Paragkamian, S.; Christakis, C. A.; Michalopoulou, V. A.; Plakogiannaki, M.; Soultatos, S.; Arapitsas, N. P.; Vaxevanopoulos, M.; Sotiriadis, Y.; Pennos, C.; Markakis, E. A.; Sarris, P. F.

Introduction: Caves represent unique, nutrient-limited windows into the deep biosphere, yet the microbiology of the deep terrestrial subsurface remains remarkably under-explored. In this work, we took advantage of a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the deepest vertical systems in the world, which had remained untouched by humans for 19 years. Methods: We performed a high-resolution vertical profiling of the microbiome by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation using various media and temperatures, we established a biobank of 820 bacterial isolates. Results: Taxonomic identification of a 362-isolate subset revealed a diverse community spanning 25 genera and 4 phyla, dominated by Pseudomonas, Bacillus, and Stenotrophomonas. Beyond characterizing diversity, we explored the biotechnological potential of these subterrane-an microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum, Verticillium dahliae, and Phytophthora nicoti-anae, uncovered a significant group of strains with potent antagonistic activity, particularly with-in the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacte-ria (Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 bio-synthetic gene clusters (BGCs); notably, over half of these showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Finally, ex vivo trials showed that the Pseudomonas sp. SRL917 isolate significantly reduced Botrytis cinerea infections on tomato leaves, even surpassing the performance of a commercial biocontrol agent. Discussion: Collectively, our results demonstrate that deep karstic systems are not just geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.