Strain degeneration has been defined as a decrease or loss in the yield of important commercial traits resulting from subsequent culture, which ultimately leads to Reactive Oxygen Species (ROS) production. Pleurotus ostreatus is a lignin-producing nematophagous edible mushroom. Mycelia for mushroom production are usually maintained in subsequent culture in solid media and frequently show symptoms of strain degeneration. The dikaryotic strain P. ostreatus (DkN001) has been used in our lab as a model organism for different purposes. Hence, different tools have been developed to uncover genetic and molecular aspects of this fungus. In this work, strain degeneration was studied in a full-sib monokaryotic progeny of the DkN001 strain with fast (F) and slow (S) growth rates by using different experimental approaches (light microscopy, malondialdehyde levels, whole-genome transcriptome analysis, and chitosan effect on monokaryotic mycelia). The results obtained showed that: (i) strain degeneration in P. ostreatus is linked to oxidative stress, (ii) the oxidative stress response in monokaryons is genotype dependent, (iii) stress and detoxifying genes are highly expressed in S monokaryons with symptoms of strain degeneration, (iv) chitosan addition to F and S monokaryons uncovered the constitutive expression of both oxidative stress and cellular detoxifying genes in S monokaryon strains which suggest their adaptation to oxidative stress, and (v) the overexpression of the cell wall genes, Uap1 and Cda1, in S monokaryons with strain degeneration phenotype indicates cell wall reshaping and the activation of High Osmolarity Glycerol (HOG) and Cell Wall Integrity (CWI) pathways. These results could constitute a hallmark for mushroom producers to distinguish strain degeneration in commercial mushrooms.

Mitonuclear interactions are crucial in governing mitochondrial function, development and responses to stress in eukaryotic organisms. In this study, we explored how varying mitochondrial haplotypes affect the phenotype and oxidative stress response using hybrids of the basidiomycete Pleurotus ostreatus ( P. ostreatus ) as a model system. By performing reciprocal crosses between monokaryotic strains with distinct nuclear and mitochondrial genomes, we identified notable differences in growth rates, accumulation of reactive oxygen species (ROS) and gene expression patterns. Hybrids with incompatible mitonuclear combinations displayed slower growth and elevated expression of genes — some showing transgressive inheritance — associated with the Electron Transport Chain (ETC) and antioxidant defences. Mitochondria-dependent heterosis was observed in hybrids sharing the same nuclear background, but differing in mitochondrial genome, suggesting that mitonuclear incompatibilities can result in oxidative imbalance and compromised fungal performance. This experimental approach opens wide possibilities for exploring mitonuclear interactions and highlights the significance of mitonuclear co-adaptation in an edible mushroom, offering valuable insights for enhancing hybrid breeding programmes by accounting for the role of mitonuclear interactions in shaping quantitative traits related to mushroom yield.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

Mitochondrial genomes play a central role in fungal physiology and adaptation, yet their evolutionary dynamics during domestication remain poorly understood. Here, we performed a comparative mitogenomic and gene-expression analysis of three Pleurotus ostreatus dikaryotic strains differing in origin and degree of adaptation to laboratory conditions: the long-term commercial strain dkN001, the laboratory-maintained wild isolate dkF515, and the recently collected wild strain dkN009. High-throughput Illumina sequencing enabled complete assembly of circular mitochondrial genomes, revealing substantial size variation among strains, where the dkN001 strain exhibited the second smallest mitogenome reported for the genus Pleurotus. Comparative analyses showed >99% sequence identity between wild isolates and ~95% identity relative to the commercial strain. Variations in genome size among strains were associated with intron dynamics in the cox1 and rnl genes, as well as intron loss predominantly in the commercial strain dkN001, consistent with mitochondrial genome streamlining during domestication. Expression profiling of mitochondrial protein-coding genes (PCGs) under multiple culture conditions revealed conserved transcriptional responses in dkN001 and dkF515 that contrasted sharply with those of dkN009. The differences observed, which affected components of the electron transport chain, suggested shifts in energy metabolism associated with long-term laboratory maintenance. Therefore, our results demonstrate that domestication in P. ostreatus involves both structural remodelling of the mitogenome and changes in regulation of mitochondrial PCGs, highlighting the importance of mitonuclear interactions in fungal adaptation to controlled environments.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots and are known for their peculiar aromas and flavors. Axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation prompts searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) has been isolated and cultured, and its transcriptome has been analyzed under different in vitro culture conditions. The results show that the best T. borchii SP1 growth was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22ºC. The transcriptome analysis of this strain cultured in different media indicated that most of the gene transcription effort is due to a limited number of genes (20% of genes account for 80% of the transcription), that the transcription profile of the central metabolism genes was similar in the different conditions analyzed with a transcription signal detected for around 80% of the annotated genes. The gene expression profile suggests that T. borchii uses a fermentative rather than respiratory metabolism, even in aerobic conditions. Finally, there is a reduced expression of genes belonging to secondary metabolite clusters, whereas there is a significative transcription of those involved in producing volatile aromatic compounds.

Mitonuclear interactions play a fundamental role in eukaryotic evolution, fitness, and hybrid viability. Despite extensive studies in animals and plants, their impact on fungi such as Pleurotus ostreatus (P. ostreatus) remains underexplored. In the dN001 (n) strain, long-term subculturing was shown to lead to degeneration, which was associated with mitonuclear incompatibilities, oxidative stress, and reduced growth. This work demonstrates how mitonuclear incompatibilities trigger strain degeneration and influence phenotypic and agronomic performance traits in P. ostreatus. These findings were supported by a divergent breeding programme derived from the commercial hybrid strain dN001 (n), which enabled the generation of fast- and slow-growing parental lines. These were then crossed with the mT1 (t) tester line to produce hybrid strains with identical nuclei, but different mitochondrial types, thereby allowing the molecular basis of mitonuclear interactions in P. ostreatus to be investigated. Strains were evaluated under different temperatures and carbon source conditions, highlighting the importance of mitonuclear–environment interactions in determining performance. RT-qPCR experiments targeting mitochondrial electron transport chain (ETC) components, antioxidant enzymes, and iron homeostasis-related genes revealed that gene expression was closely linked to mitochondrial type and nuclear compatibility. In particular, the (t) mitochondrial type was responsible for the exceptional performance of the d(NS423 × T1, t) strain, which exhibited the highest commercial performance among all strains studied. Overall, the results of this thesis demonstrate the critical role of mitonuclear interactions in growth rate and mushroom production traits. They also suggest that maintaining the two F₁ parental strains of commercial P. ostreatus hybrids in long-term storage, followed by remaking the hybrid shortly before use, may help to preserve strain performance and prevent degeneration.