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.
Keywords16S 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
Next Generation Sequencing
- PP 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.
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.
- 17.Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320PubMedPubMedCentralCrossRefGoogle Scholar
- 33.Mitter B, Pfaffenbichler N, Flavell R, Compant S, Antonielli L, Petric A, Berninger T, Naveed M, Sheibani-Tezerji R, von Maltzahn G, Sessitsch A (2017) A new approach to modify plant microbiomes and traits by introducing beneficial bacteria at flowering into progeny seeds. Front Microbiol 8:11PubMedPubMedCentralGoogle Scholar
- 46.Thomas P, Sekhar AC, Mujawar MM (2012) Non-recovery of varying proportions of viable bacteria during spread-plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU. J Appl Microbiol 113:339–350PubMedCrossRefPubMedCentralGoogle Scholar
- 54.White JF, Kingsley KL, Butterworth S, Brindisi L, Gatei JW, Elmore MT, Verma SK, Yao X, Kowalski KP (2019) Seed-vectored microbes: their roles in improving seedling fitness and competitor plant suppression. In: Verma SK, White Jr JF (eds) Seed endophytes. Springer, Cham, pp 3–20CrossRefGoogle Scholar
- 55.Xu M, Sheng J, Chen L, Men Y, Gan L, Guo S, Shen L (2014) Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings. World J Microbiol Biotechnol 30:835–845PubMedCrossRefPubMedCentralGoogle Scholar