Antibiotic-induced dominance relaxation restructures a closed in vitro grapevine endophyte community without clear secondary dominance

Technical Summary: Antibiotic-Induced Dominance Relaxation in a Closed In Vitro Grapevine Endophyte Community

Problem Statement
Latent endophytic bacteria in plant tissue culture pose a significant challenge to cryopreservation and micropropagation, often surviving surface sterilization and causing culture loss or reduced post-thaw recovery. While previous work by the authors demonstrated that targeted rifampicin treatment against a specific culturable endophyte (Leifsonia poae) in Vitis vinifera 'Ruby Seedless' improved post-thaw survival, the broader community-level consequences of this intervention remained uncharacterized. Specifically, it was unclear whether the suppression of a dominant bacterial signal would lead to "secondary dominance" (where a single resistant taxon replaces the original dominant one) or a more complex, heterogeneous restructuring of the residual microbiome. Understanding this dynamic is critical, as secondary dominance could necessitate a cycle of identifying and targeting new dominant taxa, rendering routine culture management impractical.

Methodology
The study employed a comparative amplicon sequencing approach on in vitro shoot cultures of Vitis vinifera 'Ruby Seedless'.

• Experimental Design: Six biological replicates were used (three untreated controls and three treated with rifampicin at 31 µg mL⁻¹, the minimum bactericidal concentration against L. poae). Treated explants were cultured for four weeks on rifampicin-supplemented Woody Plant Medium (WPM), followed by transfer to antibiotic-free medium for two additional subculture cycles to allow for community stabilization.
• Sequencing and Curation: DNA was extracted from pooled stem tissue, and the V3–V4 region of the 16S rRNA gene was sequenced on an Illumina MiSeq platform. A rigorous bioinformatic pipeline was applied:
  • Raw reads were processed using DADA2 to infer Amplicon Sequence Variants (ASVs).
  • Crucially, host-derived sequences (chloroplast, mitochondria, and Vitis vinifera nuclear DNA) were identified via BLASTN and removed. This step was essential because host DNA dominated the libraries (up to 99.88% in some samples), and standard rarefaction of total reads would not equalize bacterial read depth.
  • The final curated dataset consisted of 99 bacterial ASVs.
• Analytical Framework: The study utilized taxonomic profiling, ASV-level fate tracking (categorizing ASVs as Loss, Persistence, or Emergence), and alpha- and beta-diversity analyses. Diversity metrics were calculated using non-rarefied counts adjusted via Analysis of Covariance (ANCOVA) to account for varying bacterial read depths, with sensitivity analyses performed on rarefied data. Beta-diversity was assessed using Bray–Curtis dissimilarity and phylogenetic UniFrac distances.

Key Results

• Disruption of Dominance: Untreated cultures were characterized by extreme dominance of the genus Mycobacterium (mean relative abundance 71.2%), which accounted for the majority of the Actinomycetota phylum. Rifampicin treatment completely eliminated Mycobacterium signals.
• Absence of Secondary Dominance: Contrary to the hypothesis of a single successor taxon taking over, the treated community did not converge on a new dominant lineage. Instead, abundance was redistributed across multiple taxa, including Pediococcus, Leifsonia, Pseudomonas, and Levilactobacillus. While Pediococcus became abundant in some replicates (mean 44.3%), high among-replicate variability prevented the formation of a uniform, single-dominant state.
• Extensive ASV Turnover: ASV-level fate analysis revealed significant community restructuring. Of the 99 ASVs, 41 were lost (detected only in untreated), 57 emerged (detected only in treated), and only one ASV persisted in both groups. The "Emergence" fraction was taxonomically heterogeneous, with many low-abundance ASVs appearing in only single replicates.
• Alpha-Diversity Shifts: Rifampicin treatment did not reduce overall richness (observed ASVs remained comparable). However, it significantly increased evenness and diversity indices (Shannon, Gini-Simpson, Pielou's evenness) while decreasing dominance metrics (Simpson and Berger-Parker indices). This indicates a shift from a highly skewed community to a more evenly distributed assemblage.
• Beta-Diversity and Dispersion: Beta-diversity analysis (Bray–Curtis) confirmed substantial compositional restructuring between treated and untreated groups. Notably, treated cultures exhibited elevated within-group dispersion (higher Bray–Curtis dissimilarity among replicates), suggesting that post-treatment communities reassembled heterogeneously rather than converging on a shared state.

Significance and Claims
The paper claims that targeted antibiotic treatment in closed in vitro systems can induce a "dominance relaxation" and "non-convergent reassembly" of the bacterial community. The study provides a community-level framework suggesting that the practical improvement in cryopreservation outcomes observed in previous work may be attributed not just to the removal of a specific pathogen (L. poae), but to the disruption of a strongly dominant community structure (Mycobacterium-dominated) that was prone to causing contamination.

The authors emphasize that the absence of a single replacement-dominant taxon is ecologically informative, suggesting that in closed systems with limited immigration, post-disturbance assembly is driven by historical contingency and stochastic factors rather than deterministic succession toward a new dominant state. The study explicitly frames these findings as descriptive and exploratory due to the small sample size ($n=3$) and the reliance on relative abundance data without absolute quantification. Consequently, the authors caution against inferring direct functional consequences for the host or making definitive claims about the durability of this "dominance relaxation" over extended subculture cycles without further validation involving absolute bacterial loads and functional assays. The work serves primarily to characterize the ecological trajectory of the microbiome following antibiotic intervention, distinguishing between simple suppression and complex community reorganization.