A Thousand Meters Deep: Vertical Profiling of the Subterranean Microbiome of Gourgouthakas Cave

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 conducted a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the world’s deepest vertical systems, which had remained untouched by humans for 19 years. Methods We performed a high-resolution vertical profiling of the cavès microbes by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation on various media and at different temperatures, we established a biobank of 820 bacterial isolates. Results Taxonomic identification of a 374-isolate subset revealed a diverse community spanning 35 genera and 4 phyla, dominated by Pseudomonas , Aquipseudomonas , Bacillus , and Stenotrophomonas . Beyond characterizing this taxonomic diversity, we explored the biotechnological potential of these subterranean microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum , Verticillium dahliae , and Phytophthora nicotianae , uncovered a notable group of strains with potent antagonistic activity, particularly within the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacteria ( Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 biosynthetic gene clusters (BGCs), over half of which showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Pangenomic analysis of Streptomyces revealed 1,497 unique gene clusters. Finally, ex vivo trials showed that the Aquipseudomonas paracarnis (formerly Pseudomonas sp. ) isolate SRL917 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 merely geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.