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Microcosm Approaches to Investigate Multitrophic Interactions between Microbial Communities in the Rhizosphere of Plants

Part of the Rhizosphere Biology book series (RHBIO)

Abstract

Plant roots are constantly forced to interact with a multitude of root colonising and free-living microorganisms in the rhizosphere. Rhizosphere microbiology, however, is traditionally split into several sub-disciplines, focusing e.g. on mycorrhiza, other endophytic fungi, bacteria, ‘plant growth promoting bacteria’, protists, ‘root fungal pathogens’, root nematodes, etc.. Accordingly, many plant-microbe studies exist on single organism groups. Combining different microbial groups in an experimental study is technically challenging because of the complexity of the set up, timing of inoculation and risk of contaminations. This chapter is intended as a step-by-step manual for the set-up of multitrophic experiments with microorganisms and plants. Technical challenges will be discussed at each step. I will first discuss different methods of soil sterilization. Then I will present different isolation, cultivation and inoculation techniques that we developed or adapted for multitrophic interaction studies. Finally, I present different microcosm systems designed to investigate specific interactions in plant-microbe studies.

Keywords

  • Axenic plant cultivation
  • Soil microcosms
  • Split-root system
  • Systemic effects
  • Protists
  • Amoeba
  • Plant pathogens
  • Arbuscular mycorrhiza
  • Protist microbiome

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Fig. 14.1
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References

  • Agler MT, Ruhe J, Kroll S, Morhenn C, Kim ST, Weigel D, Kemen EM (2016) Microbial hub taxa link host and abiotic factors to plant microbiome variation. PLoS Biol 14:e1002352

    CrossRef  Google Scholar 

  • Alphei J, Scheu S (1993) Effects of biocidal treatments on biological and nutritional properties of a mull-structured woodland soil. Geoderma 56:435–448

    CAS  CrossRef  Google Scholar 

  • Alphei J, Bonkowski M, Scheu S (1996) Protozoa, Nematoda and Lumbricidae in the rhizosphere of Hordelymus europaeus (Poaceae): faunal interactions, response of microorganisms and effects on plant growth. Oecologia 106:111–126

    CrossRef  Google Scholar 

  • Archibald JM, Simpson AGB, Slamovits CH (2017) Handbook of the protists. Springer, Heidelberg

    CrossRef  Google Scholar 

  • Bai Y, Muller DB, Srinivas G, Garrido-Oter R, Potthoff E, Rott M, Dombrowski N, Munch PC, Spaepen S, Remus-Emsermann M, Huttel B, McHardy AC, Vorholt JA, Schulze-Lefert P (2015) Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528:364–369

    CAS  CrossRef  Google Scholar 

  • Baldrian P (2004) Increase of laccase activity during interspecific interactions of white-rot fungi. FEMS Microbiol Ecol 50:245–253

    CAS  CrossRef  Google Scholar 

  • Baldrian P, López-Mondéjar R (2014) Microbial genomics, transcriptomics and proteomics: new discoveries in decomposition research using complementary methods. Appl Microbiol Biotechnol 98:1531–1537

    CAS  CrossRef  Google Scholar 

  • Baldrian P, Kolařík M, Štursová M, Kopecký J, Valášková V, Větrovský T, Žifčáková L, Šnajdr J, Rídl J, Vlček Č, Voříšková J (2011) Active and total microbial communities in forest soil are largely different and highly stratified during decomposition. ISME J 6:248

    CrossRef  Google Scholar 

  • Barberan A, Bates ST, Casamayor EO, Fierer N (2012) Using network analysis to explore co-occurrence patterns in soil microbial communities. ISME J 6:343–351

    CAS  CrossRef  Google Scholar 

  • Berney C, Geisen S, Van Wichelen J, Nitsche F, Vanormelingen P, Bonkowski M, Bass D (2015) Expansion of the ‘Reticulosphere’: diversity of novel branching and network-forming amoebae helps to define Variosea (Amoebozoa). Protist 166:271–295

    CrossRef  Google Scholar 

  • Bonkowski M (2004) Protozoa and plant growth: the microbial loop in soil revisited. New Phytol 162:617–631

    CrossRef  Google Scholar 

  • Bonkowski M, Griffiths B, Scrimgeour C (2000) Substrate heterogeneity and microfauna in soil organic ‘hotspots’ as determinants of nitrogen capture and growth of ryegrass. Appl Soil Ecol 14:37–53

    CrossRef  Google Scholar 

  • Bukovská P, Bonkowski M, Konvalinková T, Beskid O, Hujslová M, Püschel D, Řezáčová V, Gutiérrez-Núñez MS, Gryndler M, Jansa J (2018) Utilization of organic nitrogen by arbuscular mycorrhizal fungi – is there a specific role for protists and ammonia oxidizers? Mycorrhiza 28:269–283

    CrossRef  Google Scholar 

  • Butler H, Rogerson A (1995) Temporal and spatial abundance of naked amoebae (Gymnamoebae) in marine benthic sediments of the Clyde Sea area, Scotland. J Eukaryot Microbiol 42:724–730

    CrossRef  Google Scholar 

  • Cardinale M, Grube M, Erlacher A, Quehenberger J, Berg G (2015) Bacterial networks and co-occurrence relationships in the lettuce root microbiota. Environ Microbiol 17:239–252

    CAS  CrossRef  Google Scholar 

  • Clarholm M (1985) Interactions of bacteria, protozoa and plants leading to mineralization of soil nitrogen. Soil Biol Biochem 17:181–187

    CAS  CrossRef  Google Scholar 

  • Dijkstra P, Blankinship JC, Selmants PC, Hart SC, Koch GW, Schwartz E, Hungate BA (2010) Probing carbon flux patterns through soil microbial metabolic networks using parallel position-specific tracer labeling. Soil Biol Biochem 43:126–132

    CrossRef  Google Scholar 

  • Drigo B, Pijl AS, Duyts H, Kielak A, Gamper HA, Houtekamer MJ, Boschker HTS, Bodelier PLE, Whiteley AS, van Veen JA, Kowalchuk GA (2010) Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2. Proc Natl Acad Sci U S A 107:10938–10942

    CAS  CrossRef  Google Scholar 

  • Ekelund F (1998) Enumeration and abundance of mycophagous protozoa in soil, with special emphasis on heterotrophic flagellates. Soil Biol Biochem 30:1343–1347

    CAS  CrossRef  Google Scholar 

  • Foissner W (1987) Soil protozoa: fundamental problems, ecological significance, adaptations in ciliates and testaceans, bioindicators, and guide to the literature. Progr Protistol 2:69–212

    Google Scholar 

  • Geisen S, Koller R, Hünninghaus M, Dumack K, Urich T, Bonkowski M (2016) The soil food web revisited: diverse and widespread mycophagous soil protists. Soil Biol Biochem 94:10–18

    CAS  CrossRef  Google Scholar 

  • Gkarmiri K, Mahmood S, Ekblad A, Alström S, Högberg N, Finlay R (2017) Identifying the active microbiome associated with roots and rhizosphere soil of oilseed rape. Appl Environ Microbiol 83:e01938-17

    CrossRef  Google Scholar 

  • Hartmann M, Frey B, Mayer J, Mäder P, Widmer F (2014) Distinct soil microbial diversity under long-term organic and conventional farming. ISME J 9:1177

    CrossRef  Google Scholar 

  • Henkes GJ, Jousset A, Bonkowski M, Thorpe MR, Scheu S, Lanoue A, Schurr U, Roese US (2011) Pseudomonas fluorescens CHA0 maintains carbon delivery to Fusarium graminearum-infected roots and prevents reduction in biomass of barley shoots through systemic interactions. J Exp Bot 62:4337–4344

    CAS  CrossRef  Google Scholar 

  • Henkes GJ, Kandeler E, Marhan S, Scheu S, Bonkowski M (2018) Interactions of mycorrhiza and protists in the rhizosphere systemically alter microbial community composition, plant shoot-to-root ratio and within-root system nitrogen allocation. Front Environ Sci 6:117

    Google Scholar 

  • Hensel M, Bieleit G, Meyer R, Jagnow G (1990) A reliable method for the selection of axenic seedlings. Biol Fertil Soil 9:281–282

    CrossRef  Google Scholar 

  • Hess S, Melkonian M (2013) The mystery of clade X: Orciraptor gen. nov. and Viridiraptor gen. nov. are highly specialised, algivorous amoeboflagellates (Glissomonadida, Cercozoa). Protist 164:706–747

    CrossRef  Google Scholar 

  • Hiti K, Faschinger C, Haller-Schober E, Walochnik J, Aspoeck H, Hiti B (2003) Effect of microwave treatment to Acanthamoeba: a possibility of contact lens storage case sterilization? Spektrum der Augenheilkunde 17:111–114

    CrossRef  Google Scholar 

  • Horn M, Wagner M (2004) Bacterial endosymbionts of free-living amoebae. J Eukaryot Microbiol 51:509–514

    CrossRef  Google Scholar 

  • Jacoby R, Peukert M, Succurro A, Koprivova A, Kopriva S (2017) The role of soil microorganisms in plant mineral nutrition–current knowledge and future directions. Front Plant Sci 8:1617

    CrossRef  Google Scholar 

  • Jambon I, Thijs S, Weyens N, Vangronsveld J (2018) Harnessing plant-bacteria-fungi interactions to improve plant growth and degradation of organic pollutants. J Plant Interact 13:119–130

    CAS  CrossRef  Google Scholar 

  • Jeuck A, Arndt H (2013) A short guide to common heterotrophic flagellates of freshwater habitats based on the morphology of living organisms. Protist 164:842–860

    CrossRef  Google Scholar 

  • Jousset A, Scheu S, Bonkowski M (2008) Secondary metabolite production facilitates establishment of rhizobacteria by reducing both protozoan predation and the competitive effects of indigenous bacteria. Funct Ecol 22:714–719

    CrossRef  Google Scholar 

  • Jousset A, Rochat L, Lanoue A, Bonkowski M, Keel C, Scheu S (2011) Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria. Mol Plant-Microbe Interact 24:352–358

    CAS  CrossRef  Google Scholar 

  • Kiers ET, Duhamel M, Beesetty Y, Mensah JA, Franken O, Verbruggen E, Fellbaum CR, Kowalchuk GA, Hart MM, Bago A, Palmer TM, West SA, Vandenkoornhuyse P, Jansa J, Bucking H (2011) Reciprocal rewards stabilize cooperation in the mycorrhizal symbiosis. Science 333:880–882

    CAS  CrossRef  Google Scholar 

  • Koller R, Scheu S, Bonkowski M, Robin C (2013a) Protozoa stimulate N uptake and growth of arbuscular mycorrhizal plants. Soil Biol Biochem 65:204–210

    CAS  CrossRef  Google Scholar 

  • Koller R, Rodriguez A, Robin C, Scheu S, Bonkowski M (2013b) Protozoa enhance foraging efficiency of arbuscular mycorrhizal fungi for mineral nitrogen from organic matter in soil to the benefit of host plants. New Phytol 199:203–211

    CAS  CrossRef  Google Scholar 

  • Krome K, Rosenberg K, Bonkowski M, Scheu S (2009) Grazing of protozoa on rhizosphere bacteria alters growth and reproduction of Arabidopsis thaliana. Soil Biol Biochem 41:1866–1873

    CAS  CrossRef  Google Scholar 

  • Kuikman PJ, Van Veen JA (1989) The impact of protozoa on the availability of bacterial nitrogen to plants. Biol Fertil Soils 8:13–18

    CrossRef  Google Scholar 

  • Layeghifard M, Hwang DM, Guttman DS (2017) Disentangling interactions in the microbiome: a network perspective. Trends Microbiol 25:217–228

    CAS  CrossRef  Google Scholar 

  • Lee JJ, Soldo AT, Reisser W, Lee MJ, Jeon KW, Görtz HD (1985) The extent of algal and bacterial endosymbioses in protozoa. J Eukaryot Microbiol 32:391–403

    CAS  Google Scholar 

  • Lee J, Leedale G, Bradbury P (2000) An illustrated guide to the Protozoa: organisms traditionally referred to as Protozoa, or newly discovered groups, 2nd edn. Society of Protozoologists, Lawrence

    Google Scholar 

  • Madelin MF (1990) 12 methods for studying the ecology and population dynamics of soil myxomycetes. In: Grigorova R, Norris JR (eds) Methods in microbiology. Academic Press, London, pp 405–416

    Google Scholar 

  • Marschner P, Baumann K (2003) Changes in bacterial community structure induced by mycorrhizal colonisation in split-root maize. Plant Soil 251:279–289

    CAS  CrossRef  Google Scholar 

  • Morrien E, Hannula SE, Snoek LB, Helmsing NR, Zweers H, de Hollander M, Soto RL, Bouffaud ML, Buee M, Dimmers W, Duyts H, Geisen S, Girlanda M, Griffiths RI, Jorgensen HB, Jensen J, Plassart P, Redecker D, Schmelz RM, Schmidt O, Thomson BC, Tisserant E, Uroz S, Winding A, Bailey MJ, Bonkowski M, Faber JH, Martin F, Lemanceau P, de Boer W, van Veen JA, van der Putten WH (2017) Soil networks become more connected and take up more carbon as nature restoration progresses. Nat Commun 8:14349

    CAS  CrossRef  Google Scholar 

  • Müller DB, Vogel C, Bai Y, Vorholt JA (2016) The plant microbiota: systems-level insights and perspectives. Annu Rev Genet 50:211–234

    CrossRef  Google Scholar 

  • Mummey DL, Rillig MC, Holben WE (2005) Neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by T-RFLP analysis. Plant Soil 271:83–90

    CAS  CrossRef  Google Scholar 

  • Petz W, Foissner W, Adam H (1985) Culture, food selection and growth rate in the mycophagous ciliate Grossglockneria acuta Foissner, 1980: first evidence of autochthonous soil ciliates. Soil Biol Biochem 17:871–875

    CrossRef  Google Scholar 

  • Pivato B, Bru D, Busset H, Deau F, Matejicek A, Philippot L, Moreau D (2017) Positive effects of plant association on rhizosphere microbial communities depend on plant species involved and soil nitrogen level. Soil Biol Biochem 114:1–4

    CAS  CrossRef  Google Scholar 

  • Rasche F, Lueders T, Schloter M, Schaefer S, Buegger F, Gattinger A, Hood-Nowotny RC, Sessitsch A (2009) DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes. New Phytol 181:802–807

    CAS  CrossRef  Google Scholar 

  • Rogerson A, Detwiler A (1999) Abundance of airborne heterotrophic protists in ground level air of South Dakota. Atmos Res 51:35–44

    CrossRef  Google Scholar 

  • Rosenberg K, Bertaux J, Krome K, Hartmann A, Scheu S, Bonkowski M (2009) Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana. ISME J 3:675–684

    CAS  CrossRef  Google Scholar 

  • Saleem M, Fetzer I, Dormann CF, Harms H, Chatzinotas A (2012) Predator richness increases the effect of prey diversity on prey yield. Nat Commun 3:1305

    CrossRef  Google Scholar 

  • Schuster FL (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 15:342–354

    CrossRef  Google Scholar 

  • Semenov A, van Bruggen A, Zelenev V (1999) Moving waves of bacterial populations and total organic carbon along roots of wheat. Microb Ecol 37:116–128

    CAS  CrossRef  Google Scholar 

  • Sharma S, Szele Z, Schilling R, Munch JC, Schloter M (2006) Influence of freeze-thaw stress on the structure and function of microbial communities and denitrifying populations in soil. Appl Environ Microbiol 72:2148–2154

    CAS  CrossRef  Google Scholar 

  • Shinner F, Öhlinger R, Kandeler E, Margesin R (1996) Methods in soil biology. Springer-Verlag, Berlin

    CrossRef  Google Scholar 

  • Smirnov AV (2003) Optimizing methods of the recovery of gymnamoebae from environmental samples: a test of ten popular enrichment media, with some observations on the development of cultures. Protistology 3:47–57

    Google Scholar 

  • Smirnov AV, Brown S (2004) Guide to the methods of study and identification of soil gymnamoebae. Protistology 3:148–190

    Google Scholar 

  • Smirnov AV, Chao E, Nassonova ES, Cavalier-Smith T (2011) A revised classification of naked Lobose amoebae (Amoebozoa: Lobosa). Protist 162:545–570

    CrossRef  Google Scholar 

  • Trap J, Bonkowski M, Plassard C, Villenave C, Blanchart E (2016) Ecological importance of soil bacterivores for ecosystem functions. Plant Soil 398:1–24

    CAS  CrossRef  Google Scholar 

  • Venturi V, Keel C (2016) Signaling in the rhizosphere. Trends Plant Sci 21:187–198

    CAS  CrossRef  Google Scholar 

  • Vetsigian K, Jajoo R, Kishony R (2011) Structure and evolution of streptomyces interaction networks in soil and in silico. PLoS Biol 9:e1001184

    CAS  CrossRef  Google Scholar 

  • Wei Z, Yang T, Friman VP, Xu Y, Shen Q, Jousset A (2015) Trophic network architecture of root-associated bacterial communities determines pathogen invasion and plant health. Nat Commun 6:8413

    CAS  CrossRef  Google Scholar 

  • Weidner S, Koller R, Latz E, Kowalchuk G, Bonkowski M, Scheu S, Jousset A (2015) Bacterial diversity amplifies nutrient-based plant–soil feedbacks. Funct Ecol 29:1341–1349

    CrossRef  Google Scholar 

  • Wolters V (1989) Die Wirkung der Bodenversauerung auf Protura, Diplura und Collembola (Insecta, Apterygota)- Untersuchungen am Stammfuß von Buchen. Jahersberichte des naturwissenschaftlichen Vereins Wuppertal 42:45–50

    Google Scholar 

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Acknowlegements

This work was supported by the Cluster of Excellence on Plant Sciences (CEPLAS), and the priority program of the German Science Foundation DFG-SPP2089 “Rhizosphere Spatiotemporal Organisation”. We are particularly grateful for the continuous contributions and support of Leo Leson and the whole workshop team of the Biocenter, University of Cologne.

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Correspondence to Michael Bonkowski .

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Appendix

Appendix

  1. 1.

    Neff’s modified Amoeba Salinae (NMAS):

  • Stock 1: 12 g NaCl, 0.4 g MgSO4 * 7H2O, 0.6 g CaCl2 * 6 H2O, fill up to 1000 ml with H2Odest.

  • Stock 2: 14.2 g Na2HPO4, 13.6 g KH2PO4, fill up to 1000 ml with H2Odest.

  • Mix 5 ml of stock 1 and 5 ml of stock 2 with 1000 ml of H2Odest and autoclave for 20 min at 120 °C.

  1. 2.

    NB-NMAS (Nutrient Broth – Neff’s Modified Amoebae Salinae):

  • Add 0.8 g nutrient broth (Oxoid, UK) to 1000 ml NMAS. Autoclave for 20 min at 120 °C.

  1. 3.

    Prescott’s James’s solution (PJ):

    Stock solution 1 Add 0.433 g CaCl2·2H2O and 0.162 g KCl to 100 ml H2O
    Stock solution 2 Add 0.512 g K2HPO4 to 100 ml H2O
    Stock solution 3 Add 0.280 g MgSO4 to100 ml H2O

One ml of the Stock solutions 1, 2 and 3 each is filled up to 1000 ml with H2Odest. Autoclave for 20 min at 120 °C.

  1. 4.

    Wheat Grass Medium (WG):

  • 0,15 g wheat grass powder are three times boiled up with 200 ml H2Odest, filtered and added to 1000 ml PJ solution. Autoclave for 20 min at 120 °C.

  1. 5.

    Gamborg medium (for plants):

  • Add 3.2 g of Gamborg’s B-5 basal medium with minimal organics to 1000 ml H2Odest. Autoclave for 20 min at 120 °C.

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Bonkowski, M. (2019). Microcosm Approaches to Investigate Multitrophic Interactions between Microbial Communities in the Rhizosphere of Plants. In: Reinhardt, D., Sharma, A. (eds) Methods in Rhizosphere Biology Research. Rhizosphere Biology. Springer, Singapore. https://doi.org/10.1007/978-981-13-5767-1_14

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