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Section 8 Biofilm Formation and Attachment to Roots Chapter 66 Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications for Plant Growth Annette A. Angus Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, USA Ann M. Hirsch Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, USA; Molecular Biology Institute, University of California, Los Angeles, USA 66.1 INTRODUCTION Soil fertility is intimately tied to sustaining life on our planet. An ever-increasing human population as well as mounting concerns about climate change has resulted in a correlative upsurge in attention to marginalized agricultural lands, the consequences of deforestation, and the anthropogenic activities that affect one of our most important resources, the soil. Thus, more emphasis has been placed on soil fertility and its maintenance for growing food and fuel crops even as the world around us changes. However, depending on its location, the soil environment is inherently complex and diverse. Moreover, soil can be considered to be “alive” because of the various organisms (plants, insects, fungi, bacteria, etc.) that inhabit it. Preserving these life forms is essential not only for maintaining soil fertility but also biodiversity. Previously, we reviewed various aspects of soil microbes, particularly rhizobial species, with regard to adaptations to various environmental stresses such as pH, salinity, and desiccation (Hirsch, 2010). Other reviews have also discussed the effects of stress on rhizobial viability (Zahran, 1999; Sadowsky, 2005; Vriezen et al., 2007). We have also investigated survival mechanisms for rhizobia subjected to stress by studying the formation of biofilms and describing many of the factors that mediate biofilm formation (Fujishige et al., 2006a,b, 2008; Hirsch et al., 2009). Biofilms afford a community of single or mixed species of bacteria, especially non-spore formers such as rhizobia, protection from the fluctuating and often severe conditions of the rhizosphere, such as desiccation, extreme pH levels, temperature, salt, and nutrient availability. In this chapter, we focus on the importance of multiple species biofilms composed of either synergistically acting bacteria or antagonistic species that compete for root colonization, as well as specific effects of these interactions on plant health, growth, and development. 66.2 THE SOIL Soil is a mixture of organic and inorganic materials that vary in texture, structure, and nutrients. Much of the soil is considered “bulk soil” and is inhabited by few microbes. In those areas surrounding the roots of various plants, however, microbial consortia are rich in species Molecular Microbial Ecology of the Rhizosphere, Volume 2, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc. 703 704 Chapter 66 Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications diversity and abundance, although many of them have yet to be cultured (Torvik and Øvreås, 2002). This region includes the immediate (ca. 50 µm thick) surface area directly adjacent to plant roots (the rhizoplane) followed by the next ca. 1–5 mm around the root (the rhizosphere) (Foster et al., 1983). The rhizoplane consists of both the plant root surface and secreted exudates, whereas, the rhizosphere, which is rich in plant and microbial exudates, not only contains soil particles but also bacteria, archaea, fungi, and viruses. The plant root exhibits a longitudinal developmental gradient from the apical meristem and its surrounding root cap and border cells to the root hair zone and finally to a region where root cells completely differentiate. The surface of the older parts of roots, where the metaxylem elements still conduct, often becomes encased within a tightly adhering amalgam of soil particles, plant- and microbial-derived mucilage, as well as microbial and plant cells. This so-called rhizosheath completely covers the root hairs, which are prevalent, and is particularly obvious when roots are grown under dry conditions (Watt et al., 1994) (Fig. 66.1). We consider these intimate associations between plant roots and microbes as biofilms (Fujishige et al., 2006a). 66.3 WHAT WE KNOW ABOUT BIOFILMS ON ROOTS Bacterial biofilms are most often discussed in the context of chronic mammalian infections as they frequently cause disease by circumventing innate immune responses and antibiotic therapy (Hall-Stoodley et al., Primary root 2004). Infections resulting from biofilm formation along tissues and medical devices, for example, colonization of cystic-fibrosis-affected patient’s lungs or catheters by Pseudomonas aeruginosa, poses serious threats to human health. Interestingly, the same mechanisms that allow bacteria to form biofilms within and on the human host are also at work in the plant’s environment, especially in the rhizosphere. Early biofilm reviews have centered on the structural and developmental dynamics of biofilms and gene expression of the microbial community, primarily focusing on P. aeruginosa as a model (Costerton et al., 1995; O’Toole et al., 2000). The clinical relevance of Gram-positive species biofilms (Staphylococcus epidermidis, Staphylococcus aureus, and various enterococci) as well as multispecies biofilms, especially dental plaque, has been extensively discussed. What are not yet fully understood are the implications for biofilm formation in the prokaryotic life cycle in vivo with respect to biotic and abiotic surfaces and stresses. Most studies on biofilms have been performed in vitro and under artificial conditions. Also unknown are the details of the genes involved in the interaction between different species existing in biofilms and the mechanisms used for cell-to-cell communication. Recently, more specific reviews have targeted the roles of biofilms on plant surfaces and in the rhizosphere (Fujishige et al., 2006a; Danhorn and Fuqua, 2007; Rudrappa et al., 2008a; Rinaudi and Giordano, 2010). In vivo root colonization of field-isolated wheat roots (Watt et al., 2006) and of roots in nonsterile soil (see references in Fujishige et al., 2006a) have been Rhizoplane Rhizosphere (~1–5mm) Epidermis Root Hairs Rhizosheath Figure 66.1 Schematic overview of the Cortex Endodermis and stele Soil particles Microbes Magnification Meristem q.c. Root cap Exudates Mucilage Border cells Mucilage Exudates structure of the rhizosphere/rhizosheath. The surface of primary roots, composed of various specialized and differentiated or undifferentiated cell types, provides the major biotic surface in the rhizosphere. Root hairs protruding from epidermal cells provide additional surface area to which mucilage, soil particles, and microbes adhere. Additionally, microbes attach to soil particles directly and to each other. Both plant and microbial exudates also play a role in chemical signaling, leading to colonization. After diagrams in Foster et al. (1983) and Marschner (1995). 66.3 What we know about Biofilms on Roots analyzed, and biofilm bacteria have been identified using fluorescence in situ hybridization (FISH; see Chapters 39 and 87). A number of articles have now described some of the initial studies carried out to understand the chemical components secreted by roots. One of the most encompassing studies of these early sources is a chapter on the “soil–root interface (rhizosphere) in relation to mineral nutrition” (Marschner, 1995). Also, the complexity of biofilm formation on biotic surfaces has received more attention, especially because a greater interest has been shown in understanding plant root involvement in physiological processes in the soil. Bacterial communication within and between species in addition to the communication between microbes and root systems has also been explored (Rudrappa et al., 2008a; Hirsch and Fujishige, 2012 for reviews). Lastly, root attachment mechanisms via secreted bacterial compounds (Nod factors, polysaccharides, adhesins) have also been comprehensively covered (Hirsch et al., 2009; Downie, 2010). This increase in information regarding microbial soil interactions has been an exciting venture into understanding how we can tie all of this together in the context of biofilm formation in the rhizosphere, and more importantly, what this means for the plant host. 66.3.1 Biofilm structure and phenotypes Biofilm morphology is characterized by the formation of multicellular structures enveloped by exopolysaccharides and attached to a surface through external cellular appendages (e.g., pili, flagella). In rhizobia, mutations in genes for exopolysaccharide synthesis (Fujishige et al., 2006b; Xie et al., 2012) and flagella synthesis (Fujishige et al., 2006b) negatively affect biofilm structure. Individual cells within wild-type biofilms are supplied with mechanisms of nutrient uptake and waste removal through open channels within the biofilm. The final biofilm assemblage is the end result of changes in microbial gene expression, development, and environmental factors. Largely, biofilm cells are in stationary phase (Stoodley et al., 1994). Studies on P. aeruginosa have helped define five stages of biofilm development ranging from ephemeral association, to adhesion, to microcolony development, maturation, and eventual final maturation (Sauer et al., 2002) (Fig. 66.2). Mature biofilms exhibit a variety of structures. The most commonly described are either mushroom shaped, or have some other elaborate three-dimensional shape, or are flat. The variations in morphology depend on a number of factors such as cellular motility, twitching behavior, and nutrient sources (see references in Hirsch et al., 2009). 66.3.2 Significance of biofilms in the rhizosphere We proposed that biofilm formation promotes survival in harsh environments (Fujishige et al., 2006b) to ensure that some level of bacterial carryover takes place until external conditions become favorable again. Considerable support for nondividing, quiescent cells, or persisters in such environments exists (England et al., 1993; Ratcliff and Denison, 2011), but it is not agreed on whether the persisters occur as free livers or as part of a biofilm that might be adherent to either a soil particle or remnants of root tissue. Early work described a number of factors Dispersal Planktonic cells: reversible attachment Irreversible attachment 705 Final maturation Maturation Plant root surface Figure 66.2 Schematic overview of multispecies biofilm formation. Biofilm formation includes key developmental stages for successful maturation on various surfaces. Free-swimming planktonic cells interact with plant surface cells in a transient way (black arrows indicate changes in attachment) before modification in gene expression commits cells to irreversible attachment and aggregation. Clusters, or microcolonies, develop as cells grow and begin to secrete exopolysaccharides, which provide the matrix for the nascent biofilm structure. The biofilm continues to grow and matures into a full-scale community with various members experiencing different nutrient and oxygen levels based on spatial and temporal arrangement. Water channels (blue arrows) depict how nutrients and wastes are exchanged in the mature biofilm. Finally, individuals can leave the biofilm and continue the same cycle once conditions are favorable. Illustration based on biofilm phenotypes and characteristics described by Sauer et al. (2002). 706 Chapter 66 Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications affecting rhizobial inoculant survival (see references in Vriezen et al., 2012). For example, Rhizobium. leguminosarum bv. trifolii has been detected in higher numbers in soils containing fine clay particle versus coarse sand (Marshall and Roberts, 1963; Postma et al., 1990), but it is not known whether the increased numbers are a direct or indirect effect, that is, by the soil retaining more moisture or by influencing other soil microbes that interact positively or negatively with rhizobia. Sinorhizobium meliloti persisters exhibit a polar localization of polyhydroxybutyrate (PHB) granules and are more resistant to ampicillin than low-PHB cells (Ratcliff and Denison, 2011). Interestingly, we found that S. meliloti grown in medium at pH 4.0 established better biofilms than cells grown in pH 7.0 medium, but the pH 4.0-grown bacteria stained red after staining with the Live–Dead stain (Rinaudi et al., 2006). However, it is unlikely that these cells were dead in spite of having leaky membranes, based on the uptake of propidium iodide, because they were seen to swim and twitch within the biofilm, suggesting they were being dispersed to establish a new biofilm (Fig. 66.2). We consider such cells to be persisters. A recent study (Vriezen et al., 2012) has shown that S. meliloti cells subjected to desiccation can enter a viable but nonculturable (VBNC) state. Many of these cells stain red with the Live–Dead stain. In the rhizosphere, environmental extremes are common, and with the density of various microbial species present in the soil (estimated to be up to 109 microbes per gram of soil), it is important to understand how multispecies interactions are involved in plant growth and development through the formation of biofilms. A summary of bacteria known to form biofilms and their respective characteristics is shown in Table 66.1. Of these species, many are involved in plant-growth-promoting activities, biocontrol, and protection against environmental stresses (see Chapters 53, 54). Not surprisingly, several pathogens form biofilms to elicit disease. Our goal in this chapter is to provide insights into what we have learned about mixed-species biofilms in the context of their formation on biotic and abiotic surfaces and of their use in biocontrol. 66.4 MIXED-SPECIES BIOFILMS 66.4.1 Bacterial quorum sensing: inter-/intraspecies communication via diffusible signals Successful colonization, biofilm formation, and sustained symbiotic interactions rely on initial microbial encounters. Numerous studies for a number of species have connected quorum sensing (see Section 9) to biofilm formation. Planktonic cells engage in chemical signaling before forming biofilms to coordinate their efforts. Before “settling down” on a surface, quorum sensing allows bacteria to assess the size and status of their population. The most common diffusible signals used in bacterial communication are N-acyl-homoserine lactones (AHLs), autoinducer-2 (AI-2) and 2-heptyl-3-hydroxy-4-quinoline (PQS). In P. aeruginosa, the lasI gene (upstream of quorum-sensing genes) is involved in the development of biofilms ranging from the wild-type phenotype to flat, undifferentiated mutant phenotypes (Davies et al., 1998). Although, quorum sensing was thought to be a unique species-specific communication initially, it is now known that different species “eavesdrop” on signals from different species (Pierson et al., 1994; Pierson et al., 1998; Wood and Pierson, 1996; Ahmer, 2004). In Bacillus subtilis, biofilm formation is influenced by other species, albeit, more so by members of the same genus (Shank et al., 2011). In another example, all plant-associated Burkholderia species possess a uniqueand highly conserved AHL system, BraI/R (see references in Hirsch and Fujishige, 2012). Both single-species and cross-species signaling via AI-2 has been documented and recently reviewed (Waters and Bassler 2005). 66.4.2 Root colonization and species dominance Remarkably, other species can disrupt intraspecies signaling and, in so doing, interfere with biofilm formation (see Chapter 76). Structurally similar signaling compounds of the red algae Delisea pulchra interrupt signaling and biofilm formation in P. aeruginosa (Hentzer et al., 2002). Similarly, signaling between Stenotrophomonas maltophilia and P. aeruginosa, which often reside together in environments ranging from the human lung to the rhizosphere, affect biofilm architecture and resistance to antibiotics. Ryan et al. (2008) described how a diffusible signaling factor (DSF) produced by S. maltophilia changed P. aeruginosa biofilm development from its normal growth pattern into a more extended, filamentous shape. Moreover, P. aeruginosa became more tolerant to the antimicrobial peptide polymyxin when cocultured or exposed to S. maltophilia DSF (Ryan et al., 2008). In another study, cyanogenic pseudomonads were shown to have multiple effects on the rhizosphere of Arabidopsis thaliana including the prevention of B. subtilis colonization and rhizosphere formation on roots (Rudrappa et al., 2008b). In contrast, mixed cyanobacteria-based biofilms have proven to produce more robust biofilms by serving as a matrix to support interspecies biofilms composed of Azotobacter, Pseudomonas, Serratia, and Mesorhizobium species (Prasanna et al., 2011). Characterization of the resulting biofilms revealed enhanced PGPR activity, increased 707 66.4 Mixed-Species Biofilms Table 66.1 Summary of known biofilm-forming rhizosphere bacteria Organism Phenotype Acinetobacter calcoaceticus P23 Root colonization of duckweed Agrobacterium tumefaciens Disease of pea Azorhizobium brasilense Root colonization of wheat Azorhizobium caulinodans Root colonization of rice Bacillus amyloliquefaciens Root colonization of tomato, S499 maize, and Arabidopsis thaliana Bacillus cereus and Bacillus Root colonization of wild pumilus barley found in the Evolution Canyon, Israel Bacillus polymyxa Root colonization of cucumber Bacillus subtilis Root colonization of Arabidopsis thaliana Burkholderia cepacia and Diseases of onion and wheat Burkholderia cenocepacia Cyanobacteria spp. Enterobacter agglomerans Klebsiella pneumoniae Microsphaeropsis spp. Paenibacillus lentimorbus Paenibacillus polymyxa Pantoea agglomerans Pseudomonas aeruginosa PAO1 Pseudomonas aureofaciens Pseudomonas brassicacearum Pseudomonas chlororaphis Pseudomonas fluorescens Pseudomonas putida Rhizobium alamii Rhizobium leguminosarum bv. viciae 3841 Rhizobium (Sinorhizobium) sp. strain NGR234 Shewanella putrefaciens strain CN-32 Enhanced mixed-species biofilm formation with Rhizobium, Azotobacter and Pseudomonas spp. Root colonization of cotton Root colonization of wheat Root colonization of onion Root colonization of chickpea Root colonization; prevents crown rot disease in peanut Root colonization of chickpea and wheat Disease of poplar tree Relevant Characteristics Reference PGPB, bioremediation Pathogenesis PGPR PGPR PGPB, biocontrol Yamaga et al. (2010) Hawes and Smith (1989) Kim et al. (2005) Van Nieuwenhove et al. (2000) Nihorimbere et al. (2012); Fan et al. (2011) Salt, heat, and desiccation tolerance Timmusk et al. (2011) PGPB Biocontrol Yang et al. (2004) Rudrappa and Bais (2007) Pathogenesis Ellis and Cooper (2010); Jacobs et al. (2008); Balandreau et al. (2001) Prasanna et al. (2011) Various PGPB activities and biocontrol Biocontrol Nitrogen fixation, PGPB Biocontrol Heavy metal tolerance Biocontrol Chernin et al. (1995) Iniguez et al. (2004) Carisse et al. (2001) Khan et al. (2012) Haggag and Timmusk (2008) PGPR and moisture control Pathogenesis Chauhan and Nautiyal (2010); Amellal et al. (1998) Attila et al. (2008) Root colonization of wheat Root colonization of Arabidopsis thaliana Root colonization of wheat General rhizosphere colonization Root colonization of maize and competitive colonization in the rhizosphere of Arabidopsis thaliana Root colonization of Arabidopsis thaliana and rapeseed Root colonization of various legumes Biocontrol Biocontrol Sigler et al. (2001) Lalaouna et al. (2012) Biocontrol Biocontrol Chin et al. (2000) Silby and Levy (2004) Root colonization of various legumes and competitive colonization in the rhizosphere of cowpea Biofilm formation on mineral surfaces Nitrogen fixation, PGPR Bioremediation and desiccation Matilla et al. (2011); tolerance Nilsson et al. (2011) Heavy metal tolerance Schue et al. (2011) Nitrogen fixation and desiccation tolerance, PGPR Vanderlinde et al. (2009), Janczarek and Skorupska (2011), Williams et al. (2008), Fujishige et al. (2006a) Krysciak et al. (2011) Microbial-mediated geochemistry Huang et al. (2011) 708 Chapter 66 Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications population counts, and fungal antagonism. The latter is a particularly exciting finding in the context of sustainable agricultural practices, which enhance the effectiveness of bioinoculants. With respect to the rhizosphere, it could be expected that this sort of microbial conversation may occur quite frequently because surface colonization, the first step of biofilm formation, requires changes in gene expression, which are often regulated by quorum sensing. Because plant root surfaces are prime real estate for soil microbes, efforts to thwart the efficient colonization of competing species could be an effective survival strategy. Whether it is a beneficial strategy for the plant will be discussed later. In the next section, we discuss factors involved in attachment to both biotic and abiotic surfaces by soil bacteria. 66.5 BIOFILM FORMATION ON BIOTIC VERSUS ABIOTIC SURFACES 66.5.1 Attachment via bacterial and surface secretions Because the rhizosphere is made up not only of living material but also inert soil particles, it is valuable to understand how biofilms of various species attach to both types of surfaces (Fig. 66.3). Colonization of a surface may not necessarily reflect a symbiotic interaction, but may instead be a simple means of occupying a space to survive dehydration, antimicrobials, or other environmental stresses. Therefore, one could infer that specific and possibly unique pathways exist for attachment to biotic versus abiotic surfaces. As an example, Nielsen et al. (A) (2011) described a novel exopolysaccharide involved in Pseudomonas putida biofilm-formation processes that vary under different hydration conditions. By mutant analysis, two different gene clusters were found to play distinct roles in root colonization of maize (Nielsen et al., 2011). It is interesting that specific loci producing diverse exopolysaccharides are utilized on the basis of external factors leading to optimal cell–cell and cell–surface interactions. In another case, the rhizobial adhesion protein RapA1 was shown to be critical for attachment to plant roots, but did not enhance nodulation. Results showed that even when RapA1 was overexpressed, attachment to in vitro surfaces did not increase, although when the modified rhizobia were introduced to roots, colonization was enhanced. This finding indicated that the role of this particular adhesion is specific to biotic rather than to inert surfaces and has implications in rhizosphere colonization (Mongiardini et al., 2008). Other studies have also dissected the differences in rhizobial attachment and biofilm formation on different varied surfaces, which describe a variety of phenotypes with respect to surface colonization and implications for competitive fitness in the rhizosphere (Yousef-Coronado et al., 2008; Barahona et al., 2010). Recent evidence also points to factors that influence R. leguminosarum bv. viciae’s ability to attach to nonlegume as well as legume roots. Rhizobia attach polarly to host root epidermal cells, but a recent study shows that R. leguminosarum bv. viciae attach not only to roots of other legumes in addition to its host pea, but also to wheat and Arabidopsis roots (Xie et al., 2012). Root-exudatestimulated attachment, which was dependent not only on (C) (B) Figure 66.3 Different views of biofilms. (a) Inverted microscope view of Sinorhizobium meliloti 1021 microcolonies and the beginning of larger colony formation (arrow) on glass 24 h after growth in filtered RDM medium containing 2% sucrose. Phase contrast optics. A.M. Hirsch, unpublished. Bar, 10 µm. (b) S. meliloti 1021 cells carrying a nodA-gfp transcriptional fusion attached to sand particles, 24 h postinoculation. Bar, 40 µm. Source: From Fujishige et al. (2008). (c) Confocal scanning microscopic views (top and side) of a 72-h-old biofilm of Burkholderia unamae, a nitrogen-fixing betaproteobacterium, expressing GFP. M.R. Lum and A.M. Hirsch, unpublished. Bar, 10 µm. 66.7 Concluding Remarks and Perspectives rhizobial acid exopolysaccharide production but also on an arabinogalactan protein exuded by both legumes and nonlegumes. So what do these data mean in terms of multispecies interactions? It is possible that the ability to form attachments and to colonize various surfaces successfully in the rhizosphere optimizes the encounters that soil bacteria have with each other. If a host plant is absent, the rhizosphere community dramatically changes (see references in Kent and Triplett, 2002). If different species utilize other plants or adhere to abiotic surfaces temporarily, these interactions may have the potential to be sustained in the absence of root surfaces. Ultimately, it is the health and development of the plant that benefits directly and indirectly by microbial attachment to the root. One direct benefit is that of the biocontrol properties of some bacteria, which is exerted following their colonization of plant roots. 66.6 BIOFILMS AS BIOCONTROL MECHANISMS 66.6.1 Competitive colonization Earlier it was implied that competitive colonization in the rhizosphere could be advantageous for plant hosts. By occupying available surface areas vulnerable to pathogenic microbes, symbiotic bacteria serve as biocontrol species by preventing disease initiation (Lugtenberg and Kamilova, 2009; see Chapter 54). Biofilm formation as a means of biocontrol has been well documented for Pseudomonas fluorescens for wheat, Paenibacillus polymyxa for peanut, and B. subtilis for various plants (Wei and Zhang, 2006; Haggag and Timmusk, 2008; Rudrappa and Bais, 2007; Bais et al., 2004). These reports describe how the successful colonization of the rhizosphere by the biocontrol organism defends the host roots from disease by pathogens. Quorum-sensing regulated biofilm formation of P. fluorescens allows the organism to create a niche, into which, it then secretes antifungal compounds, particularly the antibiotic phenazine, in this way protecting the wheat rhizosphere. Studies with P. polymyxa strains revealed that the more efficient biofilm former was also more efficient against fungal pathogens both in vitro and in vivo. Although a direct mechanism was not identified in the latter case, the hypothesis was proposed that competition for colonization sites and nutrients in the rhizosphere occurs, but further investigation is required to understand this system better. B. subtilis, the most widely known biocontrol organism, is known to colonize the roots of a variety of crop plants and secrete antifungal compounds. Rudrappa and Bais (2007) investigated the surface of A. 709 thaliana roots, and showed that the surface chemistry of roots regulates biofilm formation. This suggests that the host may be driving biocontrol by altering the chemical compounds on its root surfaces, which then mediates interactions with symbiotic bacteria. In another study, Bais et al. (2004) demonstrated the biocontrol ability of B. subtilis by showing that surfactin production was not only necessary for biofilm formation but also elicited antimicrobial activity. Taken together, the various biocontrol mechanisms are complex and we are just at the start of understanding the complete story. Interestingly, a recent technique has been introduced to help decipher the specific interactions between mutualists and pathogens in the rhizosphere, termed ecosystem screening (Galiana et al., 2011). This method allows one to assess the microbial community, identify, and characterize isolates that may be potential biocontrol organisms. Such advances should help to achieve efforts to improve alternative measures for pesticide use in agriculture. 66.7 CONCLUDING REMARKS AND PERSPECTIVES 66.7.1 Biofilms and implications in circumventing stressful life in the soil and climate change Survival of important yet environmentally vulnerable bacterial symbionts in the rhizosphere is an important area of research, directly impacting our ability to offer food security to people in a changing world. The physiological stress of life in the soil (with or without a host) may be alleviated through the formation of biofilms, which under ideal conditions permits enhanced symbiosis, but under environmental stress, facilitates species persistence. As an example, LPS mutants of R. leguminosarum that are impaired in biofilm formation also lack desiccation tolerance (Vanderlinde et al., 2009). With regard to mixed-species biofilms, considerable evidence has demonstrated that soil microbes live in consortia interacting with one another and facilitating survival in specific microniches. One advantage of communitybased biofilms is the preservation of the rhizosphere community itself. Whether maintaining specific plant-growthpromoting rhizobia or preventing pathogens from colonizing plant roots, multispecies biofilms provide benefits to the roots of plants that they colonize. How distant bacterial species communicate to initiate and organize biofilms or influence gene expression in other species is only barely understood (see Chapter 74). Moreover, other than the pioneering electron micrographic studies of Foster et al. (1983), the actual structure and arrangement of 710 Chapter 66 Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications components within rhizospheres have not been adequately described. Future molecular plant–microbe interactions should focus on these and related topics because singlespecies interactions do not tell the whole story of what is happening in nature. Not only is this a promising venture into the basic science of host–microbe interactions but it is also an important investment into developing novel ways to preserve one of Earth’s most important, but yet underappreciated resource—the soil beneath our feet. ACKNOWLEDGMENTS Research on plant–microbe interactions in the Hirsch laboratory is supported by the US National Science Foundation, the Sol Leshin Program for Collaboration between the Ben Gurion University of the Negev, Israel, and the University of California at Los Angeles, and the Shanbrom Family Fund. Annette A. Angus was supported by a President’s Postdoctoral Fellowship provided by the University of California Office of the President. We would like to thank Nancy Fujishige and Michelle Lum for images in Fig. 66.3. We also thank Joshua McVeigh-Schultz for thoughtful comments on a draft of the manuscript. REFERENCES Ahmer BM. Cell-to-cell signalling in Escherichia coli and Salmonella enterica. Mol Microbiol 2004;52:933–945. Amellal N, Burtin G, Bartoli F, Heulin T. Colonization of wheat roots by an exopolysaccharide-producing Pantoea agglomerans strain and its effect on rhizosphere soil aggregation. Appl Environ Microbiol 1998;64:3740–3747. Attila C, Ueda A, Cirillo SL, Cirillo JD, Chen W, Wood TK. Pseudomonas aeruginosa PAO1 virulence factors and poplar tree response in the rhizosphere. Microb Biotechnol 2008;1:17–29. Bais HP, Fall R, Vivanco JM. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol 2004;134:307–319. Balandreau J, Viallard V, Cournoyer B, Coenye T, Laevens S, Vandamme P. Burkholderia cepacia genomovar III Is a common plant-associated bacterium. Appl Environ Microbiol 2001;67:982–985. Barahona E, Navazo A, Yousef-Coronado F, Aguirre de Carcer D, Martinez-Granero F, Espinosa-Urgel M, et al. Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces. Environ Microbiol 2010;12:3185–3195. Carisse O, El Bassam S, Benhamou N. Effect of Microsphaeropsis sp. strain P130A on germination and production of sclerotia of Rhizoctonia solani and Interaction between the antagonist and the pathogen. Phytopathology 2001;91:782–791. Chauhan PS, Nautiyal CS. The purB gene controls rhizosphere colonization by Pantoea agglomerans. Lett Appl Microbiol 2010;50:205–210. Chernin L, Ismailov Z, Haran S, Chet I. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl Environ Microbiol 1995;61:1720–1726. Chin AWTF, Bloemberg GV, Mulders IH, Dekkers LC, Lugtenberg BJ. Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol Plant-Microbe Interact 2000;13:1340–1345. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annu Rev Microbiol 1995;49:711–745. Danhorn T, Fuqua C. Biofilm formation by plant-associated bacteria. Annu Rev Microbiol 2007;61:401–422. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 1998;280:295–298. Downie JA. The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev 2010;34:150–170. Ellis CN, Cooper VS. Experimental adaptation of Burkholderia cenocepacia to onion medium reduces host range. Appl Environ Microbiol 2010;76:2387–2396. England LS, Lee H, Trevors JT. Bacterial survival in soil: effect of clays and protozoa. Soil Biol Biochem 1993;25:525–531. Fan B, Chen XH, Budiharjo A, Bleiss W, Vater J, Borriss R. Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein. J Biotechnol 2011;151:303–311. Foster RC, Rovira AD, Cock TW. Ultrastructure of the Root-Soil Interface. St. Paul, MN: The American Phytopathological Society; 1983. p 5–11. Fujishige NA, Kapadia NN, Hirsch AM. A feeling for the microorganism: structure on a small scale. Biofilms on plant roots. Bot J Linn Soc 2006a;150:79–88. Fujishige NA, Kapadia NN, De Hoff PL, Hirsch AM. Investigations of Rhizobium biofilm formation. FEMS Microbiol Ecol 2006b;56:195–206. Fujishige NA, Lum MR, De Hoff PL, Whitelegge JP, Faull KF, Hirsch AM. Rhizobium common nod genes are required for biofilm formation. Mol Microbiol 2008;67:504–515. Galiana E, Marais A, Mura C, Industri B, Arbiol G, Ponchet M. Ecosystem screening approach for pathogen-associated microorganisms affecting host disease. Appl Environ Microbiol 2011;77:6069–6075. Haggag WM, Timmusk S. Colonization of peanut roots by biofilmforming Paenibacillus polymyxa initiates biocontrol against crown rot disease. J Appl Microbiol 2008;104:961–969. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2004;2:95–108. Hawes MC, Smith LY. Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants. J Bacteriol 1989;171:5668–5671. Hentzer M, Riedel K, Rasmussen TB, Heydorn A, Andersen JB, Parsek MR, et al. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 2002;148:87–102. Hirsch AM, Lum MR, Fujishige NA. 2009. Microbial encounters of a symbiotic kind—attaching to and other surfaces. In: Emons, AMC., Ketelaar, T, editors. Root Hairs. Plant Cell Monographs. Volume 12. Springer. Berlin, Heidelberg p 295–314 Hirsch AM. How rhizobia survive in the absence of a legume host, a stressful world indeed. In: Seckbach J, Grube M, editors. Symbiosis and Stress: Joint Ventures in Biology, Cellular Origin, Life in Extreme Habitats and Astrobiology. Volume 17, p 375–391. Springer Science+Business Media B.V; New York 2010. DOI: 10:1007/97890-481-9449-018. References Hirsch AM, Fujishige NA. 2012. Molecular signals and receptors: communication between nitrogen-fixing bacteria and their plant hosts. In: Wizany, G, Baluska, F, editors. Biocommunication in Plants. Springer. Berlin, Heidelberg Volume 14, 255–280, DOI: 10.1007/978-3-642-23524-5_1. Huang JH, Elzinga EJ, Brechbuehl Y, Voegelin A, Kretzschmar R. Impacts of Shewanella putrefaciens strain CN-32 cells and extracellular polymeric substances on the sorption of As(V) and As(III) on Fe(III)-(hydr)oxides. Environ Sci Technol 2011;45:2804–2810. Iniguez AL, Dong Y, Triplett EW. Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342. Mol Plant-Microbe Interact 2004;17:1078–1085. Jacobs JL, Fasi AC, Ramette A, Smith JJ, Hammerschmidt R, Sundin GW. Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil. Appl Environ Microbiol 2008;74:3121–3129. Janczarek M, Skorupska A. Modulation of rosR expression and exopolysaccharide production in Rhizobium leguminosarum bv. trifolii by phosphate and clover root exudates. Int J Mol Sci 2011;12:4132–4155. Kent AD, Triplett EW. Microbial communities and their interactions in soil and rhizosphere ecosystems. Annu Rev Microbiol 2002;56:211–236. Khan N, Mishra A, Chauhan PS, Sharma YK, Nautiyal CS. Paenibacillus lentimorbus enhances growth of chickpea (Cicer arietinum L.) in chromium-amended soil. Antonie Van Leeuwenhoek 2012;101:453–459. Kim C, Kecskes ML, Deaker RJ, Gilchrist K, New PB, Kennedy IR, et al. Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea. Can J Microbiol 2005;51:948–956. Krysciak D, Schmeisser C, Preuss S, Riethausen J, Quitschau M, Grond S, et al. Involvement of multiple loci in quorum quenching of autoinducer I molecules in the nitrogen-fixing symbiont Rhizobium (Sinorhizobium) sp. strain NGR234. Appl Environ Microbiol 2011;77:5089–5099. Lalaouna D, Fochesato S, Sanchez L, Schmitt-Kopplin P, Haas D, Heulin T, et al. Phenotypic switching involves GacS/GacAdependent Rsm small RNAs in Pseudomonas brassicacearum. Appl Environ Microbiol 2012;78:1658–1665. Lugtenberg B, Kamilova F. Plant-growth-promoting rhizobacteria. Annu Rev Microbiol 2009;63:541–556. Marschner H. Mineral Nutrition of Higher Plants. Academic Press; Waltham, Massachusetts 1995. p 537–598. Marshall KC, Roberts FJ. Influence of fine particle materials on survival of Rhizobium trifolii in sandy soil. Nature 1963;195:410–411. Matilla MA et al. Complete genome of the plant growthpromoting rhizobacterium Pseudomonas putida BIRD-1. J Bacteriol 2011;193:1290. Mongiardini EJ, Ausmees N, Perez-Gimenez J, Julia Althabegoiti M, Ignacio Quelas J, Lopez-Garcia SL, et al. The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation. FEMS Microbiol Ecol 2008;65:279–288. Nielsen L, Li X, Halverson LJ. Cell-cell and cell-surface interactions mediated by cellulose and a novel exopolysaccharide contribute to Pseudomonas putida biofilm formation and fitness under waterlimiting conditions. Environ Microbiol 2011;13:1342–1356. Nihorimbere V, Cawoy H, Seyer A, Brunelle A, Thonart P, Ongena M. Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499. FEMS Microbiol Ecol 2012;79:176–191. Nilsson M, Chiang WC, Fazli M, Gjermansen M, Givskov M, Tolker-Nielsen T. Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability. Environ Microbiol 2011;13:1357–1369. 711 O’Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol 2000;54:49–79. Pierson LS 3rd, Keppenne VD, Wood DW. Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30–84 is regulated by PhzR in response to cell density. J Bacteriol 1994;176:3966–3974. Pierson LS 3rd, Wood DW, Pierson EA. Homoserine lactonemediated gene regulation in plant-associated bacteria. Annu Rev Phytopathol 1998;36:207–225. Postma J, Hok-A-Hin CH, Oude Voshaar JH. Influence of the inoculum density on the growth and survival of Rhizobium leguminosarum bv. trifolii introduced into sterile and non-sterile loamy sand and silt loam. FEMS Microbiol Ecol 1990;73:49–58. Prasanna R, Pattnaik S, Sugitha TC, Nain L, Saxena AK. Development of cyanobacterium-based biofilms and their in vitro evaluation for agriculturally useful traits. Folia Microbiol (Praha) 2011;56:49–58. Ratcliff WC, Denison RF. Bacterial persistence and bet hedging in Sinorhizobium meliloti. Commun Integr Biol 2011;20:1740–1744. Rinaudi L, Fujishige NA, Hirsch AM, Banchio E, Zorreguieta A, Giordano W. Effects of nutritional and environmental conditions on Sinorhizobium meliloti biofilm formation. Res Microbiol 2006;157:867–875. Rinaudi LV, Giordano W. An integrated view of biofilm formation in rhizobia. FEMS Microbiol Lett 2010;304:1–11. Rudrappa T, Bais HP. Arabidopsis thaliana root surface chemistry regulates in planta biofilm formation of Bacillus subtilis. Plant Signal Behav 2007;2:349–350. Rudrappa T, Biedrzycki ML, Bais HP. Causes and consequences of plant-associated biofilms. FEMS Microbiol Ecol 2008a;64:153–166. Rudrappa T, Splaine RE, Biedrzycki ML, Bais HP. Cyanogenic pseudomonads influence multitrophic interactions in the rhizosphere. PLoS One 2008b;3:e2073. Ryan RP, Fouhy Y, Garcia BF, Watt SA, Niehaus K, Yang L, et al. Interspecies signalling via the Stenotrophomonas maltophilia diffusible signal factor influences biofilm formation and polymyxin tolerance in Pseudomonas aeruginosa. Mol Microbiol 2008;68:75–86. Sadowsky MJ. Soil stress factors influencing symbiotic nitrogen fixation. In: Werner D, Newton WE, editors. Nitrogen Fixation Research in Agriculture, Forestry, Ecology, and the Environment. Dordrecht, The Netherlands: Springer; 2005. p 89–102. Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 2002;184:1140–1154. Schue M, Fekete A, Ortet P, Brutesco C, Heulin T, SchmittKopplin P, et al. Modulation of metabolism and switching to biofilm prevail over exopolysaccharide production in the response of Rhizobium alamii to cadmium. PLoS One 2011;6:e26771. Shank EA, Klepac-Ceraj V, Collado-Torres L, Powers GE, Losick R, Kolter R. Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. Proc Natl Acad Sci U S A 2011;108:E1236–1243. Sigler WV, Nakatsu CH, Reicher ZJ, Turco RF. Fate of the biological control agent Pseudomonas aureofaciens TX-1 after application to turfgrass. Appl Environ Microbiol 2001;67:3542–3548. Silby MW, Levy SB. Use of in vivo expression technology to identify genes important in growth and survival of Pseudomonas fluorescens Pf0-1 in soil: discovery of expressed sequences with novel genetic organization. J Bacteriol 2004;186:7411–7419. Stoodley P, Debeer D, Lewandowski Z. Liquid flow in biofilm systems. Appl Environ Microbiol 1994;60:2711–2716. Timmusk S, Paalme V, Pavlicek T, Bergquist J, Vangala A, Danilas T, et al. Bacterial distribution in the rhizosphere of wild barley under contrasting microclimates. PLoS One 2011;6:e17968. Torvik V, Øvreås L. Microbial diversity and function in soil: from genes to ecosystems. Curr Opin Microbiol 2002;5:240–245. 712 Chapter 66 Biofilm Formation in the Rhizosphere: Multispecies Interactions and Implications Van Nieuwenhove C, Holm V, Kulasooriya SA, Vlassak K. Establishment of Azorhizobium caulinodans in the rhizosphere of wetland rice (Oryza sativa L.). Biol Fertil Soils 2000;31:143–149. Vanderlinde EM, Muszynski A, Harrison JJ, Koval SF, Foreman DL, Ceri H, et al. Rhizobium leguminosarum biovar viciae 3841, deficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility. Microbiology 2009;155:3055–3069. Vriezen JAC, de Bruijn FJ, Nüsslein K. Responses of rhizobia to desiccation in relation to osmotic stress, oxygen, and temperature. Appl Environ Microbiol 2007;73:3451–3459. Vriezen JAC, de Bruijn FJ, Nüsslein K. Desiccation induces viable but non-culturable cells in Sinorhizobium meliloti 1021. AMB Express 2012; 2:6. doi: 10.1186/2191-0855-2-6. Waters CM, Bassler BL. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 2005;21:319–346. Watt M, Hugenholtz P, White R, Vinall K. Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH). Environ Microbiol 2006;8:871–884. Watt M, McCully ME, Canny MJ. Formation and stabilization of rhizosheaths of Zea mays L. Plant Physiol 1994;106: 179–186. Wei HL, Zhang LQ. Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24. Antonie Van Leeuwenhoek 2006;89:267–280. Williams A, Wilkinson A, Krehenbrink M, Russo DM, Zorreguieta A, Downie JA. Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection. J Bacteriol 2008;190:4706–4715. Wood DW, Pierson LS 3rd. The phzI gene of Pseudomonas aureofaciens 30–84 is responsible for the production of a diffusible signal required for phenazine antibiotic production. Gene 1996;168: 49–53. Xie F, Williams A, Edwards A, Downie JA. A plant arabinogalactanlike glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum. Mol Plant-Microbe Interact 2012;25: 250–258. Yamaga F, Washio K, Morikawa M. Sustainable biodegradation of phenol by Acinetobacter calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna aoukikusa. Environ Sci Technol 2010;44:6470–6474. Yang J, Kharbanda PD, Mirza M. Evaluation of Paenibacillus polymyxa PKB1 for biocontrol of Pythium disease of cucumber in a hydroponic system. Acta Hortic (ISHS) 2004;635:59–6. Yousef-Coronado F, Travieso ML, Espinosa-Urgel M. Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida. FEMS Microbiol Lett 2008;288: 118–124. Zahran HH. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev 1999;63:968–989.