Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition

The ISME Journal (2015) 9, 2349–2359 © 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition Andrzej Tkacz1,2, Jitender Cheema1,3, Govind Chandra1, Alastair Grant4 and Philip S Poole1,2 1 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, UK; Department of Plant Sciences, Oxford University, Oxford, UK; 3Department of Computational and Systems Biology, John Innes Centre, Norwich Research Park, Norwich, UK and 4Earth and Life Systems Alliance, The School of Environmental Sciences, University of East Anglia, Norwich, UK 2 We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alternative community compositions rather than a single distinctive community. Organisms selected in rhizospheres can have positive or negative effects. Two abundant bacteria are shown to promote plant growth, but in Brassica the pathogen Olpidium brassicae came to dominate the fungal community. So plants exert strong selection on the rhizosphere microbiota but soil composition is critical to its stability. microbial succession/ plant–microbe interactions/rhizosphere microbiota/selection. The ISME Journal (2015) 9, 2349–2359; doi:10.1038/ismej.2015.41; published online 24 April 2015 Introduction The rhizosphere is a critical zone of soil surrounding roots that is directly influenced by the plant and into which plants secrete as much as 11% of net fixed carbon (Jones et al., 2009; Dennis et al., 2010). It is a ‘hot spot’ of microbial activity, with increased microbial numbers, microbial interactions and genetic exchange (Bulgarelli et al., 2013; Turner et al., 2013a). There is a close two-way interaction between the microbial community (microbiota) and plant host that is an essential determinant of plant health and productivity. This is very apparent in symbiotic associations between rhizobia and legumes, resulting in fixation of 40–50% of the biosphere’s nitrogen (Terpolilli et al., 2012; Udvardi and Poole, 2013) as well as widespread acquisition of nutrients such as phosphate by mycorrhizae (Lanfranco and Young, 2012; Nuccio et al., 2013). Correspondence: PS Poole, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK. E-mail: Received 6 June 2014; revised 5 February 2015; accepted 16 February 2015; published online 24 April 2015 However, interaction between plants and microbes is more general with plants shown to shape the rhizosphere microbiota in legumes, potatoes and suppressive soils (Sharma et al., 2005; Lebreton et al., 2007; Manter et al., 2010; Kinkel et al., 2011; Mendes et al., 2011; Turner et al., 2013b). Some microbial root colonists are saprophytes, which also colonise splinters of wood inserted into soil (Bulgarelli et al., 2012), but others are selected by exudation of nutrient sources and phytoalexins, such as glucosinolates and avenacins (Sonderby et al., 2007; Bressan et al., 2009; Turner et al., 2013b). Furthermore, by altering the rhizosphere microbiota, plants induce formation of suppressive soil where growth of plant pathogens is inhibited (Lebreton et al., 2007; Kinkel et al., 2011; Mendes et al., 2011). Microbes can increase plant growth by releasing phytohormones, biofertilization (Lugtenberg and Kamilova, 2009) and stimulation of both the induced systemic resistance and systemic-acquired resistance components of the plant immune system (Van der Ent et al., 2009). It has been difficult to study these complex interactions at the community level but recently high-throughput sequencing methods have been used to show that Arabidopsis preferentially Succession of the rhizosphere microbiota A Tkacz et al 2350 selects specific microbial endophytes, which colonise plant tissues, with some, albeit weaker, selection in the rhizosphere (Bulgarelli et al., 2012; Lundberg et al., 2012). Previous studies on the soil microbiota of plants grown in natural soil have shown that plant and soil are both important factors in community structure (Berg and Smalla, 2009). However, as shown by Lauber et al., 2009, soil pH also has a dramatic influence on the soil microbiota. Studying field grown plants for their influence on the microbiota is hindered by high soil complexity and local variation in pH. Moreover, most previous studies used DNA fingerprinting methods with limited replication (o4 samples). As we show here it is vital to sample at least 10–15 rhizosphere samples (biological replicates) in order to establish statistical significance. To study microbial succession we used a nutritionally defined medium that in the first generation consisted of a 10% soil inoculum mixed with either autoclaved sand or compost. In two succeeding generations, 25% of the growth medium was transferred from the preceding generation and mixed with 75% sand or compost. This allowed control of soil nutrients, as they were refreshed in each generation and separation of the effects of soil characteristics from those of the initial microbial community composition. This approach was used to examine rhizosphere succession over three generations in three model and three crop plants, belonging to distinct families. Sand and compost were used to allow assessment of the relative importance of root exudation and soil organic matter in supporting rhizosphere communities. Materials and methods Soil collection and plant growth Soil used as a microbial inoculum was collected from a naturally grassed and unfertilised part of John Innes Centre Church Farm, Bawburgh, Norfolk, UK (52°37′39.35′′N, 1°10′43′′E). Covering vegetation was stripped off and soil collected from a depth of 10–30 cm. Chemical analyses (by MacaulaySoils, James Hutton Institute, Aberdeen, UK) showed that the soil is poor in nutrients (NO−3 3.49 mg kg − 1, P − 3 120.5 mg kg − 1, K+ 168.2 mg kg − 1, Mg2+ 33.55 mg kg − 1), is pH 7.5 and contains 2.92% organic matter. According to the UK Soil Ob (...truncated)