Characterization of the microbiome from subcortical tissues of Quercus suber threatened by Diplodia corticola

Cork oak ( Quercus suber ) forests in the western Mediterranean basin are increasingly affected by decline associated with drought stress and the incidence of the Botryosphaeria canker, a disease caused by the emergent pathogen Diplodia corticola , which is considered a major concern for forest owners. In this study, we investigated whether D. corticola behaves as an endophytic fungus and how trunk-associated microbes (i.e. fungi and bacteria) are associated with cork oak decline. Subcortical tissues (phelloderm and phloem) were sampled from five managed Q. suber stands in Catalonia (north-eastern Spain) from healthy trees, asymptomatic tissue from symptomatic trees, and active necrotic cankers. Fungal and bacterial communities were characterized using DNA high-throughput sequencing (metabarcoding) followed by structural and functional characterization of the microbiome. Fungal alpha and beta diversity did not differ significantly among tissue categories, and a stable core mycobiome was detected across all health conditions, including known oak pathogens such as D. corticola and Biscogniauxia mediterranea . Their detection in visually healthy tissues supports a latent persistence. In contrast, bacterial assemblages shifted markedly with decline. Healthy tissues were dominated by Alphaproteobacteria (including Novosphingobium and Methylobacterium ), whereas necrotic tissues were enriched in Actinobacteria and Bacilli, specifically taxa associated with decomposition and anaerobic metabolism. Several fungal taxa previously reported as antagonistic or entomopathogenic with potential biocontrol activity (i.e. Clonostachys sp., Beauveria bassiana ) were detected mainly in healthy tissues. These results show that main Q. suber phytopathogens are common as endophytes even in vigorous trees, and that bacterial shifts tracked the necrotic stage of decline. These findings improve understanding of microbiome dynamics associated with cork oak decline and may contribute to future management strategies. • Cork oak decline reflects microbiome reassembly rather than single-pathogen invasion. • Diplodia corticola persists as a latent endophyte in healthy subcortical tissues. • Fungal communities are stable, while bacterial communities track tissue necrosis. • Beneficial and antagonistic taxa occur naturally in healthy cork oak trunks. • Microbiome-aware management may improve cork oak resilience without chemicals.