Culturomics Reveals Microbial Dynamics in the AppleCarposphere Across Developmental Stages, Altitude andTissue Types

Abstract

The apple carposphere harbours a diverse community of microorganisms that could play a crucial role in fruit health andpostharvest preservation. While culture‐independent techniques have advanced our understanding of the apple microbiome, asubstantial portion of this community remains unexplored due to cultivation limitations. In this study, a culturomics approachwas adopted using culture media enriched with apple‐derived nutrients to recover and characterise the epiphytic and en-dophytic bacterial and fungal communities of apple fruits. Aiming to analyse the effects of the altitude and developmental stageof the fruit on these microbial communities. To do this, the impact of altitude (Pyrenees mountain vs. Ebro valley), fruitdevelopmental stage (30 days before harvest vs. harvest) and tissue type (epiphytes vs. endophytes) was assessed on microbialdiversity and composition across four orchards in Catalonia, Spain. Using 13 distinct culture media, 50% more microbial generawere recovered than conventional laboratory media, yielding over 919 isolates, consisting of 489 bacteria, 222 filamentous fungiand 208 yeasts. The results showed that altitude, developmental stage and type of culture media influenced microbial diversityand composition. Richness of endophytic fungi was more influenced by altitude and developmental stage than epiphytic fungaland bacterial communities. On the other hand, bacterial community composition was strongly influenced by the type of culturemedium used. Functional characterisation of isolates revealed potential biocontrol agents and plant pathogens, with somegenera displaying altitude‐specific distributions. Our findings demonstrate that culturomics provides an essential tool to unlockthe hidden diversity of fruit‐associated microbiomes, paving the way for future applications in sustainable agriculture andpostharvest disease management.