Cave ecosystems impose strong environmental filters, such as permanent darkness and oligotrophy, which shape microbial communities. Although fungal diversity in Brazilian caves has been increasingly reported, integrative analyses of community composition and functional structure remain limited. In this study, culture-dependent isolation (air and sediment) and ITS2 metabarcoding were combined to characterise the fungal community of Penhasco Cave, located in the Brazilian savannah (Cerrado), and to contextualise its inferred functional profile using previously published cave records from other Brazilian biomes. Across datasets, the community was dominated by Ascomycota, followed by Basidiomycota and Mucoromycota. Within Ascomycota, Sordariomycetes and Eurotiomycetes were the most representative classes. Culturable richness was higher in sediment than in air, with only two taxa shared between substrates and marked spatial turnover among sampling points. Metabarcoding reinforced the dominance of Ascomycota and detected additional lineages not recovered by cultivation. FUNGuild-based functional assignments indicated a predominance of multitrophic categories, particularly Pathotroph–Saprotroph and Pathotroph–Saprotroph–Symbiotroph modes, suggesting diverse ecological strategies. Undefined Saprotrophs were the most frequent guild, while plant-associated groups such as Plant Pathogens and Endophytes were also well represented. This pattern is consistent with the influence of allochthonous inputs from surrounding vegetation and with surface–subsurface connectivity. However, because functional assignments are based on database annotations and literature-derived genus lists, they should be interpreted as putative associations rather than direct evidence of functional activity. Overall, integrating culture-dependent and metabarcoding approaches improves the understanding of fungal diversity and functional patterns, suggesting that tropical caves may serve as reservoirs of fungal functional potential.
Lentinula edodes is a globally popular edible mushroom prized for its distinctive flavor compounds. However, the over reliance of traditional L. edodes cultivation on woods poses adverse challenges to the ecological environment. Therefore, developing alternative substrates from agricultural waste is essential for the sustainable production of L. edodes. This study systematically investigated the effects of using Dimocarpus longan fruiting shells (DLS) as a partial replacement for wood sawdust on the growth and quality of L. edodes. Results showed that a 15% substitution level significantly promoted mycelial growth and increased crude protein and free amino acid contents of the fruiting bodies. Volatile metabolome analysis revealed that DLS supplementation reshaped the volatile profile, especially by enhancing the synthesis of characteristic sulfur-containing flavor compounds such as lenthionine and methanethiol. Integrated transcriptome and correlation analysis between two omics datasets further uncovered that this flavor improvement was driven by the upregulation of genes involved in sulfur metabolism and cysteine/methionine metabolism pathways, including genes encoding cysteine synthase and cystathionine γ-synthase. Our findings offer a mechanistic, waste-to-value strategy for improving L. edodes quality and advancing the circular bioeconomy.
Fungal metabarcoding is a vital tool for understanding plant-microbiome dynamics and assessing how natural and anthropogenic drivers influence the diversity of plant-associated fungal communities across environments. Commonly used fungal ITS primers have inherent biases, often co-amplifying off-target plant host DNA while underrepresenting ecologically important groups (such as the mycorrhizal-containing phyla Glomeromycota and Mucoromycota). Here, we conducted both in silico and experimental comparisons of commonly used fungal primer combinations against a newly designed primer developed to reduce plant host binding and provide a more balanced fungal taxonomic coverage. Primer performance was evaluated across diverse habitat types, using plant root tissue and soil samples. Metrics for species richness and community diversity varied significantly depending on the primer pair used, with performance influenced by both habitat and sample type. Despite this variability, our novel primer demonstrated the most consistent, reliable, and balanced performance. This makes it a suitable option for capturing a wide portion of the fungal community (including mycorrhizal fungi) without having to employ multiple primer pairs.
The Southern Brazilian Highland Grasslands are one of the few subtropical high-altitude grasslands in the Neotropics, featuring highly endemic biodiversity. Yet, this formation remains largely neglected in conservation policies and faces several anthropogenic pressures, despite being part of the Brazilian Atlantic Forest global biodiversity hotspot. While plant communities are well understood in this formation, its soil funga is completely unknown. For the first time, we characterized the diversity of soil fungal communities in these grasslands, using shotgun metagenomics to generate ITS and 18S profiles, assess potential composition of trophic modes, and predict functionality based on detected genes across three preserved grassland fragments. We also sought to unveil how fungal communities are shaped by different assembly processes at a local scale. Ascomycota dominated across the 16 detected phyla, while Basidiomycota and Chytridiomycota showed lower but consistent relative abundances, a pattern that holds for order and genus levels. Taxonomic profiles indicated that saprotrophs and plant pathogens have homogenous relative abundances across fragments, with genes revealing a metabolic potential consistent with this pattern. However, community structures were significantly different among fragments, which also showed distinct alpha diversity patterns. Edaphic and plant community-related variables commonly known to modulate soil fungal communities explained a small portion of the variance observed in communities. In accordance, we showed that stochastic and deterministic community assembly processes have contrasting dominance within and across fragments. This indicates asymmetric connectivity between grassland patches and complex community assembly dynamics at small scales, which can be linked to the mosaic patterns formed between grasslands and adjacent formations. Our findings establish a baseline for soil fungal diversity across local Southern Brazilian Highland Grasslands fragments and highlight the need to integrate overlooked, yet diverse, fungal communities into conservation planning for this endangered formation.
