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

  • Pious ThomasEmail author
  • Sadiq Pasha Shaik
Plant Microbe Interactions


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.


16S rRNA amplicon profiling Bacterial activation Cultivation-recalcitrant endophytic bacteria (CREB) Solanum lycopersicum L. Metagenomics Seed microbial community 



Cultivation-recalcitrant endophytic bacteria


Filter-sterilized autoclaved distilled water

MS medium

Murashige and Skoog medium


Nutrient agar


Next Generation Sequencing

PP bags

Polypropylene bags




Single plate-serial dilution spotting


Seed tissue homogenate


Trypticase soy agar



The NGS and bioinformatics support by M/s Eurofins Genomics India Pvt. Ltd., Bengaluru, is gratefully acknowledged. This study partly formed the component of the Ph.D. thesis of the co-author at the Jain University, Bengaluru, India.

Funding information

The study was funded under the ICAR-AMAAS Net-work project “Genomics-mediated taxonomic and functional analysis of endophytic microbiome in horticultural crops and plant-microbe interaction studies” by the ICAR-National Bureau of Agriculturally Important Microorganisms, Mau Nath Bhanjan, Uttar Pradesh, India.

Supplementary material

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Biotechnology, Endophytic and Molecular Microbiology LaboratoryICAR-Indian Institute of Horticultural Research, Hessaraghatta LakeBengaluruIndia
  2. 2.Thomas Biotech & Cytobacts Centre for BiosciencesBengaluruIndia
  3. 3.Department of Biotechnology, Centre for Post-Graduate StudiesJain UniversityBengaluruIndia

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