CC77DRAFT_931421, 3.1-fold), malate synthase (TCA cycle; CC77DRAFT_1056777, 4.3-fold), 4-hydroxyphenylpyruvate dioxygenase (CC77DRAFT_950966, 20.0-fold), and AaAOX (CC77DRAFT_1013694, 5.2-fold).

For the TYC8-2 vs. TYC8-1 comparison, proteins associated with biological oxidation constituted 40.3% (29/72) of nodes. Significantly upregulated genes involved in glutathione metabolism included glutathione reductase (CC77DRAFT_1096877, 3.6-fold), gamma-glutamylcyclotransferase (CC77DRAFT_1060450, 3.2-fold), and glutathione synthetase (CC77DRAFT_1021015, 2.7-fold). Additionally, a thioredoxin-like protein (CC77DRAFT_77574, 4.8-fold), tyrosine metabolism-associated 4-hydroxyphenylpyruvate dioxygenase (CC77DRAFT_950966, 4.9-fold), and AaAOX (CC77DRAFT_1013694, 1.7-fold) were also induced.

Figure 4 and Figure S9 show gene expression changes in azoxystrobin-resistant mutants TYC8-1 and TYC8-2, emphasizing divergent energy metabolism patterns between resistant and wild-type (YC8) strains. Key genes in the EMP pathway and TCA cycle, including phosphoenolpyruvate carboxykinase (CC77DRAFT_1021809) and malate synthase (CC77DRAFT_1056777) were significantly upregulated in both TYC8-1 vs. YC8 and TYC8-2 vs. YC8 comparisons. AaAOX expression (CC77DRAFT_1013694) was markedly higher in TYC8-2 than in YC8 (5.2-fold upregulation) or TYC8-1 (1.7-fold upregulation). This pattern aligned with changes in other core metabolic genes: EMP pathway hexokinase (CC77DRAFT_931421; 3.1-fold vs. YC8, 1.4-fold vs. TYC8-1), TCA cycle isocitrate lyase (CC77DRAFT_938842; 4.8-fold vs. YC8, 1.3-fold vs. TYC8-1), and glutamate dehydrogenase (CC77DRAFT_938842; 3.1-fold vs. YC8, 1.7-fold vs. TYC8-1).

3.4. Upregulated AaAOX Expression in Azoxystrobin-Resistant Mutants

RT-qPCR analysis detected significant differential expression of the AaAOX gene between mutants (TYC8-1, TYC8-2, TYC4-1, TYC4-2, TYC4-3, TJN12-1, TJN5-1, TJH1-16-1, TND1-8.2-1) and their corresponding parental strains (YC8, YC4, JN12, JN5, JH1-16, ND1-8.2) (Figure 5A). Upon treatment with 100 μg/mL azoxystrobin, the six parental strains exhibited an average AaAOX upregulation of 1.72-fold (range: 1.32–3.26), whereas the nine mutants (seven resistant and two sensitive mutants) showed a more pronounced increase (average: 3.24-fold; range: 1.67–5.47). Notably, in the azoxystrobin-treated group, AaAOX expression in resistant mutants was 3.76-fold higher than in their respective parents (range: 2.16–6.91), strengthening the evidence for a robust correlation between AaAOX upregulation and azoxystrobin resistance. By contrast, AaCyt b transcript levels remained relatively stable: only TYC8-1 (1.25-fold) and TYC4-2 (1.10-fold) displayed slight upregulation compared to their parents, with no significant differences observed in the remaining strains (Figure 5B). Collectively, these results demonstrate that azoxystrobin resistance is strongly associated with AaAOX upregulation, whereas AaCyt b expression changes slightly and contributes minimally to the resistance phenotype.

3.5. Increased Sensitivity of A. alternata to Azoxystrobin After SHAM Addition

To elucidate the mechanism underlying geographical differences in resistance, the sensitivity of 140 A. alternata strains to azoxystrobin was evaluated in the presence of 150 μg/mL salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidase (AOX). Co-treatment with SHAM significantly reduced the EC₅₀ values for all strains, confirming the involvement of AaAOX in mediating resistance. However, the extent of this reduction varied significantly among geographic origins (

Table 1. Comparative antifungal activity of azoxystrobin (Azo) against Alternaria alternata strains from geographically diverse regions, with and without SHAM supplementation.

Geographic Origin	Number of Strains	EC50 (μg/mL) (Azo)		EC50 (μg/mL) (Azo + SHAM)		Fold Decrease in EC50
		Range	Mean ± SD *	Range	Mean ± SD *
Baishan City	54	7.4811~296.7761	99.0426 ± 45.6339 a	0.0352~34.1890	8.4198 ± 15.0470 b	11.76
Dunhua City	14	2.2030~254.8740	103.5884 ± 75.2379 a	0.2379~45.4485	18.6287 ± 15.5597 a	5.56
Jilin City	7	3.3902~208.8421	115.2909 ± 71.8518 a	0.2500~9.3677	4.0582 ± 3.3024 ab	28.41
Tonghua City	36	0.8127~338.3047	99.6048 ± 83.0539 a	0.0331~32.1715	6.7790 ± 7.4204 b	14.70
Changchun City	29	0.3284~232.2230	98.8807 ± 78.0944 a	0.0016~50.5887	7.5120 ± 10.7372 b	13.16
Total	140	0.3284~338.3047	100.4206 ± 67.2701	0.0016~50.5887	8.6126 ± 12.6023	11.66

* Based on Tukey’s HSD (honestly significant difference) test (p < 0.05), values sharing the same letter are not significantly different.

). Strains from Jilin City exhibited the most dramatic decrease in EC₅₀ (28.41-fold), which was significantly higher than the 5.56-fold reduction observed in strains from Dunhua City (p < 0.05). Furthermore, the overall average reduction across all tested strains was 11.66-fold. Specifically, strains from Baishan, Jilin, Tonghua, and Changchun cities showed significantly lower post-SHAM EC₅₀ values compared to those from Dunhua City, suggesting that the reliance on AOX-mediated resistance is unevenly distributed across different geographical populations.

3.6. Effects of Alternative Oxidase Gene (AaAOX) on the Biological Functions of A. alternata

3.6.1. Screening and Verification of Transformants

To generate and validate knockout and complemented mutant strains of AaAOX gene, we used potato dextrose agar (PDA) medium supplemented with 50 μg/mL hygromycin B and 5 μg/mL azoxystrobin. Quantitative real-time PCR (qPCR) was performed to compare AaAOX expression levels among the wild-type (WT) strain, knockout mutants (ΔAOX-4, ΔAOX-6), and the complemented mutant (ΔAOX-C). Results indicated that AaAOX expression was nearly undetectable in the knockout mutants ΔAOX-4 and ΔAOX-6. In contrast, the complemented mutant ΔAOX-C displayed no significant difference in AaAOX expression compared to the WT, with its expression level reaching 98% of the WT value (Figures S4–S8).

3.6.2. Increased Sensitivity of AaAOX Gene Knockout Mutants to Azoxystrobin

Sensitivity assays demonstrated that the azoxystrobin sensitivity of ΔAOX-4 and ΔAOX-6 was 2.7296 × 10⁴ to 3.6394 × 10⁴ fold higher than that of the WT (Figure 6A;

Table 2. Sensitivity profiles of wild type, ΔAOX-4, ΔAOX-6, and ΔAOX-C strains of Alternaria alternata to azoxystrobin.

Strain	Type	Toxicity Regression Equation	EC50 (μg/mL)	r
WT	Wild	y = 0.2762x + 4.4526	98.2656	0.9695
ΔAOX-4	Mutant	y = 0.5500x + 6.3614	0.0036	0.9678
ΔAOX-6	Mutant	y = 0.6845x + 6.7738	0.0027	0.9834
ΔAOX-C	Mutant	y = 0.2773x + 4.4779	102.1629	0.9810

). Notably, the complemented mutant ΔAOX-C showed no significant difference in sensitivity compared to the WT. These results, where knockout mutants exhibited drastically increased sensitivity while complemented mutants retained wild-type sensitivity, strongly validate that the AaAOX gene plays a critical role in mediating azoxystrobin resistance, directly influencing the fungal sensitivity to this fungicide.

3.6.3. AaAOX Gene Affects the Response of Strains to Multiple Stress Conditions

Consistent with Figure 7, ΔAOX knockout mutants displayed increased sensitivity to NaCl, H₂O₂, Congo red (CR), and NaAc relative to the wild-type (WT) strain. Complemented mutants (ΔAOX-C) showed no significant sensitivity differences from the WT under these stresses. These findings suggest that beyond its established role in azoxystrobin resistance-associated metabolic pathways, the AaAOX gene is critical for mitigating osmotic, peroxide, and toxic substance stresses (Table S5).

3.6.4. Increased Sensitivity of AaAOX Gene Knockout Mutants to Different Fungicides

As shown in Figure 6B, the ΔAOX-4 knockout mutant displayed significantly increased sensitivity to four fungicides, picoxystrobin, azoxystrobin, kresoxim-methyl, and pyraclostrobin, relative to the wild-type (WT) strain. Notably, the complemented mutant (ΔAOX-C) showed no significant differences in sensitivity to these fungicides compared to the WT (p > 0.05;

Table 3. Mycelial growth of WT, ΔAOX-4, and ΔAOX-C strains of Alternaria alternata under different fungicide treatments on PDA 25 °C for 7 d.

Fungicides	Concentration (μg/mL)	Mycelial Growth (mm)
		WT *	ΔAOX-4 *	ΔAOX-C *
CK	0.0	62.50 ± 1.25 a	62.05 ± 1.50 a	61.75 ± 2.00 a
Picoxystrobin	1000.0	32.50 ± 2.00 a	8.00 ± 0.00 b	31.50 ± 1.50 a
	10.0	51.00 ± 1.75 a	16.50 ± 0.50 b	49.75 ± 2.50 a
Azoxystrobin	10.0	28.50 ± 1.75 a	8.00 ± 0.00 b	27.50 ± 2.50 a
	0.1	57.65 ± 1.50 a	14.50 ± 1.50 b	56.75 ± 1.00 a
Kresoxim-methyl	1.0	44.55 ± 2.75 a	20.75 ± 2.50 b	45.50 ± 1.75 a
Pyraclostrobin	0.1	51.50 ± 1.50 a	8.00 ± 0.00 b	52.00 ± 3.75 a

* Mycelial growth is presented as mean ± standard deviation (n = 9). Based on Tukey’s HSD (honestly significant difference) test (p < 0.05), values sharing the same letter are not significantly different.

).

3.6.5. Reduced Pathogenicity of AaAOX Gene Knockout Mutants to Ginseng Leaves

As shown in Figure 8, the ΔAOX-4 knockout mutant exhibited significantly reduced pathogenicity compared to both the wild-type (WT) and complemented (ΔAOX-C) strains. At 7 days post-inoculation (dpi), ΔAOX-4-inoculated leaves displayed only marginal chlorosis and fading at the mycelial plug edge, forming small, localized lesions. Consistently, ΔAOX-4 showed diminished mycelial growth vigor relative to the WT.

4. Discussion

QoI (quinone outside inhibitor) fungicides are widely recognized for their safety, broad-spectrum activity, and high efficacy in controlling plant diseases. However, their single-site mode of action renders plant pathogens prone to rapid resistance development. Alternaria spp., the primary causal agent of ginseng Alternaria leaf and stem blight (GALSB), readily develops azoxystrobin resistance [22], yet the underlying resistance mechanisms of A. alternata to azoxystrobin remain poorly understood. Our results demonstrate that the principal mechanism of azoxystrobin resistance in A. alternata involves a G143A mutation in the AaCyt b gene, which reduces azoxystrobin binding to its target protein. Notably, resistant strains lacking this mutation exhibited significantly elevated AaAOX expression, which markedly decreased their sensitivity to azoxystrobin. Consistently, AaAOX gene knockout drastically increased sensitivity to azoxystrobin and other QoI fungicides (kresoxim-methyl, pyraclostrobin, and picoxystrobin) while enhancing susceptibility to abiotic stresses (NaCl, H₂O₂, and CR). These findings indicate that target AaCyt b gene mutation is not the sole driver of azoxystrobin resistance in A. alternata, and AaAOX overexpression also confers resistance to QoI fungicides.

4.1. The Relationship Between Gene Point Mutations, Gene Upregulation, and QoI Resistance in A. alternata

Previous studies have established that QoI resistance commonly arises from point mutations in the Cyt b gene, particularly the glycine-to-alanine substitution at position 143 (G143A), which often confers high-level resistance [7]. Other substitutions, such as phenylalanine-to-leucine (F129L) at position 129 and glycine-to-arginine (G137R) at position 137, can mediate moderate QoI resistance in fungi [15]. In this study, we identified the G143A mutation in the AaCyt b gene of TYC8-1, a highly azoxystrobin-resistant (HR) A. alternata strain. Molecular docking analyses further revealed that the G143A mutation significantly reduces azoxystrobin binding affinity by disrupting hydrogen bonding networks and hydrophobic interactions between azoxystrobin and the pyrimidine ring of the target protein. This confirms that the G143A mutation (glycine-to-alanine substitution at AaCyt b position 143) may be the primary mechanism underlying high-level azoxystrobin resistance in A. alternata. This finding aligns with the resistance mechanism of Glomerella cingulata to pyraclostrobin [29]. Similarly, Johnson et al. (2025) reported that a G143A mutation of Cyt b gene in Colletotrichum chrysophilum significantly enhances QoI resistance [30], and Dorigan et al. (2025) observed G143A mutations in Cyt b of QoI-resistant A. alternata strains causing citrus Alternaria brown spot [31]. Collectively, these studies, combined with our work, confirm that the Cyt b G143A mutation is a key driver of azoxystrobin and QoI resistance in pathogens.

Beyond Cyt b target-site mutations, overexpression of resistance-associated genes is a major fungal fungicide resistance mechanism. In our study, azoxystrobin resistance associated with Cyt b was driven by a single point mutation, and no significant upregulation of AaCyt b gene expression was observed in azoxystrobin-resistant mutant strains. This result differs from that reported that Cyt b gene expression was upregulated in Lasiodiplodia theobromae resistant to pyraclostrobin [21]. Other non-target mechanisms, such as ABC transporters [32,33] and cytochrome P450 enzymes [34], are critical for pathogen resistance but remain uncharacterized in A. alternata. Additionally, we observed a 20-fold upregulation of 4-hydroxyphenylpyruvate dioxygenase (CC77DRAFT_950966) in TYC8-2 versus the wild type. As a key tyrosine metabolism enzyme, its role in azoxystrobin resistance warrants functional validation. Transcriptome analysis also revealed elevated EMP–TCA genes in resistant strains; whether this upregulation enhances energy metabolism to promote resistance requires further investigation.

4.2. AaAOX Overexpression Confers QoI Resistance and Environmental Stress Tolerance in A. alternata from Ginseng

Cyt b point mutation is not the only QoI resistance mechanism, activation of the alternative respiratory pathway (ARP) also reduces fungal sensitivity to QoIs [35]. Alternative oxidase (AOX), the terminal oxidase of the ARP, is conserved in mitochondria across plants, fungi, and other organisms. AOX provides an electron bypass in the mitochondrial electron transport chain (ETC), shunting electrons from coenzyme Q directly to oxygen (bypassing Complexes III and IV) to form water. While this pathway does not contribute to ATP synthesis, it mitigates oxidative stress from ETC inhibition and maintains metabolic flux [36]. In the azoxystrobin-resistant strain TYC8-2 (lacking AaCyt b mutations), transcriptome sequencing and RT-qPCR revealed significant AaAOX upregulation levels exceeding those in TYC8-1 (the G143A mutant). Notably, all nine azoxystrobin-resistant mutants lacking AaCyt b mutations exhibited elevated AaAOX expression, indicating AaAOX overexpression as an alternative resistance mechanism in A. alternata. This aligns with Dong et al. (2022), who reported AOX upregulation confers pyraclostrobin resistance in L. theobromae LDJH13107 [21], and Wu et al. (2024), who linked AOX overexpression to pyraclostrobin resistance in Fusarium graminearum PH-1 [11]. Jing et al. (2024) further demonstrated that SHAM (an AOX inhibitor) enhances QoI fungicide activity against Phytophthora litchi [37]. Consistent with these studies, we found SHAM (150 μg/mL) significantly increased azoxystrobin sensitivity in 140 geographically diverse A. alternata strains, providing broad empirical support for AaAOX involvement in azoxystrobin resistance. We further validated AaAOX function via knockout and complementation in A. alternata. The AaAOX knockout mutant exhibited a 2.7 × 10⁴-fold increase in azoxystrobin sensitivity and significantly higher sensitivity to other QoIs (kresoxim-methyl, pyraclostrobin, picoxystrobin). It also showed greater sensitivity to abiotic stresses (NaCl, H₂O₂, Congo red). While these findings implicate AaAOX in QoI resistance, the distinction between direct AaAOX-mediated resistance and general physiological impairment must be interpreted carefully. Previous studies highlight that AaAOX is integral not only to respiration but also to pathogenesis, primarily by scavenging host-derived reactive oxygen species (ROS) during infection [38,39,40]. Consistent with this, our validation demonstrated that AaAOX deletion concurrently triggered a profound reduction in fungal virulence, environmental adaptability, and stress tolerance. This suggests that the drastic hypersensitivity observed in the knockout mutants is likely a composite effect: the elimination of the direct electron-shunting bypass (the specific resistance mechanism) coupled with a broader collapse in physiological homeostasis [36,41]. Therefore, AaAOX upregulation serves a dual purpose in resistant strains; it directly circumvents AaCyt b inhibition to confer QoI resistance while simultaneously fortifying the pathogen’s physiological robustness to maintain pathogenicity. Our results indicate that under field conditions, AaAOX-mediated QoI resistance and environmental stress tolerance in A. alternata share a common molecular basis, conferring a survival advantage to resistant strains.

Although exogenous SHAM treatment effectively suppresses AaAOX-mediated QoI resistance and restores the sensitivity of A. alternata to several fungicides (azoxystrobin), its practical application for field resistance management remains to be comprehensively evaluated. Such an evaluation should encompass field efficacy, economic feasibility, potential impacts on the growth of ginseng plants, and effects on ginsenoside biosynthesis and metabolism.

4.3. Novel Findings from Transcriptome Sequencing Analysis for Resistant Strains

Transcriptome sequencing revealed that resistant strains TYC8-2 (AOX-overexpressing) and TYC8-1 (G143A mutant) share comparable energy metabolism pathways. Notably, TYC8-2 exhibited upregulated key genes in the EMP–TCA cycle relative to TYC8-1. In resistant strains, EMP pathway activation and upregulation of phosphoenolpyruvate carboxykinase (CC77DRAFT_1021809) and malate synthase (CC77DRAFT_1056777) promoted glucose-to-pyruvate conversion, partially compensating for ATP deficits caused by mitochondrial ETC inhibition. In TYC8-2, upregulated malate synthase and isocitrate dehydrogenase remodeled TCA flux, facilitated the glyoxylate shunt, reduced acetyl-CoA wastage, and improved energetic efficiency. Additionally, glutamate dehydrogenase (CC77DRAFT_938842) in fumarate metabolism was significantly upregulated (3.1-fold), suggesting increased production of reducing equivalents (e.g., FADH₂) to supply electrons to coenzyme Q. Given that azoxystrobin inhibits wild-type Cyt b more potently than the G143A variant, it is plausible that in TYC8-2, coenzyme Q transfers more electrons to AOX during oxidative phosphorylation, alleviating stress from excessive FADH₂ accumulation. This aligns with Hou et al. (2018) [42], who reported that an AOX-overexpressing Aspergillus nidulans mutant (strain 102) exhibits elevated EMP and TCA fluxes, higher citric acid production, and correlated ATP/NADH levels. Kirimura et al. (2000) [43] similarly confirmed AOX’s role in citric acid production in A. nidulans, and Tran-Ly et al. (2020) [44] reported that AOX maintains ATP/NADPH supply by regulating growth in A. nidulans. In our study, gene enrichment analysis highlighted substantial EMP pathway representation. Consistent with this, Wu et al. (2024) [11] observed that Cox gene deletion in F. graminearum PH-1 induces AOX upregulation and alters EMP/pyruvate metabolism. Collectively, these findings suggest that AOX overexpression enhances EMP and TCA activity in pathogens. Beyond electron shunting, resistant strains may mitigate QoI-induced ETC inhibition by upregulating energy metabolism. However, microbial fungicide resistance is a fundamentally complex and multifactorial biological network. While our current findings elucidate target-site mutations and AaAOX-mediated metabolic compensation, this primarily intracellular focus presents a limitation, as it does not fully account for other potential synergistic mechanisms, such as the overexpression of multidrug efflux pumps or broader epigenetic adaptations. Overcoming these intricate defense mechanisms requires a methodological shift toward the development of novel intervention targets and robust resistance management strategies [45]. Differentially expressed genes identified by transcriptomic analysis in resistant A. alternata strains were predominantly up-regulated and are hypothesized to be involved in AaAOX-mediated resistance and pathogen fitness. However, the specific biological functions of these candidate genes and their regulatory networks require further validation through targeted experimentation.

4.4. Future Perspectives

Although we obtained AaCyt b single-point mutant strains and confirmed via molecular docking that their binding energy was significantly reduced, suggesting a link between resistance and this point mutation, we nevertheless acknowledge the inherent limitations of molecular docking technology. As an in silico predictive tool used to simulate the binding affinity between the fungicide and the target protein, it cannot independently confirm the in vivo resistance mechanisms of fungi. Therefore, to avoid overinterpreting the computational simulations, our study utilized docking strictly to provide preliminary structural evidence. The actual resistance mechanisms of A. alternata to azoxystrobin should be jointly verified through a comprehensive approach integrating phenotypic assessments, gene expression profiling, and gene functional verification [46].

Building upon this foundation, in our future work, we will expand the scale of field strain collection, increase transcriptome sequencing samples of different resistance phenotypes and geographical populations, and further explore novel molecular adaptive mechanisms and multi-mechanism combined resistance genotypes of A. alternata against azoxystrobin.

5. Conclusions

In this study, sequencing analysis and molecular docking confirmed that the G143A mutation in the target gene AaCyt b significantly reduces azoxystrobin binding affinity to its target protein, representing one key resistance mechanism of Alternaria alternata (Aa) to azoxystrobin. Through transcriptome sequencing and gene knockout/complementation experiments, we further uncovered for the first time that upregulated expression of the non-target alternative oxidase (AOX) gene constitutes an additional resistance mechanism in A. alternata. Notably, these two mechanisms do not coexist in the same strain. This work not only advances our understanding of A. alternata resistance to azoxystrobin but also provides critical insights to enhance resistance monitoring, management strategies, and, ultimately, the effective control of GALSB disease.