Cultivation of microbes isolated from Agave spp. in the semiarid region
The increasing recognition of microbial diversity through molecular approaches has renewed interest in the isolation and long-term maintenance of reference strains for taxonomic and biotechnological applications. Although innovative technologies aimed at recovering previously unculturable microorganisms have demonstrated effectiveness, new isolation approaches remain at early stages of development or require costly and highly specialized equipment, limiting their use in resource-constrained laboratories. Consequently, conventional selective cultivation strategies continue to be widely applied. This study aimed to optimize and standardize accessible traditional isolation methodologies to develop a high-yield, resource-efficient cultivation protocol capable of rapidly establishing microbiological libraries with minimal infrastructure. The protocol was employed on plant growth-promotion microorganisms associated with the soil and rhizosphere of Agave spp., cultivated in a semiarid region, emphasizing the economic relevance and sustainability of bio-input development and Agave production. Ten semi-selective culture media were utilized to isolate distinct functional groups microorganisms: Pseudomonas Isolation Agar, JNFb, NFB, VERMA, HP, MacConkey Agar, Bacillus ChromoSelect Agar®, T3, Dichloran Rose-Bengal Chloramphenicol Agar, and Glucose–Yeast Extract–Peptone Agar. The protocol yielded 3,918 viable isolates after cryopreservation at -80 °C, comprising 3,567 bacterial strains (78.22% recovery) and 351 filamentous fungi and yeasts (30.79% recovery). Contamination rates assessed by 16S rRNA and ITS sequencing were 6.84% for prokaryotes and 18.42% for fungi and yeasts. The recovery of rare and unclassified taxa suggests that the protocol facilitates access to underrepresented microbial diversity, highlighting the potential of the resulting microbial library for future biotechnological and agronomic exploration.