Fungal communities on the rinds of alpine cheese from Southern Switzerland are composed of a remarkably large number of species. Statistical analyses suggest that the communities are distinct in accordance not only with the type of milk used for cheesemaking, but also with the geographic location of the cellars. This result is only in partial agreement with the outcome of a study of 137 cheese rind samples from 10 different countries, in which similar communities of bacteria and fungi were detected in geographically distinct parts of the world (Wolfe et al. 2014). Wolfe et al (2014), however, carried out their analysis only at the generic level, whereas we used the fungal species as operational taxonomic units.
Ticino, the Southern Switzerland region in which the cellars examined are situated, is small and partitioned lengthwise by four main valleys and high mountain ranges in between, with many alpine meadows where cattle, goats, and sheep graze during the summertime. It is to be expected that the microecological conditions in each valley are very distinct. We decided to investigate only cellars that complied with the standard conditions required for the “Protected Designation of Origin” label (FOAG) for Alpine cheese produced in Ticino to set a baseline for future studies. Given the homogeneity among the different cellars, as suggested by the temperature and relative humidity data collected during the cheese ripening season, we expected similar fungal communities on the AC rinds, but data show marked differences among the five cellars, as highlighted by the heatmap (Fig. 2) and MDS analyses (FIGs 1C, D). For instance, the dominance of Mucor and the paucity of yeast species separates cellar C from all the others. These differences cannot be conclusively explained: we expect that they may be caused by the type of milk (goat and cow) used for the cheese production and by the different cleaning method used. The geographic location (or perhaps the macro-and microecological conditions at the different locations), however, may also be determinant for the difference.
Cheese rind microbial communities are less complex than those seen in other natural environments, also because of the stress conditions caused by the salty environment (Irlinger et al. 2015). In this study, the cheese rinds with the highest number of species recovered by serial dilutions originated from cellars B and D, and those analyzed by metabarcoding from cellar D. Serial dilution, however, yielded only 6 species, whereas metabarcoding identified 40. For metabarcoding as well, however, the species richness is strongly reduced to at most 6 (cellars D and E), if only taxa with more than 2% reads are considered. Furtehrmore, some of the species recorded (e.g., Ustilago panici-gracilis, Peniophorella pubera, Phlebia spp., Trametes spp.) are most likely occasional contaminants that play no role in the colonization of the cheese rinds, do not modify the cheese taste or texture, and have no influence on health issues such as the production of mycotoxins. It is unlikely that rare taxa play an important role, at least when the standard ripening conditions are kept, but this needs to be verified in larger, culture-based studies.
Debaryomyces was the predominant yeast genus present on all cheese rinds sampled, although it was less frequent in cellar C. It was also often reported from natural and washed rinds in a large study investigating different cheeses from Europe and North America (Wolfe et al. 2014). D. hansenii is commonly used in the cheesemaking process (Fröhlich-Wyder et al. 2019), but its abundance in three of the five cellars studied cannot be explained, because it is not used by the local cheesemakers. D. hansenii, however, can be present in the raw milk (Quigley et al. 2013) and the favorable environmental conditions during cheese making (high salt content, low pH and temperature, lactate as carbon source) may allow colonization of the cheese surface by this yeast during the early ripening phase (Irlinger et al. 2015; Quijada et al. 2020). D. hansenii was dominant in 63% of all cheese types studied by Banjara et al. (2015), and, together with Geotrichum candidum, it was detected in 12 French cheese varieties (Dugat-Bony et al. 2016). Quijada et al. (2020) also reported Debaryomyces from all rind samples they studied.
The identification of Debaryomyces species deserves a brief comment. MALDI-TOF MS identified all isolates as D. hansenii, whereas ITS metabarcoding indicated in the samples the presence of D. prosopidis and D. coudertii, but not D. hansenii. Contrarily to MALDI-TOF MS, however, ITS sequencing does not distinguish D. coudertii from D. hansenii or D. prosopidis (Martorell et al. 2005; Nguyen et al. 2009). We thus expect all Debaryomyces isolates to belong to D. hansenii, even if we have carried out the statistical and ecological analyses with the original data.
As already reported in other studies on cheese mycobiota, Mucor spp. and Penicillium spp. were the most common filamentous fungi on the cheese rind samples examined, Mucor being the second most prominent genus after Penicillium. Mucor lanceolatus and M. racemosus, detected by serial dilution techniques and metabarcoding, were also among six Mucor species isolated from French cheeses (Hermet et al. 2012) and were detected in Saint-Nectaire cheese by Dugat-Bony et al. (2016). A species of Mucor, very likely M. racemosus, is considered a contaminant of alpine cheeses, with a negative impact on the taste and becoming problematic when its occurrence is chronical and persistent (Pillonel 2006). Mucor species were not mentioned in the Wolfe et al. (2014) study that analyzed bloomy, natural or washed rinds of European and North American cheeses. One can speculate that not enough humidity was present in the samples studied by Wolfe et al. (2014) as they reported some Scopulariopsis species that are considered xerophilic (Domsch et al. 2007). Selected Mucor strains are used to ripen some semi-soft cheeses including uncooked Saint Nectaire, Tomme de Savoie, or Taleggio (Fox and McSweeney 2004), as they produce lipases and proteases that contribute to the distinctive flavors, texture, or nutritional quality of the cheeses (Hermet et al. 2012). On some types of soft cheeses produced in Southern Switzerland, Mucor fuscus is also very common (Petrini L.E., unpublished data): this species, however, was not recorded from our samples, probably because their water content was not enough to allow this taxon to compete with other faster growing Mucor species.
As expected, P. camemberti was very frequently isolated by serial dilutions and detected by metabarcoding (Tables 3 and 4). It was comparatively rare only in samples from cellars B and C (serial dilutions, Table 3) and E (metabarcoding, Table 4). Among the Penicillium species recorded by either method, P. echinulatum, P. solitum, and P. verrucosum have already been reported from cheese; only P. oxalicum has so far been described only from soil samples (Samson et al. 2004). Penicillium species may enter production facilities or ripening rooms as contaminants and colonize the cheese surfaces (Nielsen et al. 1998; Ropars et al. 2012; Bodinaku et al. 2019). Penicillium cultures are sometimes added to the cheese curd during the cheese making process, but none was added to any of the cheeses studied, thus they most probably originated from the manufacturing or ripening environments.
MALDI-TOF MS identified the dominant species as P. camemberti, whereas the β-tubulin gene sequencing method identified it as P. biforme, another species frequently recorded from cheese. Index Fungorum (https://indexfungorum.org) considers P. biforme a synonym of P. camemberti; MycoBank re-introduced P. biforme Thom as legitimate in 2010 and this taxon is now considered distinct from P. camemberti (Giraud et al. 2010). In a whole genome-based analysis P. biforme, P. camemberti, and P. fuscoglaucum formed separate and specific genetic clusters, with P. biforme and P. camemberti, however, being sister clades (Ropars et al. 2020). These authors concluded that these three species represent closely related, but different lineages that have all evolved traits beneficial for cheese-making. For the sake of our study, however, no detailed taxonomic analyses were considered necessary, and we used the names provided by the ITS sequencing and metabarcoding in the statistical models.
Some fungal species such as Scopulariopsis flava, S. fusca or Mammaria sp. have often been observed on AC (L.E. Petrini, unpublished data) but were not recorded during this study. Probably they emerge later in the ripening process when the cheese surface offers a drier environment, hampering fast growing Mucor and Penicillium species.
In this study we used a combination of culture-dependent and independent methods (Fröhlich-Wyder et al. 2019) because both systems are considered complementary and not contradictory or exclusive (Irlinger et al. 2015). Both methods produced similar results in terms of dominant taxa present in the samples and similarity of the fungal communities inhabiting the cheese rinds, metabarcoding giving overall more detailed information (Table 3, Supplementary Table S1). Metabarcoding has some advantages over cultural methods for ecology studies. Culture-based methods are difficult to carry out in large-scale studies; they are more demanding and often require selective media. In addition, they are more expensive and time consuming compared to metabarcoding. Non-culturable species cannot be detected, and slow growing strains are easily overgrown by fast-growing ones (Banjara et al. 2015). Metabarcoding, on the other hand, is very sensitive and cost-effective, but it does not distinguish between colonizing species and occasional, dormant contaminants. The number of reads, however, may provide some clues on this issue, and an overall knowledge of the mycobiota present on the rind of cheese ripening under standard, controlled conditions will enable further investigations on the influence of changing conditions such as climate change or modification of ripening conditions in the cellars. Culture-dependent systems, however, will still be crucial for experimental studies.
Our study has some limitations. Because of economic reasons and lacking manpower, the number of cellars and rind samples studied was small. The cheese rinds were sampled only once at the early-mid ripening phase (on average 30 days after production, Table 3) and metabarcoding was carried out on only one sample, precluding the possibility to detect intra-cellar variation of the mycobiota. It is known that the dynamic of bacterial and fungal microbiota evolves throughout the ripening process (Quijada et al. 2020; Penland et al. 2021): future studies should, therefore, foresee sampling during the early and late ripening stages to gather information on the evolution of the rind communities throughout the cheese maturation period.