**Background:** True morels (Morchella spp.) are highly valued edible mushrooms with expanding global cultivation, particularly in China where cultivation reached 16,466 ha during the 2021–2022 season. Despite cultivation breakthroughs, morel production faces significant disease challenges, with an estimated 25% of cultivation area affected by fungal infections causing severe yield losses. In spring 2022, a disease manifesting as red-stipe symptom emerged, causing cessation of fruiting body growth, visible loss of viability, and eventual rot. The etiology remains debated—some studies implicate Fusarium nematophilum, others suggest bacterial pathogens, and some propose nutritional deficiencies. This study aimed to characterize the physiological and biochemical responses of M. sextelata fruiting bodies to the red-stipe symptom using integrated transcriptomic and metabolomic approaches.
**Methods:** Fresh cultivated morels were collected from a farm in Fujian Province, China (119.199865° E, 26.712312° N) in March 2022. Two groups were established: red-stipe (R) fruiting bodies showing red or yellow discoloration, and normal (N) fruiting bodies with white stipes. For transcriptomics, three samples per group underwent RNA sequencing on the Illumina Novaseq 6000 platform. Due to low alignment rates (61.51% to M. sextelata genome, ~20% to M. conica genome), de novo transcriptome assembly was performed using Trinity 2.4.0. Differentially expressed genes (DEGs) were identified using edgeR with |log2 fold change| > 1 and p-value < 0.05. For metabolomics, six samples per group were analyzed using liquid chromatography–mass spectrometry (LC–MS) with an UltiMate 3000 HPLC coupled to a Q-Exactive mass spectrometer. Metabolites were annotated using KEGG and HMDB databases. Differentially accumulated metabolites (DAMs) were identified using PLS-DA with VIP > 1.0 and Student's t-tests. Integrated pathway analysis was performed on common KEGG pathways from both datasets. Four DEGs in the tyrosine metabolism pathway were validated by qRT-PCR.
**Key Results:** Transcriptome sequencing generated 241,412,342 high-quality clean reads (33.51 GB). De novo assembly produced 22,969 unigenes (N50 length 1,585 bp), with 74.68% of unigenes matching Morchella species. PCA showed clear separation between R and N groups (PC1 explained 72.65% of variation). A total of 4,264 DEGs were identified—1,876 upregulated and 2,388 downregulated in the R group. The most upregulated gene (TRINITY_DN14301_c2_g8, log2 FC = 9.64) had unknown function. GO enrichment identified 151 significant terms, including immune-related categories. KEGG enrichment revealed 23 significantly enriched pathways, with tyrosine metabolism, riboflavin metabolism, and glycerophospholipid metabolism being most prominent. Metabolomics identified 3,829 metabolites in positive mode and 3,856 in negative mode. DAMs included 412 upregulated and 760 downregulated in positive mode, and 445 upregulated and 975 downregulated in negative mode. KEGG enrichment of DAMs identified 74 significant pathways. Integrated analysis revealed 58 common pathways between transcriptome and metabolome, with 17 significantly enriched. The three most enriched were nicotinate and nicotinamide metabolism, starch and sucrose metabolism, and tyrosine metabolism. Notably, 26 DEGs belonged to the tyrosine metabolism pathway, including di-copper center-containing proteins, PLP-dependent transferases, and copper amine oxidases. qRT-PCR validation of four selected genes confirmed RNA-Seq expression patterns. Key findings included upregulation of leucoanthocyanidin dioxygenase (log2 FC = 3.21), β,β-carotene 15,15′-dioxygenase (log2 FC = 1.95), laccase-2 (log2 FC = 6.84), and programmed cell death protein 6 (log2 FC = 1.53). Fifteen DEGs were annotated to immune-related GO terms (11 upregulated, 4 downregulated). Twenty-seven DEGs were enriched in autophagy-related pathways. Mapping to the F. nematophilum genome yielded <0.1% alignment, ruling out this pathogen as the cause.
**Clinical Implications:** This is the first integrated transcriptomic and metabolomic study of the red-stipe symptom in M. sextelata. The identification of 4,264 DEGs and over 1,100 DAMs provides a comprehensive molecular framework for understanding this economically damaging condition. The consistent enrichment of tyrosine metabolism across this study and previous work on Morchella development and pathogen response suggests this pathway is central to both normal physiology and stress responses. The upregulation of pigment-related genes (leucoanthocyanidin dioxygenase, carotenoid oxygenases) and immune-related genes (chitin recognition protein, laccase-2, programmed cell death protein) indicates that the red-stipe symptom involves both active defense responses and metabolic reprogramming. The exclusion of F. nematophilum as the causative agent in these samples highlights the need for continued pathogen identification efforts. These findings provide potential molecular targets for breeding disease-resistant morel strains and developing diagnostic markers for early detection of the red-stipe symptom in commercial cultivation.