Genotypic and phenotypic variability in Geotrichum candidum : the cheese making fungus

Abstract

Geotrichum candidum is a fungus widely associated with the production of ripened cheeses and plays a fundamental role in the Natural Bloomy Rind Artisanal Minas Cheese (NBRAMC). In this traditional product, the species naturally dominates the cheese surface during ripening, resulting in the characteristic white surface and contributing to changes in the curd, as well as to the development of desirable texture, aroma, and flavor. Despite its technological relevance, the phenotypic, genetic, and functional diversity of G. candidum are still not fully understood, particularly for Brazilian strains, since most available studies rely on strains obtained from European cheeses. In this study, strains of G. candidum obtained from cheeses, including NBRAMC, and from the environment were characterized through morphological, physiological, genomic, and functional analyses, to assess the intraspecific diversity present in strains associated with different origins. A high level of polymorphism was observed in the ITS and LSU regions of the rDNA, along with the detection of ambiguous positions in the housekeeping gene ACT. Morphological characterization revealed six distinct morphotypes, indicating that traditional classification is insufficient and highlighting the absence of dimorphism in the species. A total of 40 complete genomes were sequenced using Oxford Nanopore technology and analyzed together with 96 publicly available genomes. Comparative analysis revealed the formation of a cluster composed of strains originating from cheese/dairy products, distinct from mixed origin, indicating the existence of genetically diverse populations of G. candidum. Seven representative strains were selected for detailed structural and functional genomic analyses, resulting in high-quality assemblies with contiguity ranging from 13 to 409 contigs and completeness > 95.6%. The analyses revealed varying degrees of heterozygosity, evidence of polyploidy, and pronounced intraspecific divergence. Functional annotation showed that the environmental strain exhibited a broader predicted enzymatic repertoire, including a higher count of CAZymes and KEGG pathways, suggesting higher metabolic versatility. In contrast, cheese-associated strains displayed reduced gene repertoires, consistent with domestication within the dairy environment. Furthermore, the study demonstrated that G. candidum strains produce a wide variety of volatile compounds related to flavor development and antimicrobial activity. A total of 76 compounds were identified, six of which were produced by all strains: phenylethyl alcohol, hexanoic acid, dodecanoic acid, ethyl caprylate, ethyl decanoate, and 2-tridecanone. In microbiological assays, all tested G. candidum strains inhibited the growth of Aspergillus ochraceus and prevented the production of ochratoxin A, even under conditions favorable for mycotoxin synthesis. The results reveal intraspecific diversity of G. candidum in the Brazilian context and reinforce the functional relevance of the species in the ripening and biological protection of surface-ripened artisanal cheeses. Additionally, the findings provide an important foundation for future genomic studies involving G. candidum, emphasizing the need for sequencing and annotating new environmental strains and for deepening phylogenetic analyses.