Molecular Profiling on Surface-Disinfected Tomato Seeds Reveals High Diversity of Cultivation-Recalcitrant Endophytic Bacteria with Low Shares of Spore-Forming Firmicutes.

Seeds are known to harbor diverse microorganisms offering protective effects on them with the prospects of quick root colonization at germination, selective recruitment as endophytes, and possible vertical transmission. The study was undertaken to assess the gross seed-internal bacterial community in tomato and to confirm if spore-forming Firmicutes constituted major seed endophytes adopting cultivation versus molecular approach on surface-sterilized seeds. Testing the initial seed wash solutions of "Arka Vikas" and "Arka Abha" cultivars showed > 1000 bacterial cfu per dry seed, largely Bacillus spp. Tissue homogenates from surface-disinfected seeds did not show any cultivable bacteria on enriched media for 1-2 weeks, while 16S rRNA V3-V4 taxonomic profiling revealed a huge bacterial diversity (10-16 phyla per cultivar). Proteobacteria formed the dominant phylum (65.7-69.6% OTUs) followed by Firmicutes, Actinobacteria, Bacteroidetes, and a notable share of Euryarchaeota (1.1-3.1%). Five more phyla appeared common to both cultivars in minor shares (Acidobacteria, Planctomycetes, Chloroflexi, Spirochaetes, Verrucomicrobia) with the ten phyla together constituting 99.6-99.9% OTUs. Class level and family level, the cultivars displayed elevated bacterial diversity, but similar taxonomic profiles. Arka Vikas and Arka Abha showed 114 and 107 genera, respectively, with 63 common genera constituting 96-97% OTUs. Psychrobacter formed the dominant genus. Bacillus and related genera constituted only negligible OTU share (0.16-0.28%). KEGG functional analysis showed metabolism as the major bacterial community role. One-month-old in vitro seedlings showed the activation of some originally uncultivable bacteria uninfluenced by the OTU share. The study reveals a high diversity of cultivation-recalcitrant endophytic bacteria prevailing in tomato seeds with possible vertical transmission and significant roles in plant biology.