Combined physiological and transcriptomic analysis revealed the defense mechanism of Agaricus bisporus against Trichoderma harzianum infection

Combined physiological and transcriptomic analysis revealed the defense mechanism of Agaricus bisporus against Trichoderma harzianum infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Combined physiological and transcriptomic analysis revealed the defense mechanism of Agaricus bisporus against Trichoderma harzianum infection Jia Wang, Jiali Han, Yueyuan Li, Xiangyou Wang, Yanyin Guo, Hiroaki Kitazawa, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7749895/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Trichoderma harzianum ( T. harzianum ) infection causes black lesions on the surface of Agaricus bisporus ( A. bisporus ) and soft rot of the infected tissues, thereby severely compromising mushroom quality. To investigate the host response, T. harzianum was inoculated onto the fruiting bodies of A. bisporus . This study aimed to comprehensively elucidate the resistance mechanisms of A. bisporus by analyzing defense-related enzyme activities and transcriptomic profiles. Physiological analyses revealed that antioxidant enzymes (SOD, POD, CAT), defense-related enzymes (CHT, PAL, GLU, PRs), and glutathione-associated enzymes (GR, GST, GSH) were activated, with their activities markedly increased during the early stages of infection. Transcriptomic analysis at 0, 18, 24, and 36 h post-inoculation identified 4,530 differentially expressed genes. Notably, genes involved in the glutathione metabolism pathway (GR, GSH, GST) were up-regulated, consistent with the observed enzyme activity changes. In addition, key genes associated with glycolysis (EMP), the tricarboxylic acid cycle (TCA), oxidative phosphorylation (OXPHOS), and the mitogen-activated protein kinase (MAPK) cascade exhibited enhanced expression. Collectively, these findings indicate that A. bisporus mounts defense responses by activating glutathione metabolism, EMP, TCA, OXPHOS, and MAPK pathways, thereby enhancing antioxidant and defense enzyme activities and improving resistance. This study elucidates the molecular mechanisms underlying the response of A. bisporus to T. harzianum infection and provides a theoretical basis for developing strategies to control postharvest mushroom diseases. Agaricus bisporus Trichoderma harzianum Pathogen infection Defense response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Highlights 1. T. harzianum induced activity of defense-related enzymes in A. bisporus . 2. T. harzianum accelerated the pathways related to energy metabolism of A. bisporus . 3. A. bisporus defense responses were revealed by transcriptome analysis. 1. Introduction Agaricus bisporus (A. bisporus) , commonly known as the white mushroom, possesses diverse bio-active properties, including antimicrobial, anti-tumor, antioxidant, and immunoregulatory activities (Wang et al., 2013 ; Leong et al., 2021 ). It remains to be determined whether the antimicrobial activity of A. bisporus contributes to its intrinsic defense mechanisms against pathogenic infection. Currently, research on A. bisporus has primarily focused on delaying postharvest senescence, while relatively little attention has been paid to its disease resistance mechanisms. Elucidating the processes underlying pathogen infection in postharvest A. bisporus may provide valuable insights and guidance for developing strategies to prevent and control postharvest diseases. Trichoderma harzianum ( T. harzianum ), a ubiquitous species of the genus Trichoderma , is widely distributed in soil, plant rhizospheres, and decaying organic matter (Pfordt et al., 2023 ). It exhibits strong competitiveness against other microorganisms and pronounced mycoparasitic capacity, which has led to its widespread use in agriculture as a biocontrol agent against plant pathogens (Lotfalinezhad et al., 2024 ). However, these mycoparasitic properties also pose a serious threat to edible mushroom cultivation, in contrast to its well-documented role as an antagonist of plant pathogens (Silva et al., 2024 ; He et al., 2023 ). In our previous study, T. harzianum was isolated and identified as the primary pathogen responsible for causing disease in A. bisporus . Pathogenicity assays confirmed its high virulence toward A. bisporus (Han et al., 2024 ), consistent with findings reported by Altaf et al. ( 2022 ). While the pathogenicity of T. harzianum in edible fungi has been increasingly recognized, the molecular defense strategies employed by A. bisporus in response to infection remain poorly understood compared with the well-characterized immune responses of plants. Plants are frequently infected by pathogens in natural environments and have consequently evolved an array of defense mechanisms, collectively referred to as immune responses (Dodds & Rathjen, 2010 ; Dodds et al., 2024 ). Upon pathogen invasion, host metabolism undergoes profound reprogramming characterized by enhanced respiration, macromolecule degradation, and altered synthesis of organic compounds (Dean et al., 2012 ). Concurrently, immune responses are activated when pattern recognition receptors (PRRs) embedded in the cell membrane perceive pathogen-associated molecular patterns (PAMPs), thereby initiating PAMP-triggered immunity (PTI) to mount defense responses while maintaining cellular homeostasis (Parker, 2003 ; Bent & Mackey, 2007 ; Robert-Seilaniantz et al., 2011 ). At the early stage of infection, plants often mount a hypersensitive response (HR), in which cells at the infection site undergo rapid programmed cell death (PCD) to form localized lesions, thereby restricting pathogen spread. Simultaneously, infected tissues accumulate high levels of reactive oxygen species (ROS), which act both as antimicrobial agents and as signaling molecules to amplify host defense signaling (Kunstler et al., 2016). Although fungi differ fundamentally from plants in physiology and development, they encounter similar ecological pressures from microbial pathogens. Edible fungi such as A. bisporus are particularly vulnerable to postharvest infections, raising the critical question of whether these fungi employ defense strategies analogous to plants or instead rely on unique mechanisms shaped by their distinct biology. Understanding this transition from well-established plant immune concepts to fungal defense systems provides a valuable framework for investigating disease resistance in edible fungi. Transcriptomic analysis based on RNA sequencing (RNA-seq) is a powerful high-throughput technology that enables comprehensive investigation of transcriptomes, overcoming the inherent limitations of DNA microarrays and providing robust support for transcriptomic research. For instance, Li et al. ( 2024 ) demonstrated through RNA-seq that phenylalanine enhances disease resistance in pear by acting as a positive regulator of phenylpropanoid metabolism, WRKY transcription factors, and calcium signaling pathways. Similarly, Meline et al. ( 2023 ) employed RNA-seq to elucidate the molecular basis of tomato resistance to bacterial wilt, revealing that resistant genotypes induce PTI responses, activate additional defense mechanisms, and modulate growth processes to confer effective resistance against Ralstonia infection. These studies demonstrate that RNA-seq can elucidate host-pathogen interaction mechanisms more clearly, characterize host immune responses more systematically, and provide valuable insights for disease management strategies. Given the conceptual parallels between plant and fungal defense responses, RNA-seq provides an ideal approach for investigating whether A. bisporus employs comparable defense mechanisms against pathogenic fungi. Building on this approach, RNA-seq was employed to investigate the molecular defense mechanisms of A. bisporus against pathogen infection in this study. In this study, we focus on the defense mechanisms of A. bisporus in response to infection by T. harzianum . Enzyme activities, differentially expressed genes (DEGs), and metabolic pathways revealed through transcriptomic analysis of postharvest A. bisporus following infection were investigated, and real-time quantitative PCR (qRT-PCR) was employed to validate the differential expression of selected genes, thereby verifying the reliability of the transcriptomic data. This integrative analysis provides a comprehensive view of the effects of pathogen infection on the physiological and metabolic processes of postharvest A. bisporus . By bridging plant immune concepts with fungal defense, our findings expand the theoretical understanding of resistance mechanisms in edible fungi, offer novel insights into the regulation of postharvest disease resistance, and lay a theoretical foundation for the selection and breeding of disease-resistant A. bisporus strains. 2. Materials and methods 2.1. Fruit material Fruiting bodies of A. bisporus were obtained from Zibo City, Shandong Province, China. Healthy and uniform fruiting bodies were selected for subsequent experiments. 2.2. Pathogen strains Strains of T. harzianum were isolated and identified from diseased A. bisporus in the laboratory and stored at − 80°C (Han et al., 2024 ). The pathogen was revived and cultured on potato dextrose agar (PDA) at 25°C for 5 d. Spores were harvested, and a suspension containing 1 × 10 5 colony-forming units (CFU) mL − 1 was prepared using a hemocytometer. 2.3. Sample treatment For the experimental group, the caps of A. bisporus fruiting bodies were injected with 20 µL of T. harzianum suspension (1 × 10 5 CFU mL − 1 ) using a sterile syringe, whereas the control group was treated with sterile water. The experimental group was designated J, and the control group was designated CK. All fruiting bodies were incubated at 25°C and 85% relative humidity. Tissues within 3 cm of the inoculation site were excised and stored at − 80°C for subsequent analyses. Samples were collected every 12 h, with 10 biological replicates per treatment. 2.4. Effect of T. harzianum infection on physiological indicators of A. bisporus 2.4.1. Antioxidant-related enzyme activities The activity of superoxide dismutase (SOD, EC 1.15.1.1) was determined following the method of Wang et al. ( 2019 ), with minor modifications. Mushroom samples (1 g) were homogenized in 3 mL of 50 mM phosphate-buffered saline (PBS, pH 7.8) and centrifuged at 4°C and 12, 000 × g for 15 min. The supernatant was collected as the crude enzyme extract. The reaction mixture contained 0.3 mL methionine, 0.3 mL nitro blue tetrazolium (NBT), 0.03 mL 100 µM EDTA-Na 2 , 2 mL 50 mM PBS (pH 7.8), 0.5 mL enzyme extract, and 0.3 mL riboflavin. Absorbance was measured at 560 nm. SOD activity was expressed as 10 5 U kg − 1 s − 1 . Catalase (CAT, EC 1.11.1.6) activity was measured following the methods of Han et al. ( 2023 ) and Jiang et al. ( 2024 ). Mushroom samples (1 g) were homogenized in 3 mL of 50 mM PBS (pH 7.0) and centrifuged at 4°C and 12, 000 × g for 15 min. The supernatant was used as the crude enzyme extract. The reaction mixture consisted of 1 mL Tris-HCl buffer (pH 7.1), 1.7 mL distilled water, and 0.1 mL enzyme extract. The mixture was incubated in a 25°C water bath for 3 min, followed by the addition of 20 mM H 2 O 2 . Absorbance at 240 nm was immediately recorded at 30 s intervals for 2 min. CAT activity was expressed as U kg − 1 s − 1 . Peroxidase (POD, EC 1.11.1.7) activity was determined using a commercial POD assay kit (Beijing Solarbio Science & Technology, Beijing, China). 2.4.2. Defense-related enzyme activities Defense-related enzyme activities were assayed as follows. Frozen mushroom tissue (1 g) was homogenized in 3 mL of 0.05 M PBS (pH 6.8). The homogenate was centrifuged at 4°C and 12, 000 × g for 12 min, and the resulting supernatant was used to determine the activities of phenylalanine ammonia-lyase (PAL), chitinase (CHT), and β-1,3-glucanase (GLU). Three replicates were performed for each treatment group. The levels of pathogenesis-related proteins (PRs) were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Baiyi, Shanghai, China). CHT (EC 3.2.1.14) activity was determined according to the method of Boller et al. ( 1983 ), with slight modifications. Briefly, 0.5 mL of enzyme extract was mixed with 0.5 mL of colloidal chitin and 0.5 mL of 50 mM acetic acid - sodium acetate buffer (pH 5.2). After incubation in a 37°C water bath for 1 h, the reaction mixture was supplemented with 0.1 mL of 30 g L − 1 desalted snail enzyme and 0.2 mL of 0.6 M potassium tetraborate solution. Absorbance was measured at 585 nm, and CHT activity was expressed as 10 3 U kg − 1 s − 1 . The activity of GLU (EC 3.2.1.39) was determined according to the method of Zheng et al. ( 2011 ), with slight modifications. Briefly, 0.3 mL of crude enzyme extract was mixed with 0.3 mL of a 4 g L − 1 laminarin solution and incubated at 37°C for 40 min. Subsequently, 1.8 mL of distilled water and 1.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent were added, and the mixture was boiled for 3 min. The final reaction solution was diluted to 25 mL, and absorbance was measured at 540 nm. GLU activity was expressed as 10 3 U kg − 1 s − 1 . The activity of PAL (EC 4.3.1.5) was determined according to the method of Zheng et al. ( 2011 ), with slight modifications. Briefly, 0.5 mL of enzyme extract was mixed with 3 mL of 0.05 M borate buffer (pH 8.8) and 0.5 mL of 0.02 M L-phenylalanine. The absorbance at 290 nm was immediately recorded as OD 0 . The mixture was then incubated in a water bath at 37°C for 1 h, after which the absorbance at 290 nm was again measured (OD 1 ). One unit of PAL activity was defined as the change in absorbance at 290 nm per hour, and the activity was expressed as U kg − 1 s − 1 . 2.4.3. Glutathione-related enzyme activities Glutathione reductase (GR, EC 1.6.4.2) activity was determined according to the method of Smith et al. ( 1988 ), with minor modifications. Briefly, 0.2 mL of enzyme extract was added to 2 mL of reaction mixture containing 50 mM phosphate buffer (pH 7.5), 5 mM oxidized glutathione (GSSG), and 5 mM MgCl 2 . The reaction was initiated by the addition of 0.1 mM NADPH, and the absorbance at 340 nm was immediately recorded. The change in absorbance at 340 nm per minute was used to calculate GR activity. Reduced glutathione (GSH) and GSSG were determined with slight modifications according to the method of Feng et al. ( 2022 ). Briefly, 2 g of frozen mushroom tissue was homogenized in 10 mL of 6% 5-sulfosalicylic acid and centrifuged at 12, 000 × g for 25 min at 4°C. The supernatant was collected for subsequent analysis. For determination of total glutathione content (GSH + GSSG), the reaction mixture consisted of 20 µL of supernatant, 200 µL of 0.5 M PBS (6.3 mM EDTA, pH 7.5), 370 µL of 10 mM EDTA, 200 µL of 6 mM 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB), and 200 µL of 2.1 mM NADPH. The reaction was initiated by the addition of 2 U GR, and the change in absorbance at 412 nm was recorded. For GSSG determination, 20 µL of supernatant was incubated with 200 µL PBS and 4 µL of 2.1 mM 2-vinylpyridine to remove GSH, and the subsequent steps were carried out as described for total glutathione measurement. The GSH content was calculated as the difference between total glutathione and GSSG. The activity of glutathione S-transferase (GST, EC 2.5.1.18) was determined using a commercial GST assay kit (Beijing Solarbio Science & Technology, Beijing, China) according to the manufacturer’s instructions. 2.5. Effect of T. harzianum infection on DEGs in A. bisporus 2.5.1. RNA extraction, library construction and sequencing RNA-seq samples were collected as described in Section 2.3 . The CK group was treated with sterile water, whereas the J was inoculated with a T. harzianum suspension. Fruiting body samples of A. bisporus were collected at 0, 6, 18, 24, and 36 h post-inoculation. Total RNA was extracted according to the method of Zhou et al. ( 2023 ), and the quality of the extracted RNA was subsequently evaluated. The purified RNA was then sent to Meiji Biotechnology Co., Ltd. (Shanghai, China) for cDNA library construction and sequencing. 2.5.2. Select DEGs The raw sequencing data were filtered to remove low-quality reads, and the remaining high-quality sequences were de novo assembled to generate transcript sequences, which were subsequently clustered. The longest transcript in each cluster was defined as a Unigene. Differential gene expression analysis was performed using the DESeq software package, and DEGs were identified based on the criteria |log 2 (fold change)| ≥ 1 and P -value < 0.05. 2.5.3. GO and KEGG enrichment analysis of DEGs The differentially expressed genes were classified and functionally annotated against the GO ( http://www.geneontology.org/ ) and KEGG ( https://www.kegg.jp/kegg/ ) databases. P values were adjusted using the false discovery rate (FDR) method, and significant enrichment was determined at FDR ≤ 1.0. 2.6. qRT-PCR The validation of transcription-derived gene expression changes was performed by qRT-PCR on an Eppendorf system (Hamburg, Germany). Gene-specific primers for candidate genes were designed using Primer 5.0 software (see Table S1), with EF1-α serving as the reference gene. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Each 15 µL reaction contained 0.6 µL of 10 µmol L − 1 forward and reverse primers, 0.6 µL of cDNA template, 5.7 µL of nuclease-free water, and 7.5 µL of SYBR Green PCR Master Mix (Toyobo, Osaka, Japan). The PCR conditions were 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 10 s. Melting curve analysis was performed over a temperature range of 60–95°C after amplification. Three biological replicates were included for each gene to ensure accuracy. Relative expression levels were calculated using the 2 −ΔΔCT method. 2.7. Statistical analysis All data were presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, USA) with a completely randomized design and a minimum of three replicates per treatment. Multiple comparisons and pair-wise comparison were conducted using Tukey’s test and t -test, respectively ( P < 0.05). Differences among groups were considered statistically significant when P < 0.05, as indicated by different letters in the figures and tables. 3. Results 3.1 Growth of the wound of A. bisporus infested by T. harzianum Figure 1 depicts mushrooms inoculated with either sterile water or T. harzianum . As shown in Fig. 1 A, mild symptoms of A. bisporus appeared at 24 h post-inoculation, while pronounced browning at the inoculation site was observed at 36 h. Thereafter, the lesions gradually expanded, accompanied by deeper discoloration and tissue softening. Microscopic observation of the inoculation site at 36 h using a 3D microscope revealed that the endogenous mycelium in the CK displayed clear differentiation and a positive guaiac reaction (Fig. 1 B). In contrast, in the infected group (J), T. harzianum mycelial tissue was clearly visible at the inoculation site, and the inoculation wound in A. bisporus was enlarged with sparser and wetter mycelium (Fig. 1 C). These observations indicated that mushroom quality began to deteriorate markedly at 36 h following T. harzianum infection, with affected tissues becoming softened and decayed, thereby severely compromising overall quality. To determine whether A. bisporus exhibits a defense response at the physiological level prior to visible symptom onset, samples were collected at 0 h, 6 h (post-inoculation), 18 h (pre-symptomatic stage), 24 h (early symptomatic stage), and 36 h (fully symptomatic stage) for RNA-Seq analysis. Enzyme activity was measured every 12 h to account for the lag relative to changes in gene expression. 3.2 Antioxidant-related enzyme activities Changes in the activities of SOD, POD, and CAT in A. bisporus inoculated with T. harzianum are presented in Fig. 2 (A-C). The activities of all three antioxidant enzymes initially increased but subsequently declined as the infection progressed. In the CK group, SOD activity peaked at 60 h, whereas POD activity reached its maximum at 36 h. In contrast, the J group exhibited earlier peaks, with SOD and POD reaching maximum levels at 36 h and 24 h, respectively. Notably, at 12 h, CAT activity in the J group was 7.43-fold higher than that in the CK group. However, after 36 h, the enzyme activities in the CK group exceeded those in the J group. 3.3 Defense-related enzyme activities As shown in Fig. 3 , the activities of four defense-related enzymes (PRS, CHT, GLU, and PAL) increased to varying extents during A. bisporus infection by T. harzianum , reflecting the defense response of host. PRS activity (Fig. 3 A) exhibited a continuous increase, with both the CK and J groups peaking at 72 h. In contrast, the activities of CHT (Fig. 3 B), GLU (Fig. 3 C), and PAL (Fig. 3 D) initially increased but subsequently declined following inoculation. At 24 h, the greatest differences in CHT and PAL activities were observed, with the J group being 1.59 - and 3.84 - fold higher, respectively, than the CK group. At 36 h, GLU activity in the J group was 2.2 - fold higher than in the CK group. After 48 h, GLU activity in the CK group increased rapidly, whereas it declined in the J group. At 72 h, GLU activity reached 0.506 × 10 3 U kg − 1 s − 1 in the CK group, whereas it decreased to 0.286 × 10 3 U kg − 1 s − 1 in the J group. 3.4 Glutathione-related enzyme activities Figure 4 presents the activities of GR and GST, the levels of GSH, and the GSH/GSSG ratio in A. bisporus , all of which are involved in glutathione metabolism (GSH cycle). GR (Fig. 4 A) and GST (Fig. 4 D) activities initially increased and then declined. In the CK group, GR and GST reached their peaks at 72 h and 48 h, respectively, whereas in the J group, maximum activities occurred earlier, at 48 h and 12 h. GSH levels in the CK group decreased gradually over time. In the J group, GSH levels increased at 12 h but subsequently declined sharply, becoming lower than those in the CK group after 36 h. In the CK group, the GSH/GSSG ratio (Fig. 4 C) exhibited a continuous increase, peaking at 72 h. In contrast, the ratio in the J group peaked at 24 h, then declined and dropped below the CK group by 48 h. 3.5 Transcriptome data analysis and functional annotation To investigate the molecular responses of A. bisporus to T. harzianum infection, tissue samples were collected from the CK and J groups at 0, 6, 18, 24, and 36 h for transcriptome sequencing. After removing low-quality reads and adapter sequences, 61.38 Gb of clean data were obtained in total, with no less than 6.04 Gb per sample. The sequencing quality was high, with Q20 ≥ 97.48%, Q30 ≥ 92.31%, and GC content ranging from 49.06% to 49.66% (Table S1). These results demonstrate that the quality and robustness of sequencing data were high, providing approximately 6, 500 genes for subsequent analyses. As shown in Fig. 5 A, BLAST searches were conducted against six databases, namely GO, KEGG, EggNOG, NR, Swiss - Prot, and Pfam, to further analyze the functional significance of the identified genes and transcripts. A total of 9, 256 genes (98.4%) were functionally annotated in the GO database, while 3, 962 genes (42.77%) were mapped to the KEGG database. As shown in Fig. 5 B, transcriptome profiling of the CK and J groups revealed distinct patterns of DEGs. At 6 h, 1, 228 DEGs were detected, including 520 up-regulated and 708 down-regulated genes. At 18 h and 24 h, 626 and 980 DEGs were detected, respectively. The highest number of DEGs was observed at 36 h, with 1, 696 genes differentially expressed, including 916 up - regulated and 780 down - regulated. 3.6 GO enrichment analysis and KEGG pathway analysis of DEGs GO and KEGG enrichment analyses were performed on the DEGs from four pairwise comparisons (CK6h vs. J6h, CK18h vs. J18h, CK24h vs. J24h, and CK36h vs. J36h) with an adjusted P -value (Padj) < 0.05 and |log 2 (fold change)| ≥ 1. In the GO enrichment analysis, 154, 99, 217, and 245 terms were significantly enriched in CK6h vs. J6h, CK18h vs. J18h, CK24h vs. J24h, and CK36h vs. J36h, respectively. To further investigate the defense responses of A. bisporus to pathogen invasion, we selected GO terms associated with more than two DEGs in each of the three major categories (Biological Process, Molecular Function, and Cellular Component), ranked them by -log 10 ( P ) values, and retained the top 20 terms for each group (Fig. 6 ). Notably, DEGs at 18 h, 24 h, and 36 h showed significant enrichment in oxidoreductase activity, suggesting that oxidoreductases were activated during the early stages of pathogen infection and played a critical role in the defense response of A. bisporus . The KEGG database is a widely used resource for the systematic analysis of metabolic and signal transduction pathways in living organisms (Zhang et al., 2022a ). As shown in Fig. 7 , DEGs identified in CK6h vs. J6h, CK18h vs. J18h, CK24h vs. J24h, and CK36h vs. J36h were mapped to the KEGG database to elucidate their molecular functions in the response of A. bisporus to T. harzianum infection. The top 20 significantly enriched pathways were then retained for analysis. At 6 h, DEGs were mainly enriched in pathways related to amino acid and carbohydrate metabolism, including alanine, aspartate and glutamate metabolism; amino sugar and nucleotide sugar metabolism; glycine, serine and threonine metabolism; and GSH metabolism. At 18 h, the enriched pathways were primarily associated with the ribosome, biosynthesis of cofactors, and glycerolipid metabolism. At 24 h, the major enriched pathways included the ribosome, DNA replication, the yeast cell cycle, and GSH metabolism. At 36 h, DEGs were mainly enriched in oxidative phosphorylation (OXPHOS), glycolysis (EMP), the citrate cycle (TCA), and peroxisome-related pathways. Among these, energy metabolism-related pathways (OXPHOS, EMP, TCA), GSH metabolism, and MAPK signaling pathways were of particular interest, as they were significantly enriched. We therefore conducted further analyses of these pathways to elucidate the underlying defense mechanisms. 3.7 DGEs enrichment of metabolic pathways 3.7.1 EMP and TCA pathways In this study, 48 DEGs were enriched in the EMP and TCA pathways. Figure 8 A shows the 16 DEGs with the most significant expression differences, including homologs of key enzyme genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH), pyruvate dehydrogenase, and pyruvate kinase (PK), all of which were significantly enriched. The expression of GAPDH homologs (78276 and 115671) was up-regulated 1.61-fold and 9.21-fold at 36 h, respectively. Similarly, the PK homolog (110153) exhibited the same trend as GAPDH, reaching its highest expression level at 36 h. The PDHA and PDHB subunits of pyruvate dehydrogenase were also up-regulated as infection progressed, suggesting that T. harzianum infection may accelerate the catabolism of pyruvate, the end product of glycolysis. Likewise, ALDH homologs (80727, 108035, 108032, 46604, 86450, 65619) exhibited a consistent up-regulation trend over time. In contrast, in the J group, gene expression at 36 h was down-regulated relative to the CK group, potentially leading to acetaldehyde accumulation and subsequent cellular damage in A. bisporus . A total of 15 DEGs were identified in the TCA pathway, and transcriptome analysis revealed an overall up-regulation as infection progressed. However, the J group displayed down-regulated expression compared with the CK group, particularly at 24 h and 36 h. Expression of succinate dehydrogenase (SDH) homologs (111304, 115047, 101253, 52249, 57403) peaked at 24 h in the J group but declined thereafter, whereas expression levels in the CK group remained higher at both 24 h and 36 h. Similarly, malate dehydrogenase (MDH) homologs (119105, 115666) exhibited expression trends consistent with those of SDH, showing overall up-regulation with time, but their expression levels were consistently lower in the J group compared to the CK group. 3.7.2 OXPHOS pathway Transcriptomic analysis revealed 94 DEGs in the OXPHOS pathway. As shown in Fig. 8 B, gene expression in both CK and J groups gradually increased over time, suggesting that mechanical damage and pathogen infection activate the OXPHOS pathway, thereby enhancing energy conversion in A. bisporus and triggering a resistance response. However, in the J group, gene expression showed a down-regulation trend compared with the CK group, particularly at 36 h, when homologous genes encoding ATP synthase subunits (ATP1-5, ATP7, and ATP14-20) were significantly down-regulated. These results suggest that pathogen infection may inhibit the energy conversion required for the defense response of A. bisporus , especially during the later stages of infection, thereby reducing energy production in the OXPHOS pathway and accelerating quality deterioration. 3.7.3 GSH cycle pathway Transcriptomic analysis revealed that genes related to glutathione metabolism were differentially expressed in A. bisporus upon infection with T. harzianum (Fig. 9 ). In the glutathione metabolic pathway, the homologous gene (110335), which encodes 6-phosphogluconate dehydrogenase, was up-regulated at 6, 12, 18, and 36 h, thereby facilitating NADPH production. Similarly, homologous genes 125563, 84806/113538, and 58613/113579, encoding glutathione reductase, disulfide-bond oxidoreductase, and ribonucleoside-diphosphate reductase, respectively, were also up-regulated. Their increased expression contributes to maintaining glutathione in its reduced state, thereby enhancing ROS scavenging and strengthening host defense. At 6 h post-infection, 20 GST homologs were differentially expressed (10 up-regulated and 10 down-regulated). At 18, 24, and 36 h post-infection, 21, 21, and 22 GST homologs were differentially expressed, respectively, with differing ratios of up- and down-regulation. These expression patterns suggest that GST-related DEGs were increasingly up-regulated in response to stress as infection progressed. 3.7.4 MAPKs pathway During the early stage of infection with T. harzianum , the MAPK signaling pathway in A. bisporus was rapidly activated, mediating the pheromone response, cell wall integrity, high-osmolarity response, and filamentation pathways (Fig. 10 ). The expression levels of genes associated with pheromone signaling, cell wall stress, and filamentation progressively increased over time. They were consistently higher in the infected group than in the control, with marked differences at 36 h. In contrast, genes in the high-osmolarity pathway were initially up-regulated at 6 h but declined thereafter. No significant differences in gene expression were detected between CK and J groups during the early stage. However, after 36 h, gene expression in the infected group was significantly higher than in the control, which coincided with the onset of visible disease symptoms in A. bisporus . Thus, T. harzianum infection at later stages disrupted the osmotic balance of A.. bisporus , which explains the observed liquid exudation and tissue softening during decomposition. 3.8 qRT-PCR results Five DEGs enriched in KEGG pathways were selected for qRT-PCR analysis to validate the transcriptome results. Ef1α was used as an internal control to normalize the relative expression levels of these genes. The qRT-PCR results showed that the expression patterns of the five DEGs were highly consistent with those obtained from the transcriptome analysis, thereby validating the reliability and accuracy of the transcriptome sequencing data (Fig. 11 ). 4. Discussion Our previous study demonstrated that T. harzianum infection causes severe surface lesions accompanied by tissue decay in mushrooms, thereby posing a substantial threat to the postharvest storage of A. bisporus (Han et al., 2024). In higher plants, stress conditions often activate intrinsic defense mechanisms, particularly those associated with oxidative stress regulation. Inspired by this paradigm, the present study investigated the defense responses of A. bisporus against T. harzianum infection. Our results revealed significant alterations in antioxidant enzymes, defense enzymes, and several metabolic pathways following inoculation. Under biotic stress, plants rapidly generate ROS and trigger localized programmed cell death (PCD) at the infection site (Tian et al., 2013; Yang et al., 2024). To maintain ROS homeostasis, plants employ an enzymatic antioxidant defense system comprising SOD, CAT, and POD. SOD catalyzes the dismutation of O 2 •⁻ into H 2 O 2 , which is subsequently decomposed by CAT and POD into H 2 O and O 2 via distinct catalytic pathways. This coordinated enzymatic system detoxifies ROS and safeguards cellular macromolecules from oxidative damage (Mittler, 2002). Although fungi are phylogenetically distinct from plants, similar ROS-associated defense mechanisms may operate in mushrooms when challenged by pathogenic fungi. In our study, we observed a pronounced increase in antioxidant enzyme activities in A. bisporus during the early stage of T. harzianum infection, suggesting an enhanced and rapid defense response compared with mechanical injury. However, after 36 h of T. harzianum infection, A. bisporus experienced stress that weakened its antioxidant defense system, ultimately leading to tissue deterioration. This defense activation was transient; enzyme activities declined at later stages of infection, thereby exacerbating tissue deterioration in A. bisporus . Our observations were consistent with previous findings. Shi et al. (2024) demonstrated that SOD, CAT, and POD protect plant cells from oxidative damage and further showed that compounds such as para-coumaric acid enhance the activities of these enzymes, thereby enhancing the resistance of mushrooms to Brown Blotch disease. Likewise, Zhang et al. (2022b) reported that the application of Meyerozyma guilliermondii to pathogen-infected broccoli enhanced ROS metabolism, with the associated increase in antioxidant enzyme activity indicative of an induced immune response. These findings collectively suggested that, similar to plants, mushrooms also relied on antioxidant enzyme systems to regulate ROS metabolism under biotic stress, although the strength and persistence of these responses might be distinct due to their unique biology. GR is a flavoprotein oxidoreductase that utilizes NADPH to catalyze the reduction of GSSG to its reduced form GSH. Through this process, GR maintains the intracellular glutathione pool in a reduced state and mitigates ROS-induced damage (Pang and Wang, 2010). GSH acts as a potent ROS scavenger, eliminating peroxides generated during metabolism and environmental stress. In plants, enhanced GSH synthesis has been shown to improve stress resistance in response to external stimuli (Alscher, 1989). The GSH/GSSG couple, one of the most abundant redox pairs in plant cells, plays a central role in maintaining redox homeostasis (Hasanuzzaman et al., 2017). Under normal physiological conditions, the GSH/GSSG ratio decreases as GSH is consumed during ROS detoxification and metabolic processes. In this study, the GSH/GSSG ratio in the J group peaked at 24 h and remained higher than that of the CK group until 36 h, indicating an enhanced reducing capacity in mushrooms during the early stage of pathogen infection. This elevated redox state is likely to have preserved protein structure and function, thereby sustaining normal cellular processes under stress. GSTs, a superfamily of enzymes, play an important catalytic role in the glutathione cycle by conjugating GSH and detoxifying harmful endogenous compounds such as hydroxyalkyls and hydroperoxides, thereby conferring both antioxidant and detoxification capacities (Nahar et al., 2015). In plants, enhanced GST expression is widely recognized as a hallmark of stress responses. Consistent with this, in A. bisporus , both GST activity and GST-related gene expression were significantly induced before 36 h of T. harzianum infection, underscoring the critical role of GST in the fungal defense response. Furthermore, glutathione metabolism has been shown to be directly involved in plant-pathogen interactions (Gill & Tuteja, 2010). In plants, glutathione accumulation not only scavenges ROS but also restricts pathogen growth. Our findings suggested that edible fungi might employ a comparable strategy, with increases in glutathione metabolism contributing to defense activation. Taken together, the observed enhancement of enzyme activities and the upregulation of glutathione-related genes highlight an active defense response in A. bisporus , reinforcing the pivotal role of glutathione metabolism in fungal resistance to pathogen infection. PRs constitute a family of proteins that are closely associated with disease resistance and defense mechanisms. In plants, PRs rapidly accumulate following pathogen infection and degrade pathogen cell-wall macromolecules, thereby enhancing host resistance (Ge et al., 2015; Wang et al., 2009). Most PRs function as hydrolases; for example, GLU (PR-2 family) and CHT (PR-3 family) directly inhibit pathogens by hydrolyzing their cell-wall components (Smith et al., 2009). Normally expressed at low levels, these enzymes are strongly induced under pathogen-induced stress, where they act synergistically to reinforce host cell walls and promote the biosynthesis of antimicrobial compounds (Zhang et al., 2022c). PAL, a key enzyme in the phenylpropanoid pathway, contributes to the production of flavonoids and phenolic compounds, both of which are important for plant defense (Li et al., 2023). Although the roles of PRs and defense enzymes have been extensively characterized in plants, evidence in fungi remains limited. In our study, the activities of defense enzymes-including PRs, CHT, GLU, and PAL-increased in both the CK and J groups, most likely due to puncture-induced mechanical injury. However, their activities were consistently higher in the J group, indicating that A. bisporus responded not only to wounding but also to pathogen challenge. These results indicated that pathogen infection transiently enhanced defense-related enzyme activities in A. bisporus . Although both pathogen infection and mechanical damage could stimulate such activities, the defense response induced by pathogen infection was more rapid and pronounced. Overall, these findings suggested that edible fungi, similar to plants, activated defense enzyme systems to strengthen their resistance mechanisms.. Activation of plant defense responses requires both carbon skeletons and substantial energy input, with ATP serving as the primary energy currency (Chen et al., 2021). In plants, energy is derived mainly from EMP, TCA cycle, and the pentose phosphate pathway (Bolton et al., 2008). Upregulation of GAPDH homologs, a key rate-limiting enzyme in EMP, suggests that EMP activity may be enhanced in A. bisporus during resistance to T. harzianum infection. Similarly, PK, another critical EMP enzyme, catalyzes the irreversible conversion of phosphoenolpyruvate to pyruvate, and its high expression in infected tissues has been associated with enhanced EMP flux (Mutuku & Nose, 2012). Consistent with observations in plants (Bolton et al., 2008), our results indicate that EMP activation in A. bisporus contributes to resistance. Pyruvate generated via EMP subsequently enters the TCA cycle, where it is fully oxidized to produce ATP and reducing equivalents. SDH, a key enzyme of the TCA cycle, catalyzes the dehydrogenation of succinate while coupling with ATP generation; its activity is widely regarded as a marker of TCA efficiency (Wang et al., 2020). Upregulation of homologs encoding enzymes such as SDH and MDH further supports the activation of the TCA cycle in A. bisporus . These findings suggested that, similar to plants, fungi mobilized energy metabolism to meet the elevated ATP demand of defense responses. However, in mushrooms infected for more than 36 h, the activities of EMP and TCA cycle pathways declined, resulting in reduced ATP production and accelerated tissue deterioration. Mitochondrial OXPHOS constitutes the final stage of ATP synthesis. This process, mediated by the mitochondrial electron transport chain (ETC), couples electron transfer with oxygen consumption to generate ATP, the universal energy currency (Kummer & Ban, 2021; Meyer et al., 2019). The ETC comprises five major enzyme complexes: Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase), Complex III (cytochrome c oxidoreductase), Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) (Lu et al., 2023). In this study, homologous genes encoding subunits of these complexes were generally downregulated under T. harzianum infection (Fig. 8B), suggesting reduced ATP production through OXPHOS. Previous studies have demonstrated that inadequate ATP supply contributes to senescence and physiological disorders in harvested fruits and vegetables (Friedman & Nunnari, 2014; Nolfi-Donegan et al., 2020). Our findings extend this concept to edible fungi, indicating that energy deficiency resulting from impaired OXPHOS may be a critical factor underlying the decline in resistance and the development of disease symptoms in A. bisporus . The MAPK signaling pathway plays a central role in plant defense by regulating hormone biosynthesis, ROS production, and defense-related gene expression. This highly conserved cascade in eukaryotes is composed of three core kinases-MAPKKK, MAPKK, and MAPK-that sequentially activate one another through phosphorylation (Meng & Zhang, 2013). In plants, MAPK cascades are among the earliest pathways activated during pathogen infection, transmitting signals from membrane-localized receptors and amplifying downstream defense responses (Walls et al., 2015; Bi & Zhou, 2017). Although well characterized in plants, MAPK-mediated defense mechanisms in fungi remain less understood. In this study, transcriptomic analysis revealed that the homologous gene encoding the transmembrane receptor Sho1 was upregulated in both the hyperosmotic and filamentous growth pathways, suggesting that pathogen-derived signals were perceived and transmitted via the Hog1 and Kss1 cascades. Previous studies have shown that Hog1 regulates carotene and glycerol accumulation in Cordyceps militaris, thereby maintaining osmotic balance and enhancing antioxidant capacity (Zhao et al., 2021). In contrast, the Kss1 pathway primarily activates adenylate cyclase, promoting filamentous growth and preserving cell wall integrity (Cherkasov et al., 2003). Collectively, our results indicate that both mechanical injury and pathogen infection activated MAPK signaling in A. bisporus , with Hog1 playing a more prominent role than Kss1. These findings suggest that MAPK signaling contributes to fungal resistance by maintaining cellular homeostasis under biotic stress. 5. Conclusion A. bisporus mounted an active defense response to T. harzianum infection. In this study, pathogen challenge enhanced the activities of antioxidant, defense, and glutathione-related enzymes, and significantly upregulated the transcription of genes associated with the glutathione cycle, EMP, TCA cycle, OXPHOS, and MAPK pathways, thereby activating defense responses in A. bisporus . Specifically, key enzyme genes such as PK in the glycolysis/gluconeogenesis pathway were upregulated to accelerate energy metabolism and secure sufficient energy supply. The expression of TCA cycle-related genes was elevated, providing precursors for the biosynthesis of resistance-related metabolites. Activation of the oxidative phosphorylation pathway improved ATP production efficiency, thereby maintaining cellular homeostasis. Glutathione metabolism contributed to the scavenging of pathogen-induced ROS, while the MAPK signaling cascade was continuously activated through phosphorylation events to regulate defense-related transcription factors. Collectively, these positive regulatory processes facilitated energy redistribution, enhanced antioxidant capacity, and activated systemic resistance, as validated by qRT-PCR results consistent with transcriptomic data. These findings provide theoretical support for elucidating the defense mechanisms of antifungal resistance in mushrooms and establish a foundation for effective disease prevention and control. Declarations The authors report no declarations of interest in this paper. Author statement Jia Wang : Resources, Investigation, Methodology, Data curation, Writing-original draft. Jiali Han : Resources, Investigation, Methodology, Data curation, Writing-original draft. Yueyuan Li : Conceptualization, Formal analysis. Xiangyou Wang : Writing - review & editing. Yanyin Guo : Writing - review & editing. Hiroaki Kitazawa : Writing - review & editing. Ling Li : Conceptualization, Supervision, Resources, Funding acquisition, Writing - review & editing. Funding This research was supported by the National Natural Science Foundation of China (31901765 and 31972144). Data Availability declaration The datasets used or analyzed in the study, which can be obtained from the corresponding author upon reasonable request. Acknowledgments The authors gratefully acknowledge the valuable contributions of all collaborators who provided resources, technical support, and constructive feedback during this research. 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17:03:19","extension":"xml","order_by":51,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186643,"visible":true,"origin":"","legend":"","description":"","filename":"596eb433c098405790d76b2955c8c8a91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/ea553700c676f30d07afa28a.xml"},{"id":93707894,"identity":"84ef50c7-f08c-45e8-a234-7c9bf0525725","added_by":"auto","created_at":"2025-10-16 17:03:19","extension":"html","order_by":52,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202923,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/e67e6594c77a33423f1a5964.html"},{"id":93708626,"identity":"c627222e-7fa6-42f8-aad0-9bcd0f281f79","added_by":"auto","created_at":"2025-10-16 17:11:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":670669,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of \u003cem\u003eT. harzianum\u003c/em\u003e infection on \u003cem\u003eA. bisporus\u003c/em\u003e (A). 3D microscopic image of the wound of \u003cem\u003eA. bisporus\u003c/em\u003e, inoculated with sterile water (B), inoculated with \u003cem\u003eT. harzianum\u003c/em\u003e liquid (C).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/c3c7c73d25cb082fe32f7a55.png"},{"id":93707829,"identity":"d708fdcf-93c0-4d87-9c41-7fe416add9ba","added_by":"auto","created_at":"2025-10-16 17:03:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43985,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eT. harzianum\u003c/em\u003e on SOD (A), POD (B), CAT (C) activities of \u003cem\u003eA. bisporus\u003c/em\u003e. \u003cem\u003eA. bisporus \u003c/em\u003einoculated with sterile water was used as the control group. At any time in this study, the experimental groups differed significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in level on the same day by different lowercase letters (a, b). The experiment was repeated three times for each experimental group, and each value was expressed as the average ± standard error SE.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/68f309ba06b9242be51d7612.png"},{"id":93708849,"identity":"87c27ee8-4701-42f8-821a-f72c22e5e469","added_by":"auto","created_at":"2025-10-16 17:19:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82311,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eT. harzianum \u003c/em\u003eon PRs (A), CHT (B), GLU (C), PAL (D) activities of \u003cem\u003eA. bisporus\u003c/em\u003e. \u003cem\u003eA. bisporus\u003c/em\u003e inoculated with sterile water was used as the control group. The experiment was repeated three times for each experimental group, and each value was expressed as the mean ± (SE). \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered a significant difference, indicated by different lowercase letters (a, b).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/b556c916732e92aaf02e9cba.png"},{"id":93707835,"identity":"0e78687a-01d4-4755-b6f2-831657204845","added_by":"auto","created_at":"2025-10-16 17:03:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97669,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eT. harzianum\u003c/em\u003e on GR (A), GSH (B), GSH/GSSG (C), GST (D) activities of \u003cem\u003eA. bisporus\u003c/em\u003e. \u003cem\u003eA. bisporus\u003c/em\u003e inoculated with sterile water was used as the control group. The experiment was repeated three times for each experimental group, and each value was expressed as the mean ± SE. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered a significant difference, indicated by different lowercase letters (a, b).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/2c67346116e0a103239f92a0.png"},{"id":93708850,"identity":"8a5e895a-a336-40d2-bdcb-55d3d9ce7a2b","added_by":"auto","created_at":"2025-10-16 17:19:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":134022,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional annotation of \u003cem\u003eA. bisporus\u003c/em\u003e genes across different databases using RNA-Seq analysis. (A) Distribution of annotated sequences in each database (horizontal axis: database name; vertical axis: number of annotated sequences). (B) Number of DEGs and volcano plots showing DEGs between the CK and J groups at different time points (6, 18, 24, and 36 h).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/914bbb7a4cfca7bae62af1e1.png"},{"id":93707841,"identity":"041a5148-9aac-4ee5-8a5f-2f6ea4eca11e","added_by":"auto","created_at":"2025-10-16 17:03:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":357371,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis of DEGs between the CK and J groups at different time points: 6 h (A), 18 h (B), 24 h (C), and 36 h (D). The figure shows the top 20 GO terms ranked by -log\u003csub\u003e10\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e-value).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/392e708396ef25acfb535bee.png"},{"id":93708633,"identity":"463426df-fd74-4ba9-b7e1-22304e6f00cb","added_by":"auto","created_at":"2025-10-16 17:11:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":255433,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment analysis of DEGs between the CK and J groups at different time points: 6 h (A), 18 h (B), 24 h (C), and 36 h (D). The figure shows the top 20 KEGG terms ranked by -log\u003csub\u003e10\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e-value).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/c1f11a2818117faa3260ce59.png"},{"id":93709583,"identity":"3d64c43c-724d-44a8-8850-94ced082aecd","added_by":"auto","created_at":"2025-10-16 17:27:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":329824,"visible":true,"origin":"","legend":"\u003cp\u003e(DEGs involved in the EMP and TCA cycles (A), and the OXPHOS pathway (B) of \u003cem\u003eA. bisporus\u003c/em\u003e under \u003cem\u003eT. harzianum\u003c/em\u003e stress, with reference to KEGG pathways. Heatmaps are based on fragments per kilobase of transcript per million mapped reads (FPKM) values. The color scale ranges from green (low expression) to red (high expression), representing relative expression levels.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/a87afeadac745ff95b1ef180.png"},{"id":93708629,"identity":"fe611cbe-9b7d-4d50-b86b-f355f6034f4d","added_by":"auto","created_at":"2025-10-16 17:11:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":117320,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs involved in the GSH cycle pathway of \u003cem\u003eA. bisporus\u003c/em\u003e under \u003cem\u003eT. harzianum\u003c/em\u003estress, referenced to the KEGG pathway. The heatmap is based on FPKM values, with color intensity ranging from green (low expression) to red (high expression), indicating relative gene expression levels.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/80e0ccd2da911eaf0ddbc67f.png"},{"id":93707844,"identity":"6dec8ee9-efe4-476f-82b6-b8566da8f67d","added_by":"auto","created_at":"2025-10-16 17:03:18","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":140368,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs involved in the MAPK signaling pathway of \u003cem\u003eA. bisporus\u003c/em\u003e under \u003cem\u003eT. harzianum\u003c/em\u003e stress, referenced to the KEGG pathway. The heatmap is based on FPKM values, with a color scale from green (low expression) to red (high expression), representing the relative expression levels of each gene.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/131c17f04e9147a04813c682.png"},{"id":93707842,"identity":"eeb964c4-2aab-47da-92d3-81cd44d398e8","added_by":"auto","created_at":"2025-10-16 17:03:18","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":79944,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of five differentially expressed genes enriched in KEGG pathways during \u003cem\u003eT. harzianum\u003c/em\u003e infection of \u003cem\u003eA. bisporus\u003c/em\u003efruiting bodies, including mitochondrial ETC-related genes and OXPHOS-related genes (QCR6, SDH3, COX4, ATP2) and the EMP-related gene (STK-HK). Bar charts represent transcript abundance quantified by RNA-seq (FPKM values, left y-axis), and overlaid line charts indicate relative expression levels validated by qRT-PCR (right y-axis).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/ff257b487ef25de5b3367328.png"},{"id":93962252,"identity":"c7808bd5-e55f-43ec-8b92-ae35b04c7193","added_by":"auto","created_at":"2025-10-20 17:22:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3194485,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/6ae40545-45b1-4bd9-b39b-3a35e7203cfd.pdf"},{"id":93707830,"identity":"eb0bc80f-7246-48cc-a2e9-94753a8407c8","added_by":"auto","created_at":"2025-10-16 17:03:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16430,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1QualityofcleanreadsinA.docx","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/68b46395f6f26e052f6feb94.docx"},{"id":93707833,"identity":"c92eede5-fb0b-4d0d-aa03-05f7ca1edc3f","added_by":"auto","created_at":"2025-10-16 17:03:18","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":99502,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-7749895/v1/336c69458e5453993ea91599.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combined physiological and transcriptomic analysis revealed the defense mechanism of Agaricus bisporus against Trichoderma harzianum infection","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. \u003cem\u003eT. harzianum\u003c/em\u003e induced activity of defense-related enzymes in \u003cem\u003eA. bisporus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e2. \u003cem\u003eT. harzianum\u003c/em\u003e accelerated the pathways related to energy metabolism of \u003cem\u003eA. bisporus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e3. \u003cem\u003eA. bisporus\u003c/em\u003e defense responses were revealed by transcriptome analysis.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eAgaricus bisporus (A. bisporus)\u003c/em\u003e, commonly known as the white mushroom, possesses diverse bio-active properties, including antimicrobial, anti-tumor, antioxidant, and immunoregulatory activities (Wang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Leong et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It remains to be determined whether the antimicrobial activity of \u003cem\u003eA. bisporus\u003c/em\u003e contributes to its intrinsic defense mechanisms against pathogenic infection. Currently, research on \u003cem\u003eA. bisporus\u003c/em\u003e has primarily focused on delaying postharvest senescence, while relatively little attention has been paid to its disease resistance mechanisms. Elucidating the processes underlying pathogen infection in postharvest \u003cem\u003eA. bisporus\u003c/em\u003e may provide valuable insights and guidance for developing strategies to prevent and control postharvest diseases.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTrichoderma harzianum\u003c/em\u003e (\u003cem\u003eT. harzianum\u003c/em\u003e), a ubiquitous species of the genus \u003cem\u003eTrichoderma\u003c/em\u003e, is widely distributed in soil, plant rhizospheres, and decaying organic matter (Pfordt et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It exhibits strong competitiveness against other microorganisms and pronounced mycoparasitic capacity, which has led to its widespread use in agriculture as a biocontrol agent against plant pathogens (Lotfalinezhad et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, these mycoparasitic properties also pose a serious threat to edible mushroom cultivation, in contrast to its well-documented role as an antagonist of plant pathogens (Silva et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; He et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our previous study, \u003cem\u003eT. harzianum\u003c/em\u003e was isolated and identified as the primary pathogen responsible for causing disease in \u003cem\u003eA. bisporus\u003c/em\u003e. Pathogenicity assays confirmed its high virulence toward \u003cem\u003eA. bisporus\u003c/em\u003e (Han et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), consistent with findings reported by Altaf et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While the pathogenicity of \u003cem\u003eT. harzianum\u003c/em\u003e in edible fungi has been increasingly recognized, the molecular defense strategies employed by \u003cem\u003eA. bisporus\u003c/em\u003e in response to infection remain poorly understood compared with the well-characterized immune responses of plants.\u003c/p\u003e\u003cp\u003ePlants are frequently infected by pathogens in natural environments and have consequently evolved an array of defense mechanisms, collectively referred to as immune responses (Dodds \u0026amp; Rathjen, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Dodds et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Upon pathogen invasion, host metabolism undergoes profound reprogramming characterized by enhanced respiration, macromolecule degradation, and altered synthesis of organic compounds (Dean et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Concurrently, immune responses are activated when pattern recognition receptors (PRRs) embedded in the cell membrane perceive pathogen-associated molecular patterns (PAMPs), thereby initiating PAMP-triggered immunity (PTI) to mount defense responses while maintaining cellular homeostasis (Parker, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Bent \u0026amp; Mackey, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Robert-Seilaniantz et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). At the early stage of infection, plants often mount a hypersensitive response (HR), in which cells at the infection site undergo rapid programmed cell death (PCD) to form localized lesions, thereby restricting pathogen spread. Simultaneously, infected tissues accumulate high levels of reactive oxygen species (ROS), which act both as antimicrobial agents and as signaling molecules to amplify host defense signaling (Kunstler et al., 2016). Although fungi differ fundamentally from plants in physiology and development, they encounter similar ecological pressures from microbial pathogens. Edible fungi such as \u003cem\u003eA. bisporus\u003c/em\u003e are particularly vulnerable to postharvest infections, raising the critical question of whether these fungi employ defense strategies analogous to plants or instead rely on unique mechanisms shaped by their distinct biology. Understanding this transition from well-established plant immune concepts to fungal defense systems provides a valuable framework for investigating disease resistance in edible fungi.\u003c/p\u003e\u003cp\u003eTranscriptomic analysis based on RNA sequencing (RNA-seq) is a powerful high-throughput technology that enables comprehensive investigation of transcriptomes, overcoming the inherent limitations of DNA microarrays and providing robust support for transcriptomic research. For instance, Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated through RNA-seq that phenylalanine enhances disease resistance in pear by acting as a positive regulator of phenylpropanoid metabolism, WRKY transcription factors, and calcium signaling pathways. Similarly, Meline et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) employed RNA-seq to elucidate the molecular basis of tomato resistance to bacterial wilt, revealing that resistant genotypes induce PTI responses, activate additional defense mechanisms, and modulate growth processes to confer effective resistance against Ralstonia infection. These studies demonstrate that RNA-seq can elucidate host-pathogen interaction mechanisms more clearly, characterize host immune responses more systematically, and provide valuable insights for disease management strategies. Given the conceptual parallels between plant and fungal defense responses, RNA-seq provides an ideal approach for investigating whether \u003cem\u003eA. bisporus\u003c/em\u003e employs comparable defense mechanisms against pathogenic fungi. Building on this approach, RNA-seq was employed to investigate the molecular defense mechanisms of \u003cem\u003eA. bisporus\u003c/em\u003e against pathogen infection in this study.\u003c/p\u003e\u003cp\u003eIn this study, we focus on the defense mechanisms of \u003cem\u003eA. bisporus\u003c/em\u003e in response to infection by \u003cem\u003eT. harzianum\u003c/em\u003e. Enzyme activities, differentially expressed genes (DEGs), and metabolic pathways revealed through transcriptomic analysis of postharvest \u003cem\u003eA. bisporus\u003c/em\u003e following infection were investigated, and real-time quantitative PCR (qRT-PCR) was employed to validate the differential expression of selected genes, thereby verifying the reliability of the transcriptomic data. This integrative analysis provides a comprehensive view of the effects of pathogen infection on the physiological and metabolic processes of postharvest \u003cem\u003eA. bisporus\u003c/em\u003e. By bridging plant immune concepts with fungal defense, our findings expand the theoretical understanding of resistance mechanisms in edible fungi, offer novel insights into the regulation of postharvest disease resistance, and lay a theoretical foundation for the selection and breeding of disease-resistant \u003cem\u003eA. bisporus\u003c/em\u003e strains.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Fruit material\u003c/h2\u003e\u003cp\u003eFruiting bodies of \u003cem\u003eA. bisporus\u003c/em\u003e were obtained from Zibo City, Shandong Province, China. Healthy and uniform fruiting bodies were selected for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Pathogen strains\u003c/h2\u003e\u003cp\u003eStrains of \u003cem\u003eT. harzianum\u003c/em\u003e were isolated and identified from diseased \u003cem\u003eA. bisporus\u003c/em\u003e in the laboratory and stored at \u0026minus;\u0026thinsp;80\u0026deg;C (Han et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The pathogen was revived and cultured on potato dextrose agar (PDA) at 25\u0026deg;C for 5 d. Spores were harvested, and a suspension containing 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e colony-forming units (CFU) mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was prepared using a hemocytometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Sample treatment\u003c/h2\u003e\u003cp\u003eFor the experimental group, the caps of \u003cem\u003eA. bisporus\u003c/em\u003e fruiting bodies were injected with 20 \u0026micro;L of \u003cem\u003eT. harzianum\u003c/em\u003e suspension (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) using a sterile syringe, whereas the control group was treated with sterile water. The experimental group was designated J, and the control group was designated CK. All fruiting bodies were incubated at 25\u0026deg;C and 85% relative humidity. Tissues within 3 cm of the inoculation site were excised and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analyses. Samples were collected every 12 h, with 10 biological replicates per treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Effect of T. harzianum infection on physiological indicators of A. bisporus\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Antioxidant-related enzyme activities\u003c/h2\u003e\u003cp\u003eThe activity of superoxide dismutase (SOD, EC 1.15.1.1) was determined following the method of Wang et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), with minor modifications. Mushroom samples (1 g) were homogenized in 3 mL of 50 mM phosphate-buffered saline (PBS, pH 7.8) and centrifuged at 4\u0026deg;C and 12, 000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min. The supernatant was collected as the crude enzyme extract. The reaction mixture contained 0.3 mL methionine, 0.3 mL nitro blue tetrazolium (NBT), 0.03 mL 100 \u0026micro;M EDTA-Na\u003csub\u003e2\u003c/sub\u003e, 2 mL 50 mM PBS (pH 7.8), 0.5 mL enzyme extract, and 0.3 mL riboflavin. Absorbance was measured at 560 nm. SOD activity was expressed as 10\u003csup\u003e5\u003c/sup\u003e U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCatalase (CAT, EC 1.11.1.6) activity was measured following the methods of Han et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Jiang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mushroom samples (1 g) were homogenized in 3 mL of 50 mM PBS (pH 7.0) and centrifuged at 4\u0026deg;C and 12, 000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min. The supernatant was used as the crude enzyme extract. The reaction mixture consisted of 1 mL Tris-HCl buffer (pH 7.1), 1.7 mL distilled water, and 0.1 mL enzyme extract. The mixture was incubated in a 25\u0026deg;C water bath for 3 min, followed by the addition of 20 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Absorbance at 240 nm was immediately recorded at 30 s intervals for 2 min. CAT activity was expressed as U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePeroxidase (POD, EC 1.11.1.7) activity was determined using a commercial POD assay kit (Beijing Solarbio Science \u0026amp; Technology, Beijing, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Defense-related enzyme activities\u003c/h2\u003e\u003cp\u003eDefense-related enzyme activities were assayed as follows. Frozen mushroom tissue (1 g) was homogenized in 3 mL of 0.05 M PBS (pH 6.8). The homogenate was centrifuged at 4\u0026deg;C and 12, 000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 12 min, and the resulting supernatant was used to determine the activities of phenylalanine ammonia-lyase (PAL), chitinase (CHT), and β-1,3-glucanase (GLU). Three replicates were performed for each treatment group.\u003c/p\u003e\u003cp\u003eThe levels of pathogenesis-related proteins (PRs) were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Baiyi, Shanghai, China).\u003c/p\u003e\u003cp\u003eCHT (EC 3.2.1.14) activity was determined according to the method of Boller et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), with slight modifications. Briefly, 0.5 mL of enzyme extract was mixed with 0.5 mL of colloidal chitin and 0.5 mL of 50 mM acetic acid - sodium acetate buffer (pH 5.2). After incubation in a 37\u0026deg;C water bath for 1 h, the reaction mixture was supplemented with 0.1 mL of 30 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e desalted snail enzyme and 0.2 mL of 0.6 M potassium tetraborate solution. Absorbance was measured at 585 nm, and CHT activity was expressed as 10\u003csup\u003e3\u003c/sup\u003e U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe activity of GLU (EC 3.2.1.39) was determined according to the method of Zheng et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), with slight modifications. Briefly, 0.3 mL of crude enzyme extract was mixed with 0.3 mL of a 4 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e laminarin solution and incubated at 37\u0026deg;C for 40 min. Subsequently, 1.8 mL of distilled water and 1.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent were added, and the mixture was boiled for 3 min. The final reaction solution was diluted to 25 mL, and absorbance was measured at 540 nm. GLU activity was expressed as 10\u003csup\u003e3\u003c/sup\u003e U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe activity of PAL (EC 4.3.1.5) was determined according to the method of Zheng et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), with slight modifications. Briefly, 0.5 mL of enzyme extract was mixed with 3 mL of 0.05 M borate buffer (pH 8.8) and 0.5 mL of 0.02 M L-phenylalanine. The absorbance at 290 nm was immediately recorded as OD\u003csub\u003e0\u003c/sub\u003e. The mixture was then incubated in a water bath at 37\u0026deg;C for 1 h, after which the absorbance at 290 nm was again measured (OD\u003csub\u003e1\u003c/sub\u003e). One unit of PAL activity was defined as the change in absorbance at 290 nm per hour, and the activity was expressed as U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. Glutathione-related enzyme activities\u003c/h2\u003e\u003cp\u003eGlutathione reductase (GR, EC 1.6.4.2) activity was determined according to the method of Smith et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), with minor modifications. Briefly, 0.2 mL of enzyme extract was added to 2 mL of reaction mixture containing 50 mM phosphate buffer (pH 7.5), 5 mM oxidized glutathione (GSSG), and 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e. The reaction was initiated by the addition of 0.1 mM NADPH, and the absorbance at 340 nm was immediately recorded. The change in absorbance at 340 nm per minute was used to calculate GR activity.\u003c/p\u003e\u003cp\u003eReduced glutathione (GSH) and GSSG were determined with slight modifications according to the method of Feng et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, 2 g of frozen mushroom tissue was homogenized in 10 mL of 6% 5-sulfosalicylic acid and centrifuged at 12, 000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 25 min at 4\u0026deg;C. The supernatant was collected for subsequent analysis. For determination of total glutathione content (GSH\u0026thinsp;+\u0026thinsp;GSSG), the reaction mixture consisted of 20 \u0026micro;L of supernatant, 200 \u0026micro;L of 0.5 M PBS (6.3 mM EDTA, pH 7.5), 370 \u0026micro;L of 10 mM EDTA, 200 \u0026micro;L of 6 mM 5,5\u0026rsquo;-dithiobis (2-nitrobenzoic acid) (DTNB), and 200 \u0026micro;L of 2.1 mM NADPH. The reaction was initiated by the addition of 2 U GR, and the change in absorbance at 412 nm was recorded. For GSSG determination, 20 \u0026micro;L of supernatant was incubated with 200 \u0026micro;L PBS and 4 \u0026micro;L of 2.1 mM 2-vinylpyridine to remove GSH, and the subsequent steps were carried out as described for total glutathione measurement. The GSH content was calculated as the difference between total glutathione and GSSG.\u003c/p\u003e\u003cp\u003eThe activity of glutathione S-transferase (GST, EC 2.5.1.18) was determined using a commercial GST assay kit (Beijing Solarbio Science \u0026amp; Technology, Beijing, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Effect of T. harzianum infection on DEGs in A. bisporus\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. RNA extraction, library construction and sequencing\u003c/h2\u003e\u003cp\u003eRNA-seq samples were collected as described in Section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e. The CK group was treated with sterile water, whereas the J was inoculated with a \u003cem\u003eT. harzianum\u003c/em\u003e suspension. Fruiting body samples of \u003cem\u003eA. bisporus\u003c/em\u003e were collected at 0, 6, 18, 24, and 36 h post-inoculation. Total RNA was extracted according to the method of Zhou et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and the quality of the extracted RNA was subsequently evaluated. The purified RNA was then sent to Meiji Biotechnology Co., Ltd. (Shanghai, China) for cDNA library construction and sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Select DEGs\u003c/h2\u003e\u003cp\u003eThe raw sequencing data were filtered to remove low-quality reads, and the remaining high-quality sequences were de novo assembled to generate transcript sequences, which were subsequently clustered. The longest transcript in each cluster was defined as a Unigene. Differential gene expression analysis was performed using the DESeq software package, and DEGs were identified based on the criteria |log\u003csub\u003e2\u003c/sub\u003e (fold change)| \u0026ge; 1 and \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3. GO and KEGG enrichment analysis of DEGs\u003c/h2\u003e\u003cp\u003eThe differentially expressed genes were classified and functionally annotated against the GO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org/\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/kegg/\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases. \u003cem\u003eP\u003c/em\u003e values were adjusted using the false discovery rate (FDR) method, and significant enrichment was determined at FDR\u0026thinsp;\u0026le;\u0026thinsp;1.0.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.6. qRT-PCR\u003c/h2\u003e\u003cp\u003eThe validation of transcription-derived gene expression changes was performed by qRT-PCR on an Eppendorf system (Hamburg, Germany). Gene-specific primers for candidate genes were designed using Primer 5.0 software (see Table S1), with EF1-α serving as the reference gene. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).\u003c/p\u003e\u003cp\u003eEach 15 \u0026micro;L reaction contained 0.6 \u0026micro;L of 10 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e forward and reverse primers, 0.6 \u0026micro;L of cDNA template, 5.7 \u0026micro;L of nuclease-free water, and 7.5 \u0026micro;L of SYBR Green PCR Master Mix (Toyobo, Osaka, Japan). The PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of denaturation at 95\u0026deg;C for 5 s, annealing at 60\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 10 s. Melting curve analysis was performed over a temperature range of 60\u0026ndash;95\u0026deg;C after amplification. Three biological replicates were included for each gene to ensure accuracy. Relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, USA) with a completely randomized design and a minimum of three replicates per treatment. Multiple comparisons and pair-wise comparison were conducted using Tukey\u0026rsquo;s test and \u003cem\u003et\u003c/em\u003e-test, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Differences among groups were considered statistically significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, as indicated by different letters in the figures and tables.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Growth of the wound of A. bisporus infested by T. harzianum\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts mushrooms inoculated with either sterile water or \u003cem\u003eT. harzianum\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, mild symptoms of \u003cem\u003eA. bisporus\u003c/em\u003e appeared at 24 h post-inoculation, while pronounced browning at the inoculation site was observed at 36 h. Thereafter, the lesions gradually expanded, accompanied by deeper discoloration and tissue softening. Microscopic observation of the inoculation site at 36 h using a 3D microscope revealed that the endogenous mycelium in the CK displayed clear differentiation and a positive guaiac reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, in the infected group (J), \u003cem\u003eT. harzianum\u003c/em\u003e mycelial tissue was clearly visible at the inoculation site, and the inoculation wound in \u003cem\u003eA. bisporus\u003c/em\u003e was enlarged with sparser and wetter mycelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These observations indicated that mushroom quality began to deteriorate markedly at 36 h following \u003cem\u003eT. harzianum\u003c/em\u003e infection, with affected tissues becoming softened and decayed, thereby severely compromising overall quality.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether \u003cem\u003eA. bisporus\u003c/em\u003e exhibits a defense response at the physiological level prior to visible symptom onset, samples were collected at 0 h, 6 h (post-inoculation), 18 h (pre-symptomatic stage), 24 h (early symptomatic stage), and 36 h (fully symptomatic stage) for RNA-Seq analysis. Enzyme activity was measured every 12 h to account for the lag relative to changes in gene expression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Antioxidant-related enzyme activities\u003c/h2\u003e\u003cp\u003eChanges in the activities of SOD, POD, and CAT in \u003cem\u003eA. bisporus\u003c/em\u003e inoculated with \u003cem\u003eT. harzianum\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (A-C). The activities of all three antioxidant enzymes initially increased but subsequently declined as the infection progressed. In the CK group, SOD activity peaked at 60 h, whereas POD activity reached its maximum at 36 h. In contrast, the J group exhibited earlier peaks, with SOD and POD reaching maximum levels at 36 h and 24 h, respectively. Notably, at 12 h, CAT activity in the J group was 7.43-fold higher than that in the CK group. However, after 36 h, the enzyme activities in the CK group exceeded those in the J group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Defense-related enzyme activities\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the activities of four defense-related enzymes (PRS, CHT, GLU, and PAL) increased to varying extents during \u003cem\u003eA. bisporus\u003c/em\u003e infection by \u003cem\u003eT. harzianum\u003c/em\u003e, reflecting the defense response of host. PRS activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) exhibited a continuous increase, with both the CK and J groups peaking at 72 h. In contrast, the activities of CHT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), GLU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and PAL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) initially increased but subsequently declined following inoculation. At 24 h, the greatest differences in CHT and PAL activities were observed, with the J group being 1.59 - and 3.84 - fold higher, respectively, than the CK group. At 36 h, GLU activity in the J group was 2.2 - fold higher than in the CK group. After 48 h, GLU activity in the CK group increased rapidly, whereas it declined in the J group. At 72 h, GLU activity reached 0.506 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the CK group, whereas it decreased to 0.286 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the J group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Glutathione-related enzyme activities\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the activities of GR and GST, the levels of GSH, and the GSH/GSSG ratio in \u003cem\u003eA. bisporus\u003c/em\u003e, all of which are involved in glutathione metabolism (GSH cycle). GR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and GST (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) activities initially increased and then declined. In the CK group, GR and GST reached their peaks at 72 h and 48 h, respectively, whereas in the J group, maximum activities occurred earlier, at 48 h and 12 h. GSH levels in the CK group decreased gradually over time. In the J group, GSH levels increased at 12 h but subsequently declined sharply, becoming lower than those in the CK group after 36 h. In the CK group, the GSH/GSSG ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) exhibited a continuous increase, peaking at 72 h. In contrast, the ratio in the J group peaked at 24 h, then declined and dropped below the CK group by 48 h.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Transcriptome data analysis and functional annotation\u003c/h2\u003e\u003cp\u003eTo investigate the molecular responses of \u003cem\u003eA. bisporus\u003c/em\u003e to \u003cem\u003eT. harzianum\u003c/em\u003e infection, tissue samples were collected from the CK and J groups at 0, 6, 18, 24, and 36 h for transcriptome sequencing. After removing low-quality reads and adapter sequences, 61.38 Gb of clean data were obtained in total, with no less than 6.04 Gb per sample. The sequencing quality was high, with Q20\u0026thinsp;\u0026ge;\u0026thinsp;97.48%, Q30\u0026thinsp;\u0026ge;\u0026thinsp;92.31%, and GC content ranging from 49.06% to 49.66% (Table S1). These results demonstrate that the quality and robustness of sequencing data were high, providing approximately 6, 500 genes for subsequent analyses.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, BLAST searches were conducted against six databases, namely GO, KEGG, EggNOG, NR, Swiss - Prot, and Pfam, to further analyze the functional significance of the identified genes and transcripts. A total of 9, 256 genes (98.4%) were functionally annotated in the GO database, while 3, 962 genes (42.77%) were mapped to the KEGG database. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, transcriptome profiling of the CK and J groups revealed distinct patterns of DEGs. At 6 h, 1, 228 DEGs were detected, including 520 up-regulated and 708 down-regulated genes. At 18 h and 24 h, 626 and 980 DEGs were detected, respectively. The highest number of DEGs was observed at 36 h, with 1, 696 genes differentially expressed, including 916 up - regulated and 780 down - regulated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 GO enrichment analysis and KEGG pathway analysis of DEGs\u003c/h2\u003e\u003cp\u003eGO and KEGG enrichment analyses were performed on the DEGs from four pairwise comparisons (CK6h vs. J6h, CK18h vs. J18h, CK24h vs. J24h, and CK36h vs. J36h) with an adjusted \u003cem\u003eP\u003c/em\u003e-value (Padj)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log\u003csub\u003e2\u003c/sub\u003e (fold change)| \u0026ge; 1. In the GO enrichment analysis, 154, 99, 217, and 245 terms were significantly enriched in CK6h vs. J6h, CK18h vs. J18h, CK24h vs. J24h, and CK36h vs. J36h, respectively. To further investigate the defense responses of \u003cem\u003eA. bisporus\u003c/em\u003e to pathogen invasion, we selected GO terms associated with more than two DEGs in each of the three major categories (Biological Process, Molecular Function, and Cellular Component), ranked them by -log\u003csub\u003e10\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e) values, and retained the top 20 terms for each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, DEGs at 18 h, 24 h, and 36 h showed significant enrichment in oxidoreductase activity, suggesting that oxidoreductases were activated during the early stages of pathogen infection and played a critical role in the defense response of \u003cem\u003eA. bisporus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe KEGG database is a widely used resource for the systematic analysis of metabolic and signal transduction pathways in living organisms (Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, DEGs identified in CK6h vs. J6h, CK18h vs. J18h, CK24h vs. J24h, and CK36h vs. J36h were mapped to the KEGG database to elucidate their molecular functions in the response of \u003cem\u003eA. bisporus\u003c/em\u003e to \u003cem\u003eT. harzianum\u003c/em\u003e infection. The top 20 significantly enriched pathways were then retained for analysis. At 6 h, DEGs were mainly enriched in pathways related to amino acid and carbohydrate metabolism, including alanine, aspartate and glutamate metabolism; amino sugar and nucleotide sugar metabolism; glycine, serine and threonine metabolism; and GSH metabolism. At 18 h, the enriched pathways were primarily associated with the ribosome, biosynthesis of cofactors, and glycerolipid metabolism. At 24 h, the major enriched pathways included the ribosome, DNA replication, the yeast cell cycle, and GSH metabolism. At 36 h, DEGs were mainly enriched in oxidative phosphorylation (OXPHOS), glycolysis (EMP), the citrate cycle (TCA), and peroxisome-related pathways. Among these, energy metabolism-related pathways (OXPHOS, EMP, TCA), GSH metabolism, and MAPK signaling pathways were of particular interest, as they were significantly enriched. We therefore conducted further analyses of these pathways to elucidate the underlying defense mechanisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.7 DGEs enrichment of metabolic pathways\u003c/h2\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.7.1 \u003cem\u003eEMP and TCA\u003c/em\u003e pathways\u003c/h2\u003e\u003cp\u003eIn this study, 48 DEGs were enriched in the EMP and TCA pathways. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA shows the 16 DEGs with the most significant expression differences, including homologs of key enzyme genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase (ALDH), pyruvate dehydrogenase, and pyruvate kinase (PK), all of which were significantly enriched. The expression of GAPDH homologs (78276 and 115671) was up-regulated 1.61-fold and 9.21-fold at 36 h, respectively. Similarly, the PK homolog (110153) exhibited the same trend as GAPDH, reaching its highest expression level at 36 h. The PDHA and PDHB subunits of pyruvate dehydrogenase were also up-regulated as infection progressed, suggesting that \u003cem\u003eT. harzianum\u003c/em\u003e infection may accelerate the catabolism of pyruvate, the end product of glycolysis. Likewise, ALDH homologs (80727, 108035, 108032, 46604, 86450, 65619) exhibited a consistent up-regulation trend over time. In contrast, in the J group, gene expression at 36 h was down-regulated relative to the CK group, potentially leading to acetaldehyde accumulation and subsequent cellular damage in \u003cem\u003eA. bisporus\u003c/em\u003e. A total of 15 DEGs were identified in the TCA pathway, and transcriptome analysis revealed an overall up-regulation as infection progressed. However, the J group displayed down-regulated expression compared with the CK group, particularly at 24 h and 36 h. Expression of succinate dehydrogenase (SDH) homologs (111304, 115047, 101253, 52249, 57403) peaked at 24 h in the J group but declined thereafter, whereas expression levels in the CK group remained higher at both 24 h and 36 h. Similarly, malate dehydrogenase (MDH) homologs (119105, 115666) exhibited expression trends consistent with those of SDH, showing overall up-regulation with time, but their expression levels were consistently lower in the J group compared to the CK group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e3.7.2 OXPHOS pathway\u003c/h2\u003e\u003cp\u003eTranscriptomic analysis revealed 94 DEGs in the OXPHOS pathway. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, gene expression in both CK and J groups gradually increased over time, suggesting that mechanical damage and pathogen infection activate the OXPHOS pathway, thereby enhancing energy conversion in \u003cem\u003eA. bisporus\u003c/em\u003e and triggering a resistance response. However, in the J group, gene expression showed a down-regulation trend compared with the CK group, particularly at 36 h, when homologous genes encoding ATP synthase subunits (ATP1-5, ATP7, and ATP14-20) were significantly down-regulated. These results suggest that pathogen infection may inhibit the energy conversion required for the defense response of \u003cem\u003eA. bisporus\u003c/em\u003e, especially during the later stages of infection, thereby reducing energy production in the OXPHOS pathway and accelerating quality deterioration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.7.3 GSH cycle pathway\u003c/h2\u003e\u003cp\u003eTranscriptomic analysis revealed that genes related to glutathione metabolism were differentially expressed in \u003cem\u003eA. bisporus\u003c/em\u003e upon infection with \u003cem\u003eT. harzianum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In the glutathione metabolic pathway, the homologous gene (110335), which encodes 6-phosphogluconate dehydrogenase, was up-regulated at 6, 12, 18, and 36 h, thereby facilitating NADPH production. Similarly, homologous genes 125563, 84806/113538, and 58613/113579, encoding glutathione reductase, disulfide-bond oxidoreductase, and ribonucleoside-diphosphate reductase, respectively, were also up-regulated. Their increased expression contributes to maintaining glutathione in its reduced state, thereby enhancing ROS scavenging and strengthening host defense.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt 6 h post-infection, 20 GST homologs were differentially expressed (10 up-regulated and 10 down-regulated). At 18, 24, and 36 h post-infection, 21, 21, and 22 GST homologs were differentially expressed, respectively, with differing ratios of up- and down-regulation. These expression patterns suggest that GST-related DEGs were increasingly up-regulated in response to stress as infection progressed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.7.4 MAPKs pathway\u003c/h2\u003e\u003cp\u003eDuring the early stage of infection with \u003cem\u003eT. harzianum\u003c/em\u003e, the MAPK signaling pathway in \u003cem\u003eA. bisporus\u003c/em\u003e was rapidly activated, mediating the pheromone response, cell wall integrity, high-osmolarity response, and filamentation pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The expression levels of genes associated with pheromone signaling, cell wall stress, and filamentation progressively increased over time. They were consistently higher in the infected group than in the control, with marked differences at 36 h. In contrast, genes in the high-osmolarity pathway were initially up-regulated at 6 h but declined thereafter. No significant differences in gene expression were detected between CK and J groups during the early stage. However, after 36 h, gene expression in the infected group was significantly higher than in the control, which coincided with the onset of visible disease symptoms in \u003cem\u003eA. bisporus\u003c/em\u003e. Thus, \u003cem\u003eT. harzianum\u003c/em\u003e infection at later stages disrupted the osmotic balance of \u003cem\u003eA.. bisporus\u003c/em\u003e, which explains the observed liquid exudation and tissue softening during decomposition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.8 qRT-PCR results\u003c/h2\u003e\u003cp\u003eFive DEGs enriched in KEGG pathways were selected for qRT-PCR analysis to validate the transcriptome results. \u003cem\u003eEf1α\u003c/em\u003e was used as an internal control to normalize the relative expression levels of these genes. The qRT-PCR results showed that the expression patterns of the five DEGs were highly consistent with those obtained from the transcriptome analysis, thereby validating the reliability and accuracy of the transcriptome sequencing data (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur previous study demonstrated that \u003cem\u003eT. harzianum\u003c/em\u003e infection causes severe surface lesions accompanied by tissue decay in mushrooms, thereby posing a substantial threat to the postharvest storage of \u003cem\u003eA. bisporus\u003c/em\u003e (Han et al., 2024). In higher plants, stress conditions often activate intrinsic defense mechanisms, particularly those associated with oxidative stress regulation. Inspired by this paradigm, the present study investigated the defense responses of \u003cem\u003eA. bisporus\u003c/em\u003e against \u003cem\u003eT. harzianum\u003c/em\u003e infection. Our results revealed significant alterations in antioxidant enzymes, defense enzymes, and several metabolic pathways following inoculation.\u003c/p\u003e\n\u003cp\u003eUnder biotic stress, plants rapidly generate ROS and trigger localized programmed cell death (PCD) at the infection site (Tian et al., 2013; Yang et al., 2024). To maintain ROS homeostasis, plants employ an enzymatic antioxidant defense system comprising SOD, CAT, and POD. SOD catalyzes the dismutation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;⁻\u003c/sup\u003e into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which is subsequently decomposed by CAT and POD into H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e via distinct catalytic pathways. This coordinated enzymatic system detoxifies ROS and safeguards cellular macromolecules from oxidative damage (Mittler, 2002). Although fungi are phylogenetically distinct from plants, similar ROS-associated defense mechanisms may operate in mushrooms when challenged by pathogenic fungi. In our study, we observed a pronounced increase in antioxidant enzyme activities in \u003cem\u003eA. bisporus\u003c/em\u003e during the early stage of \u003cem\u003eT. harzianum\u003c/em\u003e infection, suggesting an enhanced and rapid defense response compared with mechanical injury. However, after 36 h of \u003cem\u003eT. harzianum\u003c/em\u003e infection, \u003cem\u003eA. bisporus\u003c/em\u003e experienced stress that weakened its antioxidant defense system, ultimately leading to tissue deterioration. This defense activation was transient; enzyme activities declined at later stages of infection, thereby exacerbating tissue deterioration in \u003cem\u003eA. bisporus\u003c/em\u003e. Our observations were consistent with previous findings. Shi et al. (2024) demonstrated that SOD, CAT, and POD protect plant cells from oxidative damage and further showed that compounds such as para-coumaric acid enhance the activities of these enzymes, thereby enhancing the resistance of mushrooms to Brown Blotch disease. Likewise, Zhang et al. (2022b) reported that the application of Meyerozyma guilliermondii to pathogen-infected broccoli enhanced ROS metabolism, with the associated increase in antioxidant enzyme activity indicative of an induced immune response. These findings collectively suggested that, similar to plants, mushrooms also relied on antioxidant enzyme systems to regulate ROS metabolism under biotic stress, although the strength and persistence of these responses might be distinct due to their unique biology.\u003c/p\u003e\n\u003cp\u003eGR is a flavoprotein oxidoreductase that utilizes NADPH to catalyze the reduction of GSSG to its reduced form GSH. Through this process, GR maintains the intracellular glutathione pool in a reduced state and mitigates ROS-induced damage (Pang and Wang, 2010). GSH acts as a potent ROS scavenger, eliminating peroxides generated during metabolism and environmental stress. In plants, enhanced GSH synthesis has been shown to improve stress resistance in response to external stimuli (Alscher, 1989). The GSH/GSSG couple, one of the most abundant redox pairs in plant cells, plays a central role in maintaining redox homeostasis (Hasanuzzaman et al., 2017). Under normal physiological conditions, the GSH/GSSG ratio decreases as GSH is consumed during ROS detoxification and metabolic processes. In this study, the GSH/GSSG ratio in the J group peaked at 24 h and remained higher than that of the CK group until 36 h, indicating an enhanced reducing capacity in mushrooms during the early stage of pathogen infection. This elevated redox state is likely to have preserved protein structure and function, thereby sustaining normal cellular processes under stress. GSTs, a superfamily of enzymes, play an important catalytic role in the glutathione cycle by conjugating GSH and detoxifying harmful endogenous compounds such as hydroxyalkyls and hydroperoxides, thereby conferring both antioxidant and detoxification capacities (Nahar et al., 2015). In plants, enhanced GST expression is widely recognized as a hallmark of stress responses. Consistent with this, in \u003cem\u003eA. bisporus\u003c/em\u003e, both GST activity and GST-related gene expression were significantly induced before 36 h of \u003cem\u003eT. harzianum\u003c/em\u003e infection, underscoring the critical role of GST in the fungal defense response. Furthermore, glutathione metabolism has been shown to be directly involved in plant-pathogen interactions (Gill \u0026amp; Tuteja, 2010). In plants, glutathione accumulation not only scavenges ROS but also restricts pathogen growth. Our findings suggested that edible fungi might employ a comparable strategy, with increases in glutathione metabolism contributing to defense activation. Taken together, the observed enhancement of enzyme activities and the upregulation of glutathione-related genes highlight an active defense response in \u003cem\u003eA. bisporus\u003c/em\u003e, reinforcing the pivotal role of glutathione metabolism in fungal resistance to pathogen infection.\u003c/p\u003e\n\u003cp\u003ePRs constitute a family of proteins that are closely associated with disease resistance and defense mechanisms. In plants, PRs rapidly accumulate following pathogen infection and degrade pathogen cell-wall macromolecules, thereby enhancing host resistance (Ge et al., 2015; Wang et al., 2009). Most PRs function as hydrolases; for example, GLU (PR-2 family) and CHT (PR-3 family) directly inhibit pathogens by hydrolyzing their cell-wall components (Smith et al., 2009). Normally expressed at low levels, these enzymes are strongly induced under pathogen-induced stress, where they act synergistically to reinforce host cell walls and promote the biosynthesis of antimicrobial compounds (Zhang et al., 2022c). PAL, a key enzyme in the phenylpropanoid pathway, contributes to the production of flavonoids and phenolic compounds, both of which are important for plant defense (Li et al., 2023). Although the roles of PRs and defense enzymes have been extensively characterized in plants, evidence in fungi remains limited. In our study, the activities of defense enzymes-including PRs, CHT, GLU, and PAL-increased in both the CK and J groups, most likely due to puncture-induced mechanical injury. However, their activities were consistently higher in the J group, indicating that \u003cem\u003eA. bisporus\u003c/em\u003e responded not only to wounding but also to pathogen challenge. These results indicated that pathogen infection transiently enhanced defense-related enzyme activities in \u003cem\u003eA. bisporus\u003c/em\u003e. Although both pathogen infection and mechanical damage could stimulate such activities, the defense response induced by pathogen infection was more rapid and pronounced. Overall, these findings suggested that edible fungi, similar to plants, activated defense enzyme systems to strengthen their resistance mechanisms..\u003c/p\u003e\n\u003cp\u003eActivation of plant defense responses requires both carbon skeletons and substantial energy input, with ATP serving as the primary energy currency (Chen et al., 2021). In plants, energy is derived mainly from EMP, TCA cycle, and the pentose phosphate pathway (Bolton et al., 2008). Upregulation of GAPDH homologs, a key rate-limiting enzyme in EMP, suggests that EMP activity may be enhanced in \u003cem\u003eA. bisporus\u003c/em\u003e during resistance to \u003cem\u003eT. harzianum\u003c/em\u003e infection. Similarly, PK, another critical EMP enzyme, catalyzes the irreversible conversion of phosphoenolpyruvate to pyruvate, and its high expression in infected tissues has been associated with enhanced EMP flux (Mutuku \u0026amp; Nose, 2012). Consistent with observations in plants (Bolton et al., 2008), our results indicate that EMP activation in \u003cem\u003eA. bisporus\u003c/em\u003e contributes to resistance. Pyruvate generated via EMP subsequently enters the TCA cycle, where it is fully oxidized to produce ATP and reducing equivalents. SDH, a key enzyme of the TCA cycle, catalyzes the dehydrogenation of succinate while coupling with ATP generation; its activity is widely regarded as a marker of TCA efficiency (Wang et al., 2020). Upregulation of homologs encoding enzymes such as SDH and MDH further supports the activation of the TCA cycle in \u003cem\u003eA. bisporus\u003c/em\u003e. These findings suggested that, similar to plants, fungi mobilized energy metabolism to meet the elevated ATP demand of defense responses. However, in mushrooms infected for more than 36 h, the activities of EMP and TCA cycle pathways declined, resulting in reduced ATP production and accelerated tissue deterioration.\u003c/p\u003e\n\u003cp\u003eMitochondrial OXPHOS constitutes the final stage of ATP synthesis. This process, mediated by the mitochondrial electron transport chain (ETC), couples electron transfer with oxygen consumption to generate ATP, the universal energy currency (Kummer \u0026amp; Ban, 2021; Meyer et al., 2019). The ETC comprises five major enzyme complexes: Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase), Complex III (cytochrome c oxidoreductase), Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) (Lu et al., 2023). In this study, homologous genes encoding subunits of these complexes were generally downregulated under \u003cem\u003eT. harzianum\u003c/em\u003e infection (Fig. 8B), suggesting reduced ATP production through OXPHOS. Previous studies have demonstrated that inadequate ATP supply contributes to senescence and physiological disorders in harvested fruits and vegetables (Friedman \u0026amp; Nunnari, 2014; Nolfi-Donegan et al., 2020). Our findings extend this concept to edible fungi, indicating that energy deficiency resulting from impaired OXPHOS may be a critical factor underlying the decline in resistance and the development of disease symptoms in \u003cem\u003eA. bisporus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe MAPK signaling pathway plays a central role in plant defense by regulating hormone biosynthesis, ROS production, and defense-related gene expression. This highly conserved cascade in eukaryotes is composed of three core kinases-MAPKKK, MAPKK, and MAPK-that sequentially activate one another through phosphorylation (Meng \u0026amp; Zhang, 2013). In plants, MAPK cascades are among the earliest pathways activated during pathogen infection, transmitting signals from membrane-localized receptors and amplifying downstream defense responses (Walls et al., 2015; Bi \u0026amp; Zhou, 2017). Although well characterized in plants, MAPK-mediated defense mechanisms in fungi remain less understood. In this study, transcriptomic analysis revealed that the homologous gene encoding the transmembrane receptor Sho1 was upregulated in both the hyperosmotic and filamentous growth pathways, suggesting that pathogen-derived signals were perceived and transmitted via the Hog1 and Kss1 cascades. Previous studies have shown that Hog1 regulates carotene and glycerol accumulation in Cordyceps militaris, thereby maintaining osmotic balance and enhancing antioxidant capacity (Zhao et al., 2021). In contrast, the Kss1 pathway primarily activates adenylate cyclase, promoting filamentous growth and preserving cell wall integrity (Cherkasov et al., 2003). Collectively, our results indicate that both mechanical injury and pathogen infection activated MAPK signaling in \u003cem\u003eA. bisporus\u003c/em\u003e, with Hog1 playing a more prominent role than Kss1. These findings suggest that MAPK signaling contributes to fungal resistance by maintaining cellular homeostasis under biotic stress.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003e\u003cem\u003eA. bisporus\u003c/em\u003e mounted an active defense response to \u003cem\u003eT. harzianum\u003c/em\u003e infection. In this study, pathogen challenge enhanced the activities of antioxidant, defense, and glutathione-related enzymes, and significantly upregulated the transcription of genes associated with the glutathione cycle, EMP, TCA cycle, OXPHOS, and MAPK pathways, thereby activating defense responses in \u003cem\u003eA. bisporus\u003c/em\u003e. Specifically, key enzyme genes such as PK in the glycolysis/gluconeogenesis pathway were upregulated to accelerate energy metabolism and secure sufficient energy supply. The expression of TCA cycle-related genes was elevated, providing precursors for the biosynthesis of resistance-related metabolites. Activation of the oxidative phosphorylation pathway improved ATP production efficiency, thereby maintaining cellular homeostasis. Glutathione metabolism contributed to the scavenging of pathogen-induced ROS, while the MAPK signaling cascade was continuously activated through phosphorylation events to regulate defense-related transcription factors. Collectively, these positive regulatory processes facilitated energy redistribution, enhanced antioxidant capacity, and activated systemic resistance, as validated by qRT-PCR results consistent with transcriptomic data. These findings provide theoretical support for elucidating the defense mechanisms of antifungal resistance in mushrooms and establish a foundation for effective disease prevention and control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors report no declarations of interest in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJia Wang\u003c/strong\u003e: Resources, Investigation, Methodology, Data curation, Writing-original draft. \u003cstrong\u003eJiali Han\u003c/strong\u003e: Resources, Investigation, Methodology, Data curation, Writing-original draft. \u003cstrong\u003eYueyuan Li\u003c/strong\u003e: Conceptualization, Formal analysis. \u003cstrong\u003eXiangyou Wang\u003c/strong\u003e: Writing - review \u0026amp; editing. \u003cstrong\u003eYanyin Guo\u003c/strong\u003e: Writing - review \u0026amp; editing. \u003cstrong\u003eHiroaki Kitazawa\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWriting - review \u0026amp; editing. \u003cstrong\u003eLing Li\u003c/strong\u003e: Conceptualization, Supervision, Resources, Funding acquisition, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (31901765 and 31972144).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed in the study, which can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the valuable contributions of all collaborators who provided resources, technical support, and constructive feedback during this research. 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Preharvest L-arginine treatment induced postharvest disease resistance to \u003cem\u003eBotrysis cinerea\u003c/em\u003e in tomato fruits. \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e, \u003cem\u003e59\u003c/em\u003e(12), 6543\u0026ndash;6549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf2000053\u003c/span\u003e\u003cspan address=\"10.1021/jf2000053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou, Z., Han, P., Bai, S., Ma, N., Fang, D., Yang, W., Hu, Q., \u0026amp; Pei, F. (2023). Transcriptome analysis reveals the mechanism of caffeic acid-grafted-chitosan/polylactic acid film packaging to delay quality deterioration in postharvest \u003cem\u003eAgaricus bisporus\u003c/em\u003e. \u003cem\u003eScientia Horticulturae\u003c/em\u003e, \u003cem\u003e309\u003c/em\u003e, 111647. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2022.111647\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2022.111647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Table S1 Quality of clean reads in \u003cem\u003eA. bisporus\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Agaricus bisporus, Trichoderma harzianum, Pathogen infection, Defense response","lastPublishedDoi":"10.21203/rs.3.rs-7749895/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7749895/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eTrichoderma harzianum\u003c/em\u003e (\u003cem\u003eT. harzianum\u003c/em\u003e) infection causes black lesions on the surface of \u003cem\u003eAgaricus bisporus\u003c/em\u003e (\u003cem\u003eA. bisporus\u003c/em\u003e) and soft rot of the infected tissues, thereby severely compromising mushroom quality. To investigate the host response, \u003cem\u003eT. harzianum\u003c/em\u003e was inoculated onto the fruiting bodies of \u003cem\u003eA. bisporus\u003c/em\u003e. This study aimed to comprehensively elucidate the resistance mechanisms of \u003cem\u003eA. bisporus\u003c/em\u003e by analyzing defense-related enzyme activities and transcriptomic profiles. Physiological analyses revealed that antioxidant enzymes (SOD, POD, CAT), defense-related enzymes (CHT, PAL, GLU, PRs), and glutathione-associated enzymes (GR, GST, GSH) were activated, with their activities markedly increased during the early stages of infection. Transcriptomic analysis at 0, 18, 24, and 36 h post-inoculation identified 4,530 differentially expressed genes. Notably, genes involved in the glutathione metabolism pathway (GR, GSH, GST) were up-regulated, consistent with the observed enzyme activity changes. In addition, key genes associated with glycolysis (EMP), the tricarboxylic acid cycle (TCA), oxidative phosphorylation (OXPHOS), and the mitogen-activated protein kinase (MAPK) cascade exhibited enhanced expression. Collectively, these findings indicate that \u003cem\u003eA. bisporus\u003c/em\u003e mounts defense responses by activating glutathione metabolism, EMP, TCA, OXPHOS, and MAPK pathways, thereby enhancing antioxidant and defense enzyme activities and improving resistance. This study elucidates the molecular mechanisms underlying the response of \u003cem\u003eA. bisporus\u003c/em\u003e to \u003cem\u003eT. harzianum\u003c/em\u003e infection and provides a theoretical basis for developing strategies to control postharvest mushroom diseases.\u003c/p\u003e","manuscriptTitle":"Combined physiological and transcriptomic analysis revealed the defense mechanism of Agaricus bisporus against Trichoderma harzianum infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 17:03:13","doi":"10.21203/rs.3.rs-7749895/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-16T20:51:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T20:55:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T20:44:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T11:56:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72101355866700884094851205290072565573","date":"2025-10-08T07:47:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207641639529471354335472408210202176202","date":"2025-10-07T22:24:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45762488931220665034828309630329389992","date":"2025-10-07T17:08:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66038196070951975886475998441712741679","date":"2025-10-07T06:05:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330506909123640281165671099815481861969","date":"2025-10-06T07:18:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-05T16:18:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T12:20:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-03T01:35:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2025-09-30T09:33:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ed501f50-9f2c-4d0b-bc7c-85b116b16396","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T00:53:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 17:03:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7749895","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7749895","identity":"rs-7749895","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}