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

  • Plant Microbe Interactions
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Abstract

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.

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Abbreviations

CREB:

Cultivation-recalcitrant endophytic bacteria

FDW:

Filter-sterilized autoclaved distilled water

MS medium:

Murashige and Skoog medium

NA:

Nutrient agar

NGS:

Next Generation Sequencing

PP bags:

Polypropylene bags

SATS:

Spotting-and-tilt-spreading

SP-SDS:

Single plate-serial dilution spotting

STH:

Seed tissue homogenate

TSA:

Trypticase soy agar

References

  1. Adam E, Bernhart M, Müller H, Winkler J, Berg G (2018) The Cucurbita pepo seed microbiome: genotype-specific composition and implications for breeding. Plant Soil 422:35–49

    CAS  Google Scholar 

  2. Alam SI, Singh L, Dube S, Reddy GSN, Shivaji S (2003) Psychrophilic planococcus maitriensis sp. nov. from Antarctica. Syst Appl Microbiol 26:505–510

    CAS  PubMed  Google Scholar 

  3. Barret M, Briand M, Bonneau S, Préveaux A, Valière S, Bouchez O, Hunault G, Simoneau P, Jacques MA (2015) Emergence shapes the structure of the seed microbiota. Appl Environ Microbiol 81:1257–1266

    PubMed  PubMed Central  Google Scholar 

  4. Berg G, Raaijmakers JM (2018) Saving seed microbiomes. ISME J 12:1167–1170

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Bergna A, Cernava T, Rändler M, Grosch R, Zachow C, Berg (2019) Tomato seeds preferably transmit plant beneficial endophytes. Phytobiomes J 2:183–193

    Google Scholar 

  6. Betts G (2006) Other spoilage bacteria. In: Blackburn CW (ed) Food spoilage microorganisms. Woodhead Publishing Series in Food Science, Technology and Nutrition, Sawston, Cambridge, pp 668–693

    Google Scholar 

  7. Bodhankar S, Grover M, Hemanth S, Reddy G, Rasul S, Yadav SK, Desai S, Mallappa M, Mandapaka M, Srinivasarao C (2017) Maize seed endophytic bacteria: dominance of antagonistic, lytic enzyme-producing Bacillus spp. 3 Biotech 7:232

    PubMed  PubMed Central  Google Scholar 

  8. Compant S, Mitter B, Colli-Mull JG, Gangl H, Sessitsch A (2011) Endophytes of grapevine flowers, berries and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microb Ecol 62:188–197

    PubMed  Google Scholar 

  9. Compant S, Saikkonen K, Mitter B, Campisano A, Mercado-Blanco J (2016) Editorial special issue: soil, plants and endophytes. Plant Soil 405:1–11

    CAS  Google Scholar 

  10. Cope-Selby N, Cookson A, Squance M, Donnison I, Flavell R, Farrar K (2017) Endophytic bacteria in Miscanthus seed: implications for germination, vertical inheritance of endophytes, plant evolution and breeding. GCB Bioenergy 9:57–77

    CAS  Google Scholar 

  11. Deng ZS, Zhang BC, Qi XY, Sun ZH, He XL, Liu YZ, Li J, Chen KK, Lin ZX (2019) Root-associated endophytic bacterial community composition of Pennisetum sinese from four representative provinces in China. Microorganisms 7:47

    CAS  PubMed Central  Google Scholar 

  12. Frank AC, Saldierna Guzmán JP, Shay JE (2017) Transmission of bacterial endophytes. Microorganisms 5:70

    PubMed Central  Google Scholar 

  13. Fürnkranz M, Lukesch B, Mueller H, Huss H, Grube M, Berg G (2012) Microbial diversity inside pumpkins: microhabitat specific communities display a high antagonistic potential against phytopathogens. Microb Ecol 63:418–428

    PubMed  Google Scholar 

  14. Glassner H, Zchori-Fein E, Compant S, Sessitsch A, Katzir N, Portnoy V, Yaron S (2015) Characterization of endophytic bacteria from cucurbit fruits with potential benefits to agriculture in melons (Cucumis melo L.). FEMS Microbiol Ecol 91:fiv074

    PubMed  Google Scholar 

  15. Glassner H, Zchori-Fein E, Yaron S, Sessitsch A, Sauer U, Compant S (2018) Bacterial niches inside seeds of Cucumis melo L. Plant Soil 422:101–113

    CAS  Google Scholar 

  16. Hardoim PR, Hardoim CC, van Overbeek LS, van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7:e30438

    CAS  PubMed  PubMed Central  Google Scholar 

  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 79:293–320

    PubMed  PubMed Central  Google Scholar 

  18. Herrera SD, Grossi C, Zawoznik M, Groppa MD (2016) Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum. Microbiol Res 186:37–43

    Google Scholar 

  19. Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6:1–22

    Google Scholar 

  20. Johnston-Monje D, Mousa WK, Lazarovits G, Raizada MN (2014) Impact of swapping soils on the endophytic bacterial communities of pre-domesticated, ancient and modern maize. BMC Plant Biol 14:233

    PubMed  PubMed Central  Google Scholar 

  21. Johnston-Monje D, Lundberg DS, Lazarovits G, Reis VM, Raizada MN (2016) Bacterial populations in juvenile maize rhizospheres originate from both seed and soil. Plant Soil 405:337–355

    CAS  Google Scholar 

  22. Kandel SL, Joubert PM, Doty SL (2017) Bacterial endophyte colonization and distribution within plants. Microorganisms 5:77

    PubMed Central  Google Scholar 

  23. Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res 32(suppl 1):D277–D280

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Khalaf EM, Raizada MN (2016) Taxonomic and functional diversity of cultured seed associated microbes of the cucurbit family. BMC Microbiol 16:131

    PubMed  PubMed Central  Google Scholar 

  25. Kim SJ, Shin SC, Hong SG, Lee YM, Choi I-G, Park H (2012) Genome sequence of a novel member of the genus psychrobacter isolated from antarctic soil. J Bacteriol 194:2403

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Klaedtke S, Jacques MA, Raggi L, Préveaux A, Bonneau S, Negri V, Chable V, Barret M (2016) Terroir is a key driver of seed-associated microbial assemblages. Environ Microbiol 18:1792–1804

    CAS  PubMed  Google Scholar 

  27. Langille MG, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, Clemente JC, Burkepile DE, Thurber RLV, Knight R, Beiko RG (2013) Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol 31:814–821

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Liu H, Carvalhais LC, Crawford M, Singh E, Dennis PG, Pieterse CM, Schenk PM (2017) Inner plant values: diversity, colonization and benefits from endophytic bacteria. Front Microbiol 8:2552

    PubMed  PubMed Central  Google Scholar 

  29. López S, Pastorino G, Franco M, Medina R, Lucentini C, Saparrat M, Balatti P (2018a) Microbial endophytes that live within the seeds of two tomato hybrids cultivated in Argentina. Agronomy 8:136

    Google Scholar 

  30. López JL, Alvarez F, Principe A, Salas ME, Lozano MJ, Draghi WO, Jofré E, Lagares A (2018b) Isolation, taxonomic analysis, and phenotypic characterization of bacterial endophytes present in alfalfa (Medicago sativa) seeds. J Biotechnol 267:55–62

    PubMed  Google Scholar 

  31. Manirajan BA, Ratering S, Rusch V, Schwiertz A, Geissler-Plaum R, Cardinale M, Schnell S (2016) Bacterial microbiota associated with flower pollen is influenced by pollination type, and shows a high degree of diversity and species-specificity. Environ Microbiol 18:5161–5174

    Google Scholar 

  32. Mano H, Tanaka F, Watanabe A, Kaga H, Okunishi S, Morisaki H (2006) Culturable surface and endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field. Microbes Environ 21:86–100

    Google 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 8:11

    PubMed  PubMed Central  Google Scholar 

  34. Mora-Ruiz MDR, Font-Verdera F, Orfila A, Rita J, Rosselló-Móra R (2016) Endophytic microbial diversity of the halophyte Arthrocnemum macrostachyum across plant compartments. FEMS Microbiol Ecol 92:fiw145

    Google Scholar 

  35. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    CAS  Google Scholar 

  36. Nelson EB (2004) Microbial dynamics and interactions in the spermosphere. Annu Rev Phytopathol 42:271–309

    CAS  PubMed  Google Scholar 

  37. Nelson EB (2018) The seed microbiome: origins, interactions and impacts. Plant Soil 422:7–34

    CAS  Google Scholar 

  38. Nelson EB, Simoneau P, Barret M, Mitter B, Compant S (2018) Editorial special issue: the soil, the seed, the microbes and the plant. Plant Soil 422:1–5

    CAS  Google Scholar 

  39. Pitzschke A (2016) Developmental peculiarities and seed-borne endophytes in quinoa: omnipresent, robust bacilli contribute to plant fitness. Front Microbiol 7:2

    PubMed  PubMed Central  Google Scholar 

  40. Shahzad R, Khan AL, Bilal S, Asaf S, Lee IJ (2018) What is there in seeds? Vertically transmitted endophytic resources for sustainable improvement in plant growth. Front Plant Sci 9:24

    PubMed  PubMed Central  Google Scholar 

  41. Shaik SP, Thomas P (2019) In vitro activation of seed-transmitted cultivation- recalcitrant endophytic bacteria in tomato and host-endophyte mutualism. Microorganisms 7:132

    CAS  PubMed Central  Google Scholar 

  42. Thomas P (2011) Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures. Plant Cell Rep 30:2313–2325

    CAS  PubMed  Google Scholar 

  43. Thomas P, Sekhar AC (2017) Cultivation versus molecular analysis of banana (Musa sp.) shoot-tip tissue reveals enormous diversity of normally uncultivable endophytic bacteria. Microb Ecol 73:885–899

    PubMed  Google Scholar 

  44. Thomas P, Soly TA (2009) Endophytic bacteria associated with growing shoot tips of banana (Musa sp.) cv. Grand Naine and the affinity of endophytes to the host. Microb Ecol 58:952–964

    CAS  PubMed  Google Scholar 

  45. Thomas P, Agrawal M, Bharathkumar CB (2019) Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications. Physiol Plant 166:729–747

    CAS  PubMed  Google 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 113:339–350

    CAS  PubMed  Google Scholar 

  47. Thomas P, Sekhar AC, Pasha SS (2017) High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction and unsuspected persistence. Planta 246:879–898

    CAS  PubMed  Google Scholar 

  48. Thomas P, Sekhar AC, Upreti R, Mujawar MM, Pasha SS (2015) Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples. Biotech Rep 8:45–55

    Google Scholar 

  49. Truyens S, Weyens N, Cuypers A, Vangronsveld J (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7:40–50

    Google Scholar 

  50. Upreti R, Thomas P (2015) Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects. Front Microbiol 6:255

    PubMed  PubMed Central  Google Scholar 

  51. Vela AI, Collins MD, Latre MV, Mateos A, Moreno MA, Hutson R, Dominguez L, Fernandez-Garayzabal JF (2003) Psychrobacter pulmonis sp. nov., isolated from the lungs of lambs. Int J Syst Evol Microbiol 53:415–419

    CAS  PubMed  Google Scholar 

  52. Verma SK, Kharwar RN, White JF (2019) The role of seed-vectored endophytes in seedling development and establishment. Symbiosis 78:107–113

    Google Scholar 

  53. Walitang DI, Kim K, Madhaiyan M, Kim YK, Kang Y, Sa T (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of rice. BMC Microbiol 17:209

    PubMed  PubMed Central  Google 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 3–20

    Google 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 30:835–845

  56. Zhang J, Zhang C, Yang J, Gao J, Zhao X, Zhao J, Zhao D, Zhang X (2018) Insights into endophytic bacterial community structures of seeds among various Oryza sativa L. rice genotypes. J Plant Growth Regul 38:93–102

    Google Scholar 

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Acknowledgments

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

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.

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Authors and Affiliations

  1. Division of Biotechnology, Endophytic and Molecular Microbiology Laboratory, ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake, Bengaluru, 560 089, India

    Pious Thomas

  2. Thomas Biotech & Cytobacts Centre for Biosciences, 318 Thalakavery Layout, Amruthahalli, Bengaluru, 560092, India

    Pious Thomas

  3. Department of Biotechnology, Centre for Post-Graduate Studies, Jain University, Bengaluru, 560011, India

    Sadiq Pasha Shaik

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  1. Pious Thomas
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Correspondence to Pious Thomas.

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Thomas, P., Shaik, S.P. Molecular Profiling on Surface-Disinfected Tomato Seeds Reveals High Diversity of Cultivation-Recalcitrant Endophytic Bacteria with Low Shares of Spore-Forming Firmicutes. Microb Ecol 79, 910–924 (2020). https://doi.org/10.1007/s00248-019-01440-5

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