Download Molecular Microbial Ecology of the Rhizosphere

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
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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).
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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.
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