Isolation of Tomato Seed-Associated Microbiota through Controlled Germination and Comparative Culturing

Surface sterilization is a critical yet frequently under-optimized step in studies aiming to isolate seed-associated microorganisms, as excessively harsh treatments can reduce or distort endogenous microbial populations. This protocol presents a reproducible approach for isolating culturable tomato seed-associated microbiota using surface-sterilization conditions based on previously validated methodologies, combined with controlled seed germination. Rather than defining universally optimal sterilization treatments, the approach applies standardized conditions that can be adjusted according to seed characteristics while minimizing bias and preserving endogenous microbial populations. To enhance recovery of culturable microorganisms associated with the seed-to-seedling transition, the protocol incorporates a controlled germination step prior to microbial isolation. In contrast to conventional approaches based on the maceration of dry, non-germinated seeds, controlled germination promotes microbial activation during the seed-to-seedling transition under sterile conditions. Comparative colony-forming unit (CFU)-based analyses of equivalent biological replicates, each consisting of pooled material from 3-5 dry seeds or 3-5 germinated seedlings, revealed a marked shift in the culturable community following germination. Genera that were weakly represented in dry seeds became enriched after germination, whereas other taxa that were more abundant in dry seeds decreased in relative representation, supporting studies of early plant-microbe interactions during seedling establishment. Across biological replicates, genera including Stutzerimonas, Stenotrophomonas, and Priestia were consistently enriched among isolates recovered from germinated seedlings, whereas Paenibacillus remained abundant across conditions but showed marked shifts in species-level composition. By integrating standardized sterilization conditions with controlled germination and comparative analysis of dry and germinated material, this protocol provides a robust and reproducible framework for the culturomic isolation of seed-associated microbiota with potential functional relevance during early seedling development and supports studies of early plant-microbe interactions during seedling establishment.