Biofilm Formation on Plant Surfaces by Rhizobacteria: Impact on Plant Growth and Ecological Significance

Abstract

Microorganisms have historically been studied as planktonic or free-swimming cells, but most exist as sessile communities attached to surfaces, in multicellular assemblies known as biofilms. Biofilms on plant surfaces are of great importance to plant health. Plant growth-promoting rhizobacteria (PGPR) not only induce plant growth but also provide protection by the process known as biocontrol, whereas other bacteria in the biofilm mode of growth can create a nuisance for plants. Recent advances show that biofilm formation on plant roots is associated with the biological and pathogenic response, but its regulation by the plant is unknown. In this chapter we describe bacterial biofilm processes, the ecological significance and the microbes that form biofilms on plant roots, and the effect of root exudates on biofilms and plant health.

Full text

© CAB International 2016. The Handbook of Microbial Bioresources (eds V.K. Gupta et al.) 81
Abstract
Microorganisms have historically been studied as planktonic or free-swimming cells, but most exist as sessile
communities attached to surfaces, in multicellular assemblies known as biolms. Biolms on plant surfaces are
of great importance to plant health. Plant growth-promoting rhizobacteria (PGPR) not only induce plant growth
but also provide protection by the process known as biocontrol, whereas other bacteria in the biolm mode of
growth can create a nuisance for plants. Recent advances show that biolm formation on plant roots is associated
with the biological and pathogenic response, but its regulation by the plant is unknown. In this chapter we
describe bacterial biolm processes, the ecological signicance and the microbes that form biolms on plant
roots, and the effect of root exudates on biolms and plant health.
5 Biolm Formation on Plant Surfaces
by Rhizobacteria: Impact on Plant Growth
and Ecological Signicance
Mohammad Musheer Altaf and Iqbal Ahmad*
Department of Agricultural Microbiology, Aligarh Muslim University, India
5.1 Introduction
One major challenge for the 21st century will be
the production of sufcient food to feed the
growing population. The United Nations Popu-
lation Fund (UNFPA) estimates that the global
human population may well reach 10 billion by
2050 (UNFPA 2010). This means increasing
agricultural productivity of food crops, as plants
form the basis of every food chain. However,
agriculture in developed countries already cre-
ates a range of serious environmental problems
through the use of chemicals, salinization and
the depletion of water resources. Furthermore,
agricultural production in developing countries
cannot be increased without further destroying
the forests and turning them into arable land,
thus threatening global biodiversity, which is al-
ready under stress from human action. There is
a serious need to boost global food production in
an environmentally sustainable manner (Angus
and Hirsch, 2013). 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 (Morrissey et al., 2004).
Similarly, progress in plant biotechnology aims
to develop new crop varieties by the introduction
of desirable traits, so that these crops: (i) can
survive better under conditions of drought or
salinity; (ii) have enhanced disease and pest re-
sistance; and (iii) have high nutritional value.
However, efforts to understand the microbiome
of the rhizosphere and its inuence on regulating
plant health is not yet fully explored (Morrissey
et al., 2004; Berendsen et al., 2012).
Root exudates are well known to exert a sig-
nicant impact on rhizospheric microorgan-
isms (Bais et al., 2006). Various aspects of the
*ahmadiqbal8@yahoo.co.in

82 Mohammad Musheer Altaf and Iqbal Ahmad
interactions between plants and microorgan-
isms have been studied (Ryu et al., 2004; Timmusk
et al., 2005; Haggag and Timmusk, 2008). Re-
searchers in the last few decades have estab-
lished that the majority of the microorganisms
often exist in well-organized structures on the
surface of plants in biolms rather than in a
planktonic state (Stanley and Lazazzera, 2005;
Rudrappa et al., 2007; Beauregard et al., 2013)
(Table 5.1). The process of biolm formation on
plant roots involves a complex mechanism which
might be understood at physiological and mo-
lecular levels (Rudrappa et al., 2007; Vlamakis
etal., 2013).
Biolm formation on both biotic and abiotic
surfaces has been the subject of research in the
past (O’Toole and Stewart, 2005). The study of
bacterial biolm formation on plant surfaces, es-
pecially root surfaces, has not yet been explored
fully (Haggag and Timmusk, 2008; Timmusk
et al., 2011). Plant growth-promoting rhizobac-
teria (PGPR) are known to promote the plant
growth and yield through both direct and indirect
mechanisms which includes phosphate solu-
bilization, siderophore production, nitrogen
xation, indole acetic acid synthesis, production
of antibiotic and lytic enzymes, induction of sys-
temic resistance and stress relief (Fujishige et al.,
2006; Trivedi et al., 2011).
Bacteria are unicellular organisms that
manifest a range of collective behaviours lead-
ing to tissue-like functions. Whereas fruiting
bodies and swarming are the most spectacular
of these, cell aggregation, microcolonies and
biolm formation are the most widespread ex-
pression of the collective behaviour of bacteria.
Such behaviour provides adaptive strategies
during severe environmental stress conditions
which can also result in the differentiation of
non-specific cells into specialized lines for
performing different unique functions not ex-
hibited by single cells (Morris and Monier,
2003; Guttenplan and Kearns, 2013). PGPR
are thought to form biolms. Biolms are as-
semblages of cells embedded in a self-produced
matrix composed of extracellular polymeric
substances (EPS), proteins and sometimes DNA
(Beauregard et al., 2013). The formation of mi-
crocolonies and biolms on plant surfaces is as-
sociated with the processes of attachment of
bacterial cells and production of EPS (Vlamakis
et al., 2013).
Bacteria associated with plant surfaces can
be found on seeds, leaves and roots. Biolm for-
mations on abiotic surfaces and bacterial colon-
ization of plant surfaces have some clear similar-
ities, and are controlled by common molecular
determinants. Plant-associated communities
may usually not be as dense and structured as
biolms formed in other environmental condi-
tions. However, they have an added element of
complexity, since the plant surface is not only an
inert support for bacterial growth, but also the
main source of nutrients for the microorgan-
isms, and an active partner in the system. Plant
surface-associated bacteria have a direct role in
plant disease and health (Molina et al., 2003;
Timmusk et al., 2011; Vlamakis et al., 2013).
In this chapter we review recent progress
on plant root-associated biolms, the impact on
the plant–bacteria interaction, the ecological
signicance, and the inuence of root exudates
and their potential use for enhancing plant
growth and protecting plant health.
5.2 Processes in Biolm Formation
Biolm formation is complex, but is generally
recognized as consisting of ve stages as de-
scribed by Stoodley et al. (2002). These stages
are: (i) reversible attachment; (ii) irreversible at-
tachment; (iii) development of microcolonies
leading to biolm establishment; (iv) formation
of a mature biolm with a three-dimensional
structure; and (v) dispersion of the biolm and
release of bacterial cells for initiation of new bio-
lm formation (Fig. 5.1).
5.2.1 Initial attachment
Bacterial cells attach to a surface as a result of a
random process, mediated by Brownian motion
and gravitational forces. Such attachment can
be active or passive (Beloin et al., 2008; Kostakioti
etal., 2013). The adhesion of cells during this
process depends on the bacterial cell surface
characteristics (Kostakioti et al., 2013). The at-
tachment process may be inuenced by various
factors such as availability of nutrients, pH, tem-
perature and ionic strength. After the initial adher-
ence there is production of extracellular adhesive
materials and adhesions.

Biofilm Formation on Plant Surfaces by Rhizobacteria 83
Table 5.1. Biofilm formation by beneficial and pathogenic bacteria associated with plant roots. (Partly
adapted from Rudrappa et al., 2008, and Angus and Hirsh, 2013.)
Bacteria
Nature of
associationaPlant name Plant part References
Acinetobater
calcoacetcius P23
PGPR Duckweed Root Yamaga et al. (2010)
Azospirillum brasilense PGPR Wheat Root Kim et al. (2005),
Shelud’ko et al.
(2010)
Azorhizobium caulinodans PGPR Rice Root Van Nieuwenhove
et al. (2004)
Azotobacter chroococcum PGPR Cotton, wheat Root Kumar et al.(2007)
Bacillus amyloliquefaciens
S499
PGPR,
biocontrol
Arabidopsis
thaliana,
maize, tomato
Root Fan et al. (2011),
Nihorimbere et al.
(2012)
Bacillus cereus Under conditions
of stress by
salt, heat or
desiccation
Wild barley Root Trivedi et al. (2011)
Bacillus pumilis Under conditions
of stress by
salt, heat or
desiccation
Wild barley Root Trivedi et al. (2011)
Bacillus polymyxa PGPR Cucumber Root Yang et al. (2004),
Nihorimbere et al.
(2012)
Bacillus subtilis Biocontrol A. thaliana Root Ellis and Cooper
(2010), Beauregard
et al. (2013)
Bacillus megaterium C4 Nitrogen fixation,
PGPR
Maize, rice Root Liu et al. (2006)
Burkholderia cepacia
strain Lu10-1
Biocontrol Mulberry Root Ji et al. (2010)
Enterobacter
agglomerans
Biocontrol Cotton Root Chernin et al. (1995)
Enterobacter cloacae PGPR Rice Root Shankar et al. (2011)
Klebsiella pneumoniae Beneficial Wheat Root Dong et al. (2004), Liu
etal. (2011)
Microsphaeropsis sp. Biocontrol Onion Root Carisse et al. (2001)
Micrococcus sp. NII-0909 PGPR Cowpea Root Dastager et al. (2010)
Paenibacillus lentimorbus Heavy metal
tolerance
Chickpea Root Khan et al. (2012)
Paenibacillus polymyxa Biocontrol Peanut Root Haggag and Timmusk
(2008)
Pantoea agglomerans PGPR Chickpea, wheat Root Chauhan and Nautiyal
(2010)
Pseudomonas
aureofaciens
Biocontrol Wheat Root Sigler et al. (2001),
Zakharchenko et al.
(2011)
Pseudomonas
brassicacearum
Biocontrol A. thaliana Root Lalaouna et al. (2012)
Pseudomonas
chlororaphis
Biocontrol Wheat Root Chin-A-Woeng et al.
(2000), Shen et al.
(2012)
Continued

84 Mohammad Musheer Altaf and Iqbal Ahmad
At rst, the adherent cells, that give rise to
biolm formation, have only a fraction of EPS
and a few are able to move freely either by twitch-
ing or gliding motility (Guttenplan and Kearns,
2013). The adhesion is reversible at this stage
since the attached microorganisms are not yet
dedicated to the differentiation process under-
going a series of morphological changes which
leads to biolm development (Hall-Stoodley etal.,
2004). The surface properties are also import-
ant in determining bacterial adhesion. Gener-
ally, any surface is susceptible to biolm develop-
ment such as plastic, glass, metal, wood and food
products. Furthermore, the surfaces that are
covered with conditioning lm that contains
macromolecules, such as organic substances,
will enhance the attachment of bacterial cells
(Tang et al., 2009; Vlamakis et al., 2013).
Bacteria
Nature of
associationaPlant name Plant part References
Pseudomonas
fluorescens
Biocontrol Crop plant General
rhizosphere
colonization
Silby and Levy (2004),
Barahona et al.
(2010)
Pseudomonas putida Draught
tolerance,
bioremediation
Maize,
sunflower,
A. thaliana
Root Sandhya et al. (2009),
Matilla et al. (2011),
Jakovleva et al.
(2012)
Pseudomonas aurantiaca
SR1
PGPR Maize, wheat Root Rosas et al. (2009)
Rhizobium alamii Heavy metal
tolerance
A. thaliana,
rapeseed
Root Schue et al. (2011)
Rhizobium
leguminosarum bv.
viciae 3841
Nitrogen fixation,
PGPR,
drought
tolerance
Various
legumes
Root Fujishige et al. (2006),
Williams et al.
(2008), Janczarek
and Skorupska
(2011)
R. leguminosarum Beneficial Rice Root Janczarek (2011)
Rhizobium sp. NGR234 Nitrogen fixation,
PGPR,
Legumes
(cowpea)
Root Krysciak et al. (2011)
Rhizobium Symbiosis Legumes Root Fujishige et al. (2006),
Robledo et al.
(2012)
Sinorhizobium Symbiosis Legumes Root Fujishige et al. (2006),
Khan et al. (2012)
Stenotrophomonas
maltophilia
Biocontrol,
PGPR
Crop plant Root Ryan et al. (2008)
Shewanella putrefaiens
CN-32
Microbial
mediated
geochemistry
Biofilm on mineral surfaces Huang et al. (2011)
Cyanobacteria spp. PGPR,
biocontrol
Enhanced mixed-species biofilm
formation with Rhizobium,
Azotobacter, Pseudomonas
spp.
Prasanna et al. (2011)
Agrobacterium
tumefaciens
Pathogenic Pea Root Abarca-Grau et al.
(2011)
Escherichia coli Pathogenic Leafy
vegetables
Root Delaquis et al. (2007),
Saldaña et al.
(2011)
Enterococcus faecalis Pathogenic A. thaliana Root Jha et al. (2005)
aPGPR, Plant growth-promoting rhizobacteria.
Table 5.1. Continued.

Biofilm Formation on Plant Surfaces by Rhizobacteria 85
12 34
5
Fig. 5.1. The development of a biofilm, depicted as a five-stage process. Stage 1: attachment of cells to
the surface; stage 2: production of extracellular polymeric substances (EPS) matrix; stage 3: develop-
ment of biofilm architecture; stage 4: maturation; stage 5: dispersion of bacterial cells from the biofilm.
(From Lasa, 2006.)
5.2.2 Irreversible attachment
Reversible bacterial cell attachment leads to at-
tachment that is irreversible in nature due to
permanent bonding in the presence of EPS
(Hall-Stoodley et al., 2004).
In this state of bacterial adherence biolm
becomes tolerant to treatment such as shear
force; even chemical treatment with enzymes,
detergent and surfactant could not dislodge
the bacterial cells (Sinde and Carballo, 2000;
Augustin et al., 2004). Vlamakis et al. (2013)
reported that the extracellular matrix of
Bacillus subtilis facilitates cell attachment and
assists in microcolony formation and biolm
maturation.
5.2.3 Microcolony formation
Microcolony formation results from simultan-
eous accumulation and growth of microorgan-
isms and is connected with the production of the
extracellular matrix (Hall-Stoodley and Stood-
ley, 2009; Vlamakis et al., 2013) that nourishes
the bond between the bacteria and the surface
and protects the colony from any environmental
stress (Hall-Stoodley et al., 2004; Vlamakis et al.,
2013). Lopez et al. (2010) found that several
species of bacteria in the ecosystem studied
showed that accumulation can activate the selec-
tion of planktonic cells from the surrounding me-
dium mediated by quorum sensing. Swimming
motility was believed to allow the microorgan-
isms to control repulsive forces at the surface
water interface and enable them to reach the
substratum and form microcolonies (Beauregard
et al., 2013). Microcolonies are thought to be
beneficial as they allow interspecies substrate
exchange and mutual end-product removal
to bacteria (Hall-Stoodley et al., 2004; Madsen
et al., 2012).
5.2.4 Maturation
The biolm maturation step results in the devel-
opment of an organized structure which can be
at or mushroom-shaped depending on the nu-
trient availability (Vlamakis et al., 2013). Its
maturation comprises adhesive processes that
link bacteria together during proliferation and
disruptive processes that form channels in the
biolm structure (Stoodley et al., 2000). During
this phase, surface contact activates responses
that lead to gene expression changes, upregu-
lating factors favouring sessility, such as those
involved in the formation of the extracellular
matrix (Kostakioti et al., 2013). Bacteria grow
under sessile form in heterogeneous complex-
enclosed microcolonies scattered with open
water channels (Stoodley et al., 2000; Vlamakis
et al., 2013). Cell division is uncommon in a ma-
ture biolm, and energy is used to produce EPS,
which the biolm cells can use as nutrients. It is
not possible to identify general molecular pro-
les for a given bacterial species because some

86 Mohammad Musheer Altaf and Iqbal Ahmad
genes are important for biolm formation under
both static and dynamic conditions, whereas
others are important only under dynamic bio-
lm conditions.
5.2.5 Dispersion
Dispersion is the nal step in the biolm formation
cycle, and it allows the cells in biolm mode to
return back into their planktonic state (Kaplan,
2010). When the biolm matures, resource limi-
tation and waste product accumulation activates
the dispersion of the biolm. Hall-Stoodley et al.
(2004) studied the role of external disturbance
such as raised uid shear stress that is involved in
biolm dispersion. Vlamakis et al. (2013) observed
that the cells in a mature biolm of B. subtilis
released a mixture of D-amino acids (D-tyrosine,
D-leucine, D-tryptophan and D-methionine),
which help in dissolution and subsequent inhib-
ition of the biolm. Disseminating bacteria have
the capability to restart the process of biolm for-
mation on availability of a suitable environm ent. In
addition to dissemination of D-amino acids, the
ageing B. subtilis biolms possess the polyamine
norspermidine, which helps in dispersion of bio-
lms. The suppressing activity of norspermidine
is harmonious with that of D-amino acids, pro-
posing that these molecules act by different mech-
anisms. It interacts directly and specically with
the EPS. This interaction causes collapse of the
EPS, a process that has been visualized by micros-
copy, and a change in polymer size, as visualized
using light scattering.
5.3 Ecological Signicance of
Biolm Formation
5.3.1 Defence
A number of benets to bacterial cells are associ-
ated with biolm formation. A microbial cell in a
biolm mode of growth is well protected due to
its EPS. The chemical composition of this self-
produced matrix includes mainly EPS, protein
and nucleic acid and some other substances
(Flemming and Wingender, 2010). EPS provide
protection against both physical (e.g. UV radi-
ation, pH shift, osmotic shock and desiccation),
chemical and biological stress (Annous et al.,
2009). The EPS matrix is also helpful in provid-
ing resistance to antimicrobial agents by control-
ling diffusion of compounds from the surrounding
environment into the biolm (Flemming and
Wingender, 2010; Flemming, 2011). Further,
EPS was found to seize metals, cations and toxins
(Flemming, 2011).
5.3.2 Availability of nutrients
to microbes
Biolm formation can also lead to establishment
of nutrient availability and development of syn-
trophic association between two different bac-
teria. Such associations have been studied in
relation to methanogenic degradation (Schink,
1997). In a study conducted by Yanhong et al.
(2009) such association was demonstrated in
two strains of Pseudomonas putida (PCL1444 and
PCL1445). P. putida PCL1444 effectively utilizes
root exudate, degrades naphthalene around the
root, protects seeds from being killed by naph-
thalene and allows the plant to grow normally.
Mutants unable to degrade naphthalene do not
protect the plant. P. putida (PCL1445) was un-
able to grow on naphthalene in pure culture in
the absence of PCL1444, which indicated that
the naphthalene degradation product produced
by PCL1444 can be utilized by PCL1445 in the
rhizosphere resulting in symbiotic relationship.
5.3.3 Colonization
Biolm formation by such rhizobacteria can
provide a mechanism for their establishment
and maintenance in favourable environments.
Thus, rhizobacteria not only benet from root
exudates but also inuence the plant directly
or indirectly. Pseudomonas is a widely studied
genus and members of the genus are fairly
widely distributed in plants, soil and water and
exhibit different associations in nature (Misas-
Villamil et al., 2013). For example, Pseudomonas
syringae is associated with aerial parts of the
host plant while P. putida and Pseudomonas
uorescens are found in the rhizosphere and
help in plant growth promotion and also pro-
tect plant health by one or other mechanism
(Jakovleva et al., 2012). Similarly P. putida was

Biofilm Formation on Plant Surfaces by Rhizobacteria 87
found to metabolize toxic aromatic compounds
and exhibit rhizosphere colonizing ability and
is therefore helpful in rhizoremediation (Kuiper
et al., 2004).
5.3.4 Acquisition of new genetic traits
Rhizosphere bacterial populations are hotspots
for microbial interaction and biolm formation.
Biolm modes of bacterial growth provide close
proximity for gene transfer through various ex-
change mechanisms (e.g. conjugation, trans-
formation and transduction) (Merkey et al., 2011).
The frequencies of gene transfer are higher in
the biolm mode of growth compared with the
planktonic mode. Bacterial plasmids are known
to be involved in biolm formation and biolms
promote plasmid stability and genetic exchange.
Plasmids and phages are known to induce the
transition to the biolm mode of growth in their
respective hosts through cell–cell interaction
(Madsen et al., 2012).
5.4 Biolms in the Rhizosphere
Bacterial biolm formation has been extensively
studied in the laboratory and in medical systems
(Hall-Stoodley et al., 2004; Hoiby et al., 2010).
Biolm-related infections on medical devices and
human tissues, such as colonization of cardiac
valves and catheters by streptococci, pose serious
threats to human health. Interestingly, the mech-
anisms of biolm formation within and on the
human host are also at work in the plant’s envir-
onment, especially in the rhizosphere (Angus
and Hirsch, 2013). Microscopy-based studies of
bacterial colonization in the rhizosphere indicate
that bacteria generally form microcolonies or ag-
gregates on root surfaces and that these colonies
have a patchy, non-uniform distribution.
Many bacteria are known to form microcol-
onies during root colonization, these include P. uo-
rescens and other closely related uorescent
pseudomonads that have potential as biocontrol
agents (Couillerot et al., 2009), other PGPR such
as B. subtilis (Vlamakis et al., 2013), free-living
nitrogen-xers such as cyanobacteria (Prasanna
et al., 2011) and Azospirillum spp. (Shelud’ko
et al., 2010).
A distinct exopolymeric matrix covering
these microcolonies and aggregates has fre-
quently been encountered (Haggag and Tim-
musk, 2008; Flemming and Wingender, 2010),
particularly when roots were observed with
microscopic techniques other than the scanning
electron microscope. Most studies of coloniza-
tion patterns of roots corroborate the notion
that bacteria on root surfaces are present pri-
marily as microcolonies at sites of root exud-
ation (Bais et al., 2006). Production of EPS or
other exopolymeric material, and consequently
the formation of biolms, may enhance bacter-
ial survival and the potential for colonization
of roots. Mutants of B. subtilis defective in EPS
matrix production showed impaired biolm for-
mation on the roots of Arabidopsis thaliana
(Beauregard et al., 2013).
5.4.1 Biolm formation by PGPR
A number of PGPR form biolms. The best studied
examples of PGPR that form benecial plant–
microbe interactions are of B. subtilis, P. uo-
rescens and Paenibacillus polymyxa (Table 5.1).
Gram-positive microbes, specically Bacillus spp.,
generally used as effective biocontrol agents, are
cosmopolitan and often colonize plants.
Colonization of A. thaliana roots by B. subtilis
requires the production of surfactin. Surfactin, a
lipopeptide antimicrobial, is involved in biolm
formation in vitro. Surfactin and other lipopep-
tides produced by Bacillus spp. are known to in-
duce systematic resistance in plants and inhibit
the growth of phytopathogens. P. syringae plant
metabolites (malic acid) in root exudates were
found to enhance biolm formation on plant
roots by B. subtilis. Root exudates from plants
infected with P. syringae induce matrix gene ex-
pression in B. subtilis. Malic acid found in tomato
root exudates at elevated concentration can in-
uence matrix gene expression and biolm for-
mation in vitro (Vlamakis et al., 2013). Adherent
cells can multiply at the site of colonization to
form multicellular assemblies. Another root-
associated PGPR is Azospirillum brasilense, which
is commonly associated with cereals (Shelud’ko
et al., 2010). Exopolysaccharides, agellar motil-
ity (swimming and swarming) and specic outer
membrane proteins are needed for effective

88 Mohammad Musheer Altaf and Iqbal Ahmad
root colonization, since non-motile and non-
chemotactic mutants are among the most im-
paired in competitive root colonization (Barahona
et al., 2010). Root hairs and the elongation zone
of the root appear to be favoured colonization
sites, and dense biolms may be formed at these
positions (Timmusk et al., 2005). Burdman et al.
(1998) reported Azospirillum inoculation with
nitrogen-xing rhizobia signicantly increased
plant growth, and suggested the role of syner-
gism within mixed communities of these mi-
crobes. Several Pseudomonas spp. and derivatives
are effective PGPR, and some are biocontrol
agents (Couillerot et al., 2009). On wheat roots, a
natural population of pseudomonads makes up a
signicant portion of the microbial community,
residing within aggregates and biolms (Watt
et al., 2006).
5.4.2 Biolm formation by
phytopathogens
The association of pathogens with roots is iden-
tical to that of benecial bacteria. However,
pathogenic pseudomonads have been reported
to form thicker, more conuent biolms on the
root surface compared with the more heteroge-
neous colonization by benecial pseudomonads
(Rudrappa et al., 2008). This difference may re-
ect the interactions that lead to disease, but
may also be the consequence of different inocu-
lation strategies, growth conditions and plant
hosts. More studies are needed to compare
pathogenic and commensal interactions on the
same plant and in mixed populations.
The ubiquitous plant disease called crown
gall is caused by Agrobacterium tumefaciens. Infec-
tion occurs at wound sites along roots, and at the
crown leads to a horizontal genetic transfer from
A. tumefaciens to the plant, directing unrestricted
growth of the tissue (the gall) and production of
nutrients specic for the infecting microbe. The
mechanisms of plant attachment have remained
elusive, although a two-step model mediated
initially by an as-yet-unidentied adhesin and
followed by rm attachment via cellulose bril
production has been widely recognized. Once
attached to root tissues, A. tumefaciens can form
thick, anatomically complicated biolms, abun-
dantly covering the epidermis and root hairs
(Matthysse et al., 2005). Comparable biolms
formed on abiotic surfaces and several mutants
and genetic variants involved in biolm forma-
tion on these surfaces, show similar pheno-
types on root tissues (Danhorn et al., 2004;
Matthysse et al., 2005). The role of biolms
during the disease process remains obscure,
but may involve proximity to the appropriate
infection site, or survival of the basal plant de-
fence response. Oxygen limitation is a common
condition in the rhizosphere and also within
biolms (Okinaka et al., 2002). An A. tumefa-
ciens mutant disrupted for the FNR (fumarate
and nitrate reductase regulatory)-type tran-
scription factor SinR develops sparse, patchy
biolms on plant roots and abiotic surfaces
(Ramey et al., 2004). This regulator is a part of
an A. tumefaciens oxygen-limitation response
pathway, suggesting a link between oxygen
levels and biolm structure. Similarly, limiting
phosphorus is common in the rhizosphere due
to plant sequestration. Phosphorous limitation
enhances biolm formation by A. tumefaciens,
compared with the decreased biolm formation
reported for Pseudomonas aureofaciens (Danhorn
et al., 2004).
5.5 Multi-species Biolms
in the Rhizosphere
There has been a signicant increase in the
knowledge and understanding of microbial bio-
lms in the last few decades. The majority of
studies conducted so far on biolms are based on
monospecies. However, under natural conditions
biolm communities involve different micro-
organisms or mixed biolms (Elias and Banin,
2012). Interspecies interactions involve cell–cell
communication, via quorum sensing, and meta-
bolic cooperation or competition. The inter-
actions in the mixed biolms have important
ecological and environmental implications.
Mixed-species biolms are certainly the domin-
ant form in the rhizosphere. Mixed biolm-based
inoculants are found to form strong biolms
(Prasanna et al., 2011; Seneviratne et al., 2011).
Prasanna et al. (2011) found that cyanobacteria
form more robust biolms when inoculated with
Azotobacter and Pseudomonas.

Biofilm Formation on Plant Surfaces by Rhizobacteria 89
5.5.1 Role of bacterial signals
in biolm formation
Successful colonization and biolm formation de-
pends on initial microbial communication. The
role of quorum sensing in biolm formation is well
known (Angus and Hirsch, 2013) and mediates
both pathogenic and benecial plant–microbe
interactions. Before biolm formation, cells in
the planktonic mode are involved in chemical
signalling. The most prevalent signals involved in
bacterial communication are N-acylhomoserine
lactones (AHL), autoinducer-2 (Al-2) and 2-heptyl-
3hydroxy-4-quinoline (PQS). In Pseudomonas
aeruginosa the lasl gene is engaged in the growth
of biolms. Although, quorum sensing is a
unique species-specic communication. Elasri
etal. (2001) found that the plant-associated bac-
teria and plant-pathogenic bacteria produce
AHL more commonly than soil-borne strains
and proposed that these signals play a signi-
cant role in biolm formation. Biolm forma-
tion by B. subtilis is inuenced by other species
mainly by the members of the same genus (Shank
et al., 2011).
5.6 Role of Plant Root Exudates
on Biolms
Plant roots ceaselessly create and release a var-
iety of compounds into the rhizosphere in the
form of root exudates. The diversity of organic
compounds released by plant roots includes
various sugars, amino acids, organic acids, fatty
acids, sterols, growth factors and vitamins, en-
zymes, avonones and purines/nucleotides and
several other compounds belonging to different
chemical groups (Curl and Truelove, 1986;
Uren, 2001; Dakora and Phillips, 2002). Root
exudation can be broadly divided into two active
processes. The rst, root excretion, involves gra-
dient-dependent output of waste materials with
unknown functions, whereas the second, secre-
tion, involves exudation of compounds with
known functions, such as lubrication and de-
fence (Bais et al., 2006). Roots release com-
pounds via at least two potential mechanisms.
Root exudates are transferred across the cellular
membrane and discharged into the surrounding
rhizosphere. Plant products are also released
from root border cells and root border-like cells,
which separate from roots as they grow (Vicre
et al., 2005). Root exudation clearly represents a
signicant carbon cost to the plant (Bais et al.,
2006), and the magnitude of photosynthates se-
creted as root exudates differ with the type of
soil, age, and physiological state of the plant,
and presence of nutrients (Doornbos et al.,
2012). Although the functions of most root ex-
udates have not been determined, several com-
pounds present in root exudates play important
roles in biological processes (Bais et al., 2006;
Doornbos et al., 2012) and are likely to have an
effect on bacterial biolms.
5.7 Biolms in Relation to Plant
Growth and Health Protection
5.7.1 Role of biolms in biocontrol
of plant diseases
The biocontrol ability of bacterial strains is de-
pendent on efcient colonization on the plant
surface. Colonization of bacteria on the plant sur-
face involves biolm formation under natural
environments. Bacterial biolms on the plant
root can protect the colonization site and act as a
sink for the nutrients, making nutrients in the
root exudates unavailable for plant pathogens
(Haggag and Timmusk 2008). Plant ro ot- a ssoc iate d
benecial rhizobacteria promote plant growth
and yield through improved mineral nutrient
uptake, production of hormone(s) and biocon-
trol activity (Trivedi et al., 2011). For example, B.
subtilis can protect plants against fungal pathogen
attack, and plays a role in the degradation of or-
ganic polymers in the soil (Vlamakis et al., 2013).
Lugtenberg and Kamilova (2009) demonstrated
that B. subtilis can be used as a biocontrol agent.
Beauregard et al. (2013) studied Arabidopsis root
surfaces inoculated with B. subtilis using con-
focal microscopy to disclose a three-dimensional
structure of the B. subtilis biolm. Similarly
plant root-associated pseudomonads such as
P.uorescens can respond rapidly to the presence
of root exudates in soils and at root colonization
sites, which results in establishment of strong bio-
lm networks (Couillerot et al., 2009). Haggag

90 Mohammad Musheer Altaf and Iqbal Ahmad
and Timmusk (2008) and Chen et al. (2012) ex-
plored the role of biolm-forming Paenibacillus
polymyxa and B. subtilis strains in managing As-
pergillus niger and Ralstonia solanacearum, respect-
ively. Therefore to establish effective biocontrol,
successful colonization and biolm formation
with the biocontrol agent should be ensured.
5.7.2 Role of biolms in mitigating
stress in the rhizosphere
Survival of agriculturally important micro-
organisms in the rhizosphere under various
stressful conditions is an interesting area of
research, directly affecting our food security.
PGPR mitigate most effectively the impact of
abiotic stresses (drought, low temperature, sal-
inity, metal toxicity and high temperatures) on
plants through biolm formation, which under
normal conditions enhance plant growth and
under stressfulconditions help in better survival
(Miloševic´ et al., 2012; Bogino et al., 2013). For
example the lipopolysaccharide (LPS) mutant of
Rhizobium leguminosarum that lacks the biolm
formation property is also unable to tolerate
drought conditions (Vanderlinde et al., 2009).
Sandhya et al. (2009) also found that colon-
ization and biolm formation by Pseudomonas
putida strain GAP-P45 will alleviate drought
stress effects.
5.8 Conclusion
Bacteria–plant interactions and their associ-
ations have been the subject of research for a
long time. These interactions may be positive or
negative in terms of plant health. In the last few
decades it has been established that the biolm
mode of bacterial growth in association with the
plant surface provides protection from predation,
improved acquisition of nutrients, gene ex-
change and protection from exposure to toxic
chemicals. Biolms on plant roots also provide a
protected environment both for the pathogen
and the PGPR. PGPR effective in biolm forma-
tion will be protected from stress conditions and
can more effectively help plant growth, and pro-
tect plant health through their enhanced sur-
vival and metabolic activities. Focused research
on the mechanism of biolm formation and their
regulation by plants and the impact of environ-
ments needs to be further explored to understand
complex microbe–plant root interactions.
Acknowledgement
The author M.M. Altaf is extremely grateful to the
University Grants Commission (UGC), New Delhi,
Government of India, for providing nancial sup-
port in the form of a fellowship through Aligarh
Muslim University, Aligarh.
References
Abarca-Grau, A.M., Penyalver, R., López, M.M. and Marco-Noales, E. (2011) Pathogenic and non-pathogenic
Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains form biofilms on abiotic as well as on
root surfaces. Plant Pathology 60, 416–425.
Angus, A.A. and Hirsch, A.M. (2013) Biofilm formation in the rhizosphere: multispecies interactions and
implications for plant growth. In: de Bruijn, F.J. (ed.) Molecular Microbial Ecology of the Rhizosphere.
Wiley, Hoboken, New Jersey, pp. 703–712.
Annous, B.A., Solomon, E.B., Cooke, P.H. and Burke, A. (2009) Biofilm formation by Salmonella spp. on
cantaloupe melons. Journal of Food Safety 25, 276–287.
Augustin, M., Ali-Vehmas, T. and Atroshi, F. (2004) Assessment of enzymatic cleaning agents and dis-
infectants against bacterial biofilms. Journal of Pharmacy and Pharmaceutical Sciences 7(1),
55–64.
Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S. and Vivanco, J.M. (2006) The role of root exudates in
rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology 5 7,
233–266.
Barahona, E., Navazo, A., Yousef-Coronado, F., de Cárcer, D.A. Martínez-Granero, F., Espinosa-Urgel, M.,
Martín, M. and Rivilla, R. (2010) Efficient rhizosphere colonization by Pseudomonas fluorescens f11 3
mutants unable to form biofilms on abiotic surfaces. Environmental Microbiology 12, 3185–3195.

Biofilm Formation on Plant Surfaces by Rhizobacteria 91
Beauregard, P.B., Chai, Y., Vlamakis, H., Losick, R. and Kolter, R. (2013) Bacillus subtilis biofilm induction
by plant polysaccharides. Proceedings of the National Academy of Sciences of the United States of
America 110(17), E1621–E1630.
Beloin, C., Roux, A. and Ghigo, J.M. (2008) Escherichia coli biofilms. Current Topics in Microbiology and
Immunology 322, 249–289.
Berendsen, R., Pieterse, C. and Bakker, P. (2012) The rhizosphere microbiome and plant health. Trends
in Plant Science 17, 478–486.
Bogino, P., Abod, A., Nievas, F. and Giordano, W. (2013) Water-limiting conditions alter the structure and
biofilm-forming ability of bacterial multispecies communities in the alfalfa rhizosphere. PLoS One
8(11), e79614.
Burdman, S., Jurkevitch, E., Schwartsburd, B., Hampel, M. and Okon, Y. (1998) Aggregation in Azospiril-
lum brasilense: effects of chemical and physical factors and involvement of extracellular components.
Microbiology 44, 1989–1999.
Carisse, O., El Bassam, S. and Benhamou, N. (2001) Effect of Microsphaeropsis sp. strain 130A on ger-
mination and production of sclerotia of Rhizoctonia solani and interaction between the antagonist and
the pathogen. Phytopathology 91, 782–791.
Chauhan, P.S. and Nautiyal, C.S. (2010) The purB gene controls rhizosphere colonization by Pantoea
agglomerans. Letters in Applied Microbiology 50, 205–210.
Chen, Y., Cao, S., Chai, Y., Clardy, J., Kolter, R., Guo, J. and Losick, R. (2012) A Bacillus subtilis sensor
kinase involved in triggering biofilm formation on the roots of tomato plants. Molecular Microbiology
85, 418–430.
Chernin, L., Ismailov, Z., Haran, S. and Chet, I. (1995) Chitinolytic Enterobacter agglomerans antagonistic
to fungal plant pathogens. Applied and Environmental Microbiology 61, 1720–1726.
Chin-A-Woeng, T.F.C., Bloemberg, G.V., Mulders, I.H.M., Dekkers, L.C. and Lugtenberg, B.J.J. (2000)
Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis
PCL1391 is essential for biocontrol of tomato foot and root rot. Molecular Plant–Microbe Interactions
13, 1340–1345.
Couillerot, O., Prigent-Combaret, C., Caballero-Mellado, J. and Moenne-Loccoz, Y. (2009) Pseudomonas
fluorescens and closely-related fluorescent pseudomonads as biocontrol gents of soil-borne phyto-
pathogens. Letters in Applied Microbiology 48, 505–512.
Curl, E.A. and Truelove, B. (1986) The Rhizosphere. Advanced Series in Agricultural Sciences, Vol. 15.
Springer, Berlin.
Dakora, F.D. and Phillips, D.A. (2002) Root exudates as mediators of mineral acquisition in low-nutrient
environments. Plant and Soil 245, 35–47.
Danhorn, T., Hentzer, M., Givskov, M., Parsek, M. and Fuqua, C. (2004) Phosphorous limitation enhances
biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regula-
tory system. Journal of Bacteriology 186, 4492–4501.
Dastager, S.G., Deepa, C.K. and Pandey, A. (2010) Isolation and characterization of novel plant growth
promoting Micrococcus sp. NII-0909 and its interaction with cowpea. Plant Physiology and Biochem-
istry 48, 987–992.
Delaquis, P., Bach, S. and Dinu, L.D. (2007) Behavior of Escherichia coli O157:H7 in leafy vegetables.
Journal of Food Protection 70, 1966–1974.
Dong, Y.M., Iniguez, A.L. and Triplett, E.W. (2004) Nitrogen fixation in wheat provided by Klebsiella pneu-
moniae 342. Molecular Plant–Microbe Interactions 17, 1078–1085.
Doornbos, R.F., van Loon, L.C. and Bakker, P.A.H.M. (2012) Impact of root exudates and plant defense
signaling on bacterial communities in the rhizosphere. A review. Agronomy for Sustainable Develop-
ment 32, 227–243.
Elasri, M., Delorme, S., Lemanceau, P., Stewart, G., Laue, B., Glickmann, E., Oger, P.M. and Dessaux, Y.
(2001) Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp.
than among soil-borne Pseudomonas spp. Applied and Environmental Microbiology 67, 1198–1209.
Elias, S. and Banin, E. (2012) Multi-species biofilms: living with friendly neighbors. FEMS Microbiology
Reviews 36, 990–1004.
Ellis, C.N. and Cooper, V.S. (2010) Experimental adaptation of Burkholderia cenocepacia to onion me-
dium reduces host range. Applied and Environmental Microbiology 76, 2387–2396.
Fan, B., Chen, X.H., Budiharjo, A., Bleiss, W., Vater, J. and Borriss, R. (2011) Efficient colonization of plant
roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to
express green fluorescent protein. Journal of Biotechnology 151, 303–311.

92 Mohammad Musheer Altaf and Iqbal Ahmad
Flemming, H.C. (2011) The perfect slime. Colloids and Surfaces B: Biointerfaces 86, 251–259.
Flemming, H.C. and Wingender, J. (2010) The biofilm matrix. Nature Reviews Microbiology 8, 623–633.
Fujishige, N.A., Kapadia, N.N., De Hoff, P.L. and Hirsch, A.M. (2006) Investigations of Rhizobium biofilm
formation. FEMS Microbiology Ecology 56, 195–206.
Guttenplan, S.B. and Kearns, D.B. (2013) Regulation of flagellar motility during biofilm formation. FEMS
Microbiology Reviews 37, 849–871.
Haggag, W.M. and Timmusk, S. (2008) Colonization of peanut roots by biofilm forming Paenibacillus poly-
myxa initiates biocontrol against crown rot disease. Journal of Applied Microbiology 104(4), 961–969.
Hall-Stoodley, L. and Stoodley, P. (2009) Evolving concepts in biofilm infections. Cellular Microbiology 11,
1034–1043.
Hall-Stoodley, L., Costerton, J.W. and Stoodley, P. (2004) Bacterial biofilms: from the natural environment
to infectious diseases. Nature Reviews Microbiology 2, 95–108.
Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S. and Ciofu, O. (2010) Antibiotic resistance of bacterial bio-
films. International Journal of Antimicrobial Agents 35, 322–332.
Huang, J.H., Elzinga, E.J., Brechbuehl, Y., Voegelin, A. and Kretzschmar, R. (2011) Impacts of Shewanel-
la putrefaciens strain CN-32 cells and extracellular polymeric substances on the sorption of As(V) and
As(III) on Fe(III)-(hydr)oxides. Environmental Science and Technology 45, 2804–2810.
Jakovleva, J., Teppo, A., Velts, A., Sauma, S., Moor, H., Kivisar, M. and Teras, R. (2012) Fis regulates the
competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation. Microbiology
158(3), 708–720.
Janczarek, M. and Skorupska, A. (2011) Modulation of rosR expression and exopolysaccharide production
in Rhizobium leguminosarum bv. trifolii by phosphate and clover root exudates. International Journal
of Molecular Sciences 12, 4132–4155.
Jha, A.K., Bais, H.P. and Vivanco, J.M. (2005) Enterococcus faecalis mammalian virulence-related fac-
tors exhibit potent pathogenicity in the Arabidopsis thaliana plant model. Infection and Immunity 73,
464–475.
Ji, X., Lu, G., Gai, Y., Gao, H., Lu, B., Kong, B. and Mu, Z. (2010) Colonization of Morus alba L. by the
plant-growth-promoting and antagonist bacterium Burkholderia cepacia strain Lu10-1. BMC Micro-
biology 10, 243–254.
Kaplan, J.B. (2010) Biofilm dispersal: mechanisms, clinical implications, and potential therapeutic uses.
Journal of Dental Research 89, 205–218.
Khan, N., Mishra, A., Chauhan, P.S., Sharma, Y.K. and Nautiyal, C.S. (2012) Paenibacillus lentimorbus
enhances growth of chickpea (Cicer arietinum L.) in chromium-amended soil. Antonie Van Leeuwen-
hoek 101, 453–459.
Kim, C., Kecskes, M.L., Deaker, R.J., Gilchrist, K., New, P.B., Kennedy, I.R., Kim, S. and Sa, T. (2005)
Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea.
Canadian Journal of Microbiology 51, 948–956.
Kostakioti, M., Hadjifrangiskou, M. and Hultgren, S.J. (2013) Bacterial biofilms: development, dispersal,
and therapeutic strategies in the dawn of the post antibiotic era. Cold Spring Harbor Perspectives in
Medicine 3, a010306.
Krysciak, D., Schmeisser, C., Preusß, S., Riethausen, J., Quitschau, M., Grond, S. and Streit, W.R. (2011)
Involvement of multiple loci in quorum quenching of autoinducer I molecules in the nitrogen-fixing
symbiont Rhizobium (Sinorhizobium) sp. strain NGR234. Applied and Environmental Microbiology
77, 5089–5099.
Kuiper, I., Lagendijk, E.L., Bloemberg, G.V. and Lugtenberg, B.J.J. (2004) Rhizoremediation: a beneficial
plant–microbe interaction. Molecular Plant–Microbe Interactions 17, 6–15.
Kumar, R., Bhatia, R., Kukreja, K., Behl, R.K., Dudeja, S.S. and Narula, N. (2007) Establishment of Az-
otobacter on plant roots: chemotactic response, development and analysis of root exudates of
cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) Journal of Basic Microbiology 47,
436–439.
Lalaouna, D., Fochesato, S., Sanchez, L., Schmitt-Kopplin, P., Haas, D., Heulin, T. and Achouak, W.
(2012) Phenotypic switching involves GacS/GacAdependent Rsm small RNAs in Pseudomonas ras-
sicacearum. Applied and Environmental Microbiology 78, 1658–1665.
Lasa, I. (2006) Towards the identification of the common features of bacterial biofilm development. Inter-
national Microbiology 9, 21–28.
Liu, X., Zhao, H. and Chen, S. (2006) Colonization of maize and rice plants by strain Bacillus megaterium
C4. Current Microbiology 52(3), 186–190.

Biofilm Formation on Plant Surfaces by Rhizobacteria 93
Liu, Y., Wang, H., Sun, X., Yang, H., Wang, Y. and Song, W. (2011) Study on mechanisms of colonization
of nitrogen-fixing PGPB, Klebsiella pneumoniae NG14 on the root surface of rice and the formation
of biofilm. Current Microbiology 62(4), 1113–1122.
Lopez, D., Vlamakis, H. and Kolter, R. (2010) Biofilms. Cold Spring Harbor Perspectives in Biology
2, a000398.
Lugtenberg, B. and Kamilova, F. (2009) Plant-growth-promoting rhizobacteria. Annual Review of Micro-
biology 63, 541–556.
Madsen, J.S., Burmolle, M., Hansen, H.L. and Sorensen, S.J. (2012) The interconnection between biofilm
formation and horizontal gene transfer. FEMS Immunology & Medical Microbiology 65, 183–195.
Matilla, M.A., Pizarro-Tobias, P., Roca, A., Fernández, M., Duque, E., Molina, L., Wu, X., Lelie, D.V.D.,
Gómez, M.J., Segura, A. and Ramos, J.L. (2011) Complete genome of the plant growth promoting
rhizobacterium Pseudomonas putida BIRD-1. Journal of Bacteriology 193, 1290.
Matthysse, A.G., Marry, M., Krall, L., Kaye, M., Ramey, B.E., Fuqua, C. and White, A.R. (2005) The effect
of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.
Molecular Plant–Microbe Interaction 18, 1002–1010.
Merkey, B.V., Lardon, L.A., Sepane, J.M., Kreft, J.U. and Smets, B.F. (2011) Growth dependence of con-
jugation explains limited plasmid invasion in biofilms: an individual based modeling study. Environ-
mental Microbiology 13, 2435–2452.
Miloševic´, N.A., Marinkovic´, J.B. and Tintor, B.B. (2012) Mitigating abiotic stress in crop plants by micro-
organisms. Proceedings for Natural Sciences, Matica Srpska Novi Sad 123, 17–26.
Misas-Villamil, J.C., Kolodziejek, I., Crabill, E., Kaschani, F., Niessen, S., Shindo, T., Kaiser, M., Alfano, J.R.
and Hoorn, R.A.L.V.D. (2013) Pseudomonas syringae pv. syringae uses proteasome inhibitor syringo-
lin A to colonize from wound infection sites. PLoS Pathogens 9(3), e1003281.
Molina, M.A., Ramos, J.L. and Espinosa-Urgel, M. (2003) Plant-associated biofilms. Reviews in Environ-
mental Science and Biotechnology 2, 99–108.
Morris, C.E. and Monier, J.M. (2003) The ecological significance of biofilm formation by plant-associated
bacteria. Annual Reviews in Phytopathology 41, 429–453.
Morrissey, J.P., Dow, J.M., Mark, G.L. and O’Gara, F. (2004) Are microbes at the root of a solution to world
food production? EMBO Reports 10, 922–926.
Nihorimbere, V., Cawoy, H., Seyer, A., Brunelle, A., Thonart, P. and Ongena, M. (2012) Impact of rhizo-
sphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefa-
ciens S499. FEMS Microbiology Ecology 79, 176–191.
Okinaka, Y., Yan, C.-H., Perna, N.T. and Keen, N.T. (2002) Microarray profiling of Erwinia chrysanthemi 3937
genes that are regulated during plant infection. Molecular Plant–Microbe Interactions 15, 619–629.
O’Toole, G.A. and Stewart, P.S. (2005) Biofilms strike back. Nature Biotechnology 23, 1378–1379.
Prasanna, R., Pattnaik, S., Sugitha, T.C., Nain, L. and Saxena, A.K. (2011) Development of cyanobacter-
ium based biofilms and their in vitro evaluation for agriculturally useful traits. Folia Microbiologica 56,
49–58.
Ramey, B.E., Matthysse, A.G. and Fuqua, C. (2004) The FNR-type transcriptional regulator SinR controls
maturation of Agrobacterium tumefaciens biofilms. Molecular Microbiology 52, 1495–1511.
Robledo, M., Rivera, L., Jiménez-Zurdo, J.I., Rivas, R., Dazzo, F., Velázquez, E., Molina, M., Hirsch, A.M.
and Mateos, P.F. (2012) Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and bio-
film formation on plant roots and abiotic surfaces. Microbial Cell Factories 11, 125.
Rosas, S.B., Avanzini, G., Carlier, E., Pasluosta, C., Pastor, N. and Rovera, M. (2009) Root colonization
and growth promotion of wheat and maize by Pseudomonas aurantiaca SR1. Soil Biology and Bio-
chemistry 4, 1802–1806.
Rudrappa, T., Quinn, W.J., Stanley-Wall, N.R. and Bais, H.P. (2007) A degradation product of the salicylic
acid pathway triggers oxidative stress resulting in down-regulation of Bacillus subtilis biofilm forma-
tion on Arabidopsis thaliana roots. Planta 226, 283–297.
Rudrappa, T., Biedrzycki, M.L. and Bais, H.P. (2008) Causes and consequences of plant-associated bio-
films. FEMS Microbiology Ecology 64, 153–166.
Ryan, R.P., Fouhy, Y., Garcia, B.F., Watt, S.A., Niehaus, K., Yang, L., Tolker-Nielsen, T. and Dow, J.M.
(2008) Interspecies signalling via the Stenotrophomonas maltophilia diffusible signal factor influences
biofilm formation and polymyxin tolerance in Pseudomonas aeruginosa. Molecular Microbiology 68,
75–86.
Ryu, C.M., Farag, M.A., Hu, C.H., Reddy, M.S., Kloepper, J.W. and Paré, P.W. (2004) Bacterial volatiles
induce systemic resistance in Arabidopsis. Plant Physiology 134, 1017–1026.

94 Mohammad Musheer Altaf and Iqbal Ahmad
Saldaña, Z., Sánchez, E., Xicohtencatl-Cortes, J., Puente, J.L. and Girón, J.A. (2011) Surface structures
involved in plant stomata and leaf colonization by shiga-toxigenic Escherichia coli O157:H7. Frontiers
in Microbiology 27(2), 119.
Sandhya, V., Ali, S.K.Z., Grover, M., Reddy, G. and Venkateswarlu, B. (2009) Alleviation of drought stress
effects in sunflower seedlings by the exopolysaccharides producing Pseudomonas putida strain
GAP-P45. Biology Fertility Soils 46, 17–26.
Schink, B. (1997) Energetics of syntrophic cooperations in methanogenic degradation. Microbiology and
Molecular Biology Reviews 61, 262–280.
Schue, M., Fekete, A., Ortet, P., Brutesco, C., Heulin, T., Schmitt-Kopplin, P., Achouak, W. and Santaella, C.
(2011) Modulation of metabolism and switching to biofilm prevail over exopolysaccharide production in
the response of Rhizobium alamii to cadmium. PLoS One 6 11, e26771.
Seneviratne, G., Weerasekara, M.L.M.A.W., Seneviratne, K.A.C.N., Zavahir, J.S., Kecskés, M.L. and
Kennedy, I.R. (2011) Importance of biofilm formation in plant growth promoting rhizobacterial action.
In: Maheshwari, D.K. (ed.) Plant Growth and Health Promoting Bacteria. Microbiology Monographs,
18. Springer, Berlin, pp. 81–95.
Shank, E.A., Klepac-Ceraj, V., Collado-Torres, L., Powers, G.E., Losick, R. and Kolter, R. (2011) Interspecies
interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own
genus. Proceedings of the National Academy of the United States of America 108, E1236–E1243.
Shankar, M., Ponraj, P., Ilakkiam, D. and Gunasekaran, P. (2011) Root colonization of a rice growth pro-
moting strain of Enterobacter cloacae. Journal of Basic Microbiology 51(5), 523–530.
Shelud’ko, A.V., Shirokov, A.A., Sokolova, M.K., Sokolov, O.I., Petrova, L.P., Matora, L.Y. and Katsy, E.I.
(2010) Wheat root colonization by Azospirillum brasilense strains with different motility. Microbiology
9(5), 688–695.
Shen, X., Chen, M., Hu, H., Wang, W., Peng, H., Xu, P. and Zhang, X. (2012) Genome sequence of
Pseudomonas chlororaphis GP72, a root-colonizing biocontrol strain. Journal of Bacteriology 194(5),
1269–1270.
Sigler, W.V., Nakatsu, C.H., Reicher, Z.J. and Turco, R.F. (2001) Fate of the biological control agent
Pseudomonas aureofaciens TX-1 after application to turfgrass. Applied Environmental Microbiology
67, 3542–3548.
Silby, M.W. and Levy, S.B. (2004) 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. Journal of Bacteriology 186, 7411–7419.
Sinde, E. and Carballo, J. (2000) Attachment of Salmonella spp. and Listeria monocytogenes to stainless
steel, rubber and polytetrafluor-ethylene: the influence of free energy and the effect of commercial
sanitizers. Food Microbiology 17, 439–447.
Stanley, N.R. and Lazazzera, B.A. (2005) Defining the genetic differences between wild and domestic
strains of Bacillus subtilis that affect poly-g-DL-glutamic acid production and biofilm formation.
Molecular Microbiology 57, 1143–1158.
Stoodley, P., Hall-Stoodley, L., Boyle, J.D., Jørgensen, F. and Lappin-Scott, H.M. (2000) Environmental
and genetic factors influencing biofilm structure. In: Allison, D.G., Gilbert, P., Lappin-Scott, H.M. and
Wilson, M. (eds) SGM Symposium 59: Community Structure and Cooperation in Biofilms. Cambridge
University Press, Cambridge, pp. 53–64.
Stoodley, P., Cargo, R., Rupp, C.J., Wilson, S. and Klapper, I. (2002) Biofilm material properties as related
to shear-induced deformation and detachment phenomena. Journal of Industrial Microbiology and
Biotechnology 29(6), 361–367.
Tang, X., Flint, S.H., Bennett, R.J., Brooks, J.D. and Morton, R.H. (2009) Biofilm growth of individual and
dual strains of Klebsiella oxytoca from the dairy industry on ultra filtration membranes. Journal of
Industrial Microbiology and Biotechnology 36(12), 1491–1497.
Timmusk, S., Grantcharova, N. and Wagner, E.G.H. (2005) Paenibacillus polymyxa invades plant roots and
forms biofilms. Applied Environmental Microbiology 71, 7292–7300.
Timmusk, S., Paalme, V., Pavlicek, T., Bergquist, J., Vangala, A., Danilas, T. and Nevo, E. (2011) Bacterial
distribution in the rhizosphere of wild barley under contrasting microclimates. PLoS One 6(3), e17968.
Trivedi, P., Spann, T.M. and Wang, N. (2011) Isolation and characterization of beneficial bacteria associated
with citrus roots in Florida. Microbial Ecology 62(2), 324–336.
United Nations Population Fund (UNFPA) (2010) United Nations Population Division, World Population
Prospects. Available at www.unfpa.org or http://esa.un.org/unpd/wpp/index.htm (accessed 18 March
2014).

Biofilm Formation on Plant Surfaces by Rhizobacteria 95
Uren, N.C. (2001) Types, amounts, and possible functions of compounds released into the rhizosphere
by soil-grown plants. In: Pinton, Z.V.R. and Nannipieri, P. (eds) The Rhizosphere: Biochemistry and
Organic Substances at the Soil–Plant Interface. Marcel Dekker, New York, pp. 19–40.
Vanderlinde, E.M., Muszynski, A., Harrison, J.J., Koval, S.F., Foreman, D.L., Ceri, H., Kannenberg, E.L.,
Russell W., Carlson, R.W. and Yost, C.K. (2009) Rhizobium leguminosarum biovar viciae 3841, de-
ficient in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance,
biofilm formation and motility. Microbiology 155, 3055–3069.
Van Nieuwenhove, C., Van Holm, L., Kulasooriya, S.A., Vlassak, K. and Van Holm, L. (2004) Establish-
ment of Azorhizobium caulinodans in the rhizosphere of wetland rice (Oryza sativa L.). Biology Fer-
tility Soils 31, 143–149.
Vicre, M., Santaella, C., Blanchet, S., Gateau, A. and Driouich, A. (2005) Root border-like cells of Arabi-
dopsis. Microscopical characterization and role in the interaction with rhizobacteria. Plant Physiology
138, 998–1008.
Vlamakis, H., Chai, Y., Beauregard, P., Losick, R. and Kolter, R. (2013) Sticking together: building a biofilm
the Bacillus subtilis way. Nature Reviews Microbiology 3, 157–168.
Watt, M., Hugenholtz, P., White, R. and Vinall, K. (2006) Numbers and locations of native bacteria on field-
grown wheat roots quantified by fluorescence in situ hybridization (FISH). Environmental Microbiol-
ogy 8, 871–884.
Williams, A., Wilkinson, A., Krehenbrink, M., Russo, D.M., Zorreguieta, A. and Downie, J.A. (2008) Gluco-
mannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competi-
tive nodule infection. Journal of Bacteriology 190, 4706–4715.
Yamaga, F., Washio, K. and Morikawa, M. (2010) Sustainable biodegradation of phenol by Acinetobacter
calcoaceticus P23 isolated from the rhizosphere of duckweed Lemna aoukikusa. Environmental Sci-
ence & Technology 44, 6470–6474.
Yang, J., Kharbanda, P.D. and Mirza, M. (2004) Evaluation of Paenibacillus polymyxa pkb1 for biocontrol
of Pythium disease of cucumber in a hydroponic system. Acta Horticulturae 635, 59–66.
Yanhong, Z.H.U., Shuzhen, Z., Honglin, H. and Bei, W. (2009) Effects of maize root exudates and organic
acids on the desorption of phenanthrene from soils. Journal of Environmental Science 21(7), 920–926.
Zakharchenko, N.S., Kochetkov, V.V., Buryanov, Y.I. and Boronin, A.M. (2011) Effect of rhizosphere bac-
teria Pseudomonas aureofaciens on the resistance of micropropagated plants to phytopathogens.
Applied Biochemistry and Microbiology 47(7), 661–666.