Targeted elimination of latent endophytes improves cryopreservation success in in vitro grapevine (Vitis vinifera)...

Latent endophytic bacteria are common in grapevine in vitro cultures and, while not always causing visible culture decline, can negatively affect downstream applications such as cryopreservation by reducing post-thaw recovery. While antibiotic treatments are widely used for microbial control, their efficacy varies with bacterial species, plant genotype, and application conditions. Few studies have directly linked targeted elimination of identified endophytes with improved post-cryopreservation recovery.

In this study, we identified Leifsonia poae in 'Ruby Seedless' and Curtobacterium oceanosedimentum in 'Merlot' using 16 S rRNA sequencing. Species-specific antibiotic susceptibility testing revealed rifampicin (minimum bactericidal concentration, MBC = 31 µg mL⁻¹) as effective against L. poae, and cefotaxime (MBC = 1000 µg mL⁻¹) as effective against C. oceanosedimentum. In vitro application of these antibiotics eliminated the respective endophytes without significant phytotoxic effects at optimal concentrations. Amplicon-based 16 S metagenomic profiling confirmed complete removal of the target bacteria and revealed substantial shifts in community composition, with reduced abundance of dominant taxa but maintenance of overall endophytic diversity. Cryopreservation experiments in 'Ruby Seedless' showed that removal of endophytes increased post-thaw survival from 31.8 to 70.9% and eliminated variability in regrowth across replicates.

This study demonstrates that targeted antibiotic elimination of dominant endophytes can restructure the in vitro microbial community and substantially improve cryopreservation outcomes in grapevine. The approach offers a reproducible sanitation strategy for clonal crops vulnerable to endophyte-related culture failures and can be readily adapted for germplasm conservation programs.